Human cytomegalovirus DNA sequences

TECHNICAL FIELD
This invention pertains to the field of virology, specifically to the diagnosis, treatment and prevention of viral infections in humans. More specifically, this invention relates to the diagnosis, treatment and prevention of human cytomegalovirus infections.
BACKGROUND
Human cytomegalovirus (HCMV) is a ubiquitous agent in human populations. Infections are generally asymptomatic, but there can be serious medical sequelae in immunocompromised individuals and in congenitally infected newborns. In immunocompromised individuals, HCMV infection can result in interstitial pneumonia, retinitis progressing to blindness and disseminated infection. Infections in newborns can be severely damaging, with multiple organ involvement including the central nervous system and may also result in auditory damage. The mechanisms of pathogenesis are not understood, although it is believed that host factors, such as cellular and/or hmunoral immune responses might be involved. See, Alford and Britt, "The Humnan Herpesviruses", eds Roizman, B., R. J. Whitley and C. Lopez, Raven Press, New York, 1993, pp 227-55. It has also been speculated that genetic variability (either structural or antigenic or both) among different strains of HCMV could be responsible for the variance in clinical manifestations observed. Pritchett, J. Virol. 36:152-61(1980), Lehner, J. Clin. Microbiol. 29:249-2502(1991); Fries, J. Infect. Dis. 169:769-74(1994).
Considerable attention has been focused recently on the analysis of strain variation among HCMV isolates. Some twenty different HCMV strains have been isolated and differentiated by restriction analysis of PCR amplified DNA fragments. Chou, J. Infect. Dis. 162:738-42(1990).
One strain, the Towne strain, has been developed into a live, attenuated vaccine and administered with some success in renal transplant patients. See Quinnan, Annals of Int. Med. 101:478-83(1984); Plotkin, Lancet 1:528-30(1984). However, Towne strain vaccines who were directly challenged by low-passaged Toledo strain wild-type virus in one study were found to resist challenge doses of only 10 plaque-forming units (pfu) or less. Plotkin, J. Infect. Dis. 159:860-65(1989). Therefore, it appears the Towne strain may be overly attenuated, i.e., genetically modified so extensively resulting from serial passage in cell culture that it has lost significant immunogenicity presumably due to the loss of genetic information during the cell passage. Advantageously however, the Towne strain has never been shown to reactivate.
DNA sequence heterogeneity between the Towne strain and another strain of HCMV, AD169, has been found. Pritchett, J. Virol. 36:152-61(1980). (A restriction map of the AD169 HCMV genome is disclosed in U.S. Pat. No. 4,762,780) Variation in the DNA content among other isolated strains of HCMV has also been detected. Huang, Yale J. Biol. and Med. 49:29-43(1976). Cleavage patterns of restriction enzyme digests of HCMV DNA of various strains has been analyzed. Kilpatrick, J. Virol. 18:1095-1105(1976); LaFemina, "Structural Organization of the DNA Molecules from Human Cytomegalovirus" in Animal Virus Genetics, eds. Field, B N and R. Jaenish, Academic Press, N.Y. (1980); Chandler, J. Gen. Virol. 67:2179-92(1986); Zaia, J. Clin. Microbiol. 28:2602-07(1990). However, although the gross structural organization of the HCMV genome has been determined and strain-to-strain restriction site polymorphism mapped for many of the strains, strain-to-strain differences in the DNA sequences of the HCMV genome have not been determined. Only partial sequences have been deduced and compared, For example, the DNA and amino acid sequences of the envelope glycoprotein B �gpUL55(gB)! of both Towne and AD169 strains have been deduced, see Spaete, Virology 167:207-25(1988), and compared with various clinical isolates, see Chou, J. Infect. Dis. 163:1229-34(1991), to identify conserved regions and regions of variability. In addition, DNA sequence analysis of certain regions of the gp58/116 gene �gpUL55(gB)!, the IMP gene and the IE-1/2 enhancer/promoter has been accomplished. Lehner, J. Clin. Microbiol. 29:2494-2502(1991).
Whereas the complete DNA sequence of the AD169 strain of HCMV has been deduced, (EMBL Accession No. X17403), the complete DNA sequence of the Towne strain has not to our knowledge been deduced. However, it has been speculated that AD169 and another laboratory strain, Davis, are missing two to four kilobase pairs (kb) of DNA sequence compared to the Towne strain at the extreme internal portions of both L repeats. LeFemina, supra, at 52-53.
The public health impact of HCMV infections has not been well controlled by current treatment strategies or available antiviral chemotherapies. Preventative vaccine strategies are likely to prove efficacious because of the observations that seropositive renal allograft recipients are protected from severe HCMV disease and maternal immunity protects the fetus from disease after intrauterine infection. Marshall and Plotkin, "Cytomegalovirus Vaccines" in The Human Herpesviruses, eds Roizman, B., R. J. Whitley and C. Lopez, Raven Press, New York, 1993, pps 381-95. However, an additional obstacle to the development of a vaccine for HCMV is the lack of an animal model system that can be used to test the safety and efficacy of vaccine candidates.
There remains a need in the art for efficacious vaccines for the prophylactic treatment of HCMV in humans.
SUMMARY OF THE INVENTION
In one aspect, the invention provides novel HCMV DNA sequences not heretofore recognized or known in the art. These novel HCMV sequences were isolated from the Toledo and Towne strains of HCMV and comprise DNA that is not shared by reference strain AD169 of HCMV. Accordingly, in this aspect the invention provides novel, isolated, Toledo strain HCMV DNA sequences. As used herein, "isolated" means substantially free from other viral DNA sequences with which the subject DNA is typically found in its native, i.e., endogenous, state. These novel Toledo HCMV DNA sequences are characterized by comprising the same or substantially the same nucleotide sequence as in FIG. 1 (SEQ ID NO:6), or active fragments thereof. The DNA sequences may include 5' and 3' non-coding sequences flanking the coding sequence. The DNA sequences may be in inverted orientation with respect to the orientation shown in FIG. 1. Segments or fragments of the DNA sequence shown in FIG. 1 (SEQ ID NO:6) may be rearranged or inverted internally. The DNA sequences of the invention also comprise nucleotide sequences capable of hybridizing under stringent conditions, or which would be capable of hybridizing under said conditions but for the degeneracy of the genetic code to a sequence corresponding to the sequence of FIG. 1. FIG. 1 (SEQ ID NO:6) illustrates the DNA sequence of the novel Toledo strain HCMV. Twenty one open reading frames (ORFs) were identified in this sequence. The putative amino acid sequences of these novel Toledo strain HCMV ORFs are enumerated in sequence identification numbers 7 through 27, pages 58 through 78, infra. In FIG. 1, the beginning and ending of the 21 ORFs are identified by the arrows and the designations "UL133", "UL134", etc. (see infra.). In rearranged sequences of the invention, novel open reading frames may be created or destroyed.
In another aspect, the invention provides additional novel HCMV DNA sequences not heretofore recognized or known in the art. These additional sequences were isolated from the Towne strain of HCMV and comprise DNA that is not shared by the AD169 strain or by the Toledo strain of HCMV. Accordingly, in this aspect the invention provides novel Towne strain HCMV sequences. These novel Towne HCMV DNA sequences are characterized by as comprising the same or substantially the same nucleotide sequence as m FIG. 2 (SEQ ID NO:1), or active fragments thereof. The DNA sequence may include 5' and 3' non-coding sequences flanking the coding sequence. The DNA sequences of the invention also comprise nucleotide sequences capable of hybridizing under stringent conditions, or which would be capable of hybridizing under said conditions but for the degeneracy of the genetic code to a sequence corresponding to the sequence of FIG. 2 (SEQ ID NO:1). FIG. 2 (SEQ ID NO:1) illustrates the DNA sequence of the novel Towne strain HCMV. Four ORFs were identified in this sequence. The putative amino acid sequences of these novel ORFs are enumerated in sequence identification numbers 2 through 5, pages 42 through 45 infra. In FIG. 2, the beginning and ending of the 4 ORFs are identified by the arrows and the designations UL147, UL152, UL153 and UL154.
It is understood that the DNA sequences of this invention may exclude some or all of the signal and/or flanking sequences. In addition, the DNA sequences of the present invention may also comprise DNA capable of hybridizing under stringent conditions, or which would be capable of hybridizing under such conditions but for the degeneracy of the genetic code, to an isolated DNA sequence of FIG. 1 or FIG. 2. (SEQ ID NOS:6 and 1). As used herein, "stringent conditions" means conditions of high stringency, for example 6.times. SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 .mu.g/ml salmon sperm DNA and 15% formamide at 68 degrees C. (See Materials and Methods, Part C, infra.)
Accordingly, the DNA sequences of this invention may contain modifications in the non-coding sequences, signal sequences or coding sequences, based on allelic variation, species or clinical isolate variation or deliberate modification. Using the sequences of FIG. 1 and 2 (SEQ ID NOS:6 and 1), it is within the skill in the art to obtain other modified DNA sequences: the sequences can be truncated at their 3'-tetmini and/or their 5'-termini, the gene can be manipulated by varying individual nucleotides, while retaining the original amino acid(s), or varying the nucleotides, so as to modify amino acid(s). Nucleotides can be substituted, inserted or deleted by known techniques, including for example, in vitro mutagenesis and primer repair. In addition, short, highly degenerate oligonucleotides derived from regions of imperfect amino acid conservation can be used to identify new members of related viral and cellular families. RNA molecules, transcribed from a DNA of the invention as described above, are an additional aspect of the invention.
