Pesticidal toxins and nucleotide sequences which encode these toxins

BACKGROUND OF THE INVENTION

The soil microbe

Bacillus thuringiensis

(B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988]

TIBTECH

6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of

B. thuringiensis

subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example,

B. thuringiensis

var. kurstaki HD-1 produces a crystalline δ-endotoxin which is toxic to the larvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely

israelensis

and

morrisoni

(a.k.a.

tenebrionis,

a.k.a. B.t. M-7, a.k.a. B.t.

san diego

), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in

Controlled Delivery of Crop Protection Agents,

R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) “Mosquito Pathogenicity of

Bacillus thuringiensis

var. israelensis,”

Developments in Industrial Microbiology

22:61-76; and Beegle, C. C. (1978) “Use of Entomogenous Bacteria in Agroecosystems,”

Developments in Industrial Microbiology

20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983)

Z. ang. Ent.

96:500-508 describe

Bacillus thuringiensis

var.

tenebrionis,

which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle,

Leptinotarsa decemlineata,

and

Agelastica alni.

More recently, new subspecies of B.t. have been identified, and genes responsible for active δ-endotoxin proteins have been isolated (Höfte, H., H. R. Whiteley [1989]

Microbiological Reviews

52(2):242-255). Höfte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were Cryl (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992]

Bio/Technology

10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996]

Appl. Environ. Microbiol

62(1):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991]

JGM

138:55-62), Shevelev et al. ([1993]

FEBS Lett.

336:79-82; and Smulevitch et al. ([1991]

FEBS Lett.

293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.

The cloning and expression of a B.t. crystal protein gene in

Escherichia coli

has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981]

Proc. Natl. Acad. Sci. USA

78:2893-2897.). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclose the expression of B.t. crystal protein in

E. coli.

U.S. Pat. Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans. PCT application WO96/05314 discloses PS86W1, PS86V1, and other B.t. isolates active against lepidopteran pests. The PCT patent applications published as WO94/24264 and WO94/05771 describe B.t. isolates and toxins active against lepidopteran pests. B.t. proteins with activity against members of the family Noctuidae are described by Lambert et al, supra. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose

B. thuringiensis

strain

tenebrionis

which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include U.S. Pat. Nos. 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; 5,439,881, 5,667,993; and 5,670,365. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.

Isolating responsible toxin genes has been a slow empirical process. Carozzi et al. (Carozzi, N. B., V. C. Kramer, G. W. Warren, S. Evola, G. Koziel (1991)

Appl. Env. Microbiol.

57(11):3057-3061) describe methods for identifying toxin genes. U.S. Pat. No. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes. That patent, however, does not describe the probes and primers of the subject invention.

WO 94/21795, WO 96/10083, and Estruch, J. J. et al. (1996)

PNAS

93:5389-5394 describe toxins obtained from Bacillus microbes. These toxins are reported to be produced during vegetative cell growth and were thus termed vegetative insecticidal proteins (VIP). These toxins were reported to be distinct from crystal-forming δ-endotoxins. Activity of these toxins against lepidopteran and coleopteran pests was reported. These applications make specific reference to toxins designated Vip1A(a), Vip1A(b), Vip2A(a), Vip2A(b), Vip3A(a), and Vip3A(b). The toxins and genes of the current invention are distinct from those disclosed in the '795 and '083 applications and the Estruch article.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods useful in the control of non-mammalian pests and, particularly, plant pests. In one embodiment, the subject invention provides novel B.t. isolates having advantageous activity against non-mammalian pests. In a further embodiment, the subject invention provides new toxins useful for the control of non-mammalian pests. In a preferred embodiment, these pests are lepidopterans and/or coleopterans. The toxins of the subject invention include δ-endotoxins as well as soluble toxins which can be obtained from the supernatant of Bacillus cultures.

The subject invention further provides nucleotide sequences which encode the toxins of the subject invention. The subject invention further provides nucleotide sequences and methods useful in the identification and characterization of genes which encode pesticidal toxins.

In one embodiment, the subject invention concerns unique nucleotide sequences which are useful as hybridization probes and/or primers in PCR techniques. The primers produce characteristic gene fragments which can be used in the identification, characterization, and/or isolation of specific toxin genes. The nucleotide sequences of the subject invention encode toxins which are distinct from previously-described toxins.

In a specific embodiment, the subject invention provides new classes of toxins having advantageous pesticidal activities. These classes of toxins can be encoded by polynucleotide sequences which are characterized by their ability to hybridize with certain exemplified sequences and/or by their ability to be amplified by PCR using certain exemplified primers.

One aspect of the subject invention pertains to the identification and characterization of entirely new families of

Bacillus thuringiensis

toxins having advantageous pesticidal properties. Specific new toxin families of the subject invention include MIS-1, MIS-2, MIS-3, MIS-4, MIS-5, MIS-6, MIS-7, MIS-8, WAR-1, and SUP-1. These families of toxins, and the genes which encode them, can be characterized in terms of, for example, the size of the toxin or gene, the DNA or amino acid sequence, pesticidal activity, and/or antibody reactivity. With regard to the genes encoding the novel toxin families of the subject invention, the current disclosure provides unique hybridization probes and PCR primers which can be used to identify and characterize DNA within each of the exemplified families.

In one embodiment of the subject invention, Bacillus isolates can be cultivated under conditions resulting in high multiplication of the microbe. After treating the microbe to provide single-stranded genomic nucleic acid, the DNA can be contacted with the primers of the invention and subjected to PCR amplification. Characteristic fragments of toxin-encoding genes will be amplified by the procedure, thus identifying the presence of the toxin-encoding gene(s).

A further aspect of the subject invention is the use of the disclosed nucleotide sequences as probes to detect genes encoding Bacillus toxins which are active against pests.

Further aspects of the subject invention include the genes and isolates identified using the methods and nucleotide sequences disclosed herein. The genes thus identified encode toxins active against pests. Similarly, the isolates will have activity against these pests. In a preferred embodiment, these pests are lepidopteran or coleopteran pests.

In a preferred embodiment, the subject invention concerns plants cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by target pests. As described herein, the toxins useful according to the subject invention may be chimeric toxins produced by combining portions of multiple toxins. In addition, mixtures and/or combinations of toxins can be used according to the subject invention.

Transformation of plants with the genetic constructs disclosed herein can be accomplished using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize expression of the toxin in plants.

Alternatively, the Bacillus isolates of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. In this regard, the invention includes the treatment of substantially intact Bacillus cells, and/or recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a forward primer, designated “the 339 forward primer,” used according to the subject invention.

SEQ ID NO. 2 is a reverse primer, designated “the 339 reverse primer,” used according to the subject invention.

SEQ ID NO. 3 is a nucleotide sequence encoding a toxin from B.t. strain PS36A.

SEQ ID NO. 4 is an amino acid sequence for the 36A toxin.

SEQ ID NO. 5 is a nucleotide sequence encoding a toxin from B.t. strain PS81F.

SEQ ID NO. 6 is an amino acid sequence for the 81F toxin.

SEQ ID NO. 7 is a nucleotide sequence encoding a toxin from B.t. strain Javelin 1990.

SEQ ID NO. 8 is an amino acid sequence for the Javelin 1990 toxin.

SEQ ID NO. 9 is a forward primer, designated “158C2 PRIMER A,” used according to the subject invention.

SEQ ID NO. 10 is a nucleotide sequence encoding a portion of a soluble toxin from B.t. PS158C2.

SEQ ID NO. 11 is a forward primer, designated “49C PRIMER A,” used according to the subject invention.

SEQ ID NO. 12 is a nucleotide sequence of a portion of a toxin gene from B.t. strain PS49C.

SEQ ID NO. 13 is a forward primer, designated “49C PRIMER B,” used according to the subject invention.

SEQ ID NO. 14 is a reverse primer, designated “49C PRIMER C,” used according to the subject invention.

SEQ ID NO. 15 is an additional nucleotide sequence of a portion of a toxin gene from PS49C.

SEQ ID NO. 16 is a forward primer used according to the subject invention.

SEQ ID NO. 17 is a reverse primer used according to the subject invention.

SEQ ID NO. 18 is a nucleotide sequence of a toxin gene from B.t. strain PS10E1.

SEQ ID NO. 19 is an amino acid sequence from the 10E1 toxin.

SEQ ID NO. 20 is a nucleotide sequence of a toxin gene from B.t. strain PS31J2.

SEQ ID NO. 21 is an amino acid sequence from the 31J2 toxin.

SEQ ID NO. 22 is a nucleotide sequence of a toxin gene from B.t. strain PS33D2.

SEQ ID NO. 23 is an amino acid sequence from the 33D2 toxin.

SEQ ID NO. 24 is a nucleotide sequence of a toxin gene from B.t. strain PS66D3.

SEQ ID NO. 25 is an amino acid sequence from the 66D3 toxin.

SEQ ID NO. 26 is a nucleotide sequence of a toxin gene from B.t. strain PS68F.

SEQ ID NO. 27 is an amino acid sequence from the 68F toxin.

SEQ ID NO. 28 is a nucleotide sequence of a toxin gene from B.t. strain PS69AA2.

SEQ ID NO. 29 is an amino acid sequence from the 69AA2 toxin.

SEQ ID NO. 30 is a nucleotide sequence of a toxin gene from B.t. strain PS168G1.

SEQ ID NO. 31 is a nucleotide sequence of a MIS toxin gene from B.t. strain PS177C8.

SEQ ID NO. 32 is an amino acid sequence from the 177C8-MIS toxin.

SEQ ID NO. 33 is a nucleotide sequence of a toxin gene from B.t. strain PS177I8.

SEQ ID NO. 34 is an amino acid sequence from the 177I8 toxin.

SEQ ID NO. 35 is a nucleotide sequence of a toxin gene from B.t. strain PS185AA2.

SEQ ID NO. 36 is an amino acid sequence from the 185AA2 toxin.

SEQ ID NO. 37 is a nucleotide sequence of a toxin gene from B.t. strain PS196F3.

SEQ ID NO. 38 is an amino acid sequence from the 196F3 toxin.

SEQ ID NO. 39 is a nucleotide sequence of a toxin gene from B.t. strain PS196J4.

SEQ ID NO. 40 is an amino acid sequence from the 196J4 toxin.

SEQ ID NO. 41 is a nucleotide sequence of a toxin gene from B.t. strain PS197T1.

SEQ ID NO. 42 is an amino acid sequence from the 197T1 toxin.

SEQ ID NO. 43 is a nucleotide sequence of a toxin gene from B.t. strain PS197U2.

SEQ ID NO. 44 is an amino acid sequence from the 197U2 toxin.

SEQ ID NO. 45 is a nucleotide sequence of a toxin gene from B.t. strain PS202E1.

SEQ ID NO. 46 is an amino acid sequence from the 202E1 toxin.

SEQ ID NO. 47 is a nucleotide sequence of a toxin gene from B.t. strain KB33.

SEQ ID NO. 48 is a nucleotide sequence of a toxin gene from B.t. strain KB38.

SEQ ID NO. 49 is a forward primer, designated “ICON-forward,” used according to the subject invention.

SEQ ID NO. 50 is a reverse primer, designated “ICON-reverse,” used according to the subject invention.

SEQ ID NO. 51 is a nucleotide sequence encoding a 177C8-WAR toxin gene from B.t. strain PS177C8.

SEQ ID NO. 52 is an amino acid sequence of a 177C8-WAR toxin from B.t. strain PS177C8.

SEQ ID NO.53 is a forward primer, designated “SUP-1A,” used according to the subject invention.

SEQ ID NO.54 is a reverse primer, designated “SUP-1B,” used according to the subject invention.

SEQ ID NOS. 55-110 are primers used according to the subject invention.

SEQ ID NO. 111 is the reverse complement of the primer of SEQ ID NO. 58.

SEQ ID NO. 112 is the reverse complement of the primer of SEQ ID NO. 60.

SEQ ID NO. 113 is the reverse complement of the primer of SEQ ID NO. 64.

SEQ ID NO. 114 is the reverse complement of the primer of SEQ ID NO. 66.

SEQ ID NO. 115 is the reverse complement of the primer of SEQ ID NO. 68.

SEQ ID NO. 116 is the reverse complement of the primer of SEQ ID NO. 70.

SEQ ID NO. 117 is the reverse complement of the primer of SEQ ID NO. 72.

SEQ ID NO. 118 is the reverse complement of the primer of SEQ ID NO. 76.

SEQ ID NO. 119 is the reverse complement of the primer of SEQ ID NO. 78.

SEQ ID NO. 120 is the reverse complement of the primer of SEQ ID NO. 80.

SEQ ID NO. 121 is the reverse complement of the primer of SEQ ID NO. 82.

SEQ ID NO. 122 is the reverse complement of the primer of SEQ ID NO. 84.

SEQ ID NO. 123 is the reverse complement of the primer of SEQ ID NO. 86.

SEQ ID NO. 124 is the reverse complement of the primer of SEQ ID NO. 88.

SEQ ID NO. 125 is the reverse complement of the primer of SEQ ID NO. 92.

SEQ ID NO. 126 is the reverse complement of the primer of SEQ ID NO. 94.

SEQ ID NO. 127 is the reverse complement of the primer of SEQ ID NO. 96.

SEQ ID NO. 128 is the reverse complement of the primer of SEQ ID NO. 98.

SEQ ID NO. 129 is the reverse complement of the primer of SEQ ID NO. 99.

SEQ ID NO. 130 is the reverse complement of the primer of SEQ ID NO. 100.

SEQ ID NO. 131 is the reverse complement of the primer of SEQ ID NO. 104.

SEQ ID NO. 132 is the reverse complement of the primer of SEQ ID NO. 106.

SEQ ID NO. 133 is the reverse complement of the primer of SEQ ID NO. 108.

SEQ ID NO. 134 is the reverse complement of the primer of SEQ ID NO. 110.

SEQ ID NO. 135 is a MIS-7 forward primer.

SEQ ID NO. 136 is a MIS-7 reverse primer.

SEQ ID NO. 137 is a MIS-8 forward primer.

SEQ ID NO. 138 is a MIS-8 reverse primer.

SEQ ID NO. 139 is a nucleotide sequence of a MIS-7 toxin gene designated 157C1-A from B.t. strain PS157C1.

SEQ ID NO. 140 is an amino acid sequence of a MIS-7 toxin designated 157C1-A from B.t. strain PS157C1.

SEQ ID NO. 141 is a nucleotide sequence of a MIS-7 toxin gene from B.t. strain PS201Z.

SEQ ID NO. 142 is a nucleotide sequence of a MIS-8 toxin gene from B.t. strain PS31F2.

SEQ ID NO. 143 is a nucleotide sequence of a MIS-8 toxin gene from B.t. strain PS185Y2.

SEQ ID NO. 144 is a nucleotide sequence of a MIS-1 toxin gene from B.t. strain PS33F1.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns materials and methods for the control of non-mammalian pests. In specific embodiments, the subject invention pertains to new

Bacillus thuringiensis

isolates and toxins which have activity against lepidopterans and/or coleopterans. The subject invention further concerns novel genes which encode pesticidal toxins and novel methods for identifying and characterizing Bacillus genes which encode toxins with useful properties. The subject invention concerns not only the polynucleotide sequences which encode these toxins, but also the use of these polynucleotide sequences to produce recombinant hosts which express the toxins. The proteins of the subject invention are distinct from protein toxins which have previously been isolated from

Bacillus thuringiensis.

B.t. isolates useful according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA. The culture repository numbers of the B.t. strains are as follows:

TABLE 1

Repository

Culture

No.

Deposit Date

Patent No.

B.t. PS11B (MT274)

NRRL B-

Apr. 18, 1996

21556

B.t. PS24J

NRRL B-

Aug. 30, 1991

18881

B.t. PS31G1 (MT278)

NRRL B-

Apr. 18, 1996

21560

B.t. PS36A

NRRL B-

Dec. 27, 1991

18929

B.t. PS33F2

NRRL B-

Jul. 28, 1987

4,861,595

18244

B.t. PS40D1

NRRL B-

Feb. 3, 1988

5,098,705

18300

B.t. PS43F

NRRL B-

Feb. 2, 1988

4,996,155

18298

B.t. PS45B1

NRRL B-

Aug. 16, 1988

5,427,786

18396

B.t. PS49C

NRRL B-

Mar. 14, 1996

21532

B.t. PS52A1

NRRL B-

Jul. 28, 1987

4,861,595

18245

B.t. PS62B1

NRRL B-

Aug. 16, 1988

4,849,217

18398

B.t. PS81A2

NRRL B-

Apr. 19, 1989

5,164,180

18484

B.t. PS81F

NRRL B-

Oct. 7, 1988

5,045,469

18424

B.t. PS81GG

NRRL B-

Oct. 11, 1988

5,169,629

18425

B.t PS81I

NRRL B-

Apr. 19, 1989

5,126,133

18484

B.t. PS85A1

NRRL B-

Oct. 11, 1988

18426

B.t. PS86A1

NRRL B-

Aug. 16, 1988

4,849,217

18400

B.t. PS86B1

NRRL B-

Feb. 2, 1988

4,966,765

18299

B.t. PS86BB1 (MT275)

NRRL B-

Apr. 18, 1996

21557

B.t. PS86Q3

NRRL B-

Feb. 6, 1991

5,208,017

18765

B.t. PS86V1 (MT276)

NRRL B-

Apr. 18, 1996

21558

B.t. PS86W1 (MT277)

NRRL B-

Apr. 18, 1996

21559

B.t. PS89J3 (MT279)

NRRL B-

Apr. 18, 1996

21561

B.t. PS91C2

NRRL B-

Feb. 6, 1991

18931

B.t. PS92B

NRRL B-

Sep. 23, 1991

5,427,786

18889

B.t. PS101Z2

NRRL B-

Oct. 1, 1991

5,427,786

18890

B.t. PS122D3

NRRL B-

Jun. 9, 1988

5,006,336

18376

B.t. PS123D1

NRRL B-

Oct. 13, 1992

5,508,032

21011

B.t. PS157C1 (MT104)

NRRL B-

Jul. 17, 1987

5,262,159

18240

B.t. PS158C2

NRRL B-

Aug. 27, 1991

5,268,172

18872

B.t. PS169E

NRRL B-

Jul. 17, 1990

5,151,363

18682

B.t. PS177F1

NRRL B-

Jul. 17, 1990

5,151,363

18683

B.t. PS177G

NRRL B-

Jul. 17, 1990

5,151,363

18684

B.t. PS185L2

NRRL B-

Mar. 14, 1996

21535

B.t. PS185U2 (MT280)

NRRL B-

Apr. 18, 1996

21562

B.t. PS192M4

NRRL B-

Dec. 27, 1991

5,273,746

18932

B.t. PS201L1

NRRL B-

Jan. 9, 1991

5,298,245

18749

B.t. PS204C3

NRRL B-

Oct. 6, 1992

21008

B.t. PS204G4

NRRL B-

Jul. 17, 1990

5,262,399

18685

B.t. PS242H10

NRRL B-

Mar. 14, 1996

21439

B.t. PS242K17

NRRB B-

Mar. 14, 1996

21540

B.t. PS244A2

NRRB B-

Mar. 14, 1996

21541

B.t. PS244D1

NRRL B-

Mar. 14, 1996

21542

B.t. PS10E1

NRRL B-

Oct. 24, 1997

21862

B.t. PS31F2

NRRL B-

Oct. 24, 1997

21876

B.t. PS31J2

NRRL B-

Oct. 13, 1992

21009

B.t. PS33D2

NRRL B-

Oct. 24, 1997

21870

B.t. PS66D3

NRRL B-

Oct. 24, 1997

21858

B.t. PS68F

NRRL B-

Oct. 24, 1997

21857

B.t. PS69AA2

NRRL B-

Oct. 24, 1997

21859

B.t. PS146D

NRRL B-

Oct. 24, 1997

21866

B.t. PS168G1

NRRL B-

Oct. 24, 1997

21873

B.t. PS175I4

NRRL B-

Oct. 24, 1997

21865

B.t. PS177C8a

NRRL B-

Oct. 24, 1997

21867

B.t. PS177I8

NRRL B-

Oct. 24, 1997

21868

B.t. PS185AA2

NRRL B-

Oct. 24, 1997

21861

B.t. PS196J4

NRRL B-

Oct. 24, 1997

21860

B.t. PS196F3

NRRL B-

Oct. 24, 1997

21872

B.t. PS197T1

NRRL B-

Oct. 24, 1997

21869

B.t. PS197U2

NRRL B-

Oct. 24, 1997

21871

B.t. PS202E1

NRRL B-

Oct. 24, 1997

21874

B.t. PS217U2

NRRL B-

Oct. 24, 1997

21864

KB33

NRRL B-

Oct. 24, 1997

21875

KB38

NRRL B-

Oct. 24, 1997

21863

KB53A49-4

NRRL B-

Oct. 24, 1997

21879

KB68B46-2

NRRL B-

Oct. 24, 1997

21877

KB68B51-2

NRRL B-

Oct. 24, 1997

21880

K1B68B55-2

NRRL B-

Oct. 24, 1997

21878

PS80JJ1

NRRL B-

Jul. 17, 1990

5,151,363

18679

PS94R1

NRRL B-

Jul. 1, 1997

21801

PS101DD

NRRL B-

Jul. 1, 1997

21802

PS202S

NRRL B-

Jul. 1, 1997

21803

PS213E5

NRRL B-

Jul. 1, 1997

21804

PS218G2

NRRL B-

Jul. 1, 1997

21805

PS33F1

NRRL B-

Apr. 24, 1998

21977

PS71G4

NRRL B-

Apr. 24, 1998

21978

PS86D1

NRRL B-

Apr. 24, 1998

21979

PS185V2

NRRL B-

Apr. 24, 1998

21980

PS191A21

NRRL B-

Apr. 24, 1998

21981

PS201Z

NRRL B-

Apr. 24, 1998

21982

PS205A3

NRRL B-

Apr. 24, 1998

21983

PS205C

NRRL B-

Apr. 24, 1998

21984

PS234E1

NRRL B-

Apr. 24, 1998

21985

PS248N10

NRRL B-

Apr. 24, 1998

21986

KB63B19-13

NRRL B-

Apr. 29, 1998

21990

KB63B19-7

NRRL B-

Apr. 29, 1998

21989

KB68B62-7

NRRL B-

Apr. 29, 1998

21991

KB68B63-2

NRRL B-

Apr. 29, 1998

21992

KB69A125-1

NRRL B-

Apr. 29, 1998

21993

KB69A125-3

NRRL B-

Apr. 29, 1998

21994

KB69A125-5

NRRL B-

Apr. 29, 1998

21995

KB69A127-7

NRRL B-

Apr. 29, 1998

21996

KB69A132-1

NRRL B-

Apr. 29, 1998

21997

KB69B2-1

NRRL B-

Apr. 29, 1998

21998

KB70B5-3

NRRL B-

Apr. 29, 1998

21999

KB71A125-15

NRRL B-

Apr. 29, 1998

30001

KB71A35-6

NRRL B-

Apr. 29, 1998

30000

KB71A72-1

NRRL B-

Apr. 29, 1998

21987

KB71A134-2

NRRL B-

Apr. 29, 1998

21988

Cultures which have been deposited for the purposes of this patent application were deposited under conditions that assure that access to the cultures is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture(s). The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

Many of the strains useful according to the subject invention are readily available by virtue of the issuance of patents disclosing these strains or by their deposit in public collections or by their inclusion in commercial products. For example, the B.t. strain used in the commercial product, Javelin, and the HD isolates are all publicly available.

Mutants of the isolates referred to herein can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

In one embodiment, the subject invention concerns materials and methods including nucleotide primers and probes for isolating, characterizing, and identifying Bacillus genes encoding protein toxins which are active against non-mammalian pests. The nucleotide sequences described herein can also be used to identify new pesticidal Bacillus isolates. The invention further concerns the genes, isolates, and toxins identified using the methods and materials disclosed herein.

The new toxins and polynucleotide sequences provided here are defined according to several parameters. One characteristic of the toxins described herein is pesticidal activity. In a specific embodiment, these toxins have activity against coleopteran and/or lepidopteran pests. The toxins and genes of the subject invention can be further defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology to certain exemplified sequences as well as in terms of the ability to hybridize with, or be amplified by, certain exemplified probes and primers. The toxins provided herein can also be identified based on their immunoreactivity with certain antibodies.

An important aspect of the subject invention is the identification and characterization of new families of Bacillus toxins, and genes which encode these toxins. These families have been designated MIS-1, MIS-2, MIS-3, MIS-4, MIS-5, MIS-6, MIS-7, MIS-8, WAR-1, and SUP-1. Toxins within these families, as well as genes encoding toxins within these families, can readily be identified as described herein by, for example, size, amino acid or DNA sequence, and antibody reactivity. Amino acid and DNA sequence characteristics include homology with exemplified sequences, ability to hybridize with DNA probes, and ability to be amplified with specific primers.

The MIS-1 family of toxins includes toxins from isolates PS68F and PS33F1. Also provided are hybridization probes and PCR primers which specifically identify genes falling in the MIS-1 family.

A second family of toxins identified herein is the MIS-2 family. This family includes toxins which can be obtained from isolates PS66D3, PS197T1, and PS31J2. The subject invention further provides probes and primers for the identification of MIS-2 toxins and genes.

A third family of toxins identified herein is the MIS-3 family. This family includes toxins which can be obtained from B.t. isolates PS69AA2 and PS33D2. The subject invention further provides probes and primers for identification of the MIS-3 genes and toxins.

Polynucleotide sequences encoding MIS-4 toxins can be obtained from the B.t. isolate designated PS197U2. The subject invention further provides probes and primers for the identification of genes and toxins in this family.

A fifth family of toxins identified herein is the MIS-5 family. This family includes toxins which can be obtained from B.t. isolates KB33 and KB38. The subject invention further provides probes and primers for identification of the MIS-5 genes and toxins.

A sixth family of toxins identified herein is the MIS-6 family. This family includes toxins which can be obtained from B.t. isolates PS196F3, PS168G1, PS196J4, PS202E1, PS10E1, and PS185AA2. The subject invention further provides probes and primers for identification of the MIS-6 genes and toxins.

A seventh family of toxins identified herein is the MIS-7 family. This family includes toxins which can be obtained from B.t. isolates PS157C1, PS205C, and PS201Z. The subject invention further provides probes and primers for identification of the MIS-7 genes and toxins.