In another aspect, the invention provides novel HCMV proteins, which are substantially free fromn other HCMV proteins with which they are typically found in their native state. These novel HCMV proteins comprise the open reading frames (ORFs) UL133 (SEQ ID NO:7), UL134 (SEQ ID NO:8), UL135 (SEQ ID NO:9), UL136 (SEQ ID NO:10), UL137 (SEQ ID NO:11), UL138 (SEQ ID NO:12), UL139 (SEQ ID NO:13), UL140 (SEQ ID NO:14), UL141 (SEQ ID NO:15), UL142 (SEQ ID NO:16), UL143 (SEQ ID NO:17), UL144 (SEQ ID NO:18), UL145 (SEQ ID NO:19), UL146 (SEQ ID NO:20), UL147 (SEQ ID NO:21), UL148 (SEQ ID NO:22), UL149 (SEQ ID NO:24), UL150 (SEQ ID NO:25), and/or UL151 (SEQ ID NO:26) identified in the novel Toledo strain DNA sequence and UL147 (SEQ ID NO:2), UL152 (SEQ ID NO:3), UL153 (SEQ ID NO:4) and/or UL154 (SEQ ID NO:5) identified in the novel Towne strain DNA sequence. Two additional HCMV ORFs were identified in the novel Toledo stain DNA sequence, UL130 and UL132 (SEQ ID NOS:23 and 27). These two sequences are also present in AD169 (see FIG. 5). The proteins may be produced by recombinant genetic engineering techniques. They may additionally be purified from cellular sources infected with HCMV. They may also be synthesized by chemical techniques. One skilled in the art could apply a combination of the above-identified methodologies to synthesize the protein. Additionally, analogs of the HCMV proteins of the invention are provided and include truncated polypeptides, e.g., mutants in which there are variations in the amino acid sequence that retain activity, as defmed below, and preferably have a homology of at least 80%, more preferably 90%; and most preferably 95%, with the corresponding regions of the HCMV Towne or Toledo amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27). Examples include polypeptides with minor amino acid variations from the native amino acid sequences of HCMV Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27); in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on activity or functionality.
Using the Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27) it is within the skill in the art to obtain other polypeptides or other DNA sequences encoding the HCMV Toledo or Towne protein from clinical isolates of HCMV. For example, the structural gene can be manipulated by varying individual nucleotides, while retaining the correct amino acid(s), or varying the nucleotides, so as to modify the amino acids, without loss of activity. Nucleotides can be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair. The structural gene can be truncated at its 3'-terminus and/or its 5'-terminus while retaining its activity. It also may be desirable to remove the region encoding the signal sequence, and/or to replace it with a heterologous sequence. It may also be desirable to ligate a portion of the HCMV Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27), particularly that which includes the amino terminal domain to a heterologous coding sequence, and thus to create a fusion peptide of HCMV Toledo or Towne.
In designing such modifications, it is expected that changes to nonconserved regions of the HCMV Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27) will have relatively smaller effects on activity, whereas changes in the conserved regions, and particularly in or near the amino terminal domain are expected to produce larger effects. Amino acid residues that are conserved between the HCMV Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 14, 15, 17, 18, 19, 21, 22, 23, 24, 25, 26, and 27) and at least two other sequences, for example, from HCMV clinical isolates are not expected to be candidates for substitution. A residue which shows conservative variations among the HCMV sequences and at least two of the other sequences is expected to be capable of similar conservative substitution of the HCMV sequences. Similarly, a residue which varies nonconservatively among the HCMV sequences and at least three of the other sequences is expected to be capable of either conservative or nonconservative substitution. When designing substitutions to the HCMV sequences, replacement by an amino acid which is found in the comparable aligned position of one of the other sequences is especially preferred.
Additionally provided by this invention is a recombinant DNA vector comprising vector DNA and a DNA sequence encoding an HCMV Toledo polypeptide or HCMV Towne polypeptide. The vector provides the HCMV Toledo or Towne DNA in operative association with a regulatory sequence capable of directing the replication and expression of an HCMV Toledo or Towne protein in a selected host cell. Host cells transformed with such vectors for use in expressing recombinant HCMV Toledo or Towne proteins are also provided by this invention. Also provided is a novel process for producing recombinant HCMV Toledo or Towne proteins or active fragments thereof. In this process, a host cell line transformed with a vector as described above containing a DNA sequence (SEQ ID NOS:1 and 6) encoding expression of an HCMV Toledo or Towne protein in operative association with a suitable regulatory sequence capable of directing replication and controlling expression of an HCMV Toledo or Towne protein is cultured under appropriate conditions permitting expression of the recombinant DNA. The expressed protein is then harvested from the host cell or culture medium using suitable conventional means. This novel process may employ various known cells as host cell lines for expression of the protein. Currently preferred cells are mammalian cell lines, yeast, insect and bacterial cells. Especially preferred are mammalian cell lines.
The practice of the invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA manipulation and production, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, Eds. 1984); Transcription and Translation (B. D. Hames and S. J. Higgins, Eds. 1984); Animal Cell Culture (R. I. Freshney, Ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, Eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology, Volumes 154 and 155 (Wu and Grossman, and Wu, Eds., respectively), (Mayer and Walker, Eds.) (1987); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), Scopes, (1987); Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.); and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, Eds 1986). All patents, patent applications and publications mentioned herein, both supra and infra, are hereby incorporated by reference.
Additionally provided by this invention are compositions for detecting HCMV infections in humans. These compositions comprise probes having at least one single-stranded fragment of at least 10 bases in length, more preferably 15 bases in length, of the novel Toledo sequence, and fragments hybridizing to these single-stranded fragments under stringent hybridization conditions and non-cross-hybridizing with human DNA. Additionally, these compositions comprise at least one single-stranded fragment of at least 10 bases in length, more preferably 15 bases in length, of the novel Towne sequence, and fragments hybridizing to these single-stranded fragments under stringent hybridizing with human DNA. Such probe compositions may additionally comprise a label, attached to the fragment, to provide a detectable signal, as is taught in U.S. Pat. No. 4,762,780.
Further provided by this invention are methods for detecting an HCMV infection in a human host. Such methods comprise combining under predetermined stringency conditions a clinical sample suspected of containing HCMV DNA with at least one single-stranded DNA fragment of the novel Toledo or Towne strain of HCMV having at least 10 bases, more preferably 15 bases, and being non-cross-hybridizing with human DNA, and detecting duplex formation between the single-stranded Toledo or Towne strain HCMV fragments and the sample DNA. Alternatively, PCR may be used to increase the viral nucleic acid copy number by amplification to facilitate the identification of HCMV in infected individuals. In such case, the single-stranded Toledo or Towne strain DNA sequence fragments of the present invention can be used to construct PCR primers for PCR-based amplification systems for the diagnosis of HCMV. Such systems are well known in the art. See for example, U.S. Pat. No. 5,008,182 (detection of AIDS associated virus by PCR) and Hedrum, PCR Methods and Applications 2:167-71(1992) (detection of Chlamydia trachomatis by PCR and immunomagnetic recovery).
The DNA sequences of this invention may also be used to prepare immunizing compositions. The novel Toledo DNA sequences are recombined into the Towne strain or AD169 strain of HCMV and these recombinant viruses tested for growth properties in endothelial cells or in human tissues transplanted into SCID mice or tested in the rat eye model. Mocarski, Proc. Nat. Acad. Sci 90:104-08(1993). Such recombinants will show increased immunogenicity over that shown by the Towne-125 strain currently in use in humans, without exhibiting the full virulence shown by the Toledo-1 strain. Therefore, a further aspect of the invention is immunizing compositions comprising either the Towne strain or the AD169 reference strain of HCMV to which the novel Toledo DNA sequence, or analogs or fragments thereof, have been added, resulting in increased immunogenicity of the recombinant virus. The invention also includes a method for the prophylactic treatment of HCMV in humans comprising administering to a human patient an immunogenically inducing effective amount of an immunizing composition of the invention in a suitable pharmaceutical carrier. Still another aspect of the invention is a method of stimulating an immune response against CMV by administering to a patient an immunogenically inducing effective amount of an immunizing composition of the invention in a suitable pharmaceutical vehicle.
Other aspects and advantages of this invention are described in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1T illustrates the novel Toledo DNA sequence of the invention isolated from the Toledo strain of HCMV. The arrows indicate the beginnings and ends of nucleotide sequences encoding the 21 putative amino acid sequences identified.
FIGS. 2A-2E illustrates the novel Towne DNA sequence of the invention isolated from the Towne strain of HCMV. The arrows indicate the beginnings and ends of the nucleotide sequences encoding the 4 putative amino acid sequences identified.
FIG. 3 is a schematic representation of a Southern blot of restriction enzyme digested Towne and Toledo HCMV strain DNA as detailed in Example 1. The arrow indicates a 5 kbp (kilobase pair) band of Toledo DNA on the BamHI digest that is lacking in the Towne DNA, signifying the presence of additional Toledo DNA sequence.
FIG. 4 illustrates a composite autoradiograph of the restriction enzyme digested DNA from AD169, Towne, Toledo and five clinical isolates of HCMV as described in Example 3.
FIG. 5 is a schematic presentation of the novel open reading frames identified in the novel Toledo and Towne DNA sequences.
FIG. 6 is a schematic illustration of the relative positions of novel sequences identified in Toledo genomic DNA, Towne genomic DNA in a comparison with AD169 strain genomic DNA.

DETAILED DESCRIPTION
A. Introduction
The invention provides two novel HCMV DNA sequences, termed Toledo sequence and Towne sequence, not heretofore recognized or known in the art. The invention also provides immunization compositions and methods using the novel HCMV DNA sequences of the invention and also provides other diagnostic and therapeutic uses for the sequences and their protein products. The new DNA sequences were originally found in the Toledo and Towne strains of HCMV. Details of the sequences and structural characteristics are provided in the Examples below.
Most desirably, HCMV immunogenic compositions are provided that comprise reference strain AD169 or Towne to which the novel Toledo DNA sequences, or analogs or fragments thereof, have been added in order to increase the immunogenicity of the overly-attenuated strain. Thus, one aspect of this invention includes isolated DNA and corresponding RNA sequences as disclosed in FIGS. 1 and 2 (SEQ ID NOS:6 and 1). As used herein, "isolated" means substantially free from other nucleotide or polypeptide sequences with which the subject nucleotide sequence or polypeptide sequence is typically found in its native, i.e., endogenous, state. In another aspect, the invention comprises isolated HCMV Towne or Toledo protein encoded by the respective HCMV Towne or Toledo DNA sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27).
Another aspect of this invention includes diagnostic assays for the detection of HCMV strain variants. In brief, such diagnostic assays include the use of DNA sequence fragments of the invention as primers for amplifying HCMV related nucleic acids in a polymerase chain reaction (PCR) or by direct detection by hybridization. The diagnostic assays of the invention may also include the use of specific antibodies against the novel ORFs encoded by the Toledo or Towne DNA sequences disclosed here. Yet another aspect of the invention is the use of the novel DNA sequences modified with a unique restriction site, to act as vaccine markers.