An eighth family of toxins identified herein is the MIS-8 family. This family includes toxins which can be obtained from B.t. isolates PS31F2 and PS185Y2. The subject invention further provides probes and primers for identification of the MIS-8 genes and toxins.

In a preferred embodiment, the genes of the MIS family encode toxins having a molecular weight of about 70 to about 100 kDa and, most preferably, the toxins have a size of about 80 kDa. Typically, these toxins are soluble and can be obtained from the supernatant of Bacillus cultures as described herein. These toxins have toxicity against non-mammalian pests. In a preferred embodiment, these toxins have activity against coleopteran pests. The MIS proteins are further useful due to their ability to form pores in cells. These proteins can be used with second entities including, for example, other proteins. When used with a second entity, the MIS protein will facilitate entry of the second agent into a target cell. In a preferred embodiment, the MIS protein interacts with MIS receptors in a target cell and causes pore formation in the target cell. The second entity may be a toxin or another molecule whose entry into the cell is desired.

The subject invention further concerns a family of toxins designated WAR-1. The WAR-1 toxins typically have a size of about 30-50 kDa and, most typically, have a size of about 40 kDa. Typically, these toxins are soluble and can be obtained from the supernatant of Bacillus cultures as described herein. The WAR-1 toxins can be identified with primers described herein as well as with antibodies. In a specific embodiment, the antibodies can be raised to, for example, toxin from isolate PS177C8.

An additional family of toxins provided according to the subject invention are the toxins designated SUP-1. Typically, these toxins are soluble and can be obtained from the supernatant of Bacillus cultures as described herein. In a preferred embodiment, the SUP-1 toxins are active against lepidopteran pests. The SUP-1 toxins typically have a size of about 70-100 kDa and, preferably, about 80 kDa. The SUP-1 family is exemplified herein by toxins from isolates PS49C and PS158C2. The subject invention provides probes and primers useful for the identification of toxins and genes in the SUP-1 family

The subject invention further provides specific Bacillus toxins and genes which did not fall into any of the new families disclosed herein. These specific toxins and genes include toxins and genes which can be obtained from PS177C8 and PS177I8.

Toxins in the MIS, WAR, and SUP families are all soluble and can be obtained as described herein from the supernatant of Bacillus cultures. These toxins can be used alone or in combination with other toxins to control pests. For example, toxins from the MIS families may be used in conjunction with WAR-type toxins to achieve control of pests, particularly coleopteran pests. These toxins may be used, for example, with δ-endotoxins which are obtained from Bacillus isolates.

Table 2 provides a summary of the novel families of toxins and genes of the subject invention. Each of the eight MIS families is specifically exemplified herein by toxins which can be obtained from particular B.t. isolates as shown in Table 2. Genes encoding toxins in each of these families can be identified by a variety of highly specific parameters, including the ability to hybridize with the particular probes set forth in Table 2. Sequence identity in excess of about 80% with the probes set forth in Table 2 can also be used to identify the genes of the various families. Also exemplified are particular primer pairs which can be used to amplify the genes of the subject invention. A portion of a gene within the indicated families would typically be amplifiable with at least one of the enumerated primer pairs. In a preferred embodiment, the amplified portion would be of approximately the indicated fragment size. Primers shown in Table 2 consist of polynucleotide sequences which encode peptides as shown in the sequence listing attached hereto. Additional primers and probes can readily be constructed by those skilled in the art such that alternate polynucleotide sequences encoding the same amino acid sequences can be used to identify and/or characterize additional genes encoding pesticidal toxins. In a preferred embodiment, these additional toxins, and their genes, could be obtained from Bacillus isolates.

TABLE 2

Probes

Primer Pairs

Fragment size

Family

Isolates

(SEQ ID NO.)

(SEQ ID NOS.)

(nt)

MIS-1

PS68F, PS33F1

26, 144

56 and 111

69

56 and 112

506

58 and 112

458

MIS-2

PS66D3, PS197T1, P531J2

24, 41, 20

62 and 113

160

62 and 114

239

62 and 115

400

62 and 116

509

62 and 117

703

64 and 114

102

64 and 115

263

64 and 116

372

64 and 117

566

66 and 115

191

66 and 116

300

66 and 117

494

68 and 116

131

68 and 117

325

70 and 117

213

MIS-3

PS69AA2, P533D2

28, 22

74 and 118

141

74 and 119

376

74 and 120

389

74 and 121

483

74 and 122

715

74 and 123

743

74 and 124

902

76 and 119

253

76 and 120

266

76 and 121

360

76 and 122

592

76 and 123

620

76 and 124

779

78 and 120

31

78 and 121

125

78 and 122

357

78 and 123

385

78 and 124

544

80 and 121

116

80 and 122

348

80 and 123

376

80 and 124

535

82 and 122

252

82 and 123

280

82 and 124

439

84 and 123

46

84 and 124

205

86 and 124

177

MIS-4

PS197U2

43

90 and 125

517

90 and 126

751

90 and 127

821

92 and 126

258

92 and 127

328

94 and 127

92

MIS-5

KB33, KB38

47, 48

97 and 128

109

97 and 129

379

97 and 130

504

98 and 129

291

98 and 130

416

99 and 130

144

MIS-6

PS196F3, P5168G1, P5196J4,

18,30,35,37,

102 and 131

66

PS202E1, PS10E1, PS185AA2

39,45

102 and 132

259

102 and 133

245

102 and 134

754

104 and 132

213

104 and 133

199

104 and 134

708

106 and 133

31

106 and 134

518

108 and 134

526

MIS-7

PS205C, PS157C1 (157C1-A),

139, 141

135 and 136

598

PS201Z

MIS-8

PS31F2, PS185Y2

142,143

137 and 138

585

SUP-1

PS49C, PS158C2

10, 12, 15

53 and 54

370

Furthermore, chimeric toxins may be used according to the subject invention. Methods have been developed for making useful chimeric toxins by combining portions of B.t. proteins. The portions which are combined need not, themselves, be pesticidal so long as the combination of portions creates a chimeric protein which is pesticidal. This can be done using restriction enzymes, as described in, for example, European Patent 0 228 838; Ge, A. Z., N. L. Shivarova, D. H. Dean (1989)

Proc. Natl. Acad. Sci. USA

86:4037-4041; Ge, A. Z., D. Rivers, R. Milne, D. H. Dean (1991)

J. Biol. Chem.

266:17954-17958; Schnepf, H. E., K. Tomczak, J. P. Ortega, H. R. Whiteley (1990)

J. Biol. Chem.

265:20923-20930; Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, B. Visser (1991)

Mol. Microbiol.

5:2799-2806. Alternatively, recombination using cellular recombination mechanisms can be used to achieve similar results. See, for example, Caramori, T., A. M. Albertini, A. Galizzi (1991)

Gene

98:37-44; Widner, W. R., H. R. Whiteley (1990)

J. Bacteriol.

172:2826-2832; Bosch, D., B. Schipper, H. van der Kliej, R. A. de Maagd, W. J. Stickema (1994)

Biotechnology

12:915-918. A number of other methods are known in the art by which such chimeric DNAs can be made. The subject invention is meant to include chimeric proteins that utilize the novel sequences identified in the subject application.

With the teachings provided herein, one skilled in the art could readily produce and use the various toxins and polynucleotide sequences described herein.

Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. Chimeric genes and toxins, produced by combining portions from more than one Bacillus toxin or gene, may also be utilized according to the teachings of the subject invention. As used herein, the terms “variants” or “variations” of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term “equivalent toxins” refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.

It is apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can be derived from Bacillus isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other Bacillus toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes which encode these toxins can then be obtained from the microorganism.

Fragments and equivalents which retain the pesticidal activity of the exemplified toxins are within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to “essentially the same” sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition.

A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. Probes provide a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures.

Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be greater than 60%, preferably be greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and can be greater than 95%. These identities are as determined using standard alignment techniques. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 3 provides a listing of examples of amino acids belonging to each class.

TABLE 3

Class of Amino Acid

Examples of Amino Acids

Nonpolar

Ala, Val, Leu, Ile, Pro, Met, Phe, Trp

Uncharged Polar

Gly, Ser, Thr, Cys, Tyr, Asn, Gln

Acidic

Asp, Glu

Basic

Lys, Arg, His

In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.

The δ-endotoxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above.

As used herein, reference to “isolated” polynucleotides and/or “purified” toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to “isolated and purified” signifies the involvement of the “hand of man” as described herein. Chimeric toxins and genes also involve the “hand of man.”

Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the production and maintenance of the pesticide. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is a control of the pest. Alternatively, the microbe hosting the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

Where the Bacillus toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as

Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,

and

Azotobacter vinlandii;

and phytosphere yeast species such as

Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae,

and

Aureobasidium pollulans.

Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing a Bacillus gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Synthetic genes which are functionally equivalent to the toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.

Treatment of cells. As mentioned above, Bacillus or recombinant cells expressing a Bacillus toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the Bacillus toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form.

Treatment of the microbial cell, e.g., a microbe containing the Bacillus toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.

Methods and formulations for control of pests. Control of pests using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of Bacillus isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.

Formulated bait granules containing an attractant and the toxins of the Bacillus isolates, or recombinant microbes comprising the genes obtainable from the Bacillus isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of Bacillus cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations that contain cells will generally have from about 10

2

to about 10

4

cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

Polynucleotide probes. It is well known that DNA possesses a fundamental property called base complementarity. In nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will be opposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or “denaturation,” of the two strands. If the DNA is then placed in conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or “hybridize,” and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only strands with a high degree of base complementarity will be able to form stable double stranded structures. The relationship of the specificity of hybridization to reaction conditions is well known. Thus, hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes of the subject invention to readily detect and characterize DNA sequences of interest.

The probes may be RNA, DNA, or PNA (peptide nucleic acid). The probe will normally have at least about 10 bases, more usually at least about 17 bases, and may have up to about 100 bases or more. Longer probes can readily be utilized, and such probes can be, for example, several kilobases in length. The probe sequence is designed to be at least substantially complementary to a portion of a gene encoding a toxin of interest. The probe need not have perfect complementarity to the sequence to which it hybridizes. The probes may be labelled utilizing techniques which are well known to those skilled in this art.

One approach for the use of the subject invention as probes entails first identifying by Southern blot analysis of a gene bank of the Bacillus isolate all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the probable activity of many new Bacillus isolates, and of the individual gene products expressed by a given Bacillus isolate. Such a probe analysis provides a rapid method for identifying potentially commercially valuable insecticidal toxin genes within the multifarious subspecies of B.t.

One hybridization procedure useful according to the subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. Either lysed bacteria or total fractionated nucleic acid isolated from bacteria can be used. Cells can be treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be cut into pieces with an appropriate restriction enzyme. The pieces can be separated by size through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane.

The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied.

The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.

In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include

32

P,

35

S, or the like. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probes may be made inherently fluorescent as described in International Application No. WO 93/16094.

Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987)

DNA Probes,

Stockton Press, New York, N.Y., pp. 169-170.

As used herein “moderate to high stringency” conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Examples of moderate and high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (Maniatis et al.). In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to the exemplified toxin genes. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983]

Methods of Enzymology,

R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).

Tm=81.5° C.+16.6 Log[Na+]+0.41 (%G+C)−0.61(%formamide)−600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at 10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula:

Tm (° C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981]

ICN

-

UCLA Symp. Dev. Biol. Using Purified Genes,

D. D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Moderate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.

Thus, mutational, insertional, and deletional variants of the disclosed nucleotide sequences can be readily prepared by methods which are well known to those skilled in the art. These variants can be used in the same manner as the exemplified primer sequences so long as the variants have substantial sequence homology with the original sequence. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant probe to function in the same capacity as the original probe. Preferably, this homology is greater than 50%; more preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%. The degree of homology needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage.

PCR technology. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,”

Science

230:1350-1354.). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium

Thermus aquaticus,

the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.

The DNA sequences of the subject invention can be used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5′ end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.

All of the U.S. patents cited herein are hereby incorporated by reference.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Culturing of Bacillus Isolates Useful According to the Invention

Growth of cells. The cellular host containing the Bacillus insecticidal gene may be grown in any convenient nutrient medium. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The Bacillus cells of the invention can be cultured using standard art media and fermentation techniques. During the fermentation cycle, the bacteria can be harvested by first separating the Bacillus vegetative cells, spores, crystals, and lysed cellular debris from the fermentation broth by means well known in the art. Any Bacillus spores or crystal δ-endotoxins formed can be recovered employing well-known techniques and used as a conventional δ-endotoxin B.t. preparation. The supernatant from the fermentation process contains toxins of the present invention. The toxins are isolated and purified employing well-known techniques.

A subculture of Bacillus isolates, or mutants thereof, can be used to inoculate the following medium, known as TB broth:

Tryptone

12

g/l

Yeast Extract

24

g/l

Glycerol

4

g/l

KH

2

PO

4

2.1

g/l

K

2

HPO

4

14.7

g/l

pH 7.4

The potassium phosphate was added to the autoclaved broth after cooling. Flasks were incubated at 30° C. on a rotary shaker at 250 rpm for 24-36 hours.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The Bacillus obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation. In a specific embodiment, Bacillus proteins useful according the present invention can be obtained from the supernatant. The culture supernatant containing the active protein(s) can be used in bioassays.

Alternatively, a subculture of Bacillus isolates, or mutants thereof, can be used to inoculate the following peptone, glucose, salts medium:

Bacto Peptone

7.5

g/l

Glucose

1.0

g/l

KH

2

PO

4

3.4

g/l

K

2

HPO

4

4.35

g/l

Salt Solution

5.0

ml/l

CaCl

2

Solution

5.0

ml/l

pH 7.2

Salts Solution (100 ml)

MgSO

4

.7H

2

O

2.46

g

MnSO

4

.H

2

O

0.04

g

ZnSO

4

.7H

2

O

0.28

g

FeSO

4

.7H

2

O

0.40

g

CaCl

2

Solution (100 ml)

CaCl

2

.2H

2

O

3.66

g

The salts solution and CaCl

2

solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The Bacillus spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

EXAMPLE 2

Isolation and Preparation of Cellular DNA for PCR

DNA can be prepared from cells grown on Spizizen's agar, or other minimal or enriched agar known to those skilled in the art, for approximately 16 hours. Spizizen's casamino acid agar comprises 23.2 g/l Spizizen's minimal salts [(NH

4

)

2

SO

4

, 120 g; K

2

HPO

4

, 840 g; KH

2

PO

4

, 360 g; sodium citrate, 60 g; MgSO

4

·7H

2

O, 12 g. Total: 1392 g]; 1.0 g/l vitamin-free casamino acids; 15.0 g/l Difco agar. In preparing the agar, the mixture was autoclaved for 30 minutes, then a sterile, 50% glucose solution can be added to a final concentration of 0.5% (1/100 vol). Once the cells are grown for about 16 hours, an approximately 1 cm

2

patch of cells can be scraped from the agar into 300 μl of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA. Proteinase K was added to 50 μg/ml and incubated at 55° C. for 15 minutes. Other suitable proteases lacking nuclease activity can be used. The samples were then placed in a boiling water bath for 15 minutes to inactivate the proteinase and denature the DNA. This also precipitates unwanted components. The samples are then centrifuged at 14,000×g in an Eppendorf microfuge at room temperature for 5 minutes to remove cellular debris. The supernatants containing crude DNA were transferred to fresh tubes and frozen at −20° C. until used in PCR reactions.

Alternatively, total cellular DNA may be prepared from plate-grown cells using the QIAamp Tissue Kit from Qiagen (Santa Clarita, Calif.) following instructions from the manufacturer.

EXAMPLE 3

Use of PCR Primers to Characterize and/or Identify Toxin Genes

Two primers useful in PCR procedures were designed to identify genes that encode pesticidal toxins. Preferably, these toxins are active against lepidopteran insects. The DNA from 95 B.t. strains was subjected to PCR using these primers. Two clearly distinguishable molecular weight bands were visible in “positive” strains, as outlined below. The frequency of strains yielding a 339 bp fragment was 29/95 (31%). This fragment is referred to herein as the “339 bp fragment” even though some small deviation in the exact number of base pairs may be observed.

GARCCRTGGA AAGCAAATAA TAARAATGC

(SEQ ID NO. 1)

AAARTTATCT CCCCAWGCTT CATCTCCATT TTG

(SEQ ID NO. 2)

The strains which were positive for the 339 bp fragment (29 strains) were: PS11B, PS31G1, PS36A, PS49C, PS81A2, PS81F, PS81GG, PS81I, PS85A1, PS86BB1, PS86V1, PS86W1, PS89J3, PS91C2, PS94R1, PS101DD, PS158C2, PS185U2, PS192M4, PS202S, PS213E5, PS218G2, PS244A2, HD29, HD110, HD129, HD525, HD573a, and Javelin 1990.

The 24 strains which gave a larger (approximately 1.2 kb) fragment were: PS24J, PS33F2, PS45B1, PS52A1, PS62B1, PS80PP3, PS86A1, PS86Q3, PS88F16, PS92B, PS101Z2, PS123D1, PS157C1, PS169E, PS177F1, PS177G, PS185L2, PS201L1, PS204C3, PS204G4, PS242H10, PS242K17, PS244A2, PS244D1.

It was found that Bacillus strains producing lepidopteran-active proteins yielded only the 339 bp fragment. Few, if any, of the strains amplifying the approximately 1.2 kb fragment had known lepidopteran activity, but rather were coleopteran-, mite-, and/or nematode-active B.t. crystal protein producing strains.

EXAMPLE 4

DNA Sequencing of Toxin Genes Producing the 339 Fragment

PCR-amplified segments of toxin genes present in Bacillus strains can be readily sequenced. To accomplish this, amplified DNA fragments can be first cloned into the PCR DNA TA-cloning plasmid vector, pCRII, as described by the supplier (Invitrogen, San Diego, Calif.). Individual pCRII clones from the mixture of amplified DNA fragments from each Bacillus strain are chosen for sequencing. Colonies are lysed by boiling to release crude plasmid DNA. DNA templates for automated sequencing are amplified by PCR using vector-specific primers flanking the plasmid multiple cloning sites. These DNA templates are sequenced using Applied Biosystems (Foster City, Calif.) automated sequencing methodologies. The polypeptide sequences can be deduced from these nucleotide sequences.

DNA from three of the 29 B.t. strains which amplified the 339 bp fragments were sequenced. A DNA sequence encoding a toxin from strain PS36A is shown in SEQ ID NO. 3. An amino acid sequence for the 36A toxin is shown in SEQ ID. NO 4. A DNA sequence encoding a toxin from strain PS81F is shown in SEQ ID NO. 5. An amino acid sequence for the 81F toxin is shown in SEQ ID. NO 6. A DNA sequence encoding a toxin from strain Javelin 1990 is shown in SEQ ID NO. 7. An amino acid sequence for the Javelin 1990 toxin is shown in SEQ ID. NO 8.

EXAMPLE 5

Determination of DNA Sequences from Additional Genes Encoding Toxins from Strains PS158C2 and PS49C

Genes encoding novel toxins were identified from isolates PS158C2 and PS49C as follows: Total cellular DNA was extracted from B.t. strains using Qiagen (Santa Clarita, Calif.) Genomic-tip 500/G DNA extraction kits according to the supplier and was subjected to PCR using the oligonucleotide primer pairs listed below. Amplified DNA fragments were purified on Qiagen PCR purification columns and were used as templates for sequencing.

For PS158C2, the primers used were as follows.

158C2 PRIMER A:

GCTCTAGAAGGAGGTAACTTATGAACAAGAATAATACTAAATTAAGC (SEQ ID NO. 9)

339 reverse:

AAARTTATCT CCCCAWGCTT CATCTCCATT TTG (SEQ ID NO. 2)

The resulting PCR-amplified DNA fragment was approximately 2 kbp in size. This DNA was partially sequenced by dideoxy chain termination using automated DNA sequencing technology (Perkin Elmer/Applied Biosystems, Foster City, Calif.). A DNA sequence encoding a portion of a soluble toxin from PS158C2 is shown in SEQ ID NO. 10.

For PS49C, two separate DNA fragments encoding parts of a novel toxin gene were amplified and sequenced. The first fragment was amplified using the following primer pair:

49C PRIMER A:

CATCCTCCCTACACTTTCTAA (SEQ ID NO. 11)

339 reverse:

AAARTTATCT CCCCAWGCTT CATCTCCATT TTG (SEQ ID NO. 2)

The resulting approximately 1 kbp DNA fragment was used as a template for automated DNA sequencing. A sequence of a portion of a toxin gene from strain PS49C is shown in SEQ ID NO. 12.

The second fragment was amplified using the following primer pair:

49C PRIMER B:

AAATTATGCGTAAGTCTGC (SEQ ID NO. 13)

49C PRIMER C:

TTGATCCGGACATAATAAT (SEQ ID NO. 14)

The resulting approximately 0.57 kbp DNA fragment was used as a template for automated DNA sequencing. An additional sequence of a portion of the toxin gene from PS49C is shown in SEQ ID NO. 15.

EXAMPLE 6

Additional Primers Useful for Characterizing and/or Identifying Toxin Genes

The following primer pair can be used to identify and/or characterize genes of the SUP-1 family:

SUP-1A:

GGATTCGTTATCAGAAA (SEQ ID NO. 53)

SUP-1B:

CTGTYGCTAACAATGTC (SEQ ID NO. 54)

These primers can be used in PCR procedures to amplify a fragment having a predicted size of approximately 370 bp. A band of the predicted size was amplified from strains PS158C2 and PS49C.

EXAMPLE 7

Additional Primers Useful for Characterizing and/or Identifying Toxin Genes

Another set of PCR primers can be used to identify and/or characterize additional genes encoding pesticidal toxins. The sequences of these primers were as follows:

GGRTTAMTTGGRTAYTATTT (SEQ ID NO. 16)

ATATCKWAYATTKGCATTTA (SEQ ID NO. 17)

Redundant nucleotide codes used throughout the subject disclosure are in accordance with the IUPAC convention and include:

R=A or G

M=A or C

Y=C or T

K=G or T

W=A or T

EXAMPLE 8

Identification and Sequencing of Genes Encoding Novel Soluble Protein Toxins from Bacillus Strains

PCR using primers SEQ ID NO. 16 and SEQ ID NO. 17 was performed on total cellular genomic DNA isolated from a broad range of Bt strains. Those samples yielding an approximately 1 kb band were selected for characterization by DNA sequencing. Amplified DNA fragments were first cloned into the PCR DNA TA-cloning plasmid vector, pCR2.1, as described by the supplier (Invitrogen, San Diego, Calif.). Plasmids were isolated from recombinant clones and tested for the presence of an approximately 1 kbp insert by PCR using the plasmid vector primers, T3 and T7.

The following strains yielded the expected band of approximately 1000 bp, thus indicating the presence of a MIS-type toxin gene: PS10E1, PS31J2, PS33D2, PS66D3, PS68F, PS69AA2, PS168G1, PS177C8, PS177I8, PS185AA2, PS196F3, PS196J4, PS197T1, PS197U2, PS202E1, KB33, KB38, PS33F1, PS157C1 (157C1-A), PS201Z, PS31F2, and PS185Y2.

Plasmids were then isolated for use as sequencing templates using QIAGEN (Santa Clarita, Calif.) miniprep kits as described by the supplier. Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems. Sequencing reactions were run on a ABI PRISM 377 Automated Sequencer. Sequence data was collected, edited, and assembled using the ABI PRISM 377 Collection, Factura, and AutoAssembler software from PE ABI.

DNA sequences were determined for portions of novel toxin genes from the following isolates: PS10E1, PS31J2, PS33D2, PS66D3, PS68F, PS69AA2, PS168G1, PS177C8, PS177I8, PS185AA2, PS196F3, PS196J4, PS197T1, PS197U2, PS202E1, KB33, KB38, PS33F1, PS157C1 (157C1-A), PS201Z, PS31F2, and PS185Y2. Polypeptide sequences were deduced for portions of the encoded, novel soluble toxins from the following isolates: PS10E1, PS31J2, PS33D2, PS66D3, PS68F, PS69AA2, PS177C8, PS177I8, PS185AA2, PS196F3, PS196J4, PS197T1, PS197U2, PS202E1, and PS157C1 (toxin 157C1-A). These nucleotide sequences and amino acid sequences are shown in SEQ ID NOS. 18 to 48 and SEQ ID NOS. 139-144.

EXAMPLE 9

Restriction Fragment Length Polymorphism (RFLP) of Toxins from

Bacillus thuringiensis

Strains

Total cellular DNA was prepared from various

Bacillus thuringiensis

(B.t.) strains grown to an optical density of 0.5-0.8 at 600 nm visible light. DNA was extracted using the Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Set according to protocol for Gram positive bacteria (Qiagen Inc.; Valencia, Calif.).

Standard Southern hybridizations using

32

P-labeled probes were used to identify and characterize novel toxin genes within the total genomic DNA preparations. Prepared total genomic DNA was digested with various restriction enzymes, electrophoresed on a 1% agarose gel, and immobilized on a supported nylon membrane using standard methods (Maniatis et al.).

PCR-amplified DNA fragments 1.0-1.1 kb in length were gel purified for use as probes. Approximately 25 ng of each DNA fragment was used as a template for priming nascent DNA synthesis using DNA polymerase I Klenow fragment (New England Biolabs), random hexanucleotide primers (Boehringer Mannheim) and

32

PdCTP.

Each

32

P-labeled fragment served as a specific probe to its corresponding genomic DNA blot. Hybridizations of immobilized DNA with randomly labeled

32

p probes were performed in standard aqueous buffer consisting of 5×SSPE, 5×Denhardt's solution, 0.5% SDS, 0.1 mg/ml at 65° C. overnight. Blots were washed under moderate stringency in 0.2×SSC, 0.1% SDS at 65° C. and exposed to film. RFLP data showing specific hybridization bands containing all or part of the novel gene of interest was obtained for each strain.

TABLE 4

(Strain)/

Probe Seq I.D.