It is anticipated that the invention will enable the production of vaccines that offer advantages over the current HCMV vaccine, which is overly attenuated and therefore not consistently effective in eliciting an immune response. More specifically, the introduction or insertion of the novel Toledo strain sequences of the present invention into the Towne strain or into the AD169 strain will result in the introduction of specific DNA sequences in the HCMV Towne genome that are not possible using the cell passage vaccines. Importantly for vaccine production, this enables precise measurement of the degree of attenuation introduced by different fragments of the DNA sequences of the invention, thereby enabling the controlled modification in the attenuation of the Towne strain that is needed in the art to correct the Towne's strain's overly attenuated characteristic and improve its function as an immunogenic composition.
B. Recombinant AD169 or Towne HCMV
Recombinant AD169 or Towne DNA is derived by co-transfecting a plasmid containing the novel Toledo sequence, or analogs or fragments thereof, and a selectable marker such as gpt or .beta.-galactosidase in primary fibroblast cells, or other cell lines known to be permissive for growth of CMV. Recombinant viruses are selected by growth in media containing mycophenolic acid or identified by blue plaque phenotypes after applying a chromogenic substrate such as X-gal. Recombinant viruses are plaque purified and characterized by restriction enzyme analysis and Southern blotting procedures. The novel HCMV Toledo sequence, or analogs or fragments thereof, may be used unmodified with respect to the endogenous promoter and transcription termination signals. Alternatively, the HCMV Toledo strain DNA coding region can be placed under transcriptional control of a promoter such as the CMV (cytomegalovirus) major immediate early promoter, the SV40 early promoter or some other viral or cellular promoter that generates adequate levels of expression, as discussed herein.
Modified Towne or AD169 strain HCMV is grown in tissue culture cells. For experiments with mammals, not including humans, cells such as human foreskin fibroblasts (HF) or MRC-5 cells are used to propagate the virus. The virus is harvested from cultures of these cells and the isolated recombinant virus is then be further studied for its ability to elicit an immune response and provide protection against HCMV infection.
For use in humans, the recombinant virus is produced from an FDA approved cell line in large scale amounts. Such cells include MRC-5 or WI-38 cells (both are primary human diploid fibroblasts). The recombinant virus is generated in the production cell line by transfection of viral DNA or capsids prepared from recombinant virus isolated from another cell line. The method of transfection should prevent the contamination of FDA approved cells with adventitious agents or contaminants from a non-qualified cell line. A HCMV virus produced from the above cell lines will be used to infect progressively larger flasks of tissue culture cells. Infected cells will be used as subsequent inoculums. Viable infected tissue culture cells are removed from the tissue culture vessels using trypsin and added to a 1 to 100 fold (or more) excess of uninfected cells to accomplish progressively larger inoculations. Once an optimal yield is obtained the virus will be harvested from the tissue culture cells. This process can be repeated until a large scale production is achieved. Infected cells will be removed from the tissue culture vessel and disrupted using for example, sonication, dounce homogenization or some combination of the above. The viruses are then isolated from cellular material using centrifugation techniques known in the art. Once the virus is isolated a stabilizing agent is added, such as a carbohydrate or carbohydrate derivative and the virus is then aliquoted and lyophilized.
C. Immunogenic Compositions
Immunogenic compositions can be administered to subjects to prevent HCMV infections. The immnunogenic compositions prevent HCMV infections by stimulating the immune system with an attenuated virus incapable of fully manifesting the disease. A major advantage of the HCMV immunogenic compositions provided herein is that its increased degree of immunogenicity will result in move effective prevention of an HCMV infection in the population.
The Towne strain of HCMV will preferably serve as the parent strain due to its proven inability to reactivate. To make HCMV immunogenic compositions, full, truncated and/or modified novel Toledo DNA sequences are introduced into a HCMV AD169 or Towne strain virus as discussed herein. The effectiveness of the immunogenic composition in preventing HCMV infections will be measured in humans. Humans will be first inoculated with PFU's ranging from 100-20,000 PFU of mutant virus per inoculation, PFUs are measured as discussed herein. After the first inoculation, a second booster injection of similar or increased dosage usually may be given. Subjects will be exposed to wild-type HCMV after the first or second inoculation and the occurrence of CMV infections observed. Potential side effects of the vaccine will be monitored in volunteer adults previously exposed to CMV, before inoculating subjects that have not ever developed CMV infections. Attenuated virus is used without an adjuvant and with a physiologically suitable carrier.
As is known in the art and discussed herein, the novel DNA is inserted into the Towne or AD169 viral genome using, for example, homologous recombination techniques. The insertion is generally made into a gene which is non-essential in nature. Plasmid shuttle vectors that greatly facilitate the construction of recombinant viruses have been described. See, for example, Spaete and Mocarski, Proc. Nat. Acad. Sci 84:7213-17(1987). Expression of the polypeptide encoded by the novel Toledo DNA then occurs in cells or individuals which are immunized with the live recombinant virus.
Alternatively, the purified novel HCMV proteins may be employed in therapeutic and/or subunit immunogenic compositions for preventing and treating HCMV related conditions. Such pharmaceutical compositions comprise an immunogenically-inducing effective amount of one or more of the proteins of the present invention in admixture with a pharmaceutically acceptable carrier, for example an adjuvant/antigen presentation system such as alum. Other adjuvant/antigen presentation systems, for instance, MF59 (Chiron Corp.), QS-21 (Cambridge Biotech Corp.), 3-DMPL (3-Deacyl-Monophosphoryl Lipid A) (RibiImmunoChem Research, Inc.), clinical grade incomplete Freund's adjuvant (IFA), fusogenic liposomes, water soluble polymers or Iscoms (Iminune stimulating complexes) may also be used. Other exemplary pharmaceutically acceptable carriers or solutions are aluminum hydroxide, saline and phosphate buffered saline. The composition can be systemically administered, preferably subcutaneously or intramuscularly, in the form of an acceptable subcutaneous or intramuscular solution. Also inoculation can be effected by surface scarification or by inoculation of a body cavity. The preparation of such solutions, having due regard to pH, isotonicity, stability and the like is within the skill in the art. The dosage regimen will be determined by the attending physician considering various factors known to modify the action of drugs such as for example, physical condition, body weight, sex, diet, severity of the condition, time of administration and other clinical factors. Exemplary dosage ranges comprise between about 1 .mu.g to about 1000 .mu.g of protein.
In practicing the method of treatment of this invention, an immunologically-inducing effective amount of protein is administered to a human patient in need of therapeutic or prophylactic treatment. An immunologically inducing effective amount of a composition of this invention is contemplated to be in the range of about 1 microgram to about 1 milligram per dose administered. The number of doses administered may vary, depending on the above mentioned factors.
D. Diagnostic Assays and Use as a Vaccine Marker
The novel Toledo and Towne DNA sequences of the present invention can be used in diagnostic assays to detect HCMV in a sample, to detect Toledo and Towne--like sequences and to detect strain differences in clinical isolates of HCMV using either chemically synthesized or recombinant Toledo or Towne DNA fragments. Additionally, the novel sequences can be used as a vaccine marker to differentiate between an individual or sample infected with or containing wild type HCMV and an individual or sample infected with or containing a HCMV vaccine, i.e., a live attenuated HCMV vaccine currently in use such as the Towne vaccine. In yet another embodiment, fragments of the DNA sequences can also be linked to secondary nucleic acids with sequences that either bind a solid support or other detection probes for use in diagnostic assays. In one aspect of the invention, fragments of the novel Toledo or Towne DNA sequences (SEQ ID NOS:1 and 3) comprising at least between 10 and 20 nucleotides can be used as primers to amplify nucleic acids using polymerase chain reaction (PCR) methods well known in the art and as probes in nucleic acid hybridization assays to detect target genetic material such as HCMV DNA in clinical specimens (with or without PCR). See for example, U.S. Pat. Nos. 4,683,202; 4,683,195; 5,091,310; 5,008,182 and 5,168,039. In an exemplary assay, a conserved region of the novel DNA sequence among virus variants is selected as the sequence to be amplified and detected in the diagnostic assay. Oligonucleotide primers at least substantially complementary to (but preferably identical with) the sequence to be amplified are constructed and a sample suspected of containing a HCMV nucleic acid sequence to be detected is treated with primers for each strand of HCMV nucleic acid sequence to be detected, four different deoxynucleotide triphosphates and a polymerization agent under appropriate hybridization conditions such that an extension product of each primer is synthesized that is complementary to the HCMV nucleic acid sequences suspected in the sample, which extension products synthesized from one primer, when separated from its complement can serve as a template for synthesis of the extension product of the other primer in a polymerase chain reaction. After amplification, the product of the PCR can be detected by the addition of a labeled probe, likewise constructed from the novel DNA sequence, capable of hybridizing with the amplified sequence as is well known in the art. See, e.g. U.S. Pat. No. 5,008,182.
In another embodiment the probes or primers can be used in a vaccine marker assay to detect a vaccine or wild type infection. Alternatively, introduction of a restriction site into the novel DNA sequence will provide a vaccine marker that can be used with PCR fragments to detect such differences in a restriction digest. Such procedures and techniques for detecting sequence variants, such as, point mutations with the expected location or configuration of the mutation, are known in the art and have been applied in the detection of sickle cell anemia, hemoglobin C disease, diabetes and other diseases and conditions as disclosed in U.S. Pat. No. 5,137,806. These methods are readily applied by one skilled in the art to detect and differentiate between wild type and vaccine infections in HCMV.
In another embodiment the novel Toledo or Towne DNA sequences can be used in their entirety or as fragments to detect the presence of DNA sequences, related sequences, or transcription products in cells, tissues, samples and the like using hybridization probe techniques known in the art or in conjunction with one of the methods discussed herein. When used as a hybridization probe, fragments of the novel DNA sequences of the invention are preferably 50-200 nucleotides long, more preferably 100-300 nucleotides long and most preferably greater than 300 nucleotides long.
E. Vectors and Chimeric Virus Production
The novel DNA sequences of the invention can be expressed in different vectors using different techniques known in the art resulting in the generation of chimeric virus. Useful and known techniques include marker transfer or homologous recombination, direct in vitro ligation, defective vector technology and amplicon generation (see, e.g., Frenkel, N. et al., Gene Transfer and Cancer, edited by M. L. Pearson and N. L. Sternberg (1984), Kwong, A. D. and Frenkel, Virology 142,421-425(1985); U.S. patent (Ser. No. 07/923,015 by Roizman). Vectors used in such techniques include cosmids, plasmids, and infective or defective viruses. Such vectors are known in the art. (A cosmid as used herein is a plasmid containing a lambda bacteriophage cos site. The cos site is the cis signal for packaging lambda DNA. Therefore, a cosmid, unlike a plasmid, can be packaged with high efficiency into a lambda head in vitro. This technique allows cloning of very large (30-45 kbp) fragments of DNA.) The vectors can be either single stranded or double stranded and made of either DNA or RNA.
Generally, the DNA sequence is inserted into the vector alone or linked to other HCMV genomic DNA. In direct in vitro ligation applications, the isolated sequence alone is used. In homologous recombination and marker transfer flanking nucleic acid sequences are required to effect transfer of the sequence into a HCMV viral genome. For use in viral complementation using cosmids and other vectors discussed herein the sequence (or a fragment thereof) in a vector is preferably operatively linked to at least 1 kb of HCMV genomic nucleic acid and more preferably at least 5 kb of HCMV nucleic acid. The HCMV genomic nucleic acid can be on one side or both sides of the open reading frame. If only a specific region of the open reading frame is to be used to generate a mutant virus, an open reading frame or fragment thereof is inserted into a vector.
F. Novel Toledo and Towne Protein
Another aspect of the invention includes the isolated proteins encoded by the Toledo or Towne DNA sequence as taught herein. The proteins can be used to study and modify the life cycle of HCMV because they may encode surface glycoproteins that may be immunogenic and responsible for tissue tropism or influence the immune response in an infected individual. Such proteins could therefore be used in the production of a subunit vaccine against CMV. The construction of such CMV subunits vaccine candidates is known in the art. See, for example, Spaete, Virology 167:207-25(1988).
Twenty-one novel Toledo and four novel Towne proteins have been identified by ORF analysis. The novel Toledo proteins include UL130 (SEQ ID NO:23), UL132 (SEQ ID NO:27), UL133 (SEQ ID NO:7), UL134 (SEQ ID NO:8), UL135 (SEQ ID NO:9), UL136 (SEQ ID NO:10), UL137 (SEQ ID NO:11), UL138 (SEQ ID NO:12), UL139 (SEQ ID NO:13), UL140 (SEQ ID NO:14), UL141 (SEQ ID NO:15), UL142 (SEQ ID NO:16), UL143 (SEQ ID NO:17), UL144 (SEQ ID NO:18), UL145 (SEQ ID NO:19), UL146 (SEQ ID NO:20), UL147 (SEQ ID NO:21), UL148 (SEQ ID NO:22), UL149 (SEQ ID NO:24), UL150 (SEQ ID NO:25), and/or UL151 (SEQ ID NO:26). UL130 is encoded by nucleotides 13109 through 13753, as shown in FIG. 1. UL132 is encoded by nucleotides 11673 through 12485, as shown in FIG. 1. UL133 is encoded by nucleotides 51 through 824, as shown in FIG. 1. UL134 is encoded by nucleotides 541 through 1068, as shown in FIG. 1. UL135 is encoded by nucleotides 941 through 1927, as shown in FIG. 1. UL136 is encoded by nucleotides 2018 through 2740, as shown in FIG. 1. UL137 is encoded by nucleotides 2599 through 2890, as shown in FIG. 1. UL138 is encoded by nucleotides 2823 through 3332, as shown in FIG. 1. UL139 is encoded by nucleotides 3895 through 4302, as shown in FIG. 1. UL140 is encoded by nucleotides 4484 through 4828, as shown in FIG. 1. UL141 is encoded by nucleotides 5098 through 6375, as shown in FIG. 1. UL142 is encoded by nucleotides 6448 through 7368, as shown in FIG. 1. UL143 is encoded by nucleotides 7353 through 7631, as shown in FIG. 1. UL144 is encoded by nucleotides 8008 through 8538, as shown in FIG. 1. UL145 is encoded by nucleotides 8867 through 9169, as shown in FIG. 1. UL146 is encoded by nucleotides 9450 through 9803, as shown in FIG. 1. UL147 is encoded by nucleotides 9868 through 10347, as shown in FIG. 1. UL148 is encoded by nucleotides 10646 through 11596, as shown in FIG. 1. UL149 is encoded by nucleotides 15756 through 16124, as shown in FIG. 1. UL150 is encoded by nucleotides 15874 through 17802, as shown in FIG. 1. UL151 is encoded by nucleotides 17289 through 18299, as shown in FIG. 1.
The novel Towne proteins include UL147, UL152, UL153 and UL154 (SEQ ID NOS:2, 3, 4 and 5, respectively). UL147 is encoded by nucleotides 841 through 1321, as shown in FIG. 2. UL152 is encoded by nucleotides 1365 through 1721, as shown in FIG. 2. UL153 is encoded by nucleotides 2501 through 3337, as shown in FIG. 2. UL154 is encoded by nucleotides 3512 through 4711, as shown in FIG. 2.
"Toledo and/or Towne protein or proteins" as used herein refer to the above sequences, also enumerated in the sequence listing. "Toledo and/or Towne protein or proteins" also refers to an homologous protein from any strain or clinical isolate of HCMV, including HCMV proteins that are at least 90% homologous to the Toledo or Towne amino acid sequences (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27). The Toledo or Towne protein can be modified to affect HCMV life cycle by deletion, insertion and substitution into the DNA sequence, as discussed herein, or by chemical synthesis of different amino acid sequence or by chemical modification. Truncated proteins can be formed by deletion of a portion of the DNA sequence or the introduction of termination signal(s) into the DNA sequence. Preferred deletions to the protein correspond to deleted amino acid sequence or sequences that contain at least one amino acid selected from the group consisting of Glu, Asp, Arg, Lys, Cys and Pro. More preferably at the deleted amino acid sequence or sequences contain at least two amino acids selected from the group consisting of Glu, Asp, Arg, Lys, Cys and Pro. More preferably the deleted amino acid sequence or sequences contain at least two prolines.
Other mutations of the protein useful in modifying HCMV life cycle include, but are not limited to, modification of cAMP phosphorylation (Arg/Lys-Arg/Lys-X-X-Asp/Glu) and/or, myristylization sites (Glycine-X1-X2-X3-Ser/Thr-X-X-Asp/Glu; where X1 is not Glu, Asp, Arg, Lys, His Pro, Phe, Tyr, Trp, where X2 is any amino acid and where X3 is not Pro), or modification of the PKC phosphorylation sites (Ser/Thr-X-Arg/Lys) and/or N-linked glycosylation sites (Asn-X-Ser/Thr; where X is not Pro).
The Toledo or Towne DNA sequences, analogs or fragments thereof can be expressed in a mammalian, insect, or microorganism host. The polynucleotide is inserted into a suitable expression vector compatible with the type of host cell employed and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Site-specific DNA cleavage involved in such construction is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. A suitable expression vector is one that is compatible with the desired function (e.g., transient expression, long term expression, integration, replication, amplification) and in which the control elements are compatible with the host cell.
Mammalian Cell Expression
Vectors suitable for replication in mammalian cells are known in the art, and can include viral replicons, or sequences that ensure integration of the sequence encoding the Toledo or Towne DNA into the host genome. Exemplary vectors include those derived from SV40, retroviruses, bovine papilloma virus, vaccinia virus, other herpesviruses and adenovirus.
Such suitable mammalian expression vectors contain a promoter to mediate transcription of foreign DNA sequences and, optionally, an enhancer. Suitable promoters are known in the art and include viral promoters such as those from SV40, cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV).
The optional presence of an enhancer, combined with the promoter described above, will typically increase expression levels. An enhancer is any regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to endogenous or heterologous promoters, with synthesis beginning at the normal mRNA start site. Enhancers are also active when placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter. See Maniatis, Science 236:1237(1987), Alberts, Molecular Biology of the Cell, 2nd Ed. (1989). Enhancers derived from viruses may be particularly useful, because they typically have a broader host range. Examples include the SV40 early gene enhancer (see Dijkema, EMBO J. 4:761(1985)) and the enhancer/promoters derived from the long terminal repeat (LTR) of the RSV (see Gorman, Proc. Natl. Acad. Sci. 79:6777(1982b)) and from human cytomegalovirus (see Boshart, Cell 41:521(1985)). Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion (see Sassone-Corsi and Borelli, Trends Genet. 2:215(1986)); Maniatis, Science 236:1237(1987)). In addition, the expression vector can and will typically also include a termination sequence and poly(A) addition sequences which are operably linked to the Toledo or Towne coding sequence.
Sequences that cause amplification of the gene may also be desirably included in the expression vector or in another vector that is co-translated with the expression vector containing a Towne or Toledo DNA sequence, as are sequences which encode selectable markers. Selectable markers for mammalian cells are known in the art, and include for example, thymidine kinase, dihydrofolate reductase (together with methotrexate as a DHFR amplifier), aminoglycoside phosphotransferase, hygromycin B phosphotransferase, asparagine synthetase, adenosine deaminase, metallothionien, and antibiotic resistant genes such as neomycin.
The vector that encodes a novel Toledo or Towne protein or polypeptide of this invention can be used for transformation of a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotide into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus. The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotide into marmalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fuision, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLA cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
Insect Cell Expression
The components of an insect cell expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media. Exemplary transfer vectors for introducing foreign genes into insect cells include pAc373 and pVL985. See Luckow and Summers, Virology 17:31(1989).
The plasmid can also contains the polyhedron polyadenylation signal and a procaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli. See Miller, Ann, Rev. Microbiol. 42:177(1988).
Baculovirus transfer vectors usually contain a baculovirus promoter, i.e., a DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5' to 3') transcription of a coding sequence (e.g., structural gene) into mRNA. The promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence and typically includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector can also have an enhancer, which, if present, is usually distal to the structural gene. Expression can be either regulated or constitutive.
Yeast And Bacteria Expression
A yeast expression system can typically include one or more of the following: a promoter sequence, fusion partner sequence, leader sequence, transcription termination sequence. A yeast promoter, capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA, will have a transcription initiation region usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site (a "TATA Box") and a transcription initiation site. The yeast promoter can also have an upstream activator sequence, usually distal to the structural gene. The activator sequence permits inducible expression of the desired heterologous DNA sequence. Constitutive expression occurs in the absence of an activator sequence. Regulated expression can be either positive or negative, thereby either enhancing or reducing transcription.