Gene Name

Number

RFLP Data (approximate band sizes)

(PS)10E1

18

EcoRI: 4 and 9 kbp, EcoRV: 4.5 and 6

kbp, KpnI: 12 and 24 kbp, SacI: 13 and

24 kbp, SalI: >23 kbp, XbaI: 5 and 15 kbp

(PS)31J2

20

ApaI: >23 kbp, BgIII: 6.5 kbp, PstI: >23

kbp, SacI: >23 kbp, SalI: >23 kbp, XbaI:

5 kbp

(PS)33D2

22

EcoRI: 10 kbp, EcoRV: 15 kbp, HindIII:

18 kbp, KpnI: 9.5 kbp, PstI: 8 kbp

(PS)66D3

24

BamHI: 4.5 kbp, HindIII: >23 kbp, KpnI:

23 kbp, PstI: 15 kbp, XbaI: >23 kbp

(PS)68F

26

EcoRI: 8.5 and 15 kbp, EcoRV: 7 and 18

kbp, HindIII: 2.1 and 9.5 kbp, PstI: 3 and

18 kbp, XbaI: 10 and 15 kbp

(PS)69AA2

28

EcoRV: 9.5 kbp, HindIII: 18 kbp, KpnI:

23 kbp, NheI: >23 kbp, PstI: 10 kbp, SalI:

>23 kbp

(PS)168G1

30

EcoRI: 10 kbp, EcoRV: 3.5 kbp, NheI: 20

kbp, PstI: 20 kbp, SalI: >23 kbp, XbaI: 15

kbp

(PS)177I8

33

BamHI: >23 kbp, EcoRI: 10 kbp, HindIII:

2 kbp, SalI:>23 kbp, XbaI: 3.5 kbp

(PS)185AA2

35

EcoRI: 7 kbp, EcoRV: 10 kbp (&3.5

kbp?), NheI: 4 kbp, PstI: 3 kbp, SalI: >23

kbp, XbaI: 4 kbp

(PS)196F3

37

EcoRI: 8 kbp, EcoRV: 9 kbp, NheI: 18

kbp, PstI: 18 kbp, SalI: 20 kbp, XbaI: 7

kbp

(PS)196J4

39

BamHI: >23 kbp, EcoRI: 3.5 and 4.5 kbp,

PstI: 9 and 24 kbp, SalI: >23 kbp, XbaI:

2.4 kbp and 12 kbp

(PS)197T1

41

HindIII: 10 kbp, KpnI: 20 kbp, PstI: 20

kbp, SacI: 20 kbp, SpeI: 15 kbp, XbaI: 5

kbp

(PS)197U2

43

EcoRI: 5 kbp, EcoRV: 1.9 kbp, NheI: 20

kbp, PstI: 23 kbp, SalI: >23 kbp, XbaI: 7

kbp

(PS)202E1

45

EcoRV: 7 kbp, KpnI: 12 kbp, NheI: 10

kbp, PstI: 15 kbp, SalI: 23 kbp, XbaI: 1.8

kbp

KB33

47

EcoRI: 9 kbp, EcoRV: 6 kbp, HindIII: 8

kbp, KpnI: >23 kbp, NheI: 22 kbp, SalI:

>23 kbp

KB38

48

BamHI: 5.5 kbp, EcoRV: 22 kbp, HindIII:

2.2 kbp, NheI: 20 kbp PstI: >23 kbp

In separate experiments, alternative probes for MIS and WAR genes were used to detect novel toxin genes on Southern blots of genomic DNA by

32

P autoradiography or by non-radioactive methods using the DIG nucleic acid labeling and detection system (Boehringer Mannheim; Indianapolis, Ind.). DNA fragments approximately 2.6 kbp (PS177C8 MIS toxin gene; SEQ ID NO. 31) and 1.3 kbp (PS177C8 WAR toxin gene; SEQ ID NO. 51) in length were PCR amplified from plasmid pMYC2450 and used as the probes for all strains listed. Fragments were gel purified and approximately 25 ng of each DNA fragment was randomly labeled with

32

P for radioactive detection or approximately 300 ng of each DNA fragment was randomly labeled with the DIG High Prime kit for nonradioactive detection. Hybridization of immobilized DNA with randomly labeled

32

P probes were performed in standard formamide conditions: 50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 0.1 mg/ml sonicated sperm DNA at 42° C. overnight. Blots were washed under low stringency in 2×SSC, 0.1% SDS at 42° C. and exposed to film. RFLP data showing DNA bands containing all or part of the novel gene of interest was obtained for each strain.

RFLP data using Probe 177C8-MIS (SEQ ID NO. 31) were as follows:

TABLE 5

RFLP

RFLP Data (approximate band size in

Class

Strain Name(s)

base pairs)

A

177C8, 74H3, 66D3

HindIII: 2,454; 1,645

XbaI: 14,820; 9,612; 8,138; 5,642;

1,440

B

177I8

HindIII: 2,454

XbaI: 3,500 (very faint 7,000)

C

66D3

HindIII: 2,454 (faint 20,000)

XbaI: 3,500 (faint 7,000)

D

28M, 31F2, 71G5,

HindIII: 11,738; 7,614

71G7, 71I1, 71N1,

XbaI: 10,622; 6,030

146F, 185Y2, 201JJ7,

KB73, KB68B46-2,

KB71A35-4,

KB71A116-1

D

1

70B2, 71C2

HindIII: 11,738; 8,698; 7,614

XbaI: 11,354; 10,622; 6,030

E

KB68B51-2, KB68B55-2

HindIII: 6,975; 2,527

XbaI: 10,000; 6,144

F

KB53A49-4

HindIII: 5,766

XbaI: 6,757

G

86D1

HindIII: 4,920

XbaI: 11,961

H

HD573B, 33F1, 67B3

HindIII: 6,558; 1,978

XbaI: 7,815; 6,558

I

205C, 40C1

HindIII: 6,752

XbaI: 4,618

J

130A3, 143A2, 157C1

HindIII: 9,639; 3,943, 1,954; 1,210

XbaI: 7,005; 6,165; 4,480; 3,699

K

201Z

HindIII: 9,639; 4,339

XbaI: 7,232; 6,365

L

71G4

HindIII: 7,005

XbaI: 9.639

M

KB42A33-8,

HindIII: 3,721

KB71A72-1,

XbaI: 3,274

KB71A133-11

N

KB71A134-2

HindIII: 7,523

XbaI: 10,360; 3,490

O

KB69A125-3,

HindIII: 6,360; 3,726; 1,874; 1,098

KB69A127-7,

XbaI: 6,360; 5,893; 5,058; 3,726

KB69A136-2,

KB71A20-4

RFLP data using Probe 177C8-WAR (SEQ ID NO. 51) were as follows:

TABLE 6

RFLP

RFLP Data (approximate band

Class

Strain Name(s)

size in base pairs)

A

177C8, 74H3

HindIII: 3,659, 2,454, 606

XbaI: 5,457, 4,469, 1,440,

966

B

17718, 66D3

data unavailable

C

28M, 31F2, 71G5, 71G7, 71I1,

HindIII: 7,614

71N1, 146F, 185Y2, 201JJ7,

XbaI: 10,982, 6,235

KB73, KB68B46-2, KB71A35-4,

KB71A116-1

C

1

70B2, 71C2

HindIII: 8,698, 7,614

XbaI: 11,354, 6,235

D

KB68B51-2, KB68B55-2

HindIII: 7,200

Xbal: 6,342 (and 11,225 for

51-2)(and 9,888 for 55-2)

E

KB53A49-4

HindIII: 5,766

XbaI: 6,757

F

HD573B, 33F1, 67B3

HindIII: 3,348, 2,037

(and 6,558 for HDS73B only)

XbaI: 6,953 (and7,815, 6,185

for HD573B only)

G

205C, 40C1

HindIII: 3,158

XbaI: 6,558, 2,809

H

130A3, 143A2, 157C1

HindIII: 4,339, 3,361, 1,954,

660, 349

XbaI: 9.043, 4,203, 3,583,

2,958, 581, 464

I

201Z

HindIII: 4,480, 3,819, 703

XbaI: 9,336, 3,256, 495

I

71G4

HindIII: 7,005

XbaI: 9,639

K

KB42A33-8, K1B71A72-1,

no hybridization signal

KB71A133-11

L

KB71A134-2

HindIII: 7,523

XbaI: 10,360

M

KB69A125-3, KB69A127-7,

HindIII: 5,058; 3,726; 3,198;

KB69A136-2,

2,745; 257

KB71A20-4

XbaI: 5,255; 4,341; 3,452;

1,490; 474

EXAMPLE 10

Use of Additional PCR Primers for Characterizing and/or Identifying Novel Genes

Another set of PCR primers can be used to identify additional novel genes encoding pesticidal toxins. The sequences of these primers were as follows:

ICON-forward:

CTTGAYTTTAAARATGATRTA (SEQ ID NO. 49)

ICON-reverse:

AATRGCSWATAAATAMGCACC (SEQ ID NO. 50)

These primers can be used in PCR procedures to amplify a fragment having a predicted size of about 450 bp.

Strains PS177C8, PS177I8, and PS66D3 were screened and were found to have genes amplifiable with these ICON primers. A sequence of a toxin gene from PS177C8 is shown in SEQ ID NO. 51. An amino acid sequence of the 177C8-ICON toxin is shown in SEQ ID NO. 52.

EXAMPLE 11

Use of Mixed Primer Pairs to Characterize and/or Identify Toxin Genes

Various combinations of the primers described herein can be used to identify and/or characterize toxin genes. PCR conditions can be used as indicated below:

SEQ ID NO.

SEQ. ID NO.

SEQ ID NO. 16/17

49/50

49/17

Pre-denature

94° C. 1 min.

94° C. 1 min.

94° C. 1 min.

Program

94° C. 1 min.

94° C. 1 min.

94° C. 1 min.

Cycle

42° C. 2 min.

42° C. 2 min.

42° C. 2 min.

72° C. 3 min. +

72° C. 3 min. +

72° C. 3 min. +

5 sec/cycl

5 sec/cycl

5 sec/cycl

Repeat cycle

Repeat cycle

Repeat cycle

times

29 times

29 times

Hold 4° C.

Hold 4° C.

Hold 4° C.

Using the above protocol, a strain harboring a MIS-type of toxin would be expected to yield a 1000 bp fragment with the SEQ ID NO. 16/17 primer pair. A strain harboring a WAR-type of toxin would be expected to amplify a fragment of about 475 bp with the SEQ ID NO. 49/50 primer pair, or a fragment of about 1800 bp with the SEQ ID NO. 49/17 primer pair. The amplified fragments of the expected size were found in four strains. The results are reported in Table 7.

TABLE 7

Approximate Amplified Fragment Sizes (bp)

SEQ ID NO.

SEQ ID NO.

Strain

16/17

SEQ ID NO. 49/50

49/17

PS66D3

1000

900, 475

1800

PS177C8

1000

475

1800

PS17718

1000

900, 550, 475

1800

PS217U2

1000

2500, 1500, 900, 475

no band detected

EXAMPLE 12

Characterization and/or Identification of WAR Toxins

In a further embodiment of the subject invention, pesticidal toxins can be characterized and/or identified by their level of reactivity with antibodies to pesticidal toxins exemplified herein. In a specific embodiment, antibodies can be raised to WAR toxins such as the toxin obtainable from PS177C8a. Other WAR toxins can then be identified and/or characterized by their reactivity with the antibodies. In a preferred embodiment, the antibodies are polyclonal antibodies. In this example, toxins with the greatest similarity to the 177C8a-WAR toxin would have the greatest reactivity with the polyclonal antibodies. WAR toxins with greater diversity react with the 177C8a polyclonal antibodies, but to a lesser extent. Toxins which immunoreact with polyclonal antibodies raised to the 177C8a WAR toxin can be obtained from, for example, the isolates designated PS177C8a, PS177I8, PS66D3, KB68B55-2, PS185Y2, PS146F, KB53A49-4, PS175I4, KB68B51-2, PS28K1, PS31F2, KB58B46-2, PS146D, PS74H3, PS28M, PS71G6, PS71G7, PS71I1, PS71N1, PS201JJ7, KB73, KB68B46-2, KB71A35-4, KB71A116-1, PS70B2, PS71C2, PS86D1, HD573B, PS33F1, PS67B3, PS205C, PS40C1, PS130A3, PS143A2, PS157C1, PS201Z, PS71G4, KB42A33-8, KB71A72-1, KB71A133-11, KB71A134-2, KB69A125-3, KB69A127-7, KB69A136-2, and KB71A20-4. Such diverse WAR toxins can be further characterized by, for example, whether or not their genes can be amplified with ICON primers. For example, the following isolates do not have polynucleotide sequences which are amplified by ICON primers: PS177C8a, PS177I8, PS66D3, KB68B55-2, PS185Y2, PS146F, KB53A49-4, PS175I4, KB68B51-2, PS28K1, PS31F2, KB58B46-2, and PS146D. Of these, isolates PS28K1, PS31F2, KB68B46-2, and PS146D show the weakest antibody reactivity, suggesting advantageous diversity.

EXAMPLE 13

Bioassays for Activity Against Lepidopterans and Coleopterans

Biological activity of the toxins and isolates of the subject invention can be confirmed using standard bioassay procedures. One such assay is the budworm-bollworm (

Heliothis virescens

[Fabricius] and

Helicoverpa zea

[Boddie]) assay. Lepidoptera bioassays were conducted with either surface application to artificial insect diet or diet incorporation of samples. All Lepidopteran insects were tested from the neonate stage to the second instar. All assays were conducted with either toasted soy flour artificial diet or black cutworm artificial diet (BioServ, Frenchtown, N.J.).

Diet incorporation can be conducted by mixing the samples with artificial diet at a rate of 6 mL suspension plus 54 mL diet. After vortexing, this mixture is poured into plastic trays with compartmentalized 3-ml wells (Nutrend Container Corporation, Jacksonville, Fla.). A water blank containing no B. t. serves as the control. First instar larvae (USDA-ARS, Stoneville, Miss.) are placed onto the diet mixture. Wells are then sealed with sheeting (MYLAR; ClearLam Packaging, IL) using a tacking iron, and several pinholes are made in each well to provide gas exchange. Larvae were held at 25° C. for 6 days in a 14:10 (light:dark) holding room. Mortality and stunting are recorded after six days.

Bioassay by the top load method utilizes the same sample and diet preparations as listed above. The samples are applied to the surface of the insect diet. In a specific embodiment, surface area ranged from 0.3 to approximately 0.8 cm

2

depending on the tray size, 96 well tissue culture plates were used in addition to the format listed above. Following application, samples are allowed to air dry before insect infestation. A water blank containing no B. t. can serve as the control. Eggs are applied to each treated well and were then sealed with sheeting (MYLAR; ClearLam Packaging, IL) using a tacking iron, and pinholes are made in each well to provide gas exchange. Bioassays are held at 25° C. for 7 days in a 14:10 (light:dark) or 28° C. for 4 days in a 14:10 (light:dark) holding room. Mortality and insect stunting are recorded at the end of each bioassay.

Another assay useful according to the subject invention is the Western corn rootworm assay. Samples can be bioassayed against neonate western corn rootworm larvae (

Diabrotica virgifera virgifera

) via top-loading of sample onto an agar-based artificial diet at a rate of 160 ml/cm

2

. Artificial diet can be dispensed into 0.78 cm

2

wells in 48-well tissue culture or similar plates and allowed to harden. After the diet solidifies, samples are dispensed by pipette onto the diet surface. Excess liquid is then evaporated from the surface prior to transferring approximately three neonate larvae per well onto the diet surface by camel's hair brush. To prevent insect escape while allowing gas exchange, wells are heat-sealed with 2-mil punched polyester film with 27HT adhesive (Oliver Products Company, Grand Rapids, Mich.). Bioassays are held in darkness at 25 ° C., and mortality scored after four days.

Analogous bioassays can be performed by those skilled in the art to assess activity against other pests, such as the black cutworm (

Agrotis ipsilon

).

Results are shown in Table 8.

TABLE 8

Genetics and function of concentrated B.t. supernatants screened for lepidopteran and coleopteran activity

Approx.

ca. 80-100

339 bp PCR

Total Protein

kDa protein

H. virescens

H. Zen

Diabrotica

Strain

fragment

(μg/cm

2

)

(μg/cm

2

)

% mortality

Stunting

% mortality

Stunting

% mortality

PS31G1

+

8.3

2.1

70

yes

39

yes

NT

PS49C

+

13.6

1.5

8

yes

8

no

NT

PS80JJ1

—

8.0

NT

18

no

13

no

NT

PS80JJ1 (#2)

—

35

NT

—

—

—

—

43

PS81A2 (#1)

+

30.3

2.3

100

yes

38

yes

NT

PS81A2 (#2)

+

18.8

1.6

38

yes

13

no

NT

PS81F

++

26

5.2

100

yes

92

yes

NT

PS81I

+

10.7

1.7

48

yes

13

no

NT

PS86B1 (#1)

—

23.2

4.5

17

no

13

no

—

PS86B1 (#2)

—

90

17.5

—

—

—

—

35

PS86B1 (#3)

—

35

6.8

—

—

—

—

10

PS122D3 (#1)

—

33.2

1.8

21

no

21

no

—

PS122D3 (#2)

—

124

6.7

—

—

—

—

45

PS122D3 (#3)

—

35

1.9

—

—

—

—

16

PS123D1 (#1)

—

10.7

NT

0

no

0

no

—

PS123D1 (#2)

—

69

NT

—

—

—

—

54

PS123D1 (#3)

—

35

NT

—

—

—

—

21

PS123D1 (#4)

—

17.8

NT

5

no

4

no

NT

PS149B1 (#1)

NT

9

NT

0

no

0

yes

NT

PS149B1 (#2)

NT

35

NT

—

—

—

—

50

PS157C1 (#1)

—

24

2

43

yes

13

yes

—

PS157C1 (#2)

—

93

8

—

—

—

—

40

PS157C1 (#3)

—

35

3

—

—

—

—

18

PS185L2 (#1)

—

2

NT

8

no

0

no

NT

PS185L2 (#2)

—

3

NT

10

no

25

no

NT

PS185U2

+

23.4

2.9

100

yes

100

yes

NT

PS192M4

+

10.7

2.0

9

no

4

yes

NT

HD129

+

44.4

4.9

100

yes

50

yes

NT

Javelin 1990

++

43.2

3.6

100

yes

96

yes

NT

water

0-8

—

0-4

—

12

*NT = not tested

EXAMPLE 14

Results of Western Corn Rootworm Bioassays and Further Characterization of the Toxins

Concentrated liquid supernatant solutions, obtained according to the subject invention, were tested for activity against Western corn rootworm (WCRW). Supernatants from the following isolates were found to cause mortality against WCRW: PS10E1, PS31F2, PS31J2, PS33D2, PS66D3, PS68F, PS80JJ1, PS146D, PS175I4, PS177I8, PS196J4, PS197T1, PS197U2, KB33, KB53A49-4, KB68B46-2, KB68B51-2, KB68B55-2, PS177C8, PS69AA2, KB38, PS196F3, PS168G1, PS202E1, PS217U2 and PS185AA2.

Supernatants from the following isolates were also found to cause mortality against WCRW: PS205A3, PS185V2, PS234E1, PS71G4, PS248N10, PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2, KB69A125-1, KB69A125-3, KB69A125-5, KB69A127-7, KB69A132-1, KB69B2-1, KB70B5-3, KB71A125-15, and KB71A35-6; it was confirmed that this activity was heat labile. Furthermore, it was determined that the supernatants of the following isolates did not react (yielded negative test results) with the WAR antibody (see Example 12), and did not react with the MIS (SEQ ID NO. 31) and WAR (SEQ ID NO. 51) probes: PS205A3, PS185V2, PS234E1, PS71G4, PS248N10, PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2, KB69A125-1, KB69A125-5, KB69A132-1, KB69B2-1, KB70B5-3, KB71A125-15, and KB71A35-6; the supernatants of isolates KB69A125-3 and KB69A127-7 yielded positive test results.

EXAMPLE 15

Results of Budworm/Bollworm Bioassays

Concentrated liquid supernatant solutions, obtained according to the subject invention, were tested for activity against

Heliothis virescens

(H.v.) and

Helicoverpa zea

(H.z.). Supernatants from the following isolates were tested and were found to cause mortality against H.v.: PS157C1, PS31G1, PS49C, PS81F, PS81I, Javelin 1990, PS158C2, PS202S, PS36A, HD110, and HD29. Supernatants from the following isolates were tested are were found to cause significant mortality against H.z.: PS31G1, PS49C, PS81F, PS81I, PS157C1, PS158C2, PS36A, HD110, and Javelin 1990.

EXAMPLE 16

Target Pests

Toxins of the subject invention can be used, alone or in combination with other toxins, to control one or more non-mammalian pests. These pests may be, for example, those listed in Table 9. Activity can readily be confirmed using the bioassays provided herein, adaptations of these bioassays, and/or other bioassays well known to those skilled in the art.

TABLE 9

Target pest species

ORDER/Common Name

Latin Name

LEPIDOPTERA

European Corn Borer

Ostrinia nubilalis

European Corn Borer resistant to Cryl A

Ostrinia nubilalis

Black Cutworm

Agrotis ipsilon

Fall Armyworm

Spodoptera frugiperda

Southwestern Corn Borer

Diatraea grandiosella

Corn Earworm/Bollworm

Helicoverpa zea

Tobacco Budworm

Heliothis virescens

Tobacco Budworm Rs

Heliothis virescens

Sunflower Head Moth

Homeosoma ellectellum

Banded Sunflower Moth

Cochylis hospes

Argentine Looper

Rachiplusia nu

Spilosoma

Spilosoma virginica

Bertha Armyworm

Mamestra configurata

Diamondback Moth

Plutella xylostells

COLEOPTERA

Red Sunflower Seed Weevil

Smicronyx fulvus

Sunflower Stem Weevil

Cylindrocopturus adspersus

Sunflower Beetle

Zygoramma exclamationis

Canola Flea Beetle

Phyllotreta cruciferae

Western Corn Rootworm

Diabrotica virgifera virgifera

DIPTERA

Hessian Fly

Mayetiola destructor

HOMOPTERA

Greenbug

Schizaphis graminum

HEMIPTERA

Lygus Bug

Lygus lineolaris

NEMATODA

Heterodera glycines

EXAMPLE 17

Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants with genes encoding the insecticidal toxin of the present invention. The transformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in

E. coli

and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Bacillus toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into

E. coli.

The

E. coli

cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In:

The Binary Plant Vector System,

Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al.,

Crit. Rev. Plant Sci.

4:1-46; and An et al. (1985)

EMBO J.

4:277-287.

Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using

Agrobacterium tumefaciens

or

Agrobacterium rhizogenes

as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into

Agrobacterium tumefaciens

by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in

E. coli

and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978]

Mol. Gen. Genet.

163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with

Agrobacterium tumefaciens

or

Agrobacterium rhizogenes

for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives. In biolistic transformation, plasmid DNA or linear DNA can be employed.

The transformed cells are regenerated into morphologically normal plants in the usual manner. If a transformation event involves a germ line cell, then the inserted DNA and corresponding phenotypic trait(s) will be transmitted to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic Bacillus genes for use in plants are known in the art.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

144

29 base pairs

nucleic acid

single

linear

DNA (genomic)

1
GARCCRTGGA AAGCAAATAA TAARAATGC 29

33 base pairs

nucleic acid

single

linear

DNA (genomic)

2
AAARTTATCT CCCCAWGCTT CATCTCCATT TTG 33

2375 base pairs

nucleic acid

single

linear

DNA (genomic)

36a

3
ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT60
AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAAC120
GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGA180
ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAA240
TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTT300
AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAA360
ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGA420
TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGT480
CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAA540
GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGG600
TCTCCTGCAA ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAAC660
AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGG720
AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAA780
GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGC840
CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGA900
ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGT960
AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAG1020
AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GATTGGGT1080
GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAA1140
TATCAAGTCG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTAT1200
TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAAT1260
GTAATTACTA AAATTGATTT CACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAG1320
AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAA1380
GAAGCGGAGT ATAAAACGTT AAGTGCTAAT GATGATGGGG TGTATATGCC GTTAGGTG1440
ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATT1500
AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACT1560
AGCAATAAAG AAACTAAATT GATCGTCCCG CCAAGTGGTT TTATTAGCAA TATTGTAG1620
AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGCGT1680
GTAGATCATA CAGGCGGAGT GAATGGAACT AAAGCTTTAT ATGTTCATAA GGACGGAG1740
ATTTCACAAT TTATTGGAGA TAATTTAAAA CCGAAAACTG AGTATGTAAT CCAATATA1800
GTTAAAGGAA AACCTTCTAT TCATTTAATA GATGAAAATA CTGGATATAT TCATTATG1860
GATACAAATA ATAATTTAGA AGATTATCAA ACTATTAATA AACGTTTTAC TACAGGAA1920
GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAG1980
AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTA2040
ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTT2100
CAGGGAGGAC GAGGGATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATA2160
GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGT2220
TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATGTTCAC TACAAAAT2280
GAGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTATATGG TGGTCCTA2340
GTACATTTTT ACGATGTCTC TATTAAGTAA CCCAA 2375

790 amino acids

amino acid

single

linear

protein

36a

4
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Ph
1 5 10 15
Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys As
20 25 30
Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Le
35 40 45
Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Ly
50 55 60
Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly As
65 70 75 80
Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gl
85 90 95
Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Th
100 105 110
Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Va
115 120 125
Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Ly
130 135 140
Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Va
145 150 155 160
Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Il
165 170 175
Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Th
180 185 190
Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asn Ile Leu Asp Gl
195 200 205
Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Va
210 215 220
Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gl
225 230 235 240
Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Il
245 250 255
Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Ty
260 265 270
Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Th
275 280 285
Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Th
290 295 300
Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Va
305 310 315 320
Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Al
325 330 335
Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Ly
340 345 350
Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Th
355 360 365
Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val As
370 375 380
Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Le
385 390 395 400
Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Ph
405 410 415
Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Ly
420 425 430
Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gl
435 440 445
Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Ty
450 455 460
Lys Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Va
465 470 475 480
Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Al
485 490 495
Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Ar
500 505 510
Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Il
515 520 525
Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Il
530 535 540
Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Ty
545 550 555 560
Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val Hi
565 570 575
Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Asn Leu Lys Pro Ly
580 585 590
Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile Hi
595 600 605
Leu Ile Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn As
610 615 620
Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Th
625 630 635 640
Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Gl
645 650 655
Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Ly
660 665 670
Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gl
675 680 685
Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Ar
690 695 700
Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Ar
705 710 715 720
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Se
725 730 735
Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Va
740 745 750
Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Gl
755 760 765
Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Ty
770 775 780
Asp Val Ser Ile Lys Pro
785 790

2370 base pairs

nucleic acid

single

linear

DNA (genomic)

81F

5
ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT60
AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAAC120
GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGA180
ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAA240
TTAAATACAG AATTATCTAA AGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTT300
AATGATGTTG ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAA360
ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGA420
TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGT480
CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAA540
GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGG600
TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAAC660
AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGG720
AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAA780
GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGC840
CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGA900
ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGT960
AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAG1020
AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GGTTGGGT1080
GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAA1140
TATCAAGTTG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTAT1200
TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAAT1260
GTAATTACTA AAATTGATTT TACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAG1320
AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAA1380
GAAGCGGAGT ATAGAACGTT AAGTGCTAAT GATGATGGAG TGTATATGCC GTTAGGTG1440
ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATT1500
AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACT1560
AGCAATAAAG AAACTAAATT GATCGTCCCG CCCAGTGGTT TTATTAAAAA TATTGTAG1620
AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGAGT1680
GTAGATCATA CAGGCGGAGT GAATGGRACT AAAGCTTTAT ATGTTCATAA GGACGGAG1740
ATTTCACAAT TTATTGGAGA TAAGTTAAAA CCGAAAACTG AGTATGTAAT CCAATATA1800
GTTAAAGGAA AACCTTCTAT TCATTTAAAA GATGAAAATA CTGGATATAT TCATTATG1860
GATACAAATA ATAATTTAGA AGATTATCAA ACTATTACTA AACGTTTTAC TACAGGAA1920
GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAG1980
AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTA2040
ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTT2100
CAGGGAGGAC GAGGAATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATA2160
GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGT2220
TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATTTTCAC TACAAAAT2280
GGGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTAAATGG TGGCCCTA2340
GTACAGTTTC CCGATGTCTC TATTAAGTAA 2370

789 amino acids

amino acid

single

linear

protein

81F

6
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Ph
1 5 10 15
Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys As
20 25 30
Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Le
35 40 45
Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Ly
50 55 60
Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly As
65 70 75 80
Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gl
85 90 95
Asn Gln Val Leu Asn Asp Val Asp Asn Lys Leu Asp Ala Ile Asn Th
100 105 110
Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Va
115 120 125
Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Ly
130 135 140
Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Va
145 150 155 160
Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Il
165 170 175
Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Th
180 185 190
Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Gl
195 200 205
Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Va
210 215 220
Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gl
225 230 235 240
Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Il
245 250 255
Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Ty
260 265 270
Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Th
275 280 285
Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Th
290 295 300
Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Va
305 310 315 320
Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Al
325 330 335
Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Ly
340 345 350
Pro Gly His Ala Leu Val Gly Phe Glu Ile Ser Asn Asp Ser Ile Th
355 360 365
Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val As
370 375 380
Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Le
385 390 395 400
Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Ph
405 410 415
Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Ly
420 425 430
Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gl
435 440 445
Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Ty
450 455 460
Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Va
465 470 475 480
Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Al
485 490 495
Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Ar
500 505 510
Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Il
515 520 525
Val Pro Pro Ser Gly Phe Ile Lys Asn Ile Val Glu Asn Gly Ser Il
530 535 540
Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Glu Ty
545 550 555 560
Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val Hi
565 570 575
Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Ly
580 585 590
Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile Hi
595 600 605
Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn As
610 615 620
Asn Leu Glu Asp Tyr Gln Thr Ile Thr Lys Arg Phe Thr Thr Gly Th
625 630 635 640
Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Gl
645 650 655
Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Ly
660 665 670
Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gl
675 680 685
Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Ar
690 695 700
Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Ar
705 710 715 720
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Se
725 730 735
Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Va
740 745 750
Ser Glu Ile Phe Thr Thr Lys Phe Gly Lys Asp Asn Phe Tyr Ile Gl
755 760 765
Leu Ser Gln Gly Asn Asn Leu Asn Gly Gly Pro Ile Val Gln Phe Pr
770 775 780
Asp Val Ser Ile Lys
785

2375 base pairs

nucleic acid

single

linear

DNA (genomic)

Jav90

7
ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT60
AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAAC120
GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGA180
ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAA240
TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTT300
AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAA360
ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGA420
TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGT480
CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAA540
GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGG600
TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAAC660
AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGG720
AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAA780
GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGC840
CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGA900
ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGT960
AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAG1020
AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GATTGGGT1080
GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAA1140
TATCAAGTCG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTAT1200
TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAAT1260
GTAATTACTA AAATTGATTT CACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAG1320
AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAA1380
GAAGCGGAGT ATAGAACGTT AAGTGCTAAT GATGATGGGG TGTATATGCC GTTAGGTG1440
ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATT1500
AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACT1560
AGCAATAAAG AAACTAAATT GATYGTCCCG CCAAGTGGTT TTATTAGCAA TATTGTAG1620
AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGCGT1680
GTAGATCATA CAGGCGGAGT GAATGGAACT AAAGCTTTAT ATGTTCATAA GGACGGAG1740
ATTTCACAAT TTATTGGAGA TAAGTTAAAA CCGAAAACTG AGTATGTAAT CCAATATA1800
GTTAAAGGAA AACCTTCTAT TCATTTAAAA GATGAAAATA CTGGATATAT TCATTATG1860
GATACAAATA ATAATTTAGA AGATTATCAA ACTATTAATA AACGTTTTAC TACAGGAA1920
GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAG1980
AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTA2040
ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTT2100
CAGGGAGGAC GAGGGATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATA2160
GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGT2220
TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATGTTCAC TACAAAAT2280
GAGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTATATGG TGGTCCTA2340
GTACATTTTT ACGATGTCTC TATTAAGTAA CCCAA 2375

790 amino acids

amino acid

single

linear

protein

Jav90

8
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Ph
1 5 10 15
Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys As
20 25 30
Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Le
35 40 45
Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Ly
50 55 60
Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly As
65 70 75 80
Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gl
85 90 95
Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Th
100 105 110
Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Va
115 120 125
Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Ly
130 135 140
Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Va
145 150 155 160
Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Il
165 170 175
Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Th
180 185 190
Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Gl
195 200 205
Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Va
210 215 220
Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gl
225 230 235 240
Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Il
245 250 255
Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Ty
260 265 270
Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Th
275 280 285
Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Th
290 295 300
Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Va
305 310 315 320
Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Al
325 330 335
Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Ly
340 345 350
Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Th
355 360 365
Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val As
370 375 380
Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Le
385 390 395 400
Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Ph
405 410 415
Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Ly
420 425 430
Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gl
435 440 445
Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Ty
450 455 460
Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Va
465 470 475 480
Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Al
485 490 495
Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Ar
500 505 510
Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Il
515 520 525
Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Il
530 535 540
Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Ty
545 550 555 560
Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val Hi
565 570 575
Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Ly
580 585 590
Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile Hi
595 600 605
Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn As
610 615 620
Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Th
625 630 635 640
Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Gl
645 650 655
Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Ly
660 665 670
Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gl
675 680 685
Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Ar
690 695 700
Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Ar
705 710 715 720
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Se
725 730 735
Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Va
740 745 750
Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Gl
755 760 765
Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Ty
770 775 780
Asp Val Ser Ile Lys Pro
785 790

47 base pairs

nucleic acid

single

linear

DNA (genomic)

9
GCTCTAGAAG GAGGTAACTT ATGAACAAGA ATAATACTAA ATTAAGC 47

2035 base pairs

nucleic acid

single

linear

DNA (genomic)

158C2

10
ATGAACAAGA ATAATACTAA ATTAAGCGCA AGGGCCTACC GAGTTTTATT GATTATTTTA60
ATGGCATTTA TGGATTTGCC ACTGGTATCA AAGACATTAT GAATATGATT TTTAAAACG120
ATACAGGTGG TAATCTAACC TTAGACGAAA TCCTAAAGAA TCAGCAGTTA CTAAATGAG180
TTTCTGGTAA ATTGGATGGG GTAAATGGGA GCTTAAATGA TCTTATCGCA CAGGGAAAC240
TAAATACAGA ATTAGCTAAG CAAATCTTAA AAGTTGCAAA TGAACAAAAT CAAGTTTTA300
ATGATGTTAA TAACAAACTA GACTGCGATA AATACGATGC TTAAAATATA TCTACCTAA360
ATTCACATCT ATGTTAAGTG ATGTACTGAA GCCAAAATTA TGTGCTTAAG TCTTGCAAA420
TGGAATTACC TTTAAGTAAC ATCTGCACCT TGGCAAGAAA TCTCCGACAA GCTAGATAT480
ATTAACGTAA ATGTGCTTAT TAACTCTACG CTTACTGAAA TTACACCTGC GTATCAACG540
ATTAAATATG TGAATGAAAA ATTTGACGAT TTAACTTTTG CTACAGAAAA CACTTTAAA600
GTAAAAAAGG ATAGCTCTCC TGCTGATATT CTTGACGAGT TAACTGAATT AACTGAACT660
GCGAAAAGTG TTACAAAAAA TGACGTGGAT GGTTTTGAAT TTTACCTTAA TACATTCCA720
GATGTAATGG TGGGAAATAA TTTATTCGGT CGTTCAGCTT TAAAAACTGC TTCGGAATT780
ATTGCTAAAG AAAATGTGAA AACAAGTGGC AGTGAAGTAG GAAATGTTTA TAATTTCTT840
ATTGTATTAA CAGCTCTACA AGCAAAAGCT TTTCTTACTT TAACAACATG CCGAAAATT900
TTAGGCTTAG CAGATATTGA TTATACTTCT ATCATGAATG AGCATTTAAA TAAGGAAAA960
GAGGAATTTA GAGTAAACAT CCTTCCCACA CTTTCTAATA CCTTTTCTAA TCCTAATT1020
GCAAAAGCTA AGGGAAGTAA TGAAGATACA AAGATGATTG TGGAAGCTAA ACCAGGAT1080
GTTTTGGTTG GATTTGAAAT GAGCAATAAT TCAATTACAG TATTAAAAGC ATATCAAG1140
AAGCTAAAAA AAGATTATCA AATTGATAAG GATTCGTTAT CAGAAATAAT ATATAGTA1200
TGATACGGAT AAATTATTAT GTCCGGATCA ATCTGAACAA TATATTATAC AAAGAACA1260
GCATTTCCAA ATGAATATGT TATTACTAAA ATTGCTTTTA CTAAAAAAAT GAACAGTT1320
AGGTATGAGG CGACAGCGAA TTTTTATGAT TCTTCTACAG GGGATATTGA TCTAAATA1380
ACAAAAGTAG AATCAAGTGA AGCGGAGTAT AGTATGCTAA AAGCTAGTGA TGATGAAG1440
TACATGCCGC TAGGTCTTAT CAGTGAAACA TTTTTAAATC CAATTAATGG ATTTAGGC1500
GCAGTCGATG AAAATTCCAG ACTAGTAACT TTAACATGTA GATCATATTT AAGAGAGA1560
TTGTTAGCGA CAGATTTAAA TAATAAAGAA ACTAAATTGA TTGTCCCACC TAATGTTT1620
ATTAGCAATA TTGTAGAGAA TGGAAATATA GAAATGGACA CCTTAGAACC ATGGAAGG1680
AATAATGAGA ATGCGAATGT AGATTATTCA GGCGGAGTGA ATGGAACTAG AGCTTTAT1740
GTTCATAAGG ATGGTGAATT CTCACATTTT ATTGGAGACA AGTTGAAATC TAAAACAG1800
TACTTGATTC GATATATTGT AAAAGGAAAA GCTTCTATTT TTTTAAAAGA TGAAAGAA1860
GAAAATTACA TTTACGAGGA TACAAATAAT AATTTAGAAG ATTATCAAAC TATTACTA1920
CGTTTTACTA CAGGAACTGA TTCGACAGGA TTTTATTTAT TTTTTACTAC TCAAGATG1980
AATGAAGCTT GGGGAGACAC TTTTTTTCTC TAGAAAGAGG TAACTTATGA ACAAG 2035

21 base pairs

nucleic acid

single

linear

DNA (genomic)

11
CATCCTCCCT ACACTTTCTA A 21

950 base pairs

nucleic acid

single

linear

DNA (genomic)

49C-ptl

12
AAACTAGAGG GAGTGATAAG GATGCGAAAA TCATTATGGA AGCTAAACCT GGATATGCTT60
TAGTTGGATT TGAAATAAGT AAGGATTCAA TTGCAGTATT AAAAGTTTAT CAGGCAAAG120
TAAAACACAA CTATCAAATT GATAAGGATT CGTTATCAGA AATTGTTTAT GGTGATATA180
ATAAATTATT ATGTCCGGAT CAATCTGAAC AAATGTATTA TACAAATAAA ATAGCATTT240
CAAATGAATA TGTTATCACT AAAATTGCTT TTACTAAAAA ACTGAACAGT TTAAGATAT300
AGGTCACAGC GAATTTTTAT GACTCTTCTA CAGGAGATAT TGATCTAAAT AAGAAAAAA360
TAGAATCAAG TGAAGCGGAG TTTAGTATGC TAAATGCTAA TAATGATGGT GTTTATATG420
CGATAGGTAC TATAAGTGAA ACATTTTTGA CTCCAATTAA TGGATTTGGC CTCGTAGTC480
ATGAAAATTC AAGACTAGTA ACTTTGACAT GTAAATCATA TTTAAGAGAG ACATTGTTA540
CAACAGACTT AAGTAATAAA GAAACTAAAC TGATTGTCCC ACCTAATGGT TTTATTAGC600
ATATTGTAGA AAATGGGAAC TTAGAGGGAG AAAACTTAGA GCCGTGGGAA AGCAAATAA660
AAAAATGCGT ATGTAGATCA TACCGGAGGT GTAAATGGAA CTAAAGTTTT ATATGTTCA720
GAGGATGGTG AGTTCTCACA ATTTATTGGG GATAAATTGA AATTGAAAAC AGAATATGT780
ATTCCATATA TTGTAAAGGG GAAAGCTGCT ATTTATTTAA AAGATGAAAA AAATGGGGA840
TACATATCAT GAAGAAACAT CATAATGCAA TTGAAGATTT TTCCAGCTGT AACTTCAAT900
ATGATTTTCG CATCCTTATC ATCCCTCTAG CTTTTTCATA ATAGGATAGA 950

20 base pairs

nucleic acid

single

linear

DNA (genomic)

13
AAATTATGCG CTAAGTCTGC 20

19 base pairs

nucleic acid

single

linear

DNA (genomic)

14
TTGATCCGGA CATAATAAT 19

176 base pairs

nucleic acid

single

linear

DNA (genomic)

49C-pt2

15
GTAAATTATG CGCTAAGTCT GCACCTTTTT TCACTGTTAC TAAACATCAC TTTTCCTATA60
TCCCCTTAGC TCTTATGGAT TATTGAGCAA ACTTATCTTG TTAATTACTA CTCCCCATC120
TATGCTAAAC AAAAACCAAA CAAACATTAT CTATTATATG TCCGGATCAA AATGTA 176

20 base pairs

nucleic acid

single

linear

DNA (genomic)

16
GGRTTAMTTG GRTAYTATTT 20

20 base pairs

nucleic acid

single

linear

DNA (genomic)

17
ATATCKWAYA TTKGCATTTA 20

1076 base pairs

nucleic acid

single

linear

DNA (genomic)

10E1

18
TGGGATTACT TGGATATTAT TTCCAGGATC AAAAGTTTCA GCAACTTGCT TTGATGGCAC60
ATAGACAAGC TTCTGATTTG GAAATCCCGA AAGATGACGT GAAACAGTTA CTATCCAAG120
AGCAGCAACA CATTCAATCT GTTAGATGGC TTGGCTATAT TCAGCCACCT CAAACAGGA180
ACTATGTATT GTCAACCTCA TCCGACCAAC AGGTCGTGAT TGAACTCGAT GGAAAAACC240
TTGTCAATCA AACTTCTATG ACAGAACCGA TTCAACTCGA AAAAGATAAG CTCTATAAA300
TTAGAATTGA ATATGTCCCA GAAGATACAA AAGAACAAGA GAACCTCCTT GACTTTCAG360
TCAACTGGTC GATTTCAGGA TCAGAGATAG AACCAATTCC GGAGAATGCT TTCCATTTA420
CAAATTTTTC TCGTAAACAA GATCAAGAGA AAATCATCCC TGAAACCAGT TTGTTTCAG480
AACAAGGAGA TGAGAAAAAA GTATCTCGCA GTAAGAGATC TTTAGCTACA AATCCTATC540
GTGATACAGA TGATGATAGT ATTTATGATG AATGGGAAAC GGAAGGATAC ACGATACGG600
AACAAATAGC AGTGAAATGG GACGATTCTA TGAAGGATAG AGGTTATACC AAATATGTG660
CAAACCCCTA TAAGTCTCAT ACAGTAGGAG ATCCATACAC AGATTGGGAA AAAGCGGCT720
GCCGTATCGA TAACGGTGTC AAAGCAGAAG CCAGAAATCC TTTAGTCGCG GCCTATCCA780
CTGTTGGTGT ACATATGGAA AGATTAATTG TCTCCGAAAA ACAAAATATA TCAACAGGG840
TTGGAAAAAC TGTATCTGCG TCTATGTCCG CAAGCAATAC CGCAGCGATT ACGGCAGGT900
TTGATGCAAC AGCCGGTGCC TCTTTACTCG GGCCATCTGG AAGTGTCACG GCTCATTTT960
CTTATACAGG ATCTAGTACA TCCACCGTTG AAGATAGCTC CAGCCGGAAT TGGAGTCA1020
ACCTTGGGAT CGATACGGGA CAATCTGCAT ATTTAAATGC CAAATGTACG ATATAA 1076

357 amino acids

amino acid

single

linear

peptide

10E1

19
Gly Leu Leu Gly Tyr Tyr Phe Gln Asp Gln Lys Phe Gln Gln Leu Al
1 5 10 15
Leu Met Ala His Arg Gln Ala Ser Asp Leu Glu Ile Pro Lys Asp As
20 25 30
Val Lys Gln Leu Leu Ser Lys Glu Gln Gln His Ile Gln Ser Val Ar
35 40 45
Trp Leu Gly Tyr Ile Gln Pro Pro Gln Thr Gly Asp Tyr Val Leu Se
50 55 60
Thr Ser Ser Asp Gln Gln Val Val Ile Glu Leu Asp Gly Lys Thr Il
65 70 75 80
Val Asn Gln Thr Ser Met Thr Glu Pro Ile Gln Leu Glu Lys Asp Ly
85 90 95
Leu Tyr Lys Ile Arg Ile Glu Tyr Val Pro Glu Asp Thr Lys Glu Gl
100 105 110
Glu Asn Leu Leu Asp Phe Gln Leu Asn Trp Ser Ile Ser Gly Ser Gl
115 120 125
Ile Glu Pro Ile Pro Glu Asn Ala Phe His Leu Pro Asn Phe Ser Ar
130 135 140
Lys Gln Asp Gln Glu Lys Ile Ile Pro Glu Thr Ser Leu Phe Gln Gl
145 150 155 160
Gln Gly Asp Glu Lys Lys Val Ser Arg Ser Lys Arg Ser Leu Ala Th
165 170 175
Asn Pro Ile Arg Asp Thr Asp Asp Asp Ser Ile Tyr Asp Glu Trp Gl
180 185 190
Thr Glu Gly Tyr Thr Ile Arg Glu Gln Ile Ala Val Lys Trp Asp As
195 200 205
Ser Met Lys Asp Arg Gly Tyr Thr Lys Tyr Val Ser Asn Pro Tyr Ly
210 215 220
Ser His Thr Val Gly Asp Pro Tyr Thr Asp Trp Glu Lys Ala Ala Gl
225 230 235 240
Arg Ile Asp Asn Gly Val Lys Ala Glu Ala Arg Asn Pro Leu Val Al
245 250 255
Ala Tyr Pro Thr Val Gly Val His Met Glu Arg Leu Ile Val Ser Gl
260 265 270
Lys Gln Asn Ile Ser Thr Gly Leu Gly Lys Thr Val Ser Ala Ser Me
275 280 285
Ser Ala Ser Asn Thr Ala Ala Ile Thr Ala Gly Ile Asp Ala Thr Al
290 295 300
Gly Ala Ser Leu Leu Gly Pro Ser Gly Ser Val Thr Ala His Phe Se
305 310 315 320
Tyr Thr Gly Ser Ser Thr Ser Thr Val Glu Asp Ser Ser Ser Arg As
325 330 335
Trp Ser Gln Asp Leu Gly Ile Asp Thr Gly Gln Ser Ala Tyr Leu As
340 345 350
Ala Lys Cys Thr Ile
355

1045 base pairs

nucleic acid

single

linear

DNA (genomic)

31J2

20
TGGGTTACTT GGGTATTATT TTAAAGGAAA AGATTTTAAT AATCTTACTA TATTTGCTCC60
AACACGTGAG AATACTCTTA TTTATGATTT AGAAACAGCG AATTCTTTAT TAGATAAGC120
ACAACAAACC TATCAATCTA TTCGTTGGAT CGGTTTAATA AAAAGCAAAA AAGCTGGAG180
TTTTACCTTT CAATTATCGG ATGATGAGCA TGCTATTATA GAAATCGATG GGAAAGTTA240
TTCGCAAAAA GGCCAAAAGA AACAAGTTGT TCATTTAGAA AAAGATAAAT TAGTTCCCA300
CAAAATTGAA TATCAATCTG ATAAAGCGTT AAACCCAGAT AGTCAAATGT TTAAAGAAT360
GAAATTATTT AAAATAAATA GTCAAAAACA ATCTCAGCAA GTGCAACAAG ACGAATTGA420
AAATCCTGAA TTTGGTAAAG AAAAAACTCA AACATATTTA AAGAAAGCAT CGAAAAGCA480
CTTGTTTAGC AATAAAAGTA AACGAGATAT AGATGAAGAT ATAGATGAGG ATACAGATA540
AGATGGAGAT GCCATTCCTG ATGTATGGGA AGAAAATGGG TATACCATCA AAGGAAGAG600
AGCTGTTAAA TGGGACGAAG GATTAGCTGA TAAGGGATAT AAAAAGTTTG TTTCCAATC660
TTTTAGACAG CACACTGCTG GTGACCCCTA TAGTGACTAT GAAAAGGCAT CAAAAGATT720
GGATTTATCT AATGCAAAAG AAACATTTAA TCCATTGGTG GCTGCTTTTC CAAGTGTCA780
TGTTAGCTTG GAAAATGTCA CCATATCAAA AGATGAAAAT AAAACTGCTG AAATTGCGT840
TACTTCATCG AATAATTGGT CCTATACAAA TACAGAGGGG GCATCTATTG AAGCTGGAA900
TGGACCAGAA GGTTTGTTGT CTTTTGGAGT AAGTGCCAAT TATCAACATT CTGAAACAG960
GGCCAAAGAG TGGGGTACAA CTAAGGGAGA CGCAACACAA TATAATACAG CTTCAGCA1020
ATATCTAAAT GCCAATGTAC GATAT 1045

348 amino acids

amino acid

single

linear

peptide

31J2

21
Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Th
1 5 10 15
Ile Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp Leu Glu Th
20 25 30
Ala Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln Ser Ile Ar
35 40 45
Trp Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe Thr Phe Gl
50 55 60
Leu Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly Lys Val Il
65 70 75 80
Ser Gln Lys Gly Gln Lys Lys Gln Val Val His Leu Glu Lys Asp Ly
85 90 95
Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pr
100 105 110
Asp Ser Gln Met Phe Lys Glu Leu Lys Leu Phe Lys Ile Asn Ser Gl
115 120 125
Lys Gln Ser Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu Ph
130 135 140
Gly Lys Glu Lys Thr Gln Thr Tyr Leu Lys Lys Ala Ser Lys Ser Se
145 150 155 160
Leu Phe Ser Asn Lys Ser Lys Arg Asp Ile Asp Glu Asp Ile Asp Gl
165 170 175
Asp Thr Asp Thr Asp Gly Asp Ala Ile Pro Asp Val Trp Glu Glu As
180 185 190
Gly Tyr Thr Ile Lys Gly Arg Val Ala Val Lys Trp Asp Glu Gly Le
195 200 205
Ala Asp Lys Gly Tyr Lys Lys Phe Val Ser Asn Pro Phe Arg Gln Hi
210 215 220
Thr Ala Gly Asp Pro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Le
225 230 235 240
Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Ph
245 250 255
Pro Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Gl
260 265 270
Asn Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Ty
275 280 285
Thr Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gl
290 295 300
Leu Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr Va
305 310 315 320
Ala Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln Tyr Asn Th
325 330 335
Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr
340 345

1641 base pairs

nucleic acid

single

linear

DNA (genomic)