Particularly useful yeast promoters include alcohol dehydrogenase (ADH) (EP Patent Pub. No. 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK)(EP Patent Pub. No. 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences. See Myanohara, Proc. Natl. Acad. Sci. USA 80:1(1983).
A Toledo or Towne DNA sequence, analog or an active fragment thereof can be expressed intracellularly in yeast. A promoter sequence can be directly linked with the sequence or fragment, in which case the first amino acid at the N-terninus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus can be cleaved from the protein by in vitro incubation with cyanogen bromide.
Intracellularly expressed fusion proteins provide an alternative to direct expression of a sequence. Typically, a DNA sequence encoding the N-terminal portion of a stable protein, a fusion partner, is fused to the 5' end of heterologous DNA encoding the desired polypeptide. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5' terminus of a sequence and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a clearable site. See, e.g., EP Patent Pub. No. 196 056. Alternatively, the polypeptides can also be secreted from the cell into the growth media by creating a fusion protein comprised of a leader sequence fragment that provides for secretion in yeast or bacteria of the polypeptides. Preferably, there are processing sites encoded between the leader fragment and the sequence that can be cleaved either in vivo or in vitro. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP Patent Pub. No. 12 873) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, can be used to provide for secretion in yeast (EP Patent Pub. No. 60057). Transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon. Together with the promoter they flank the desired heterologous coding sequence. These flanking sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.
Typically, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together in plasmids capable of stable maintenance in a host, such as yeast or bacteria. The plasmid can have two replication systems, so it can be maintained as a shuttle vector, for example, in yeast for expression and in a procaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 (see Botstein, Gene 8:17-24 (1979)), pCl/1 (see Brake, Proc. Natl. Acad. Sci. USA 81:4642-4646(1984)), and YRp17 (see Stinchcomb, J. Mol. Biol. 158:157(1982)). In addition, the plasmid can be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Either a high or low copy number vector may be selected, depending upon the effect on the host of the vector and the polypeptides. See, e.g., Brake, et al., supra.
Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors typically contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. See Orr-Weaver, Methods In Enzymol. 101:228-245(1983) and Rine, Proc. Natl. Acad. Sci. USA 80:6750(1983).
Typically, extrachromosomal and integrating expression vectors can contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers can include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker can also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions. See Butt, Microbiol. Rev. 51:351(1987).
Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are typically comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above. Expression and transformation vectors, either extrachromosomal or integrating, have been developed for transformation into many yeasts. Exemplary yeasts cell lines are Candida albicans (Kurtz, Mol. Cell. Biol. 6:142(1986), Candida maltosa (Kunze, J. Basic Microbiol. 25:141(1985), Hansenula polymorpha (Gletson, J. Gen. Microbiol. 132:3459(1986) and Roggenkamp, Mol. Gen. Genet. 202:302(1986), Kluyveromyces frailis (Das, J. Bacteriol. 158:1165(1984), Kluyveromyces lactis (De Louvencourt, J. Bacteriol. 154:737(1983) and Van den Berg, Bio/Technology 8:135(1990), Pichia guillerimondii (Kunze, J. Basic Microbiol. 25:141(1985), Pichia pastoris (Cregg, Mol. Cell. Biol. 5:3376 (1985), Saccharomyces cerevisiae (Hinnen, Proc. Natl. Acad. Sci. USA 75:1929(1978) and Ito, J. Bacteriol. 153:163(1983), Schizosaccharomyces pombe (Beach and Nurse, Nature 300:706(1981), and Yarrowia lipolytica (Davidow, Curr. Genet. 10:380471(1985) and Gaillardin, Curr. Genet. 10:49(1985).
Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See the publications listed in the foregoing paragraph for appropriate transformation techniques.
Additionally, the gene or fragment thereof can be expressed in a bacterial system. In such system, a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. a desired heterologous gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter can also have a second domain called an operator, that can overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein can bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation can be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli). See Raibaud, Ann. Rev. Genet. 18:173(1984). Regulated expression can therefore be either positive or negative, thereby either enhancing or reducing transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (see Chang, Nature 198:1056(1977), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (see Goeddel, Nuc. Acids Res. 8:4057(1981), Yelverton, Nuc. Acids Res. 9:731(1981), U.S. Pat. No. 4,738,921 and EP Patent Pub. Nos. 36 776 and 121 775). The lactomase (bla) promoter system (see Weissmann, Interferon 3 (ed. I. Gresser), the bacteriophage lambda PL promoter system (see Shimatake, Nature 292:128(128) and the T5 promoter system (U.S. Pat. No. 4,689,406) also provides useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter can be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter such as the tac promoter (see U.S. Pat. No. 4,551,433, Amann, Gene 25:167(1983) and de Boer, Proc. Natl. Acad. Sci. 80:21(1983)). A bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is exemplary. (see Studier, J. Mol. Biol. 189:113(1986) and Tabor, Proc. Natl. Acad. Sci. 82:1074(1985)).
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of the DNA sequence or fragment thereof in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (see Shine, Nature 254:34(1975). The SD sequence is thought to promote binding of MRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA (see Steitz, Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)(1979)).
The novel Toledo or Towne proteins of the invention can be expressed intracellularly. A promoter sequence can be directly linked with a novel Toledo or Towne DNA sequence, analog or a fragment thereof, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus can be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase. See EP Patent Pub. No. 219 237.
Fusion proteins provide an alternative to direct expression. Typically, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5' terminus of an sequence fragment thereof and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the sequence or fragment thereof (see Nagai, Nature 309:810(1984). Fusion proteins can also be made with sequences from the lacZ gene (Jia, Gene 60:197(1987),the trpE gene (Allen, J. Biotechnol. 5:93(1987) and Makoff, J. Gen. Microbiol. 135:11(1989), and the Chey gene (EP Patent Pub. No. 324 647) genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a clearable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g., ubiquitin specific processing-protease) to cleave the ubiquitin from the polypeptide. Through this method, mature Towne or Toledo polypeptides can be isolated. See Miller, Bio/Technology 7:698(1989).
Alternatively, proteins or polypeptides can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the proteins or polypeptides in bacteria. (See, for example, U.S. Pat. No. 4,336,336). The signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the protein or polypeptide.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui, Experimental Manipulation of Gene Expression (1983) and Ghrayeb, EMBO J. 3:2437(1984)) and the E. coli alkaline phosphatase signal sequence (phoA) (see Oka, Proc. Natl. Acad. Sci. 82:7212(1985). The signal sequence of the alpha-amylase gene from various Bacilus strains can be used to secrete heterologous proteins from B. subtilis (see Palva, Proc. Natl. Acad. Sci. 79:5582(1982) and EP Patent Pub. No. 244 042).
Transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon. Together with the promoter they flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the Towne or Toledo protein or polypeptide encoded by the DNA sequence. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
Typically, the promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence are maintained in an extrachromosomal element (e.g., a plasmid) capable of stable maintenance in the bacterial host. The plasmid will have a replication system, thus allowing it to be maintained in the bacterial host either for expression or for cloning and amplification. In addition, the plasmid can be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids.
Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors typically contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. See e.g., EP Patent Pub. No. 127 328.
Typically, extrachromosomal and integrating expression constructs can contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and can include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (see Davies, Ann. Rev. Microbiol. 32:469(1978). Selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are typically comprised of a selectable marker that is either maintained in an extrachromosal vector or an integrating vector, as described above.
Expression and transformation vectors, either extra-chromosomal or integrating, have been developed for transformation into many bacteria. Exemplary are the expression vectors disclosed in Palva, Proc. Natl. Acad. Sci. 79:5582(1982), EP Patent Pub. Nos. 036 259 and 063 953 and PCT Patent Publication WO 84/04541 (for B.subtilis); in Shimatake, Nature 292:128(1981), Amann, Gene 40:183(1985), Studier, J. Mol. Biol. 189:113(1986) and EP Patent Pub. Nos. 036 776, 136 829 and 136 907 (for E. coli); in Powell, Appl. Environ. Microbiol. 54:655(1988) and U.S. Pat. No. 4,745,056 (for Streptococcus).
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and typically include either the transformation of bacteria treated with CaCl.sub.2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Exemplary methodologies can be found in Masson, FEMS Microbiol. Let. 60:273(1989), Palva, Proc. Natl. Acad. Sci. 79:5582(1982), EP Patent Pub. Nos. 036 259 and 063 953 and PCT Patent Pub. WO 84/04541 for Bacillus transformation. For campylobacter transformation, see e.g., Miller, Proc. Natl. Acad. Sci. 85:856(1988) and Wang, J. Bacteriol. 172:949(1990). For E. coli, see e.g., Cohen, Proc. Natl. Acad. Sci. 69:2110(1973), Dower, Nuc. Acids Res. 16:6127(1988), Kushner, Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia), Mandel, J. Mol. Biol. 53:159(1970) and Taketo, Biochem. Biophys. Acta 949:318(1988). For Lactobacillus and Pseudomonas, see e.g., Chassy, FEMS Microbiol. Let. 44:173(1987) and Fiedler, Anal. Biochem. 170:38(1988), respectively. For Streptococcus, see e.g., Augustin, FEMS Microbiol. Let. 66:203(1990), Barany, J. Bacteriol. 144:698(1980), Harlander, Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III)(1987), Perry, Infec. Immun. 32:1295(1981), Powell, Appl. Environ. Microbiol, 54:655(1988) and Somkuti, Proc. 4th Evr. Cons. Biotechnology 1:412(1987).
The present invention is illustrated by the following examples.