33D2

22
CCAAAGGGGG NTTAAACCNG GANGGTTNNN TNNTTNNTTN TNGAANCCCA NTTGGAAACC60
CNATNAAATT CNTGGTTANT GGTNGTGAGT GNNTNTTTTA NCNGAGNTTG CCCNTTTGN120
TACCNGGATT TNAAGGCAGA ANTTNTTNNT NGCTNNTTAA AGGTTNTGNT TNTNANTGA180
TTTTTTNGGN TTTGCCCAAA AAACAAGGAT GAATCCTGTT ATTCCNCCCT NGAAAAAAT240
GAAACGGAAC AACGTGAGTA TGATAAACAT CTTTTACAAA CTGCGACATC TTGTTGAAA300
TGCCTTTTTT GAAAANNTAA AAGGTTTCGT GGCATTGCCA CACGTTATAC AAAAACCAC360
TCTGCTTTTA GAGGGGCTGT TACCTTGGCT GCTATTTCTC TGTGGTTGAA TCTCGTATA420
ACACTATCTA GTCTATACAT CTTATCTTTT CATCATGATT CCAGTCGTAC ATTTACTCA480
AAATAGAAAG GATGACCCCT ATGCAATTAA AAAATGTATA CAAATGTTTA ACCATTACA540
CGCTTTTGGC TCAAATCGCC GCCTTCCCGT CTTCCTCTTT TGCGGAAGAC GGGAAGAAA600
AAGAAGAAAA TACAGCTAAA ACAGAACATC AACAGAAAAA AGAAACAAAA CCAGTTGTG660
GATTAATTGG TCACTATTTT ACTGATGATC AGTTTACTAA CACAGCATTT ATTCAAGTA720
GAGAAAAAAG TAAATTACTA GATTCAAAAA TAGTAAAGCA AGATATGTCC AATTTGAAA780
CCATTCGATG GGAAGGAAAT GTGAAACCTC CTGAAACAGG AGAATATCTA CTTTCCACG840
CCTCTAATGA AAATGTTACA GTAAAAGTAG ATGGAGAAAC TGTTATTAAC AAAGCTAAC900
TGGAAAAAGC AATGAAACTC GAAAAAGATA AACCACACTC TATTGAAATT GAATATCAT960
TTCCTGAGAA CGGGAAGGAA CTACAATTAT TTTGGCAAAT AAATGACCAG AAAGCTGT1020
AAATCCCAGA AAAAAACATA CTATCACCAA ATCTTTCTGA ACAGATACAA CCGCAACA1080
GTTCAACTCA ATCTCAACAA AATCAAAATG ATAGGGATGG GGATAAAATC CCTGATAG1140
TAGAAGAAAA TGGCTATACA TTTAAAGACG GTGCGATTGT TGCCTGGAAC GATTCCTA1200
CAGCACTAGG CTATAAAAAA TACATATCCA ATTCTAATAA GGCTAAAACA GCTGCTGA1260
CCTATACGGA CTTTGAAAAA GTAACAGGAC ACATGCCGGA GGCAACTAAA GATGAAGT1320
AAGATCCACT AGTAGCCGCT TATCCCTCGG TAGGTGTTGC TATGGAAAAA TTTCATTT1380
CTAGAAATGA AACGGTCACT GAAGGAGACT CAGGTACTGT TTCAAAAACC GTAACCAA1440
CAAGCACAAC AACAAATAGC ATCGATGTTG GGGGATCCAT TGGATGGGGA GAAAAAGG1500
TTTCTTTTTC ATTCTCTCCC AAATATACGC ATTCTTGGAG TAATAGTACC GCTGTTGC1560
ATACTGAAAG TAGCACATGG TCTTCACAAT TAGCGTATAA TCCTTCAGAA CGTGCTTT1620
TAAATGCCAA TATACGATAT A 1641

327 amino acids

amino acid

single

linear

peptide

33D2

23
Gly Leu Ile Gly His Tyr Phe Thr Asp Asp Gln Phe Thr Asn Thr Al
1 5 10 15
Phe Ile Gln Val Gly Glu Lys Ser Lys Leu Leu Asp Ser Lys Ile Va
20 25 30
Lys Gln Asp Met Ser Asn Leu Lys Ser Ile Arg Trp Glu Gly Asn Va
35 40 45
Lys Pro Pro Glu Thr Gly Glu Tyr Leu Leu Ser Thr Ser Ser Asn Gl
50 55 60
Asn Val Thr Val Lys Val Asp Gly Glu Thr Val Ile Asn Lys Ala As
65 70 75 80
Met Glu Lys Ala Met Lys Leu Glu Lys Asp Lys Pro His Ser Ile Gl
85 90 95
Ile Glu Tyr His Val Pro Glu Asn Gly Lys Glu Leu Gln Leu Phe Tr
100 105 110
Gln Ile Asn Asp Gln Lys Ala Val Lys Ile Pro Glu Lys Asn Ile Le
115 120 125
Ser Pro Asn Leu Ser Glu Gln Ile Gln Pro Gln Gln Arg Ser Thr Gl
130 135 140
Ser Gln Gln Asn Gln Asn Asp Arg Asp Gly Asp Lys Ile Pro Asp Se
145 150 155 160
Leu Glu Glu Asn Gly Tyr Thr Phe Lys Asp Gly Ala Ile Val Ala Tr
165 170 175
Asn Asp Ser Tyr Ala Ala Leu Gly Tyr Lys Lys Tyr Ile Ser Asn Se
180 185 190
Asn Lys Ala Lys Thr Ala Ala Asp Pro Tyr Thr Asp Phe Glu Lys Va
195 200 205
Thr Gly His Met Pro Glu Ala Thr Lys Asp Glu Val Lys Asp Pro Le
210 215 220
Val Ala Ala Tyr Pro Ser Val Gly Val Ala Met Glu Lys Phe His Ph
225 230 235 240
Ser Arg Asn Glu Thr Val Thr Glu Gly Asp Ser Gly Thr Val Ser Ly
245 250 255
Thr Val Thr Asn Thr Ser Thr Thr Thr Asn Ser Ile Asp Val Gly Gl
260 265 270
Ser Ile Gly Trp Gly Glu Lys Gly Phe Ser Phe Ser Phe Ser Pro Ly
275 280 285
Tyr Thr His Ser Trp Ser Asn Ser Thr Ala Val Ala Asp Thr Glu Se
290 295 300
Ser Thr Trp Ser Ser Gln Leu Ala Tyr Asn Pro Ser Glu Arg Ala Ph
305 310 315 320
Leu Asn Ala Asn Ile Arg Tyr
325

1042 base pairs

nucleic acid

single

linear

DNA (genomic)

66D3

24
TTAATTGGGT ACTATTTTAA AGGAAAAGAT TTTAATAATC TTACTATATT TGCTCCAACA60
CGTGAGAATA CTCTTATTTA TGATTTAGAA ACAGCGAATT CTTTATTAGA TAAGCAACA120
CAAACCTATC AATCTATTCG TTGGATCGGT TTAATAAAAA GCAAAAAAGC TGGAGATTT180
ACCTTTCAAT TATCGGATGA TGAGCATGCT ATTATAGAAA TCGATGGGAA AGTTATTTC240
CAAAAAGGCC AAAAGAAACA AGTTGTTCAT TTAGAAAAAG ATAAATTAGT TCCCATCAA300
ATTGAATATC AATCTGATAA AGCGTTAAAC CCAGATAGTC AAATGTTTAA AGAATTGAA360
TTATTTAAAA TAAATAGTCA AAAACAATCT CAGCAAGTGC AACAAGACGA ATTGAGAAA420
CCTGAATTTG GTAAAGAAAA AACTCAAACA TATTTAAAGA AAGCATCGAA AAGCAGCCT480
TTTAGCAATA AAAGTAAACG AGATATAGAT GAAGATATAG ATGAGGATAC AGATACAGA540
GGAGATGCCA TTCCTGATGT ATGGGAAGAA AATGGGTATA CCATCAAAGG AAGAGTAGC600
GTTAAATGGG ACGAAGGATT AGCTGATAAG GGATATAAAA AGTTTGTTTC CAATCCTTT660
AGACAGCACA CTGCTGGTGA CCCCTATAGT GACTATGAAA AGGCATCAAA AGATTTGGA720
TTATCTAATG CAAAAGAAAC ATTTAATCCA TTGGTGGCTG CTTTTCCAAG TGTCAATGT780
AGCTTGGAAA ATGTCACCAT ATCAAAAGAT GAAAATAAAA CTGCTGAAAT TGCGTCTAC840
TCATCGAATA ATTGGTCCTA TACAAATACA GAGGGGGCAT CTATTGAAGC TGGAATTGG900
CCAGAAGGTT TGTTGTCTTT TGGAGTAAGT GCCAATTATC AACATTCTGA AACAGTGGC960
AAAGAGTGGG GTACAACTAA GGGAGACGCA ACACAATATA ATACAGCTTC AGCAGGAT1020
CTAAATGCCA ATGTACGATA TA 1042

347 amino acids

amino acid

single

linear

peptide

66D3

25
Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr Il
1 5 10 15
Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp Leu Glu Thr Al
20 25 30
Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln Ser Ile Arg Tr
35 40 45
Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe Thr Phe Gln Le
50 55 60
Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly Lys Val Ile Se
65 70 75 80
Gln Lys Gly Gln Lys Lys Gln Val Val His Leu Glu Lys Asp Lys Le
85 90 95
Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pro As
100 105 110
Ser Gln Met Phe Lys Glu Leu Lys Leu Phe Lys Ile Asn Ser Gln Ly
115 120 125
Gln Ser Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu Phe Gl
130 135 140
Lys Glu Lys Thr Gln Thr Tyr Leu Lys Lys Ala Ser Lys Ser Ser Le
145 150 155 160
Phe Ser Asn Lys Ser Lys Arg Asp Ile Asp Glu Asp Ile Asp Glu As
165 170 175
Thr Asp Thr Asp Gly Asp Ala Ile Pro Asp Val Trp Glu Glu Asn Gl
180 185 190
Tyr Thr Ile Lys Gly Arg Val Ala Val Lys Trp Asp Glu Gly Leu Al
195 200 205
Asp Lys Gly Tyr Lys Lys Phe Val Ser Asn Pro Phe Arg Gln His Th
210 215 220
Ala Gly Asp Pro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Leu As
225 230 235 240
Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pr
245 250 255
Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Glu As
260 265 270
Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Tyr Th
275 280 285
Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gly Le
290 295 300
Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr Val Al
305 310 315 320
Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln Tyr Asn Thr Al
325 330 335
Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr
340 345

1278 base pairs

nucleic acid

single

linear

DNA (genomic)

68F

26
TGGATTACTT GGGTACTATT TTAAAGGGAA AGATTTTAAT GATCTTACTG TATTTGCACC60
AACGCGTGGG AATACTCTTG TATATGATCA ACAAACAGCA AATACATTAC TAAATCAAA120
ACAACAAGAC TTTCAGTCTA TTCGTTGGGT TGGTTTAATT CAAAGTAAAG AAGCAGGCG180
TTTTACATTT AACTTATCAG ATGATGAACA TACGATGATA GAAATCGATG GGAAAGTTA240
TTCTAATAAA GGGAAAGAAA AACAAGTTGT CCATTTAGAA AAAGGACAGT TCGTTTCTA300
CAAAATAGAA TATCAAGCTG ATGAACCATT TAATGCGGAT AGTCAAACCT TTAAAAATT360
GAAACTCTTT AAAGTAGATA CTAAGCAACA GTCCCAGCAA ATTCAACTAG ATGAATTAA420
AAACCCTGAA TTTAATAAAA AAGAAACACA AGAATTTCTA ACAAAAGCAA CAAAAACAA480
CCTTATTACT CAAAAAGTGA AGAGTACTAG GGATGAAGAC ACGGATACAG ATGGAGATT540
TATTCCAGAC ATTTGGGAAG AAAATGGGTA TACCATCCAA AATAAGATTG CCGTCAAAT600
GGATGATTCA TTAGCAAGTA AAGGATATAC GAAATTTGTT TCAAACCCAC TAGATACTC660
CACGGTTGGA GATCCTTATA CAGATTATGA AAAAGCAGCA AGGGATTTAG ATTTGTCAA720
TGCAAAAGAA ACATTTAACC CATTAGTTGC GGCTTTTCCA AGTGTGAATG TGAGTATGG780
AAAAGTGATA TTGTCTCCAG ATGAGAACTT ATCAAATAGT ATCGAGTCTC ATTCATCTA840
GAATTGGTCG TATACGAATA CAGAAGGGGC TTCTATTGAA GCTGGTGGGG GAGCATTAG900
CCTATCTTTT GGTGTAAGTG CAAACTATCA ACATTCTGAA ACAGTTGGGT ATGAATGGG960
AACATCTACG GGAAATACTT CGCAATTTAA TACAGCTTCA GCGGGGTATT TAAATGCG1020
TGTTCGCTAC AATAACGTGG GAACGGGTGC AATCTATGAT GTAAAGCCAA CAACGAGT1080
TGTATTAAAT AAAGATACCA TCGCAACGAT AACAGCAAAA TCGAATACGA CTGCATTA1140
TATCTCACCA GGACAAAGTT ATCCGAAACA AGGTCAAAAT GGAATCGCGA TCACATCG1200
GGATGATTTT AACTCACATC CGATTACATT GAATAAGCAA CAGGTAGGTC AACTGTTA1260
TAATACCCAA TTAATCCA 1278

425 amino acids

amino acid

single

linear

peptide

68F

27
Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asp Leu Th
1 5 10 15
Val Phe Ala Pro Thr Arg Gly Asn Thr Leu Val Tyr Asp Gln Gln Th
20 25 30
Ala Asn Thr Leu Leu Asn Gln Lys Gln Gln Asp Phe Gln Ser Ile Ar
35 40 45
Trp Val Gly Leu Ile Gln Ser Lys Glu Ala Gly Asp Phe Thr Phe As
50 55 60
Leu Ser Asp Asp Glu His Thr Met Ile Glu Ile Asp Gly Lys Val Il
65 70 75 80
Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly Gl
85 90 95
Phe Val Ser Ile Lys Ile Glu Tyr Gln Ala Asp Glu Pro Phe Asn Al
100 105 110
Asp Ser Gln Thr Phe Lys Asn Leu Lys Leu Phe Lys Val Asp Thr Ly
115 120 125
Gln Gln Ser Gln Gln Ile Gln Leu Asp Glu Leu Arg Asn Pro Glu Ph
130 135 140
Asn Lys Lys Glu Thr Gln Glu Phe Leu Thr Lys Ala Thr Lys Thr As
145 150 155 160
Leu Ile Thr Gln Lys Val Lys Ser Thr Arg Asp Glu Asp Thr Asp Th
165 170 175
Asp Gly Asp Ser Ile Pro Asp Ile Trp Glu Glu Asn Gly Tyr Thr Il
180 185 190
Gln Asn Lys Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gl
195 200 205
Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Thr His Thr Val Gly As
210 215 220
Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser As
225 230 235 240
Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val As
245 250 255
Val Ser Met Glu Lys Val Ile Leu Ser Pro Asp Glu Asn Leu Ser As
260 265 270
Ser Ile Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Gl
275 280 285
Gly Ala Ser Ile Glu Ala Gly Gly Gly Ala Leu Gly Leu Ser Phe Gl
290 295 300
Val Ser Ala Asn Tyr Gln His Ser Glu Thr Val Gly Tyr Glu Trp Gl
305 310 315 320
Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Ty
325 330 335
Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile Ty
340 345 350
Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Lys Asp Thr Ile Al
355 360 365
Thr Ile Thr Ala Lys Ser Asn Thr Thr Ala Leu Ser Ile Ser Pro Gl
370 375 380
Gln Ser Tyr Pro Lys Gln Gly Gln Asn Gly Ile Ala Ile Thr Ser Me
385 390 395 400
Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Gln Gln Val Gl
405 410 415
Gln Leu Leu Asn Asn Thr Gln Leu Ile
420 425

983 base pairs

nucleic acid

single

linear

DNA (genomic)

69AA2

28
TGGATTACTT GGGTACTATT TTACTGATGA TCAGTTTACT AACACAGCAT TTATTCAAGT60
AGGAGAAAAA AGTAAATTAC TAGATTCAAA AATAGTAAAA CAAGATATGT CCAATTTGA120
ATCCATTCGA TGGGAAGGAA ATGTGAAACC TCCTGAAACA GGAGAATATC TACTTTCCA180
GTCCTCTAAT GAAAATGTTA CAGTAAAAGT AGATGGAGAA ACTGTTATTA ACAAAGCTA240
CATGGAAAAA GCAATGAAAC TCGAAAAAGA TAAACCACAC TCTATTGAAA TTGAATATC300
TGTTCCTGAG AACGGGAAGG AACTACAATT ATTTTGGCAA ATAAATGACC AGAAAGCTG360
TAAAATCCCA GAAAAAAACA TACTATCACC AAATCTTTCT GAACAGATAC AACCGCAAC420
GCGTTCAACT CAATCTCAAC AAAATCAAAA TGATAGGGAT GGGGATAAAA TCCCTGATA480
TTTAGAAGAA AATGGCTATA CATTTAAAGA CGGTGCGATT GTTGCCTGGA ACGATTCCT540
TGCAGCACTA GGCTATAAAA AATACATATC CAATTCTAAT AAGGCTAAAA CAGCTGCTG600
CCCCTATACG GACTTTGAAA AAGTAACAGG ACACATGCCG GAGGCAACTA AAGATGAAG660
AAAAGATCCA CTAGTAGCCG CTTATCCCTC GGTAGGTGTT GCTATGGAAA AATTTCATT720
TTCTAGAAAT GAAACGGTCA CTGAAGGAGA CTCAGGTACT GTTTCAAAAA CCGTAACCA780
TACAAGCACA ACAACAAATA GCATCGATGT TGGGGGATCC ATTGGATGGG GAGAAAAAG840
ATTTTCTTTT TCATTCTCTC CCAAATATAC GCATTCTTGG AGTAATAGTA CCGCTGTTG900
TGATACTGAA AGTAGCACAT GGTCTTCACA ATTAGCGTAT AATCCTTCAG AACGTGCTN960
CTTAAATGCC AATAKACGAT NTA 983

327 amino acids

amino acid

single

linear

peptide

69AA2

29
Gly Leu Leu Gly Tyr Tyr Phe Thr Asp Asp Gln Phe Thr Asn Thr Al
1 5 10 15
Phe Ile Gln Val Gly Glu Lys Ser Lys Leu Leu Asp Ser Lys Ile Va
20 25 30
Lys Gln Asp Met Ser Asn Leu Lys Ser Ile Arg Trp Glu Gly Asn Va
35 40 45
Lys Pro Pro Glu Thr Gly Glu Tyr Leu Leu Ser Thr Ser Ser Asn Gl
50 55 60
Asn Val Thr Val Lys Val Asp Gly Glu Thr Val Ile Asn Lys Ala As
65 70 75 80
Met Glu Lys Ala Met Lys Leu Glu Lys Asp Lys Pro His Ser Ile Gl
85 90 95
Ile Glu Tyr His Val Pro Glu Asn Gly Lys Glu Leu Gln Leu Phe Tr
100 105 110
Gln Ile Asn Asp Gln Lys Ala Val Lys Ile Pro Glu Lys Asn Ile Le
115 120 125
Ser Pro Asn Leu Ser Glu Gln Ile Gln Pro Gln Gln Arg Ser Thr Gl
130 135 140
Ser Gln Gln Asn Gln Asn Asp Arg Asp Gly Asp Lys Ile Pro Asp Se
145 150 155 160
Leu Glu Glu Asn Gly Tyr Thr Phe Lys Asp Gly Ala Ile Val Ala Tr
165 170 175
Asn Asp Ser Tyr Ala Ala Leu Gly Tyr Lys Lys Tyr Ile Ser Asn Se
180 185 190
Asn Lys Ala Lys Thr Ala Ala Asp Pro Tyr Thr Asp Phe Glu Lys Va
195 200 205
Thr Gly His Met Pro Glu Ala Thr Lys Asp Glu Val Lys Asp Pro Le
210 215 220
Val Ala Ala Tyr Pro Ser Val Gly Val Ala Met Glu Lys Phe His Ph
225 230 235 240
Ser Arg Asn Glu Thr Val Thr Glu Gly Asp Ser Gly Thr Val Ser Ly
245 250 255
Thr Val Thr Asn Thr Ser Thr Thr Thr Asn Ser Ile Asp Val Gly Gl
260 265 270
Ser Ile Gly Trp Gly Glu Lys Gly Phe Ser Phe Ser Phe Ser Pro Ly
275 280 285
Tyr Thr His Ser Trp Ser Asn Ser Thr Ala Val Ala Asp Thr Glu Se
290 295 300
Ser Thr Trp Ser Ser Gln Leu Ala Tyr Asn Pro Ser Glu Arg Ala Xa
305 310 315 320
Leu Asn Ala Asn Xaa Arg Xaa
325

1075 base pairs

nucleic acid

single

linear

DNA (genomic)

168G1

30
TGGGTTAATT GGATATTATT TCCAGGATCA AAAATTTCAA CAACTCGCTT TAATGGTACA60
TAGGCAAGCT TCTGATTTAA AAATACTGAA AGATGACGTG AAACATTTAC TATCCGAAG120
TCAACAACAC ATTCAATCAG TAAGGTGGAT AGGCTATATT AAGCCACCTA AAACAGGAG180
CTACGTATTG TCAACCTCAT CCGACCAACA GGTCATGATT GAACTAGATG GTAAAGTCA240
TCTCAATCAG GCTTCTATGA CAGAACCTGT TCAACTTGAA AAAGATAAAC CGTATAAAA300
TAAAATTGAA TATGTTCCGG AACAAACAGA AACACAAGAT ACGCTTCTTG ATTTTAAAC360
GAACTGGTCT TTTTCAGGCG GAAAAACAGA AACGATTCCA GAAAATGCAT TTCTATTAC420
AGACCTTTCT CGTAAACAAG ATCAAGAAAA GCTTATTCCT GAGGCAAGTT TATTTCAGA480
ACCTGGAGAC GAGAAAAAAA TATCTCGAAG TAAACGGTCC TTTAACTACA GATTCTCTA540
ATGATACAAG ATGATGATGG GATTTCGGAT GCGTGGGAAA CAGAAGGATA CACGATACA600
AGACAACTGG CAGTGAAATG GGACGATTCT ATGAAGGATC GAGGGTATAC CAAATATGT660
TCTAATCCCT ATAATTCCCA TACAGTAGGG GATCCATACA CAGATTGGGA AAAAGCGGC720
GGACGTATTG ATAAGGCGAT CAAAGGAGAA GCTAGGAATC CTTTAGTCGC GGCCTATCC780
ACCGTTGGTG TACATATGGA AAAACTGATT GTCTCCGAGA AACAAAACAT ATCAACTGG840
CTCGGAAAAA CAATATCTGC GTCAATGTCT GCAAGTAATA CCGCAGCGAT TACAGCGGG900
ATTGATACGA CGGCTGGTGC TTCTTTACTT GGACCGTCTG GAAGCGTCAC GGCTCATTT960
TCTGATACAG GATCCAGTAC ATCCACTGTT GAAAATAGCT CAAGTAATAA TTGGAGTC1020
GATCTTGGAA TCGATACGGG ACAATCTGCA TATTTAAATG CCAATGTACG ATATA 1075

2645 base pairs

nucleic acid

single

linear

DNA (genomic)

PS177C8

31
ATGAAGAAGA AGTTAGCAAG TGTTGTAACG TGTACGTTAT TAGCTCCTAT GTTTTTGAAT60
GGAAATGTGA ATGCTGTTTA CGCAGACAGC AAAACAAATC AAATTTCTAC AACACAGAA120
AATCAACAGA AAGAGATGGA CCGAAAAGGA TTACTTGGGT ATTATTTCAA AGGAAAAGA180
TTTAGTAATC TTACTATGTT TGCACCGACA CGTGATAGTA CTCTTATTTA TGATCAACA240
ACAGCAAATA AACTATTAGA TAAAAAACAA CAAGAATATC AGTCTATTCG TTGGATTGG300
TTGATTCAGA GTAAAGAAAC GGGAGATTTC ACATTTAACT TATCTGAGGA TGAACAGGC360
ATTATAGAAA TCAATGGGAA AATTATTTCT AATAAAGGGA AAGAAAAGCA AGTTGTCCA420
TTAGAAAAAG GAAAATTAGT TCCAATCAAA ATAGAGTATC AATCAGATAC AAAATTTAA480
ATTGACAGTA AAACATTTAA AGAACTTAAA TTATTTAAAA TAGATAGTCA AAACCAACC540
CAGCAAGTCC AGCAAGATGA ACTGAGAAAT CCTGAATTTA ACAAGAAAGA ATCACAGGA600
TTCTTAGCGA AACCATCGAA AATAAATCTT TTCACTCAAA AAATGAAAAG GGAAATTGA660
GAAGACACGG ATACGGATGG GGACTCTATT CCTGACCTTT GGGAAGAAAA TGGGTATAC720
ATTCAAAATA GAATCGCTGT AAAGTGGGAC GATTCTYTAG CAAGTAAAGG GTATACGAA780
TTTGTTTCAA ATCCGCTAGA AAGTCACACA GTTGGTGATC CTTATACAGA TTATGAAAA840
GCAGCAAGAG ACCTAGATTT GTCAAATGCA AAGGAAACGT TTAACCCATT GGTAGCTGC900
TTTCCAAGTG TGAATGTTAG TATGGAAAAG GTGATATTAT CACCAAATGA AAATTTATC960
AATAGTGTAG AGTCTCATTC ATCCACGAAT TGGTCTTATA CAAATACAGA AGGTGCTT1020
GTTGAAGCGG GGATTGGACC AAAAGGTATT TCGTTCGGAG TTAGCGTAAA CTATCAAC1080
TCTGAAACAG TTGCACAAGA ATGGGGAACA TCTACAGGAA ATACTTCGCA ATTCAATA1140
GCTTCAGCGG GATATTTAAA TGCAAATGTT CGATATAACA ATGTAGGAAC TGGTGCCA1200
TACGATGTAA AACCTACAAC AAGTTTTGTA TTAAATAACG ATACTATCGC AACTATTA1260
GCGAAATCTA ATTCTACAGC CTTAAATATA TCTCCTGGAG AAAGTTACCC GAAAAAAG1320
CAAAATGGAA TCGCAATAAC ATCAATGGAT GATTTTAATT CCCATCCGAT TACATTAA1380
AAAAAACAAG TAGATAATCT GCTAAATAAT AAACCTATGA TGTTGGAAAC AAACCAAA1440
GATGGTGTTT ATAAGATAAA AGATACACAT GGAAATATAG TAACTGGCGG AGAATGGA1500
GGTGTCATAC AACAAATCAA GGCTAAAACA GCGTCTATTA TTGTGGATGA TGGGGAAC1560
GTAGCAGAAA AACGTGTAGC GGCAAAAGAT TATGAAAATC CAGAAGATAA AACACCGT1620
TTAACTTTAA AAGATGCCCT GAAGCTTTCA TATCCAGATG AAATAAAAGA AATAGAGG1680
TTATTATATT ATAAAAACAA ACCGATATAC GAATCGAGCG TTATGACTTA CTTAGATG1740
AATACAGCAA AAGAAGTGAC CAAACAATTA AATGATACCA CTGGGAAATT TAAAGATG1800
AGTCATTTAT ATGATGTAAA ACTGACTCCA AAAATGAATG TTACAATCAA ATTGTCTA1860
CTTTATGATA ATGCTGAGTC TAATGATAAC TCAATTGGTA AATGGACAAA CACAAATA1920
GTTTCAGGTG GAAATAACGG AAAAAAACAA TATTCTTCTA ATAATCCGGA TGCTAATT1980
ACATTAAATA CAGATGCTCA AGAAAAATTA AATAAAAATC GTACTATTAT ATAAGTTT2040
ATATGAAGTC AGAAAAAAAC ACACAATGTG AGATTACTAT AGATGGGGAG ATTTATCC2100
TCACTACAAA AACAGTGAAT GTGAATAAAG ACAATTACAA AAGATTAGAT ATTATAGC2160
ATAATATAAA AAGTAATCCA ATTTCTTCAA TTCATATTAA AACGAATGAT GAAATAAC2220
TATTTTGGGA TGATATTTCT ATAACAGATG TAGCATCAAT AAAACCGGAA AATTTAAC2280
ATTCAGAAAT TAAACAGATT TATAGTAGGT ATGGTATTAA GTTAGAAGAT GGAATCCT2340
TTGATAAAAA AGGTGGGATT CATTATGGTG AATTTATTAA TGAAGCTAGT TTTAATAT2400
AACCATTGCA AAATTATGTG ACAAAATATA AAGTTACTTA TAGTAGTGAG TTAGGACA2460
ACGTGAGTGA CACACTTGAA AGTGATAAAA TTTACAAGGA TGGGACAATT AAATTTGA2520
TTACAAAATA TAGTRAAAAT GAACAAGGAT TATTTTATGA CAGTGGATTA AATTGGGA2580
TTAAAATTAA TGCTATTACT TATGATGGTA AAGAGATGAA TGTTTTTCAT AGATATAA2640
AATAG 2645

881 amino acids

amino acid

single

linear

peptide

PS177C8

32
Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pr
1 5 10 15
Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys Th
20 25 30
Asn Gln Ile Ser Thr Thr Gln Lys Asn Gln Gln Lys Glu Met Asp Ar
35 40 45
Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Le
50 55 60
Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu Ile Tyr Asp Gln Gl
65 70 75 80
Thr Ala Asn Lys Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Il
85 90 95
Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Ph
100 105 110
Asn Leu Ser Glu Asp Glu Gln Ala Ile Ile Glu Ile Asn Gly Lys Il
115 120 125
Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gl
130 135 140
Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe As
145 150 155 160
Ile Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Se
165 170 175
Gln Asn Gln Pro Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Gl
180 185 190
Phe Asn Lys Lys Glu Ser Gln Glu Phe Leu Ala Lys Pro Ser Lys Il
195 200 205
Asn Leu Phe Thr Gln Lys Met Lys Arg Glu Ile Asp Glu Asp Thr As
210 215 220
Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Th
225 230 235 240
Ile Gln Asn Arg Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Ly
245 250 255
Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His Thr Val Gl
260 265 270
Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Se
275 280 285
Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Va
290 295 300
Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Se
305 310 315 320
Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Th
325 330 335
Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly Ile Ser Ph
340 345 350
Gly Val Ser Val Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Tr
355 360 365
Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gl
370 375 380
Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala Il
385 390 395 400
Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asp Thr Il
405 410 415
Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn Ile Ser Pr
420 425 430
Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly Ile Ala Ile Thr Se
435 440 445
Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Lys Gln Va
450 455 460
Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr Asn Gln Th
465 470 475 480
Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile Val Thr Gl
485 490 495
Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Lys Ala Lys Thr Ala Se
500 505 510
Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala Al
515 520 525
Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Ly
530 535 540
Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gl
545 550 555 560
Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met Th
565 570 575
Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gln Leu Asn As
580 585 590
Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys Le
595 600 605
Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu Tyr Asp As
610 615 620
Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn Thr Asn Il
625 630 635 640
Val Ser Gly Gly Asn Asn Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pr
645 650 655
Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gln Glu Lys Leu Asn Ly
660 665 670
Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu Lys Asn Th
675 680 685
Gln Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile Thr Thr Ly
690 695 700
Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp Ile Ile Al
705 710 715 720
His Asn Ile Lys Ser Asn Pro Ile Ser Ser Ile His Ile Lys Thr As
725 730 735
Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr Asp Val Al
740 745 750
Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys Gln Ile Ty
755 760 765
Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys Ly
770 775 780
Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn Il
785 790 795 800
Glu Pro Leu Gln Asn Tyr Val Thr Lys Tyr Lys Val Thr Tyr Ser Se
805 810 815
Glu Leu Gly Gln Asn Val Ser Asp Thr Leu Glu Ser Asp Lys Ile Ty
820 825 830
Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser Xaa Asn Gl
835 840 845
Gln Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys Ile As
850 855 860
Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr As
865 870 875 880
Lys

1022 base pairs

nucleic acid

single

linear

DNA (genomic)

177I8

33
TGGATTAATT GGGTATTATT TCAAAGGAAA AGATTTTAAT AATCTTACTA TGTTTGCACC60
GACACGTGAT AATACCCTTA TGTATGACCA ACAAACAGCG AATGCATTAT TAGATAAAA120
ACAACAAGAA TATCAGTCCA TTCGTTGGAT TGGTTTGATT CAGAGTAAAG AAACGGGCG180
TTTCACATTT AACTTATCAA AGGATGAACA GGCAATTATA GAAATCGATG GGAAAATCA240
TTCTAATAAA GGGAAAGAAA AGCAAGTTGT CCATTTAGAA AAAGAAAAAT TAGTTCCAA300
CAAAATAGAG TATCAATCAG ATACGAAATT TAATATTGAT AGTAAAACAT TTAAAGAAC360
TAAATTATTT AAAATAGATA GTCAAAACCA ATCTCAACAA GTTCAACTGA GAAACCCTG420
ATTTAACAAA AAAGAATCAC AGGAATTTTT AGCAAAAGCA TCAAAAACAA ACCTTTTTA480
GCAAAAAATG AAAAGAGATA TTGATGAAGA TACGGATACA GATGGAGACT CCATTCCTG540
TCTTTGGGAA GAAAATGGGT ACACGATTCA AAATAAAGTT GCTGTCAAAT GGGATGATT600
GCTAGCAAGT AAGGGATATA CAAAATTTGT TTCGAATCCA TTAGACAGCC ACACAGTTG660
CGATCCCTAT ACTGATTATG AAAAGGCCGC AAGGGATTTA GATTTATCAA ATGCAAAGG720
AACGTTCAAC CCATTGGTAG CTGCTTTYCC AAGTGTGAAT GTTAGTATGG AAAAGGTGA780
ATTATCACCA AATGAAAATT TATCCAATAG TGTAGAGTCT CATTCATCCA CGAATTGGT840
TTATACGAAT ACAGAAGGAG CTTCCATTGA AGCTGGTGGC GGTCCATTAG GCCTTTCTT900
TGGAGTGAGT GTTAATTATC AACACTCTGA AACAGTTGCA CAAGAATGGG GAACATCTA960
AGGAAATACT TCACAATTCA ATACGGCTTC AGCGGGATAT TTAAATGCCA ATATACGA1020
TA 1022

340 amino acids

amino acid

single

linear

peptide

177I8

34
Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Th
1 5 10 15
Met Phe Ala Pro Thr Arg Asp Asn Thr Leu Met Tyr Asp Gln Gln Th
20 25 30
Ala Asn Ala Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile Ar
35 40 45
Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe As
50 55 60
Leu Ser Lys Asp Glu Gln Ala Ile Ile Glu Ile Asp Gly Lys Ile Il
65 70 75 80
Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Glu Ly
85 90 95
Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe Asn Il
100 105 110
Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Ser Gl
115 120 125
Asn Gln Ser Gln Gln Val Gln Leu Arg Asn Pro Glu Phe Asn Lys Ly
130 135 140
Glu Ser Gln Glu Phe Leu Ala Lys Ala Ser Lys Thr Asn Leu Phe Ly
145 150 155 160
Gln Lys Met Lys Arg Asp Ile Asp Glu Asp Thr Asp Thr Asp Gly As
165 170 175
Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gln Asn Ly
180 185 190
Val Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Ly
195 200 205
Phe Val Ser Asn Pro Leu Asp Ser His Thr Val Gly Asp Pro Tyr Th
210 215 220
Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Gl
225 230 235 240
Thr Phe Asn Pro Leu Val Ala Ala Xaa Pro Ser Val Asn Val Ser Me
245 250 255
Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Gl
260 265 270
Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Se
275 280 285
Ile Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe Gly Val Ser Va
290 295 300
Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp Gly Thr Ser Th
305 310 315 320
Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Al
325 330 335
Asn Ile Arg Tyr
340

1073 base pairs

nucleic acid

single

linear

DNA (genomic)

185AA2

35
TGGATTAATT GGGTATTATT TCCAGGAGCA AAACTTTGAG AAACCCGCTT TGATAGCAAA60
TAGACAAGCT TCTGATTTGG AAATACCGAA AGATGACGTG AAAGAGTTAC TATCCAAAG120
ACAGCAACAC ATTCAATCTG TTAGATGGCT TGGCTATATT CAGCCACCTC AAACAGGAG180
CTATGTATTG TCAACCTCAT CCGACCAACA GGTCGTGATT GAACTCGATG GAAAAACCA240
TGTCAATCAA ACTTCTATGA CAGAACCGAT TCAACTAGAA AAAGATAAAC GCTATAAAA300
TAGAATTGAA TATGTCCCAG GAGATACACA AGGACAAGAG AACCTTCTGG ACTTTCAAC360
GAAGTGGTCA ATTTCAGGAG CCGAGATAGA ACCAATTCCG GATCATGCTT TCCATTTAC420
AGATTTTTCT CATAAACAAG ATCAAGAGAA AATCATCCCT GAAACCAATT TATTTCAGA480
ACAAGGAGAT GAGAAAAAAG TATCACGCAG TAAGAGATCT TCAGATAAAG ATCCTGACC540
TGATACAGAT GATGATAGTA TTTCTGATGA ATGGGAAACG AGTGGATATA CCATTCAAA600
ACAGGTGGCA GTGAAATGGG ACGATTCTAT GAAGGAGCTA GGTTATACCA AGTATGTGT660
TAACCCTTAT AAGTCTCGTA CAGTAGGAGA TCCATACACA GATTGGGAAA AAGCGGCTG720
CAGTATCGAT AATGCTGTCA AAGCAGAAGC CAGAAATCCT TTAGTCGCGG CCTATCCAA780
TGTTGGTGTA CATATGGAAA GATTAATTGT CTCCGAACAA CAAAATATAT CAACAGGGC840
TGGAAAAACC GTATCTGCGT CTACGTCCGC AAGCAATACC GCAGCGATTA CGGCAGGTA900
TGATGCAACA GCTGGTGCCT CTTTACTTGG GCCATCTGGA AGTGTCACGG CTCATTTTT960
TTACACGGGA TCTAGTACAG CCACCATTGA AGATAGCTCC AGCCGTAATT GGAGTCGA1020
CCTTGGGATT GATACGGGAC AAGCTGCATA TTTAAATGCC AATATACGAT ATA 1073

357 amino acids

amino acid

single

linear

peptide

185AA2

36
Gly Leu Ile Gly Tyr Tyr Phe Gln Glu Gln Asn Phe Glu Lys Pro Al
1 5 10 15
Leu Ile Ala Asn Arg Gln Ala Ser Asp Leu Glu Ile Pro Lys Asp As
20 25 30
Val Lys Glu Leu Leu Ser Lys Glu Gln Gln His Ile Gln Ser Val Ar
35 40 45
Trp Leu Gly Tyr Ile Gln Pro Pro Gln Thr Gly Asp Tyr Val Leu Se
50 55 60
Thr Ser Ser Asp Gln Gln Val Val Ile Glu Leu Asp Gly Lys Thr Il
65 70 75 80
Val Asn Gln Thr Ser Met Thr Glu Pro Ile Gln Leu Glu Lys Asp Ly
85 90 95
Arg Tyr Lys Ile Arg Ile Glu Tyr Val Pro Gly Asp Thr Gln Gly Gl
100 105 110
Glu Asn Leu Leu Asp Phe Gln Leu Lys Trp Ser Ile Ser Gly Ala Gl
115 120 125
Ile Glu Pro Ile Pro Asp His Ala Phe His Leu Pro Asp Phe Ser Hi
130 135 140
Lys Gln Asp Gln Glu Lys Ile Ile Pro Glu Thr Asn Leu Phe Gln Ly
145 150 155 160
Gln Gly Asp Glu Lys Lys Val Ser Arg Ser Lys Arg Ser Ser Asp Ly
165 170 175
Asp Pro Asp Arg Asp Thr Asp Asp Asp Ser Ile Ser Asp Glu Trp Gl
180 185 190
Thr Ser Gly Tyr Thr Ile Gln Arg Gln Val Ala Val Lys Trp Asp As
195 200 205
Ser Met Lys Glu Leu Gly Tyr Thr Lys Tyr Val Ser Asn Pro Tyr Ly
210 215 220
Ser Arg Thr Val Gly Asp Pro Tyr Thr Asp Trp Glu Lys Ala Ala Gl
225 230 235 240
Ser Ile Asp Asn Ala Val Lys Ala Glu Ala Arg Asn Pro Leu Val Al
245 250 255
Ala Tyr Pro Thr Val Gly Val His Met Glu Arg Leu Ile Val Ser Gl
260 265 270
Gln Gln Asn Ile Ser Thr Gly Leu Gly Lys Thr Val Ser Ala Ser Th
275 280 285
Ser Ala Ser Asn Thr Ala Ala Ile Thr Ala Gly Ile Asp Ala Thr Al
290 295 300
Gly Ala Ser Leu Leu Gly Pro Ser Gly Ser Val Thr Ala His Phe Se
305 310 315 320
Tyr Thr Gly Ser Ser Thr Ala Thr Ile Glu Asp Ser Ser Ser Arg As
325 330 335
Trp Ser Arg Asp Leu Gly Ile Asp Thr Gly Gln Ala Ala Tyr Leu As
340 345 350
Ala Asn Ile Arg Tyr
355

1073 base pairs

nucleic acid

single

linear

DNA (genomic)

196F3

37
TGGGTTACNT GGGTATTAYT TTCAGGATAC TAAATTTCAA CAACTTGCTT TAATGGCACA60
TAGACAAGCC TCAGATTTAG AAATAAACAA AAATGAMGTC AAGGATTTAC TATCAAAGG120
TCAACAACAC ATTCAAGCAG TGAGATGGAT GGGCTATATT CAGCCACCTC AAACAGGAG180
TTATGTATTG TCAACTTCAT CCGACCAACA GGTCTTCACC GAACTCNATG GAAAAATAA240
TCTCAATCAA TCTTCTATGA CCGAACCCAT TCGATTAGAA AAAGATAAAC AATATAMAA300
TAGAATTGAA TATGTATCAK AAAGTAAAAC AGAAAAAGAG ACGCTCCTAG ACTTTCAAC360
CAACTGGTCG ATTTCAGGTG CTACGGTAGA ACCAATTCCA GATAATGCTT TTCAGTTAC420
AGATCTTTCT CGGGAACAAG NTAAAGATAA AATCATCCCT GAAACAAGTT TATTGCAGG480
TCAAGGAGAA GGGAAACAAG TATCTCGAAG TAAAAGATCT CTAGCTGTGA ATCCTCTAC540
CGATACAGAT GATGATGGGA TTTACGATGA ATGGGAAACA AGCGGCTATA CGATTCAAA600
ACAATTGGCA GTAAGATGGA ACGATTCTAT GAAGGATCAA GGCTATACCA AATATGTGT660
TAATCCTTAT AAGTCTCATA CTGTAGGAGA TCCATACACA GACTGGGAAA AAGCAGCTG720
ACGTATCGAC CAAGCTGTGA AAATAGAAGC CAGAAACCCA TTAGTTGCAG CATATCCAA780
AGTTGGCGTA CATATGGAAA GACTGATTGT CTCTGAAAAA CAAAATATAG CAACAGGAC840
GGGAAAAACA GTATCTGCGT CTACATCTGC AAGTAATACA GCGGGGATTA CAGCGGGAA900
CGATGCAACG GTTGGTGCCT CTTTACTTGG ACCTTCGGGA AGTGTCACCG CCCATTTTT960
TTATACGGGT TCGAGTACAT CCACTGTTGA AAATAGCTCG AGTAATAATT GGAGTCAA1020
TCTTGGTATT GATACCAGCC AATCTGCGTA CTTAAATGCC AATGTAAGAT ATA 1073

357 amino acids

amino acid

single

linear

peptide

196F3

38
Gly Leu Xaa Gly Tyr Xaa Phe Gln Asp Thr Lys Phe Gln Gln Leu Al
1 5 10 15
Leu Met Ala His Arg Gln Ala Ser Asp Leu Glu Ile Asn Lys Asn Xa
20 25 30
Val Lys Asp Leu Leu Ser Lys Asp Gln Gln His Ile Gln Ala Val Ar
35 40 45
Trp Met Gly Tyr Ile Gln Pro Pro Gln Thr Gly Asp Tyr Val Leu Se
50 55 60
Thr Ser Ser Asp Gln Gln Val Phe Thr Glu Leu Xaa Gly Lys Ile Il
65 70 75 80
Leu Asn Gln Ser Ser Met Thr Glu Pro Ile Arg Leu Glu Lys Asp Ly
85 90 95
Gln Tyr Xaa Ile Arg Ile Glu Tyr Val Ser Xaa Ser Lys Thr Glu Ly
100 105 110
Glu Thr Leu Leu Asp Phe Gln Leu Asn Trp Ser Ile Ser Gly Ala Th
115 120 125
Val Glu Pro Ile Pro Asp Asn Ala Phe Gln Leu Pro Asp Leu Ser Ar
130 135 140
Glu Gln Xaa Lys Asp Lys Ile Ile Pro Glu Thr Ser Leu Leu Gln As
145 150 155 160
Gln Gly Glu Gly Lys Gln Val Ser Arg Ser Lys Arg Ser Leu Ala Va
165 170 175
Asn Pro Leu His Asp Thr Asp Asp Asp Gly Ile Tyr Asp Glu Trp Gl
180 185 190
Thr Ser Gly Tyr Thr Ile Gln Arg Gln Leu Ala Val Arg Trp Asn As
195 200 205
Ser Met Lys Asp Gln Gly Tyr Thr Lys Tyr Val Ser Asn Pro Tyr Ly
210 215 220
Ser His Thr Val Gly Asp Pro Tyr Thr Asp Trp Glu Lys Ala Ala Gl
225 230 235 240
Arg Ile Asp Gln Ala Val Lys Ile Glu Ala Arg Asn Pro Leu Val Al
245 250 255
Ala Tyr Pro Thr Val Gly Val His Met Glu Arg Leu Ile Val Ser Gl
260 265 270
Lys Gln Asn Ile Ala Thr Gly Leu Gly Lys Thr Val Ser Ala Ser Th
275 280 285
Ser Ala Ser Asn Thr Ala Gly Ile Thr Ala Gly Ile Asp Ala Thr Va
290 295 300
Gly Ala Ser Leu Leu Gly Pro Ser Gly Ser Val Thr Ala His Phe Se
305 310 315 320
Tyr Thr Gly Ser Ser Thr Ser Thr Val Glu Asn Ser Ser Ser Asn As
325 330 335
Trp Ser Gln Asp Leu Gly Ile Asp Thr Ser Gln Ser Ala Tyr Leu As
340 345 350
Ala Asn Val Arg Tyr
355

1073 base pairs

nucleic acid

single

linear

DNA (genomic)

196J4

39
TGGGTTAATT GGGTATTATT TCCAGGATCA AAAGTTTCAA CAACTTGCTT TAATGGCACA60
TAGACAAGCT TCTAATTTAA ACATACCAAA AAATGAAGTG AAACAGTTAT TATCCGAAG120
TCAACAACAT ATTCAATCCG TTAGGTGGAT CGGATATATC AAATCACCTC AAACGGGAG180
TTATATATTG TCAACTTCAG CCGATCGACA TGTCGTAATT GAACTTGACG GAAAAACCA240
TCTTAATCAA TCTTCTATGA CAGCACCCAT TCAATTAGAA AAAGATAAAC TTTATAAAA300
TAGAATTGAA TATGTCCCAG AAGATACAAA AGGACAGGAA AACCTCTTTG ACTTTCAAC360
GAATTGGTCA ATTTCAGGAG ATAAGGTAGA ACCAATTCCG GAGAATGCAT TTCTGTTGC420
AGACTTTTCT CATAAACAAG ATCAAGAGAA AATCATCCCT GAAGCAAGTT TATTCCAGG480
ACAAGAAGAT GCAAACAAAG TCTCTCGAAA TAAACGATCC ATAGCTACAG GTTCTCTGT540
TGATACAGAT GATGATGCTA TTTATGATGA ATGGGAAACA GAAGGATACA CGATACAAC600
TCAAATAGCG GTGAAATGGG ACGATTCTAT GAAGGAGCGA GGTTATACCA AGTATGTGT660
TAACCCCTAT AATTCGCATA CAGTAGGAGA TCCCTACACA GATTGGGAAA AAGCGGCTG720
ACGCATTGAT CAGGCAATCA AAGTAGAAGC TAGGAATCCA TTAGTTGCAG CCTATCCAA780
AGTTGGTGTA CATATGGAAA AACTGATTGT TTCTGAGAAA CAAAATATAT CAACTGGGG840
TGGAAAAACA GTATCTGCGG CTATGTCCAC TGGTAATACC GCAGCGATTA CGGCAGGAA900
TGATGCGACC GCCGGGGCAT CTTTACTTGG ACCTTCTGGA AGTGTGACGG CTCATTTTT960
TTATACAGGG TCTAGTACAT CTACAATTGA AAATAGTTCA AGCAATAATT GGAGTAAA1020
TCTGGGAATC GATACGGGGC AATCTGCTTA TTTAAATGCC AATGTACGAT ATA 1073

357 amino acids

amino acid

single

linear

peptide

196J4

40
Gly Leu Ile Gly Tyr Tyr Phe Gln Asp Gln Lys Phe Gln Gln Leu Al
1 5 10 15
Leu Met Ala His Arg Gln Ala Ser Asn Leu Asn Ile Pro Lys Asn Gl
20 25 30
Val Lys Gln Leu Leu Ser Glu Asp Gln Gln His Ile Gln Ser Val Ar
35 40 45
Trp Ile Gly Tyr Ile Lys Ser Pro Gln Thr Gly Asp Tyr Ile Leu Se
50 55 60
Thr Ser Ala Asp Arg His Val Val Ile Glu Leu Asp Gly Lys Thr Il
65 70 75 80
Leu Asn Gln Ser Ser Met Thr Ala Pro Ile Gln Leu Glu Lys Asp Ly
85 90 95
Leu Tyr Lys Ile Arg Ile Glu Tyr Val Pro Glu Asp Thr Lys Gly Gl
100 105 110
Glu Asn Leu Phe Asp Phe Gln Leu Asn Trp Ser Ile Ser Gly Asp Ly
115 120 125
Val Glu Pro Ile Pro Glu Asn Ala Phe Leu Leu Pro Asp Phe Ser Hi
130 135 140
Lys Gln Asp Gln Glu Lys Ile Ile Pro Glu Ala Ser Leu Phe Gln Gl
145 150 155 160
Gln Glu Asp Ala Asn Lys Val Ser Arg Asn Lys Arg Ser Ile Ala Th
165 170 175
Gly Ser Leu Tyr Asp Thr Asp Asp Asp Ala Ile Tyr Asp Glu Trp Gl
180 185 190
Thr Glu Gly Tyr Thr Ile Gln Arg Gln Ile Ala Val Lys Trp Asp As
195 200 205
Ser Met Lys Glu Arg Gly Tyr Thr Lys Tyr Val Ser Asn Pro Tyr As
210 215 220
Ser His Thr Val Gly Asp Pro Tyr Thr Asp Trp Glu Lys Ala Ala Gl
225 230 235 240
Arg Ile Asp Gln Ala Ile Lys Val Glu Ala Arg Asn Pro Leu Val Al
245 250 255
Ala Tyr Pro Thr Val Gly Val His Met Glu Lys Leu Ile Val Ser Gl
260 265 270
Lys Gln Asn Ile Ser Thr Gly Val Gly Lys Thr Val Ser Ala Ala Me
275 280 285
Ser Thr Gly Asn Thr Ala Ala Ile Thr Ala Gly Ile Asp Ala Thr Al
290 295 300
Gly Ala Ser Leu Leu Gly Pro Ser Gly Ser Val Thr Ala His Phe Se
305 310 315 320
Tyr Thr Gly Ser Ser Thr Ser Thr Ile Glu Asn Ser Ser Ser Asn As
325 330 335
Trp Ser Lys Asp Leu Gly Ile Asp Thr Gly Gln Ser Ala Tyr Leu As
340 345 350
Ala Asn Val Arg Tyr
355

1046 base pairs

nucleic acid

single

linear

DNA (genomic)

197T1

41
TGGATTAATT GGGTATTATT TTAAAGGAAA AGATTTTAAT AATCTTACTA TATTTGCTCC60
AACACGTGAG AATACTCTTA TTTATGATTT AGAAACAGCG AATTCTTTAT TAGATAAGC120
ACAACAAACC TATCAATCTA TTCGTTGGAT CGGTTTAATA AAAAGCAAAA AAGCTGGAG180
TTTTACCTTT CAATTATCGG ATGATGAGCA TGCTATTATA GAAATCGATG GGAAAGTTA240
TTCGCAAAAA GGCCAAAAGA AACAAGTTGT TCATTTAGAA AAAGATAAAT TAGTTCCCA300
CAAAATTGAA TATCAATCTG ATAAAGCGTT AAACCCAGAC AGTCAAATGT TTAAAGAAT360
GAAATTATTT AAAATAAATA GTCAAAAACA ATCTCAGCAA GTGCAACAAG ACGAATTGA420
AAATCCTGAA TTTGGTAAAG AAAAAACTCA AACATATTTA AAGAAAGCAT CGAAAAGCA480
CTTGTTTAGC AATAAAAGTA AACGAGATAT AGATGAAGAT ATAGATGAGG ATACAGATA540
AGATGGAGAT GCCATTCCTG ATGTATGGGA AGAAAATGGG TATACCATCA AAGGAAGAG600
AGCTGTTAAA TGGGACGAAG GATTAGCTGA TAAGGGATAT AAAAAGTTTG TTTCCAATC660
TTTTAGACAG CACACTGCTG GTGACCCCTA TAGTGACTAT GAAAAGGCAT CAAAAGATT720
GGATTTATCT AATGCAAAAG AAACATTTAA TCCATTGGTG GCTGCTTTTC CAAGTGTCA780
TGTTAGCTTG GAAAATGTCA CCATATCAAA AGATGAAAAT AAAACTGCTG AAATTGCGT840
TACTTCATCG AATAATTGGT CCTATACAAA TACAGAGGGG GCATCTATTG AAGCTGGAA900
TGGACCAGAA GGTTTGTTGT CTTTTGGAGT AAGTGCCAAT TATCAACATT CTGAAACAG960
GGCCAAAGAG TGGGGTACAA CTAAGGGAGA CGCAACACAA TATAATACAG CTTCAGCA1020
ATATCTAAAT GCCAATGTAC GATATA 1046