MATERIALS AND METHODS
A. Cells and Virus
Human CMV strains AD169, Towne and Toledo were obtained from E. S. Mocarski (Stanford University) and were used for all experiments. Two of these strains are also available through the ATCC, Accession Nos. VR-538 (AD169) and VR-977 (Towne). Virus was grown in cultures of human foreskin fibroblast (HF) cells with Dulbecco's modified Eagle's medium (DME) (JRH Biosciences, Lenexa, KS) as previously described in Spaete and Mocarski, J. Virol 56:135-43(1985), but supplemented with 10% fetal calf serum (FCS) (JRH Biosciences, Lenexa, Kans.), L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (0.1 mg/ml) and pyruvate (1 mM). To prepare AD169, Towne and Toledo strain CMV DNAs by centrifugation to equilibrium on NaI gradients as previously described in Spaete and Mocarski, J. Virol 54:817-24(1985), roller bottles were infected with the CMV strains at a multiplicity of infection (MOI) of 0.001 plaque forming units (pfu)/cell to minimize the production of defective virus particles. The infected cells were refed at four days post infection with medium. At eight days post infection when the monolayer was well infected, cells were scraped into a 50 ml conical tube in 10 mls media per roller bottle and pelleted at 1000 revolutions per minute (rpm) for 10 minutes. Pellets were resuspended in 2.0 ml 0.01M Tris and 0.01 EDTA (TE) (pH 7.4) with 1% NP40, 1% deoxycholate and incubated on ice until all cellular nuclei were lysed when viewed under a microscope. Lysates were transferred to a 2059 tube (Falcon) and spun at 2600 rpm for 5 minutes at 4.degree. C. Supernatants were transferred to another 2059 tube and RNAse (Worthington-DNase free) was added at 50 .mu.g/ml followed immediately by Proteinase K (200 .mu.mg/ml) and 1% sodium dodecyl sulfate (SDS). Supernatants were incubated in a 65.degree. C. water bath for 60 minutes, brought to 16 ml with TE, pH 7.4, added to 24 mls of saturated NaI and 0.15 ml ethidium bromide (5 mg/ml). Samples were centrifuged to equilibrium at 55,000 rpm at 20.degree. C. for 24 hours in a Beckman Ti70 rotor. Fractions containing the viral DNA were extracted with butanol equilibrated with TE with gentle rocking followed by centrifugation at 3,000 rpm for 10 min at 20.degree. C. and further extracted 2 to 3 times with butanol to reduce volume. Samples were extracted with an equal volume of isoamyl alcohol equilibrated with TE, spun and re-extracted. DNA was dialyzed against three changes of TE with 1% phenol and 1M NaCl. The OD.sub.260 and OD.sub.280 were read to determine purity of the AD169, Toledo and Towne DNA.
Clinical isolates were obtained from M. Fiala (Rancho Mirage, Calif.), and S. Chou (Oregon Health Sciences University). Rapid isolation of HCMV infected cell viral DNA was carried out as previously described in Spaete and Frenkel, Cell 30:295-304(1982), except that DNA was not radiolabeled before purification. Briefly, infected cell monolayers (25 cm.sup.2 flasks) were rinsed twice with phosphate-buffered saline (PBS) and lysed in a 1.0 ml solution of 0.1M NaCl, TE, pH 8.0, 0.05% SDS and 0.1 mg/ml Proteinase K. Lysates were incubated 2-24 hours at 37.degree. C., extracted twice with 1 volume of phenol, 1 volume of chloroform followed by centrifugation at 2500 rpm for 5 minutes to separate phases. The aqueous phase was extracted twice with 1 volume of ether and the DNA was precipitated with 0.1 volume 3M NaAC and two volumes of ethanol or isopropanol. DNA was chilled, collected by centrifugation or spooled on a glass rod, dried and resuspended in TE.
B. Plasmid DNA
Plasmids pXbaI E, pXbaI T and pXbaI Q (Thomsen and Stinski, 1981), representing Towne strain map units 0.69 to 0.8, were obtained from M. Stinski (University of Iowa).
Clone 65 was derived by cloning a gel extracted BamHI digested Toledo DNA fragment into the BamHI site of plasmid, pGEM.RTM.-3Zf+ (Promega, Madison, Wis.). Briefly, five .mu.g of Toledo DNA was digested with 40 units of BamHI and electrophoresed in a preparative 1% low-melting-point agarose gel for 490 volt hours in 1.times. TAE buffer. Toledo DNA migrating at ca. 5 kilobase pairs (kbp) was excised and the agarose was digested with 2 units of .beta.-agarase I (New England BioLabs, Beverly, Mass.). This DNA fragment was precipitated with 2 volumes of isopropanol, chilled to -20.degree. C., spun in an Eppendorf centrifuge for 15 minutes, dried and resuspended in 50 .mu.l TE. The gel extracted fragment was ligated to BamHI digested pGEM.RTM.-3Zf+ using T4 DNA ligase (New England BioLabs, Berverly, Mass.), and an aliquot of the ligation mixture was used to transform competent Escherichia coli XL-1, Blues (Stratagene, La Jolla, Calif.) by the calcium shock method (Mandel and Higa, 1970), or by electroporation using methods as written in the Pulse Controller Guide published by BioRad (Richmond, Calif.).
Cosmid 1 is a ca. 53 kbp partially digested HindIII fragment of Toledo DNA spanning 0.69 to 0.87 map units cloned into cosmid pHC79 (Hohn and Collins, 1980) obtained from E. S. Mocarski (Stanford University). Subcloned from cosmid 1 were the following:
Clones 4 and C1300 were derived by cloning BamH1 digested fragments from Cosmid 1 cloned into a Bluescript M13+ plasmid vector. As such, these clones represent Toledo DNA sequence spanning portions of Cosmid 1.
Clone C23K was derived as a complete BanH1 digested fragment of Cosmid 1 DNA and circularized by ligation.
C. Preparation of Radioactively Labeled Probes and Hybridization.
Plasmid or viral DNA was radioactively labeled in vitro by nick translation (Rigby et al., 1977) with a kit (Boebringer Mannheim), and using �.alpha..sup.32 P!dCTP (Amersham Corp.). Hybridizations to immobilized CMV DNA were performed essentially as described by Spaete and Mocarski, J. Virol 54:817-24 (1985), but at 68.degree. C. in a solution of 6.times. SSC (1.times. SSC is 0.15M NaCl plus 0.015M sodium citrate), 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, and 0.1% sodium dodecyl sulfate, with the amount of salmon sperm DNA being changed from 25 .mu.g/ml to 100 .mu.g/ml and 30% formamide being reduced to 15%.
DNA was transferred to Hybond-N+ nylon transfer membranes (Amershan Corp.), after restriction enzyme digestion and electrophoresis in 1% agarose gels by standard techniques (Maniatis et al., 1982). DNA was cross-linked to the membrane with 120,000 microjoules/cm.sup.2 of UV irradiation using a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, Calif.). Membranes were prehybridized 1 hour at 68.degree. C. in solution A (6.times. SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 .mu.g/ml salmon sperm DNA and 15% formamide), then nick-translated �.alpha..sup.32 P!-labeled probe in a solution containing 100 .mu.g/ml salmon sperm DNA was denatured by boiling for five minutes, snap-cooled on ice, added to the membrane and allowed to hybridize overnight at 68.degree. C. After hybridization, unannealed probe was removed by rinsing the membrane 3.times. with 2.times. SSC followed by reincubation in solution A lacking salmon sperm DNA at 68.degree. C. for 15 minutes. The washing procedure was repeated, the blot was rinsed in a large volume of 2.times. SSC at room temperature, the membrane was air dried and autoradiographed using Kodak X-AR film.
D. Nucleotide Sequence Determination and Analysis.
All nucleic acid sequences were determined by the dideoxynucleotide chain termination method (Sanger et al., 1977). A variety of templates were prepared for sequencing; they included single-stranded phage DNA, double-stranded plasmid and cosmid DNA, viral genomic DNA, and PCR products. Manual and automated sequencing (with an ABI 373A instrument) were employed. Both one-cycle and multi-cycle sequencing protocols were used. The sequence was determined for both strands. Ambiguous regions were corrected by additional sequencing after proofreading. The primers used for sequencing were synthesized on an ABI 392 instrument (Applied Biosystems). The contig and analysis of the sequence were performed using MacDNASIS (Hitachi). The homology searches were performed using the BLAST program through NCBI services.
EXAMPLE 1
Identification of Novel Sequences in the Genomes of CMV Towne and Toledo Strain Isolates.
To determine the cross representation of DNA sequences in the Towne and Toledo strains of CMV, viral DNA from each strain was digested to completion with XbaI, ClaI, BamHI, BglII, EcoRI, and HindIII. After electrophoresis through a 1% agarose gel, the CMV DNAs were denatured in 0.2M NaCl/0.6M NaOH, neutralized in 0.6M NaCl/1M Tris, pH 7.5, in situ, and the gel was soaked in 20.times. SSC for 30 minutes. Stereo blots were prepared by placing identically sized Hybond-N+ nylon membranes (Amersham Corp.), on either side of the gel and transferring the DNAs to the membranes in both directions using the capillary action of paper towels. After blotting overnight in 20.times. SSC, the membranes were washed in 2.times. SSC and the DNA was immobilized on the membrane by UV irradiation as described above.
DNA probes of Towne and Toledo DNA with an average size of 500 bp were prepared by sonicating 10 .mu.g of each DNA in a 2063 tube (Falcon Plastics) using 4 pulses of 10 seconds each at a setting of 3 on a Heat Systems, Inc. sonicator (Farmingdale, N.Y.). Following sonication, the viral DNAs were digested with the restriction enzymes AvaI, BanI and B.function.aI, to further reduce the size complexity of the probe DNA. These enzymes were chosen because a search of the AD169 DNA database sequences (EMBL accession number X17403), revealed abundant cut sites (326, 386, and 341, respectively); their restriction enzyme digestion buffers are compatible; and their sites do not overlap. Ethidium bromide stained gels of the sheared viral DNAs prepared in this manner revealed a range of DNA sizes from 1300 bp to less than 100 bp, with the majority of DNA migrating at approximately 300 bp as judged by comigration with a HaeIII digested .O slashed.X174 DNA standard marker (New England BioLabs, Beverly, Mass.). The Towne and Toledo sheared probe DNA was then nick translated using �.alpha..sup.32 P!dCTP (Amersham Corp.) as described above and each probe was applied to stereo blots of immobilized, restriction enzyme digested, Towne and Toledo DNAs. After hybridization and autoradiography, the hybridization patterns were analyzed to determine the fragments on each DNA profile which did not hybridize with the heterologous strain probe but did hybridize with the homologous strain probe. For example, the loss of a signal for a prominent 5 kbp band on the BamHI digest of Toledo DNA when using the Towne probe, which was present when the Toledo DNA was used to probe itself, revealed a region of sequence divergence between the two isolates (see FIG. 3).