348 amino acids

amino acid

single

linear

peptide

197T1

42
Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Th
1 5 10 15
Ile Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp Leu Glu Th
20 25 30
Ala Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln Ser Ile Ar
35 40 45
Trp Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe Thr Phe Gl
50 55 60
Leu Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly Lys Val Il
65 70 75 80
Ser Gln Lys Gly Gln Lys Lys Gln Val Val His Leu Glu Lys Asp Ly
85 90 95
Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pr
100 105 110
Asp Ser Gln Met Phe Lys Glu Leu Lys Leu Phe Lys Ile Asn Ser Gl
115 120 125
Lys Gln Ser Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu Ph
130 135 140
Gly Lys Glu Lys Thr Gln Thr Tyr Leu Lys Lys Ala Ser Lys Ser Se
145 150 155 160
Leu Phe Ser Asn Lys Ser Lys Arg Asp Ile Asp Glu Asp Ile Asp Gl
165 170 175
Asp Thr Asp Thr Asp Gly Asp Ala Ile Pro Asp Val Trp Glu Glu As
180 185 190
Gly Tyr Thr Ile Lys Gly Arg Val Ala Val Lys Trp Asp Glu Gly Le
195 200 205
Ala Asp Lys Gly Tyr Lys Lys Phe Val Ser Asn Pro Phe Arg Gln Hi
210 215 220
Thr Ala Gly Asp Pro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Le
225 230 235 240
Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Ph
245 250 255
Pro Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Gl
260 265 270
Asn Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Ty
275 280 285
Thr Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gl
290 295 300
Leu Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr Va
305 310 315 320
Ala Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln Tyr Asn Th
325 330 335
Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr
340 345

1002 base pairs

nucleic acid

single

linear

DNA (genomic)

197U2

43
TGGGTTAATT GGGTATTATT TTACGGATGA GCAGCATAAG GAAGTAGCTT TTAYTCAATT60
AGGTGAAAAA AMTACATTAG CAGATTCAGC GAAAATGAAG AAAAACGACA AAAAGATTC120
TTCAGCGCAA TGGATTGGWA ATATACAGGT ACCTCAAACA GGGGAATATA CGTTTTCCA180
CTCTTCTGAT AAAGATACTA TTTTAAAACT CAATGGGGAA ACGATTATTC AAAAATCTA240
TATGGAGAAA CCCATATATT TAGAAAAAGA TAAAGTATAC GAAATTCAAA TCGAGCATA300
CAACCCGAAT AGTGAGAAAA CTTTACGATT ATCTTGGAAA ATGGGGGGCA CCAATTCAG360
GCTCATCCCA GAAAAATACA TTCTGTCTCC CGATTTTTCT AAAATAGCAG ATCAAGAAA420
TGARAAAAAA GACGCATCGA GACATTTATT ATTTACTAAG GATGAATTGA AAGATTCTG480
TAAGGACCTT ATCCCAGATG AATTTGAAAA AAATGGGTAT ACATTCAATG GGATTCAAA540
TGTTCCTTGG GATGAATCTC TTCAAGAACA GGGCTTTAAA AAATATATTT CCAATCCAT600
TCAATCGCGT ACAGCGCAGG ATCCATATAC AGATTTTGAA AAAGTAACCG GATATATGC660
TGCCGAAACA CAACTGGAAA CGCGTGACCC TTTAGTTGCG GCTTATCCGG CTGTAGGGG720
TACGATGGAA CAGTTTATTT TCTCTAAAAA TGATAATGTG CAGGAATCTA ATGGTGGAG780
AACTTCAAAA AGTATGACAG AAAGTTCTGA AACGACTTAC TCTGTTGAGA TAGGAGGGA840
ATTTACATTG AATCCATTCG CACTGGCGGA AATTTCTCCT AAATATTCTC ACAGTTGGA900
AAATGGAGCA TCTACAACAG AGGGAGAAAG TACTTCCTGG AGCTCACAAA TTGGTATTA960
CACGGCTGAA CGCGCGTTTT TTAAATGCCA ATATTCGATA TA 1002

333 amino acids

amino acid

single

linear

peptide

197U2

44
Gly Leu Ile Gly Tyr Tyr Phe Thr Asp Glu Gln His Lys Glu Val Al
1 5 10 15
Phe Xaa Gln Leu Gly Glu Lys Xaa Thr Leu Ala Asp Ser Ala Lys Me
20 25 30
Lys Lys Asn Asp Lys Lys Ile Leu Ser Ala Gln Trp Ile Xaa Asn Il
35 40 45
Gln Val Pro Gln Thr Gly Glu Tyr Thr Phe Ser Thr Ser Ser Asp Ly
50 55 60
Asp Thr Ile Leu Lys Leu Asn Gly Glu Thr Ile Ile Gln Lys Ser As
65 70 75 80
Met Glu Lys Pro Ile Tyr Leu Glu Lys Asp Lys Val Tyr Glu Ile Gl
85 90 95
Ile Glu His Asn Asn Pro Asn Ser Glu Lys Thr Leu Arg Leu Ser Tr
100 105 110
Lys Met Gly Gly Thr Asn Ser Glu Leu Ile Pro Glu Lys Tyr Ile Le
115 120 125
Ser Pro Asp Phe Ser Lys Ile Ala Asp Gln Glu Asn Xaa Lys Lys As
130 135 140
Ala Ser Arg His Leu Leu Phe Thr Lys Asp Glu Leu Lys Asp Ser As
145 150 155 160
Lys Asp Leu Ile Pro Asp Glu Phe Glu Lys Asn Gly Tyr Thr Phe As
165 170 175
Gly Ile Gln Ile Val Pro Trp Asp Glu Ser Leu Gln Glu Gln Gly Ph
180 185 190
Lys Lys Tyr Ile Ser Asn Pro Tyr Gln Ser Arg Thr Ala Gln Asp Pr
195 200 205
Tyr Thr Asp Phe Glu Lys Val Thr Gly Tyr Met Pro Ala Glu Thr Gl
210 215 220
Leu Glu Thr Arg Asp Pro Leu Val Ala Ala Tyr Pro Ala Val Gly Va
225 230 235 240
Thr Met Glu Gln Phe Ile Phe Ser Lys Asn Asp Asn Val Gln Glu Se
245 250 255
Asn Gly Gly Gly Thr Ser Lys Ser Met Thr Glu Ser Ser Glu Thr Th
260 265 270
Tyr Ser Val Glu Ile Gly Gly Lys Phe Thr Leu Asn Pro Phe Ala Le
275 280 285
Ala Glu Ile Ser Pro Lys Tyr Ser His Ser Trp Lys Asn Gly Ala Se
290 295 300
Thr Thr Glu Gly Glu Ser Thr Ser Trp Ser Ser Gln Ile Gly Ile As
305 310 315 320
Thr Ala Glu Arg Ala Phe Phe Lys Cys Gln Tyr Ser Ile
325 330

1073 base pairs

nucleic acid

single

linear

DNA (genomic)

202E1

45
TGGGTTAATT GGGTACTATT TTCAGGATCA AAAGTTTCAA CAACTCGCTT TGATGGCACA60
TAGACAAGCT TCAGATTTAG AAATACCTAA AAATGAAGTG AAGGATATAT TATCTAAAG120
TCAACAACAT ATTCAATCAG TGAGATGGAG GGGGTATATT AAGCCACCTC AAACAGGAG180
CTATATATTG TCAACCTCAT CCGACCAACA GGTCGTGATT GAACTCGATG GAAAAAACA240
TGTCAATCAA ACTTCTATGA CAGAACCAAT TCAACTCGAA AAAGATAAAC TCTATAAAA300
TAGAATTGAA TATGTCCCAG GAGATACAAA AGGACAAGAG AGCCTCCTTG ACTTTCAAC360
TAACTGGTCA ATTTCAGGAG ATACGGTGGA ACCAATTCCG GAGAATGCAT TTCTGTTAC420
AGACTTTTCT CATCAACAAG ATCAAGAGAA ACTCATCCCT GAAATCAGTC TATTTCAGG480
ACAAGGAGAT GAGAAAAAAG TATCTCGTAG TAAGAGGTCT TTAGCTACAA ACCCTCTCC540
TGATACAGAT GATGATGGTA TTTATGATGA ATGGGAAACG GAAGGATACA CAATACAGG600
ACAACTAGCG GTGAAATGGG ACGATTCTAT GAAGGAGCGA GGTTATACTA AGTATGTGT660
TAACCCTTAC AAGGCTCATA CAGTAGGAGA TCCCTACACA GATTGGGAAA AAGCGGCTG720
CCGTATCGAT AACGCTGTCA AAGCAGAAGC TAGGAATCCT TTAGTCGCGG CCTATCCAA780
TGTTGGTGTA CATATGGAAA GACTAATTGT CTCCGAAAAA CAAAATATAT CAACAGGAC840
TGGAAAAACC GTATCTGTGT CTATGTCCGC AAGCAATACC GCAGCGATTA CGGCAGGAA900
TAATGCAACA GCCGGTGCCT CTTTACTTGG GCCATCTGGA AACGTCACGG CTCATTTTT960
TTATACAGGA TCTAGTACAT CCACTGTTGA AAATAGCTCA AGTAATAATT GGAGTCAA1020
TCTTGGAATC GATACGGGAC AATCTGCGTA TTTAAATGCC AATGTAAGAT ATA 1073

357 amino acids

amino acid

single

linear

peptide

202E1

46
Gly Leu Ile Gly Tyr Tyr Phe Gln Asp Gln Lys Phe Gln Gln Leu Al
1 5 10 15
Leu Met Ala His Arg Gln Ala Ser Asp Leu Glu Ile Pro Lys Asn Gl
20 25 30
Val Lys Asp Ile Leu Ser Lys Asp Gln Gln His Ile Gln Ser Val Ar
35 40 45
Trp Arg Gly Tyr Ile Lys Pro Pro Gln Thr Gly Asp Tyr Ile Leu Se
50 55 60
Thr Ser Ser Asp Gln Gln Val Val Ile Glu Leu Asp Gly Lys Asn Il
65 70 75 80
Val Asn Gln Thr Ser Met Thr Glu Pro Ile Gln Leu Glu Lys Asp Ly
85 90 95
Leu Tyr Lys Ile Arg Ile Glu Tyr Val Pro Gly Asp Thr Lys Gly Gl
100 105 110
Glu Ser Leu Leu Asp Phe Gln Leu Asn Trp Ser Ile Ser Gly Asp Th
115 120 125
Val Glu Pro Ile Pro Glu Asn Ala Phe Leu Leu Pro Asp Phe Ser Hi
130 135 140
Gln Gln Asp Gln Glu Lys Leu Ile Pro Glu Ile Ser Leu Phe Gln Gl
145 150 155 160
Gln Gly Asp Glu Lys Lys Val Ser Arg Ser Lys Arg Ser Leu Ala Th
165 170 175
Asn Pro Leu Leu Asp Thr Asp Asp Asp Gly Ile Tyr Asp Glu Trp Gl
180 185 190
Thr Glu Gly Tyr Thr Ile Gln Gly Gln Leu Ala Val Lys Trp Asp As
195 200 205
Ser Met Lys Glu Arg Gly Tyr Thr Lys Tyr Val Ser Asn Pro Tyr Ly
210 215 220
Ala His Thr Val Gly Asp Pro Tyr Thr Asp Trp Glu Lys Ala Ala Gl
225 230 235 240
Arg Ile Asp Asn Ala Val Lys Ala Glu Ala Arg Asn Pro Leu Val Al
245 250 255
Ala Tyr Pro Thr Val Gly Val His Met Glu Arg Leu Ile Val Ser Gl
260 265 270
Lys Gln Asn Ile Ser Thr Gly Leu Gly Lys Thr Val Ser Val Ser Me
275 280 285
Ser Ala Ser Asn Thr Ala Ala Ile Thr Ala Gly Ile Asn Ala Thr Al
290 295 300
Gly Ala Ser Leu Leu Gly Pro Ser Gly Asn Val Thr Ala His Phe Se
305 310 315 320
Tyr Thr Gly Ser Ser Thr Ser Thr Val Glu Asn Ser Ser Ser Asn As
325 330 335
Trp Ser Gln Asp Leu Gly Ile Asp Thr Gly Gln Ser Ala Tyr Leu As
340 345 350
Ala Asn Val Arg Tyr
355

967 base pairs

nucleic acid

single

linear

DNA (genomic)

KB33

47
TGGATTACTT GGGTACTATT TTGAAGAACC AAACTTTAAT GACCTTCTAT TAATCACACA60
AAAAAACAAC AGTAATTTAT CTCTAGAAAA AGAACATATT TCATCGTTAT CTAGTATTA120
AAATAAAGGC ATTCAATCTG CTAGATGGTT AGGTTTTTTA AAACCAAAGC AAACGGATG180
ATATGTTTTT TTTAGTCCTT CCAACCATGA AATCATGATT CAAATCGATA ACAAAATTA240
TGTAATGGGT AGAAAAATTA TGTTAGAAGA AGGAAAGGTA TATCCAATTC GAATTGAAT300
CCGCTTTGAA AAAACAAATA ATCTAGATAT AAACTGCGAA CTACTTTGGA CGCATTCTG360
TACAAAAGAA ATCATTTCTC AAAACTGTTT GCTGGCACCT GATTATCATA ATACAGAAT420
TTACCCAAAA ACAAATTTAT TTGGGGATGT ATCTACTACG ACTAGTGATA CTGATAATG480
TGGAATACCA GATGACTGGG AAATTAATGG TTATACGTTT GATGGTACAA ATATAATTC540
ATGGAATCCT GCTTATGAAG GGTTATATAC TAAATATATT TCTAACCCTA AACAAGCAA600
TACAGTAGGT GATCCATATA CAGATTTAGA GAACGTMCAA AGCTAAAKGG ATCAAAGAA660
CARGAAAYCC TTKTAGCAGA AGCTWATCCG AAAAATTGGA BTTAGCATGG AAGAATTAC720
CRTCTCTKTA WAARTGKTGA TKTWTTCAAA TGCTCAAGAA AATKACTACT TACTTCTAG780
AGRACAGAAG GCACTTCASG TAGYGCAGGC ATTGAGGGAG GAGCAGAAGG AAAAAAACC840
ACAGGATTGG TTTCAGCCTC CTTTTCGCAT TCATCTTCAA CAACAAACAC AACGGAACA900
ATGAATGGAA CAATGATTCA TCTTGATACA GGAGAATCAG CGTATTTAAA TGCCAATGT960
AGATATA 967

972 base pairs

nucleic acid

single

linear

DNA (genomic)

KB38

48
TGGATTACTT GGGTATTATT TTGAAGAACC AAACTTTAAT AACCTTCTAT TAATCACACA60
AAAAAACAAC AGTAATTTAT CTCTAGAAAA AGAACATATT TCATCGTTAT CTAGTATTA120
AAATAAAGGC ATTCAATCTG CTAGATGGTT AGGTTTTTTA AAACCAGAGC AAACGGATG180
ATATGTTTTT TTTAGTCCTT CCAACCATGA AATTATGATT CAAATCGATA ACAAAATTA240
TGTAATGGGT AGAAAAATTA TGTTAGAAAA AGGAAAGGTA TATCCAATTC GAATTGAAT300
CCGCTTTGAA AAAACAAATA ATATAGATAT AAACTGCGAA CTACTTTGGA CGCACTCTG360
TACAAAAGAA ATCATTTCTC AAAACTTTTT GCTGGCACCT GATTATAACA ATACAGAAT420
TTATCCAAAA ACAAATTTAT TTGGAGATGT ATCTACTACG ACTWAGTGAT ACTGATAAT480
ATGGAATACC AGATGACTGG GAAATTAATG GTTATACCTT TGATGGTACA AATATAATT540
AGTGGAATTC TGCTTATGAA GGGTTATATA CTAAATATGT TTCTAATCCT AAACAAGCA600
GTACAGTAGG TGATCCATAT ACAGATTTAG AGAAAGTAAC AGCTCAAATG GATCGAGCA660
CCTCTCTAGA AGCAAGGAAT CCTTTAGTAG CAGCTTATCC AAAAATTGGA GTTAGCATG720
AAGAATTACT CATCTCTTTA AATGTTGATT TTTCAAATGC TCAAGAAAAT ACTACTTCT780
CTAGTAGAAC AGAAGGCACT TCACGTAGCG CAGGCATTGA GGGAGGAGCA GAAGGAAAA840
AACCTACAGG ATTGGTTTCA GCCTCCTTTT CGCATTCATC TTCAACAACA AACACAACG900
AACAAATGAA TGGAACAATG ATTCATCTTG ATACAGGAGA ATCAGCGTAT TTAAATGCC960
ATGTAAGATA TA 972

21 base pairs

nucleic acid

single

linear

DNA (genomic)

49
CTTGAYTTTA AARATGATRT A 21

21 base pairs

nucleic acid

single

linear

DNA (genomic)

50
AATRGCSWAT AAATAMGCAC C 21

1341 base pairs

nucleic acid

single

linear

DNA (genomic)

PS177C8

51
ATGTTTATGG TTTCTAAAAA ATTACAAGTA GTTACTAAAA CTGTATTGCT TAGTACAGTT60
TTCTCTATAT CTTTATTAAA TAATGAAGTG ATAAAAGCTG AACAATTAAA TATAAATTC120
CAAAGTAAAT ATACTAACTT GCAAAATCTA AAAATCACTG ACAAGGTAGA GGATTTTAA180
GAAGATAAGG AAAAAGCGAA AGAATGGGGG AAAGAAAAAG AAAAAGAGTG GAAACTAAC240
GCTACTGAAA AAGGAAAAAT GAATAATTTT TTAGATAATA AAAATGATAT AAAGACAAA300
TATAAAGAAA TTACTTTTTC TATGGCAGGC TCATTTGAAG ATGAAATAAA AGATTTAAA360
GAAATTGATA AGATGTTTGA TAAAACCAAT CTATCAAATT CTATTATCAC CTATAAAAA420
GTGGAACCGA CAACAATTGG ATTTAATAAA TCTTTAACAG AAGGTAATAC GATTAATTC480
GATGCAATGG CACAGTTTAA AGAACAATTT TTAGATAGGG ATATTAAGTT TGATAGTTA540
CTAGATACGC ATTTAACTGC TCAACAAGTT TCCAGTAAAG AAAGAGTTAT TTTGAAGGT600
ACGGTTCCGA GTGGGAAAGG TTCTACTACT CCAACAAAAG CAGGTGTCAT TTTAAATAA660
AGTGAATACA AAATGCTCAT TGATAATGGG TATATGGTCC ATGTAGATAA GGTATCAAA720
GTGGTGAAAA AAGGGGTGGA GTGCTTACAA ATTGAAGGGA CTTTAAAAAA GAGTCTTGA780
TTTAAAAATG ATATAAATGC TGAAGCGCAT AGCTGGGGTA TGAAGAATTA TGAAGAGTG840
GCTAAAGATT TAACCGATTC GCAAAGGGAA GCTTTAGATG GGTATGCTAG GCAAGATTA900
AAAGAAATCA ATAATTATTT AAGAAATCAA GGCGGAAGTG GAAATGAAAA ACTAGATGC960
CAAATAAAAA ATATTTCTGA TGCTTTAGGG AAGAAACCAA TACCGGAAAA TATTACTG1020
TATAGATGGT GTGGCATGCC GGAATTTGGT TATCAAATTA GTGATCCGTT ACCTTCTT1080
AAAGATTTTG AAGAACAATT TTTAAATACA ATCAAAGAAG ACAAAGGATA TATGAGTA1140
AGCTTATCGA GTGAACGTCT TGCAGCTTTT GGATCTAGAA AAATTATATT ACGATTAC1200
GTTCCGAAAG GAAGTACGGG TGCGTATTTA AGTGCCATTG GTGGATTTGC AAGTGAAA1260
GAGATCCTAC TTGATAAAGA TAGTAAATAT CATATTGATA AAGTAACAGA GGTAATTA1320
AAGGTGTTAA GCGATATGTA G 1341

446 amino acids

amino acid

single

linear

peptide

PS177C8

52
Met Phe Met Val Ser Lys Lys Leu Gln Val Val Thr Lys Thr Val Le
1 5 10 15
Leu Ser Thr Val Phe Ser Ile Ser Leu Leu Asn Asn Glu Val Ile Ly
20 25 30
Ala Glu Gln Leu Asn Ile Asn Ser Gln Ser Lys Tyr Thr Asn Leu Gl
35 40 45
Asn Leu Lys Ile Thr Asp Lys Val Glu Asp Phe Lys Glu Asp Lys Gl
50 55 60
Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Th
65 70 75 80
Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn Lys Asn As
85 90 95
Ile Lys Thr Asn Tyr Lys Glu Ile Thr Phe Ser Met Ala Gly Ser Ph
100 105 110
Glu Asp Glu Ile Lys Asp Leu Lys Glu Ile Asp Lys Met Phe Asp Ly
115 120 125
Thr Asn Leu Ser Asn Ser Ile Ile Thr Tyr Lys Asn Val Glu Pro Th
130 135 140
Thr Ile Gly Phe Asn Lys Ser Leu Thr Glu Gly Asn Thr Ile Asn Se
145 150 155 160
Asp Ala Met Ala Gln Phe Lys Glu Gln Phe Leu Asp Arg Asp Ile Ly
165 170 175
Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala Gln Gln Val Ser Se
180 185 190
Lys Glu Arg Val Ile Leu Lys Val Thr Val Pro Ser Gly Lys Gly Se
195 200 205
Thr Thr Pro Thr Lys Ala Gly Val Ile Leu Asn Asn Ser Glu Tyr Ly
210 215 220
Met Leu Ile Asp Asn Gly Tyr Met Val His Val Asp Lys Val Ser Ly
225 230 235 240
Val Val Lys Lys Gly Val Glu Cys Leu Gln Ile Glu Gly Thr Leu Ly
245 250 255
Lys Ser Leu Asp Phe Lys Asn Asp Ile Asn Ala Glu Ala His Ser Tr
260 265 270
Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Ser Gl
275 280 285
Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gln Asp Tyr Lys Glu Ile As
290 295 300
Asn Tyr Leu Arg Asn Gln Gly Gly Ser Gly Asn Glu Lys Leu Asp Al
305 310 315 320
Gln Ile Lys Asn Ile Ser Asp Ala Leu Gly Lys Lys Pro Ile Pro Gl
325 330 335
Asn Ile Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe Gly Tyr Gl
340 345 350
Ile Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu Gln Phe Le
355 360 365
Asn Thr Ile Lys Glu Asp Lys Gly Tyr Met Ser Thr Ser Leu Ser Se
370 375 380
Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys Ile Ile Leu Arg Leu Gl
385 390 395 400
Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala Ile Gly Gly Ph
405 410 415
Ala Ser Glu Lys Glu Ile Leu Leu Asp Lys Asp Ser Lys Tyr His Il
420 425 430
Asp Lys Val Thr Glu Val Ile Ile Lys Val Leu Ser Asp Met
435 440 445

17 base pairs

nucleic acid

single

linear

DNA (genomic)

53
GGATTCGTTA TCAGAAA 17

17 base pairs

nucleic acid

single

linear

DNA (genomic)

54
CTGTYGCTAA CAATGTC 17

8 amino acids

amino acid

single

linear

peptide

55
Ala Asp Glu Pro Phe Asn Ala Asp
1 5

21 base pairs

nucleic acid

single

linear

DNA (genomic)

56
GCTGATGAAC CATTTAATGC C 21

8 amino acids

amino acid

single

linear

peptide

57
Leu Phe Lys Val Asp Thr Lys Gln
1 5

22 base pairs

nucleic acid

single

linear

DNA (genomic)

58
CTCTTTAAAG TAGATACTAA GC 22

9 amino acids

amino acid

single

linear

peptide

59
Pro Asp Glu Asn Leu Ser Asn Ile Glu
1 5

24 base pairs

nucleic acid

single

linear

DNA (genomic)

60
GATGAGAACT TATCAAATAG TATC 24

12 amino acids

amino acid

single

linear

peptide

61
Ala Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr
1 5 10

33 base pairs

nucleic acid

single

linear

DNA (genomic)

62
CGAATTCTTT ATTAGATAAG CAACAACAAA CCT 33

8 amino acids

amino acid

single

linear

peptide

63
Val Ile Ser Gln Lys Gly Gln Lys
1 5

24 base pairs

nucleic acid

single

linear

DNA (genomic)

64
GTTATTTCGC AAAAAGGCCA AAAG 24

11 amino acids

amino acid

single

linear

peptide

65
Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pro Asp
1 5 10

31 base pairs

nucleic acid

single

linear

DNA (genomic)

66
GAATATCAAT CTGATAAAGC GTTAAACCCA G 31

9 amino acids

amino acid

single

linear

peptide

67
Ser Ser Leu Phe Ser Asn Lys Ser Lys
1 5

23 base pairs

nucleic acid

single

linear

DNA (genomic)

68
GCAGCYTGTT TAGCAATAAA AGT 23

8 amino acids

amino acid

single

linear

peptide

69
Ile Lys Gly Arg Val Ala Val Lys
1 5

20 base pairs

nucleic acid

single

linear

DNA (genomic)

70
CAAAGGAAGA GTAGCTGTTA 20

9 amino acids

amino acid

single

linear

peptide

71
Val Asn Val Ser Leu Glu Asn Val Thr
1 5

25 base pairs

nucleic acid

single

linear

DNA (genomic)

72
CAATGTTAGC TTGGAAAATG TCACC 25

8 amino acids

amino acid

single

linear

peptide

73
Thr Ala Phe Ile Gln Val Gly Glu
1 5

20 base pairs

nucleic acid

single

linear

DNA (genomic)

74
AGCATTTATT CAAGTAGGAG 20

7 amino acids

amino acid

single

linear

peptide

75
Tyr Leu Leu Ser Thr Ser Ser
1 5

19 base pairs

nucleic acid

single

linear

DNA (genomic)