This 5 kbp fragment was cloned by gel extraction as described above and designated clone 65. The clone 65 Toledo DNA was sequenced in its entirety and compared to Towne DNA sequence generated from the pXbaI T clone which was shown to be divergent from AD169 DNA sequences (see Example 2 below). The full sequence of clone 65 is shown in FIG. 1. In FIG. 1, Clone 65 begins with nucleotide 4664 and ends with nucleotide 9327. Surprisingly, the DNA from the pXbaI T clone of Towne DNA (1,856 bp) and clone 65 of Toledo DNA (4,668 bp) shared 104 bp of sequence identity. This small stretch of sequence homology allowed mapping of the region of Toledo DNA divergence to the boundary of the Unique Long (U.sub.1) component and the inverted repeats (alternatively termed IRL or b' sequences) on the AD169 and Towne DNA maps. These newly isolated Toledo strain nucleotide sequences from clone 65 were not represented in the reference laboratory strain, AD169, which has been sequenced in its entirety by Chee and colleagues (EMBL accession number X17403).
EXAMPLE 2
Identification of Novel Sequences in the Genome of CMV Towne Not Found in Reference Strain AD169.
DNA sequence heterogeneity between the Towne strain and the AD169 strain has been found. See, Pritchett, J. Virology 36:152-61 (1980). However, although the gross structural organization of the CMV genome has been determined and strain to strain restriction site polymorphisms have been mapped for many strains, strain-to-strain differences on the nucleotide level have not been determined. The laboratory strain AD169 was the first CMV isolate to be sequenced and has served as the reference strain in defining the genetic complexity of the CMV genome.
In order to examine nucleotide sequence differences between Towne and AD169, we focused on the region shown to be divergent in the Toledo strain, i.e. the boundary between the U.sub.L component and the b' sequences, as explained in detail in Example 1. Plasmid pXbaI T was labeled using the NEBlot.TM. Phototope.TM. Detection Kit (New England Biolabs, Beverly, Mass.), and used as a probe on blots of immobilized restriction enzyme digested Towne, Toledo and AD169 DNAs. Briefly, pXbaI T was linearized with PvuII, ethanol precipitated and resuspended in 34 .mu.l of nuclease free water. The plasmid was denatured in boiling water for five minutes, snap cooled on ice for five minutes and centrifuged briefly at 4.degree. C. The following reagents were added to the tube in the order listed: 10 .mu.l of 5.times. labeling mix, 5 .mu.l of dNTP mix, 1 .mu.l of DNA polymerase I (Klenow fragment). The mix was incubated at 37.degree. C. for 6 hours and the reaction was terminated by adding 5 .mu.l of 0.2M EDTA, pH. 8.0. The probe was precipitated by adding 5 .mu.l of 4M LiCl and 150 .mu.l of ethanol, chilling to -80.degree. C. for 30 minutes, pelleted in an Eppendorf centrifuge, washed with 70% ethanol and resuspended in 20 .mu.l of Resuspension Buffer as supplied by the kit. The hybridization reaction was essentially as described above except that after hybridization the membrane was washed twice in 2.times. SSC, 0.1% SDS at room temperature for 5 minutes each followed by two washes in 0.1.times. SSC, 0.1% SDS at 68.degree. C. for 15 minutes. The detection reactions link the biotinylated probes to alkaline phosphatase through a strepavidin bridge and the hybridized probe was visualized by cleavage of the Lurnigen-PPD substrate. The blocking steps, strepavidin incubation, alkaline phosphatase incubation and Lumigen-PPD reaction were carried out as described in the kit manual. Exposure of the blots to Kodak XAR film revealed that, as expected, (i) an XbaI digested fragment of sized 1.85 kbp (XbaI T) was hybridized on Towne DNA probed with pXbaI T and (ii) a comigrating XbaI digested fragment was present in Toledo DNA. The AD169 DNA failed to show any hybridization signal on any of the restriction enzyme digestion patterns. Nucleotide sequence of pXbaI T confirmed the total lack of identity of the Towne DNA and AD169 DNA. Nucleotide sequencing of cosmid 1 DNA (see B. Plasmid DNA in Material and Methods, above) from Toledo revealed extensive sequence identity between the newly identified Towne DNA and the Toledo DNA of cosmid 1 in this region. Surprisingly, the orientation of the sequence was reversed in Toledo relative to Towne.
EXAMPLE 3
Identification of Novel Toledo DNA Sequences in the Genomes of Recent Clinical Isolates and not Found in Reference Strain AD169.
To determine the penetrance of sequences represented by clone 65 in recent clinical isolates, five representative clinical isolates (HCMVF, C128, C354, C793 and C980) were digested with restriction enzymes BamHI and XbaI along with the Toledo, Towne and AD169 DNAs prepared as described in the Materials and Methods section above, electrophoresed through agarose, transferred to a Hybond-N+ nylon transfer membrane, and probed with nick-translated �.alpha..sup.32 P!-labeled clone 65 according to the procedures outlined in the Materials and Methods section. As can be seen in FIG. 4, the autoradiographs revealed that homology was detected in all of the clinical isolates. In FIG. 4, a band at ca. 5 kbp is visible in lane 1 (the Toledo DNA), appears in Towne DNA (lane 2), is missing from lane 3 (the AD169 DNA), and visible in lanes 4 through 8 (the clinical isolates HCMVF, C128, C354, C793 and C980). These results demonstrate that the newly isolated sequence found in the Toledo strain of HCMV is also present in the recent clinical isolates but is not present in the AD169 reference strain. Nucleotide sequence analysis reveals the reason for the weak hybridization signal to the Towne DNA fragment is due to the existence of only 151 nucleotides of sequence identity with Towne DNA. The shared 104 bp sequence identity in Example 1 is responsible for a weak hybridization signal to XbaI "T" sized fragments from both Towne and Toledo DNAs seen in the XbaI digests (lanes 9 and 10). The XbaI digest of the clinical isolates (lanes 12 through 16) also reveals hybridization to multiple high molecular weight bands. Analysis of these and other clinical isolate genomes with other probes in the region has revealed that the shared sequences may be in inverted orientation in some isolates relative to the orientation in the Toledo strain.
FIG. 6 is a schematic illustration of the relative positions of novel sequences identified in Toledo genomic DNA, Towne genomic DNA in a comparison with AD169 strain genomic DNA. The dashed lines delimit the region of the genome where homologous and divergent sequences are found. The top line illustrates a Toledo DNA restriction map showing BamHII (indicated by "B") and XbaI (indicated by "X") restriction enzyme sites extending between the homology breakpoints identified by inverted triangles at nucleotides 175068 and 188843 (numbered with reference to the AD169 DNA sequence--EMBL accession number X17403). Subclones 4, 1300, C23K and 65 of the Toledo DNA sequence are shown in boxes above the map. An inverted region of homology with respect to Towne is shown by the inverted triangles between nucleotides 178221 and 175082. Unique sequences are shown by a thin line, and inverted repeat sequences denoted by thick lines, b'a'c'. The end of the c' repeats is shown with an arrow at nucleotide 191412. The middle line illustrates a Towne DNA restriction map showing BamHI (B) and XbaI (X) restriction enzyme sites as described above for Toledo and showing XbaI clones E, T. and Q in boxes below. Shaded area refers to homologous regions shared with Toledo DNA but inverted in orientation. Nucleotide numbers shown are with reference to the AD169 DNA sequence. Undetermined extent of b' repeat sequences in the Towne strain is shown by thin lines at AD169 strain nucleotide reference 180034. The bottom line illustrates the AD169 genome displayed in the prototype orientation. Unique sequences are displayed by a thin line, and inverted repeats of the long (U.sub.L) and short (U.sub.S) components are denoted by boxes, ab-b'a', and a'c'-ca. The a sequence, is a terminal direct repeat with an inverted copy (a'), at the junction of the long and short components. The length of the AD169 DNA sequence is indicated as 229354 nucleotides and the map position of the internal repeats are shown with the nucleotide reference numbers and arrows.
EXAMPLE 4
Open Reading Frame Analysis of the Novel Toledo and Towne DNA Sequences
The novel Toledo and Towne sequences encoded potential open reading frames (ORFs). Using an arbitrarily chosen parameter of 10 kiloDaltons as the minimum calculated protein molecular weight, a total of 36 ORFs were identified in the novel Toledo sequence and a total of 4 ORFs were identified in the novel Towne sequence. The putative amino acid sequences of these ORFs are set forth in the sequence listing (SEQ ID NOS:2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27). FIG. 5 shows the schematic presentation of these ORFs in the novel Toledo and Towne DNA sequences, together with previously reported AD169 ORFs of the corresponding region. Names were assigned for these ORFs starting from UL133 as the first ORF at the left side of the UL in Toledo sequence. The first ORF in the novel Towne sequence was assigned as UL147, which was determined to be present in the novel Toledo sequence disclosed here. UL130 and UL132 in AD169 were determined to be present in the novel Toledo sequence. Additionally, UL153 and UL154 exhibited regions of homology to IRL14 and IRL12, respectively. All ORFs were searched for homologous sequence in the nonredundant databases of NCBI using the BLASTP program. Among all ORFs searched, only UL132 identified a homologue in the database, which was HCMV mtrIII (GenBank Accession No. X75606), exhibiting 76% identity at the amino acid level. The solid circle identified the ORFs that contained the potential N-linked glycosylation site sequence, N-X(-P)-S/T. These potential glycoproteins may be biologically significant as antigenic or immunogenic molecules.