76
TCTACTTTCC ACGTCCTCT 19

7 amino acids

amino acid

single

linear

peptide

77
Gln Ile Gln Pro Gln Gln Arg
1 5

19 base pairs

nucleic acid

single

linear

DNA (genomic)

78
CAGATACAAC CGCAACAGC 19

8 amino acids

amino acid

single

linear

peptide

79
Pro Gln Gln Arg Ser Thr Gln Ser
1 5

23 base pairs

nucleic acid

single

linear

DNA (genomic)

80
CCGCAACAGC GTTCAACTCA ATC 23

7 amino acids

amino acid

single

linear

peptide

81
Asp Gly Ala Ile Val Ala Trp
1 5

21 base pairs

nucleic acid

single

linear

DNA (genomic)

82
GACGGTGCGA TTGTTGCCTG G 21

7 amino acids

amino acid

single

linear

peptide

83
Glu Gly Asp Ser Gly Thr Val
1 5

19 base pairs

nucleic acid

single

linear

DNA (genomic)

84
GAAGGAGACT CAGGTACTG 19

6 amino acids

amino acid

single

linear

peptide

85
Thr Val Thr Asn Thr Ser
1 5

19 base pairs

nucleic acid

single

linear

DNA (genomic)

86
CCGTAACCAA TACAAGCAC 19

9 amino acids

amino acid

single

linear

peptide

87
Ser Ser Gln Leu Ala Tyr Asn Pro Ser
1 5

25 base pairs

nucleic acid

single

linear

DNA (genomic)

88
CTTCACAATT AGCGTATAAT CCTTC 25

7 amino acids

amino acid

single

linear

peptide

89
Glu Gln His Lys Glu Val Ala
1 5

19 base pairs

nucleic acid

single

linear

DNA (genomic)

90
GAGCAGCATA AGGAAGTAG 19

8 amino acids

amino acid

single

linear

peptide

91
Phe Asn Gly Ile Gln Ile Val Pro
1 5

25 base pairs

nucleic acid

single

linear

DNA (genomic)

92
CATTCAATGG GATTCAAATT GTTCC 25

8 amino acids

amino acid

single

linear

peptide

93
Val Gln Glu Ser Asn Gly Gly Gly
1 5

23 base pairs

nucleic acid

single

linear

DNA (genomic)

94
GTGCAGGAAT CTAATGGTGG AGG 23

9 amino acids

amino acid

single

linear

peptide

95
Glu Ile Gly Gly Lys Phe Thr Leu Asn
1 5

22 base pairs

nucleic acid

single

linear

DNA (genomic)

96
GATAGGAGGG AAATTTACAT TG 22

19 base pairs

nucleic acid

single

linear

DNA (genomic)

97
CGAATTGAAT GCCGCTTTG 19

22 base pairs

nucleic acid

single

linear

DNA (genomic)

98
CTCAAAACTK TTTGCTGGCA CC 22

20 base pairs

nucleic acid

single

linear

DNA (genomic)

99
GGATCRAGCA ACCTCTCTAG 20

18 base pairs

nucleic acid

single

linear

DNA (genomic)

100
ACTACTTACT TCTAGTAG 18

8 amino acids

amino acid

single

linear

peptide

101
Ser Asp Gln Gln Val Val Ile Glu
1 5

21 base pairs

nucleic acid

single

linear

DNA (genomic)

102
CCGAYCRACA KGTCRTRATT G 21

7 amino acids

amino acid

single

linear

peptide

103
Asn Gln Thr Ser Met Thr Glu
1 5

21 base pairs

nucleic acid

single

linear

DNA (genomic)

104
TCARDCTTCT ATGACAGMAC C 21

8 amino acids

amino acid

single

linear

peptide

105
Gln Asp Gln Glu Lys Ile Ile Pro
1 5

24 base pairs

nucleic acid

single

linear

DNA (genomic)

106
CAAGATCAAG ARAARMTYAT YCCT 24

7 amino acids

amino acid

single

linear

peptide

107
Ser His Lys Gln Asp Gln Glu
1 5

18 base pairs

nucleic acid

single

linear

DNA (genomic)

108
CTCRTMAACA AGATCAAG 18

7 amino acids

amino acid

single

linear

peptide

109
Ser Gly Ser Val Thr Ala His
1 5

18 base pairs

nucleic acid

single

linear

DNA (genomic)

110
CTGGAARYGT SACGGCTC 18

22 base pairs

nucleic acid

single

linear

DNA (genomic)

111
GCTTAGTATC TACTTTAAAG AG 22

24 base pairs

nucleic acid

single

linear

DNA (genomic)

112
GATACTATTT GATAAGTTCT CATC 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

113
CTTTTGGCCT TTTTGCGAAA TAAC 24

31 base pairs

nucleic acid

single

linear

DNA (genomic)

114
CTGGGTTTAA CGCTTTATCA GATTGATATT C 31

23 base pairs

nucleic acid

single

linear

DNA (genomic)

115
ACTTTTATTG CTAAACARGC TGC 23

20 base pairs

nucleic acid

single

linear

DNA (genomic)

116
TAACAGCTAC TCTTCCTTTG 20

25 base pairs

nucleic acid

single

linear

DNA (genomic)

117
GGTGACATTT TCCAAGCTAA CATTG 25

19 base pairs

nucleic acid

single

linear

DNA (genomic)

118
AGAGGACGTG GAAAGTAGA 19

19 base pairs

nucleic acid

single

linear

DNA (genomic)

119
GCTGTTGCGG TTGTATCTG 19

23 base pairs

nucleic acid

single

linear

DNA (genomic)

120
GATTGAGTTG AACGCTGTTG CGG 23

21 base pairs

nucleic acid

single

linear

DNA (genomic)

121
CCAGGCAACA ATCGCACCGT C 21

19 base pairs

nucleic acid

single

linear

DNA (genomic)

122
CAGTACCTGA GTCTCCTTC 19

19 base pairs

nucleic acid

single

linear

DNA (genomic)

123
GTGCTTGTAT TGGTTACGG 19

25 base pairs

nucleic acid

single

linear

DNA (genomic)

124
GAAGGATTAT ACGCTAATTG TGAAG 25

25 base pairs

nucleic acid

single

linear

DNA (genomic)

125
GGAACAATTT GAATCCCATT GAATG 25

23 base pairs

nucleic acid

single

linear

DNA (genomic)

126
CCTCCACCAT TAGATTCCTG CAC 23

22 base pairs

nucleic acid

single

linear

DNA (genomic)

127
CAATGTAAAT TTCCCTCCTA TC 22

22 base pairs

nucleic acid

single

linear

DNA (genomic)

128
GGTGCCAGCA AAMAGTTTTG AG 22

20 base pairs

nucleic acid

single

linear

DNA (genomic)

129
CTAGAGAGGT TGCTYGATCC 20

18 base pairs

nucleic acid

single

linear

DNA (genomic)

130
CTACTAGAAG TAAGTAGT 18

21 base pairs

nucleic acid

single

linear

DNA (genomic)

131
GGTKCTGTCA TAGAAGHYTG A 21

24 base pairs

nucleic acid

single

linear

DNA (genomic)

132
AGGRATRAKY TTYTCTTGAT CTTG 24

18 base pairs

nucleic acid

single

linear

DNA (genomic)

133
CTTGATCTTG TTKAYGAG 18

18 base pairs

nucleic acid

single

linear

DNA (genomic)

134
GAGCCGTSAC RYTTCCAG 18

21 base pairs

nucleic acid

single

linear

DNA (genomic)

135
CCAGTCCAAT GAACCTCTTA C 21

21 base pairs

nucleic acid

single

linear

DNA (genomic)

136
AGGGAACAAA CCTTCCCAAC C 21

20 base pairs

nucleic acid

single

linear

DNA (genomic)

137
CARMTAKTAA MTAGGGATAG 20

22 base pairs

nucleic acid

single

linear

DNA (genomic)

138
AGYTTCTATC GAAGCTGGGR ST 22

1035 base pairs

nucleic acid

single

linear

DNA (genomic)

139
GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA60
ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCA120
CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGA180
TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGT240
TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAAT300
AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCT360
AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAA420
ATCCTGAATT TAATAAAAAG GAAACGAAAG AGCTTTTATC AAAAACAGCA AAAAGAACC480
TTTCTCTTCA AAACGGGGTT GAGAAGCGAT GAGGATGATG ATCTAGGATA CAGATGGTG540
TAGCATTCCT GGATAATTGG GAAATGAATG GATATACCAT TCAAACGAAA AATGGCAGT600
AAATGGGATG ATTCATTTGC AGAAAAAGGA TATACAAAAT TTGTTTCGAA TCCATATGA660
GCCCATACAG CAGGAGATCC TTATACCGAT TATGAAAAAG CAGCAAAAGA TATTCCTTT720
TCGAACGCAA AAGAAGCCTT TAATCCTCTT GTAGCTGCTT TTCCATCTGT CAATGTAGG780
TTAGAAAAAG TAGTAATTTC TAAAAATGAG GATATGAGTC AGGGTGTATC ATCCAGCAC840
TCGAATAGTG CCTCTAATAC AAATTCAATT GGTGTTACCG TAGATGCTGG TTGGGAAGG900
TTGTTCCCTA AATTTGGTAT TTCAACTAAT TATCAAAACA CATGGACCAC TGCACAAGA960
TGGGGCTCTT CTAAAGAAGA TTCTACCCAT ATAAATGGAG CACAATCAGC CTTTTTAA1020
GCAAATGTAC GATAT 1035

345 amino acids

amino acid

single

linear

protein

140
Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Th
1 5 10 15
Met Phe Ala Pro Thr Ile Asn Asn Thr Leu Ile Tyr Asp Arg Gln Th
20 25 30
Ala Asp Thr Leu Leu Asn Lys Gln Gln Gln Glu Phe Asn Ser Ile Ar
35 40 45
Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Gl
50 55 60
Leu Ser Asp Asp Lys Asn Ala Ile Ile Glu Ile Asp Gly Lys Val Va
65 70 75 80
Ser Arg Arg Gly Glu Asp Lys Gln Thr Ile His Leu Glu Lys Gly Ly
85 90 95
Met Val Pro Ile Lys Ile Glu Tyr Gln Ser Asn Glu Pro Leu Thr Va
100 105 110
Asp Ser Lys Val Phe Asn Asp Leu Lys Leu Phe Lys Ile Asp Gly Hi
115 120 125
Asn Gln Ser His Gln Ile Gln Gln Asp Asp Leu Lys Ile Leu Asn Le
130 135 140
Ile Lys Arg Lys Arg Lys Ser Phe Tyr Gln Lys Gln Gln Lys Glu Pr
145 150 155 160
Phe Leu Phe Lys Thr Gly Leu Arg Ser Asp Glu Asp Asp Asp Leu Gl
165 170 175
Tyr Arg Trp Xaa Xaa His Ser Trp Ile Ile Gly Lys Xaa Met Asp Il
180 185 190
Pro Phe Lys Arg Lys Met Ala Val Lys Trp Asp Asp Ser Phe Ala Gl
195 200 205
Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Tyr Glu Ala His Thr Al
210 215 220
Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Lys Asp Ile Pro Le
225 230 235 240
Ser Asn Ala Lys Glu Ala Phe Asn Pro Leu Val Ala Ala Phe Pro Se
245 250 255
Val Asn Val Gly Leu Glu Lys Val Val Ile Ser Lys Asn Glu Asp Me
260 265 270
Ser Gln Gly Val Ser Ser Ser Thr Ser Asn Ser Ala Ser Asn Thr As
275 280 285
Ser Ile Gly Val Thr Val Asp Ala Gly Trp Glu Gly Leu Phe Pro Ly
290 295 300
Phe Gly Ile Ser Thr Asn Tyr Gln Asn Thr Trp Thr Thr Ala Gln Gl
305 310 315 320
Trp Gly Ser Ser Lys Glu Asp Ser Thr His Ile Asn Gly Ala Gln Se
325 330 335
Ala Phe Leu Asn Ala Asn Val Arg Tyr
340 345

1037 base pairs

nucleic acid

single

linear

DNA (genomic)

141
GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA60
ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCA120
CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGA180
TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGT240
TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAAT300
AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCT360
AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAA420
AATCCTGAAT TTAATAAAAA AGAAACGAAA GAGCTTTTAT CAAAAACAGC AAAAAGRAA480
CTTTTCTCTT CAAACGRRGT KGAGAAGCGA TGAGGATGAT RATCYTAGAT ACAGGTGGK540
ATAGCATTCC YKGATAATTG GGGAAATGAA WGGRTATACC ATTCAACSGA AAAATGGSA600
TCAAATGGGA TGATTCATTT GCGGAAAAAG GATATACAAA ATTTGTTTCG AATCCATAT660
AAGCCCATAC AGCAGGAGAT CCTTATACCG ATTATGAAAA AGCAGCAAAA GATATTCCT720
TATCGAACGC AAAAGAAGCC TTTAATCCTC TTGTAGCTGC TTTTCCATCT GTCAATGTA780
GATTAGAAAA AGTAGTAATT TCTAAAAATG AGGATATGAG TCAGGGTGTA TCATCCAGC840
CTTCGAATAG TGCCTCTAAT ACAAATTCAA TTGGTGTTAC CGTAGATGCT GGTTGGGAA900
GTTTGTTCCC TAAATTTGGT ATTTCAACTA ATTATCAAAA CACATGGACC ACTGCACAA960
AATGGGGCTC TTCTAAAGAA GATTCTACCC ATATAAATGG AGCACAATCA GCCTTTTT1020
ATGCAAATGT ACGATAT 1037

1048 base pairs

nucleic acid

single

linear

DNA (genomic)

142
TGGGTTAATT GGGTATTATT TTAAAGGGCA AGAGTTTAAT CATCTTACTT TGTTCGCACC60
AACACGTGAT AATACCCTTA TTTATGATCA ACAAACAGCG AATTCCTTAT TAGATACCA120
GCAACAAGAA TATCAATCTA TTCGCTGGAT TGGTTTAATT CAAAGTAAAG AAACGGGTG180
TTTCACATTT AACTTATCAG ATGATCAACA TGCAATTATA GAAATCGATG GCAAAATCA240
TTCGCATAAA GGACAGAATA AACAAGTTGT TCACTTAGAA AAAGGAAAGT TAGTCCCGA300
AAAAATTGAG TATCAATCAG ATCAACTATT AAATAGGGAT AGTAACATCT TTAAAGAGT360
TAAATTATTC AAAGTAGATA GTCAGCAACA CGCTCACCAA GTTCAACTAG ACGAATTAA420
AAACCCTGCG TTTAATAAAA AGGAAACACA ACAATCTTAA GAAAAAGCAT CCAAAAACA480
TCTTTTTACA CCAGGGACAT TAAAAGGAAG ATACTGATGA TGATGATAAG GATAACAGG540
TGGGAGATTC TATTCCTGGA CCTTTTGGGG GAAGAAAATG GGTATACCAA TCCCAAAAT600
AAATAGCTGG TCCAAGTGGG ATGTTCATTC GCCGCGAAAG GGTATACAAA TTTGTTTCT660
AATCCACTTG ATAGTCATAC AGTTGGAGAT CCCTATACGG ATTATGAAAA AGCAGCAAG720
GATTTAGACT TGGCCCAATG CAAAAGAAAC ATTTAACCCA TTAGTAGCTG CTTTTCCAA780
TGTGAATGTG AATTTGGAAA AAGTCATTTT ATCTAAAGAT GAAAATCTAT CCAATAGTG840
AGAGTCACAT TCCTCCACCA ACTGGTCTTA TACGAATACA GAAGGAGCTT CTATCGAAG900
TGGGGCTAAA CCAGAGGGTC CTACTTTTGG AGTGAGTGCT ACTTATCAAC ACTCTGAAA960
AGTTGCAAAA GAATGGGGAA CATCTACAGG AAATACCTCG CAATTTAATA CAGCTTCA1020
AGGATATTTA AATGCAAATG TACGATAT 1048

1175 base pairs

nucleic acid

single

linear

DNA (genomic)

143
ACCTCTAGAT GCANGCTCGA GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGATTA60
CTTGGGTATT ATTTTAAAGG GAAAGAGTTT AATCATCTTA CTTTGTTCGC ACCAACACG120
GATAATACCC TTATTTATGA TCAACAAACA GCGAATTCCT TATTAGATAC CAAACAACA180
GAATATCAAT CTATTCGCTG GATTGGTTTG ATTCAAAGTA AAGAAACAGG TGATTTCAC240
TTTAACTTAT CTGATGATCA AAATGCAATT ATAGAAATAG ATGGCAAAAT CATTTCGCA300
AAAGGACAGA ATAAACAAGT TGTTCACTTA GAAAAAGGAA AGTTAGTCCC GATAAAAAT360
GAGTATCAAT CAGATCAGAT ATTAACTAGG GATAGTAACA TCTTTAAAGA GTTCAATTA420
TCAAAGTAGA TAGTCAAGCA ACACTCTCAC CAAAGTTCAA CTTAGGNCNG AATTAAGNA480
CCCTNGGATT TTAANTTNAA AAAAAGGAAC CCNCANCATT CTTTAGGAAA AAGCAGCAA540
AACCAAATCC TTTTTTACCA CAGGATATTG AAAAGGAGAT ACGGGNTNGA TGATGGATT600
ATACCGGGAT ACCAGTTGGG GNTTCTANTC CCTGACCTTT GGGGAAAGAA AATNGGTAT660
CCNATCCCAA AANTTAAGCC AGCTGTCCAG GTGGGATGAT TCAATTCGCC CGCGAAAGG720
TATACCAAAA TTTGTTTCTT AATCCACTTG AGAGTCATAC AGTTGGAGAT CCCTATACG780
ATTATGAAAA AGCAGCAAGA GATTTAGACT TGGCCAATGC AAAAGAAACA TTTAACCCA840
TAGTAGCTGC TTTTCCAAGT GTGAATGTGA ATTTGGAAAA AGTAATATTA TCCCCAGAT900
AGAATTTATC TAACAGTGTA GAATCTCATT CGTCTACAAA TTGGTCTTAT ACGAATACT960
AAGGAGCTTC TATCGAAGCT GGGGGTGGTC CATTAGGTAT TTCATTTGGA GTGAGTGC1020
ATTATCAACA CTCTGAAACA GTTGCAAAAG AATGGGGAAC ATCTACAGGA AATACCTC1080
AATTTAATAC AGCTTCAGCA GGATATTTAA ATGCCAATGG TCGATNTAAG CCGAATNC1140
NCACACTGNC GGCCGTTAGT AGTGGCACCG AGCCC 1175

1030 base pairs

nucleic acid

single

linear

DNA (genomic)

144
GGRTTAMTTG GGTATTATTT TAAAGGGAAA GATTTTAATG ATCTTACTGT ATTTGCACCA60
ACGCGTGGGA ATACTCTTGT ATATGATCAA CAAACAGCAA ATACATTACT AAATCAAAA120
CAACAAGACT TTCAGTCTAT TCGTTGGGTT GGTTTAATTC AAAGTAAAGA AGCAGGCGA180
TTTACATTTA ACTTATCAGA TGATGAACAT ACGATGATAG AAATCGATGG GAAAGTTAT240
TCTAATAAAG GGAAAGAAAA ACAAGTTGTC CATTTAGAAA AAGGACAGTT CGTTTCTAT300
AAAATAGAAT ATCAAGCTGA TGAACCATTT AATGCGGATA GTCAAACCTT TAAAAATTT360
AAACTCYTTA AAGTAGATAC TAAGCAACAG TCCCAGCAAA TTCAACTAGA TGAATTAAG420
AACCCTGRAA TTTAATAAAA AAGAAACACA AGAATTTCTA ACAAAAGCAA CAAAAACAA480
CCTTATTACT CAAAAAGTGA AGAGTACTAG GGATGAAGAC ACGGATACAG ATGGAGATT540
TATTCCAGAC ATTTGGGAAG AAAATGGGTA TACCATCCAA AATAAGATTG CCGTCAAAT600
GGATGATTCA TTAGCAAGTA AAGGATATAC GAAATTTGTT TCAAACCCAC TAGATACTC660
CACGGTTGGA GATCCTTATA CAGATTATGA AAAAGCAGCA AGGGATTTAG ATTTGTCAA720
TGCAAAAGAA ACATTTAACC CATTAGTTGC GGCTTTTCCA AGTGTGAATG TGAGTATGG780
AAAAGTGATA TTGTCTCCAG ATGAGAACTT ATCAAATAGT ATCGAGTCTC ATTCATCTA840
GAATTGGTCG TATACGAATA CAGAAGGGGC TTCTATTGAA GCTGGTGGGG GAGCATTAG900
CCTATCTTTT GGTGTAAGTG CAAACTATCA ACATTCTGAA ACAGTTGGGT ATGAATGGG960
AACATCTACG GGAAATACTT CGCAATTTAA TACAGCTTCA GCGGGGTATT TAAATGCC1020
TRTAMGATAT 1030

Number	Name	Date	Kind
4448885	Schnepf et al.	May 1984	A
4467036	Schnepf et al.	Aug 1984	A
4797276	Herrnstadt et al.	Jan 1989	A
4853331	Herrnstadt et al.	Aug 1989	A
4918006	Ellar et al.	Apr 1990	A
4948734	Edwards et al.	Aug 1990	A
4990332	Payne et al.	Feb 1991	A
5039523	Payne et al.	Aug 1991	A
5093120	Edwards et al.	Mar 1992	A
5126133	Payne et al.	Jun 1992	A
5151363	Payne	Sep 1992	A
5164180	Payne et al.	Nov 1992	A
5169629	Payne et al.	Dec 1992	A
5204237	Gaertner et al.	Apr 1993	A
5236843	Narva et al.	Aug 1993	A
5262399	Hickle et al.	Nov 1993	A
5270448	Payne	Dec 1993	A
5281530	Sick et al.	Jan 1994	A
5322932	Narva et al.	Jun 1994	A
5350577	Payne	Sep 1994	A
5426049	Sick et al.	Jun 1995	A
5439881	Narva et al.	Aug 1995	A
5667993	Feitelson et al.	Sep 1997	A
5670365	Feitelson	Sep 1997	A
5770696	Warren et al.	Jun 1998	A
5840868	Warren et al.	Nov 1998	A
5849870	Warren et al.	Dec 1998	A
5866326	Warren et al.	Feb 1999	A
5872212	Warren et al.	Feb 1999	A
5877012	Estruch et al.	Mar 1999	A
5888801	Warren et al.	Mar 1999	A
5889174	Warren et al.	Mar 1999	A

Number	Date	Country
0 359 472	Mar 1990	EP
WO 9404684	Mar 1994	WO
WO 9405771	Mar 1994	WO
WO 9421795	Sep 1994	WO
WO 9424264	Oct 1994	WO
WO 9605314	Feb 1996	WO
WO 9610083	Apr 1996	WO
WO 9818932	May 1998	WO

	Number	Date	Country
Parent	09/073898	May 1998	US
Child	09/850351		US

	Number	Date	Country
Parent	08/960780	Oct 1997	US
Child	09/073898		US

Pesticidal toxins and nucleotide sequences which encode these toxins

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

US Referenced Citations (32)

Foreign Referenced Citations (8)

Non-Patent Literature Citations (16)

Provisional Applications (1)

Continuations (1)

Continuation in Parts (1)

Entry
Asano, Shoji et al., “A Unique Activity in Bacillus thuringiensis Growth Medium,” Appl. Entomol. Zool., 1994, pp. 39-45, vol. 29(1).
Beegle, C.C., “Use of Entomogenous Bacteria in Agroecosystems,” Developments in Industrial Microbiology, 1978, pp. 97-104, vol. 20.
Carozzi, N.B. et al., “Prediction of Insecticidal Activity of Bacillus thuringiensis Strains by Polymerase Chain Reaction Product Profiles,” Applied and Environmental Microbiology, 1991, pp. 3057-3061, vol. 57(11).
Couch, T.L., “Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,” Developments in Industrial Microbiology, 1980, pp. 61-76, vol. 22.
Estruch, J.J. et al., “Vip3A, A Novel Bacillus thuringiensis Vegetative Insecticidal Protein with a Wide Spectrum of Activities Against Lepidopteran Insects,” Proc. Natl. Acad. Sci. USA, pp. 5389-5394, vol. 93.
Feitelson, J.S. et al., “Bacillus thuringiensis: Insects and Beyond,” Bio/Technology, 1992, pp. 271-275, vol. 10.
Gaertner, F.H. and Leo Kim, “Current Applied Recombinant DNA Projects,” TIBTECH, 1988, pp. 54-57, vol. 6(4).
Gaertner, F.H., “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” Controlled Delivery of Crop Protection Agents, R.M. Wilkins, ed., 1989, pp. 245-255, Taylor and Francis, New York and London.
Gleave, A.P. et al., “Identification of an Insecticidal Crystal Protein from Bacillus thuringiensis DSIR517 with Significant Sequence Differences from Previously Described Toxins,” Journal of General Microbiology, 1992, pp. 55-62, vol. 138.
Hofte, H. and H.R. Whiteley, “Insecticidal Crystal Proteins of Bacillus thuringiensis,” Microbiological Reviews, 1989, pp. 242-255, vol. 53(2).
Krieg, V.A. et al., “Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of Coleoptera,” Z. Ang. Ent., 1983, pp. 500-508, vol. 96, Abstract.
Lambert, B. et al., “A Bacillus thuringiensis Insecticidal Crystal Protein with a High Activity against Members of the Family Noctuidae,” Applied and Environmental Microbiology, 1996, pp. 80-86, vol. 62(1).
Li, Jade, “Bacterial Toxins,” Current Opinion in Structural Biology, 1992, pp. 545-556, vol. 2.
Schnepf, H.E. and H.R. Whiteley, “Cloning and Expression of the Bacillus thuringiensis Crystal Protein Gene in Escheria coli,” Proc. Natl. Acad. Sci. USA, 1981, pp. 2893-2897, vol. 78(5).
Shevelev, A.B. et al., “Primary Structure of cryX**, the Novel δ-endotoxin-related Gene from Bacillus thuringiensis spp. galleriae,”FEBS, 1993, pp. 79-82, vol. 336(1).
Smulevitch, S.V. et al., “Nucleotide Sequence of a Novel δ-Endotoxin Gene crylg of Bacillus thuringiensis ssp. galleriae,” FEBS, 1991, pp. 25-28, vol. 293(1-2).