The present investigation is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and any constructs, viruses or enzymes which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 27- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 4711 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: Human CMV (B) STRAIN: Towne- (ix) FEATURE: (A) NAME/KEY: CDS#(845..1321)) LOCATION: complement#/product= "UL147"R INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS#(1368..1721) LOCATION: complement#/product= "UL152"R INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS#(2504..3337) LOCATION: complement#/product= "UL153"R INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS#(3515..4711) LOCATION: complement#/product= "UL154"R INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- ATCGGGCGCC AGAGCTAGAT CAGGCGTATC AAATTCCACT GCCAGGCGAC CT - #GATTCTAA 60- CGGTTCCACG ATCCGGGAGA GCGTTTCTAG ATATAGAGCA AAGCGTACCA CG - #TCTACCTG 120- CGGTGTAAAA AACTGTTGTG GGCGTTCACC GTCGTTGACC ACGTAAGCCA CG - #TAGAGGCC 180- AACATTTTCC ACCACGGGTT CTAGCTGCAG GCGGCACGTA AAGCTTAGAA AC - #GACGGCTG 240- TACGGTTTGG TTCCCGTGAA GCTGAAGCGT CACTTCCTTG CCGGGGCTCA CC - #GTGCTGTA 300- ACGCCGCACC GAGTCGGTCA TCTGCTCCAG ATCGGTAGAC CAGAAGGGCG TG - #CAATGCAT 360- ACTGTCCCAG TCGCGACACG CAGCCCAGCC TAGCTCGGTG AAGGGTCGAC GC - #ACACCCGA 420- AAAAGTGTGC TTGAAGACCA GGGGGTCGCC TCGGTAGCTC AGTAGCCGAA CA - #TGCACATA 480- GTCGCGGCTA CGTTGACAGA CGGCCCGTAG ACAGGCAGGA CAAGCGTGAA CA - #GCAAGCGC 540- AACATGCTGC GGGTTAGAAA ATGCGGCGTG CCGGCCACCG CCCGACTCAT AA - #ACGCTACC 600- AGCATGACGT CTCAGATCAC ACAAGTGACG AGGAGCGTAC CGCAAATCAC TA - #GGGAAAAG 660- GCCAGCAGAG CCCGATAGTC TTGCTCTTCG CGAACGATCT CGTCCGGTTC CT - #CGCAGTCT 720- TCGTGGTCCA CAGAAGATGA GGAGCAGGAT TCTTCGTTAA TTTCTGCCAG GA - #TACTAGTG 780- CTGTACCACA CCAGAGCGCT CAGCGTGCCC AGGGCTACCG CACGGTAAAA TA - #GGGACATG 840- ATCACCAGCG CAATCTGAAG TGGTGGTAGT TCAGTTTCTT GGCGTATTTC CA - #GAGAAAGG 900- CTTTGTAGGC CGTAGGGACT GGCCAGGCAC CGAACTCAAT ATTGGTAGAC AC - #TACGTCGT 960- AAATGCGTTG TTCCTCGTCT AAGATTAACC GAAAAAATAG CCGGTTGATG TG - #ACGACGCA1020- CGGCTTGCGC GTTAGGATTG AGACACTTGG TGCCCTTGTC CTTTAAAATA GC - #CAGCACTT1080- CCTGACGATT GCAGCTTTCG CTCGCCGCGA TTGGCTTAAG CAATTCAGTT CC - #GATTGGCA1140- GAGTATTCAA CAGAATTTGG TTGTTACAAC GACAGCGTTT GTCGTAATCT TC - #CAATTCTA1200- AAAGATGGAC GGCTAGGGGA CATACGACAA ATAACATGTA TGCAGTCAAT TG - #CATATATC1260- GTACCGATAA AATGTTAGTG TGCGGATTCA GAATCGGATG ATGCAACCGT CT - #TAGCATCA1320- TATCGAAAAA GTATACATAT TACCGATTCA TTATAATTAG GGAATTATTT CC - #AACGCGGA1380- CGTTTGTTAG TGACAGCGTT TTCTTCTACA TGCGGTCCAT TACTATCCTT TA - #CTTTTACC1440- AATACTCTGT GCCATGAGTT GTCTTTTTTA CCATCCAGCC ATTTGGACAA AT - #GATGATCG1500- GGAGCTAAAC ATACAGGTTT ACCTCGAGGA GGCAATAGAT AATGTTGAGG TT - #TGTCACAC1560- TCAGGAGGAT TGGGAGGGTC ACGACCAACC CAAAATAAGC CACCTATAGG AT - #GATGTAAA1620- GCTTTGTGGG TACACGGACA ACGCAATTCT CTACTGTGAA CCCCATGGTA AT - #ACATAAAT1680- GCCATCAAAA GACTAATCAG CGAACCAAAA ATTAATCGCA TTCTAATTTT AT - #TAACTACG1740- TCACTATCAG TAATTCGTAA TATCCGGTAT TCCCGGAAAA TCACTCAAAA CT - #GCGTCCAT1800- GACACATCAA TTCCCGATAA GTACCCCCCT TTGAAATCGG ATCCCCCCAC AT - #ACCAATCA1860- ATCACACAAC ACACAGGTTT AAAAATCGAT CACACGTCAA TTAGGTTTCA AA - #ATCGATAC1920- TGTTTATTAT CAGGAATCTA GACTAATTCT ACAATGACAG CTCTGAATTT CT - #CTCTCGTC1980- TTTCTTGTCA GGTTCTCATC ATCAATCTTC ACTTCCACCC ATCGAGGAGT CA - #TCGTCGCT2040- CCAAAACCCT TTGGGGTCGC TGGTTGGAAA AGTCTCTGAC ACGATCCAGG CA - #CCCCGTAC2100- CCAGTCCGAC TGATCTAGCT TACGGAGCAT CTCAACAGGC ATGAGCTGCA GG - #GCCACGGC2160- TGTCACGGCA GGGATTATTA CTACCGTTCA GGTAAACTGT ATCTCCCTGA GT - #TACCGTGA2220- TGGGTCTTTC TACATGTTGA CTTTGCGTAA AAAATCGCCG GTAAAATGTT TT - #TTCTTGTT2280- CATGTAAAAG TACCGGAACT AAAATGCTAG TTAGAATGGT TGCAGTTGCT AT - #TAGCGCGG2340- CTAGTAACAG TAGTTTAGTG TTACATTGTA TACCCATGTT TTTAATAACT AT - #GAATATTC2400- TGCTTCACAC CATAAGTGCT TAACCCACAA AAACCACACG GAGACATTAT TG - #GCTAAAAA2460- TAAAAACAAA AGTTTATTGA TGTGCATGTT AGGTTTTAGT CTAAAATTCA TC - #TGGGTCGT2520- ATTTGGGAAG TTTTGTATAA CGCGGTCTTC TGGGGACGCG ACGGCTACCC AT - #GTATAAGG2580- CTATAAGTGC CACAGATACC ACTATACCCG CCCATACAGC ATGAATTCCC AG - #GGGAATGT2640- TAGTGTTTTT TACAGTTTTT ATTACATTGT CCCACGTTCT GCTATTATGC TG - #GTCTGATT2700- CCTCTTTTGT TTTACATTTA TCAGGTATAG GAGACGATGT TGCAGTTCCT GA - #TAACACGG2760- TTAAATAGTA GTTTTCCTTT TTACCGTCAC TGTAACGTTG CAAAACGTAT TT - #TCCAGCGT2820- GTTCGGTAGT TACGTTGTAT ATAGTGAGAG AGGTCTTATT GCAGTCTAAA CA - #CATGCCGT2880- TCAGTGGGGA AGTTGAATAA TAATGTCCAA TGCTGCACAG TTGGTGTGCG CG - #AGGTCCAT2940- ATTTTATCCA TTCTATATCG TGCCATACAT CCGTTCTACT GCAGTTTTTC AA - #AGTGACGT3000- ATCCACCGAC ATATCCTGTT ACATTAATTA CTTCGTAATT TAAATTAGAG TG - #TTTATAAA3060- CGGTGTACAA ACTGCCATTG CAAGTTATGT TGCTGGTATT CAACCAGGGA GT - #AGTACTAT3120- GAATGGTAGA AAACGTTAAT GTTGGCGTAG CGCTTGACGA TGATTTTGAA AG - #CGTTGAAG3180- TGGTTGCTGA TGCGACTGAA GAAGCGGTAG AGGGTTTGTG CGTGGTTCCA TT - #TGCGATAG3240- CTGAAGTGCT GTTAGCATCG GTGACAGAGT TAGAAGAATT TGTGATAGTG GA - #GGCGGTGG3300- AGGTAAAGGC AATTGCACGG ACAGGAGCAC GTGTCATTGC AACCTTCAGA TA - #TCGTAATC3360- ATCAGTAACG TCCACTTAAC CGTAAATCTC CAGTCCATAA CGTTATTAAA TT - #TCGGTTAA3420- CGGGCATTGA TGTTTCTTCG GACGTTGTTG ATCTTTCTTG CCCGTTTATT TT - #CTGATATG3480- GTCTCATAAG ACATTTATCC GGAAACGTTG CTTAGTCCTC GTGCTCAGGA TT - #GTATCGAA3540- CTATGAATTC TGATTCACTT ATATCGTCAC TTAATGGATG ATATTTTTTA TT - #TAGAGCTC3600- GTCGGACGAA AAATAGGAGA ATGCAGGCTA CACAAATTAA TGCTAACGTC CA - #CGTAGTGC3660- GTCTGCCGTG TGATGTGTTA GAATGATTGT TATAGCGGTA TAAATGATCT AT - #AGATGATG3720- TGGCTGTATT GTCTTCATAA TTGGTCGGTT TATGAGAAGT GTCCCATTCG TG - #CTTTGGTT3780- CTTCACATAC CCAGGGATTC ACGTGTGTCC CGTTTGTGTT GTTTCTAGGA TG - #TATTTGCA3840- GATTAAAGTT TTGATTTTGT TCGGAGGGAT GCCCAGTTTT ATAACATCGA AA - #GCTATATT3900- TACCAGAATG AGTAAAATTA AGACCGTACA GAGATAAAGA TAAATTACGA TC - #GCATGTAA3960- AACATAAATC ATAGTGATGT TTTAGATAAT TTGTGTGCCA CTCACATAGT AT - #ACGCGAAT4020- GGAGGATTTT CAATGAATGG TTATGATATT TTCCATTTCT TATGTTGGGA TG - #GGTGTATT4080- TTCCGTGTGT GGATATATTA AAATGTCTAA GCCAGGCTGT TTTGTAGCAC GA - #TGTGATGG4140- TTAGGTTGTG TGTTATAGTA ATATTGTCTC CTTGTGCCGC CTCCAATAAT GT - #TTCAGATT4200- CTTTTGATAT CGTATTATTT GTACTGTTAG GCGATGAGCA AGTTGGAAGC GG - #TGTAGTGA4260- CGTTTTCATT TGCATTTATC ATAGTAGTAG TGTTGGTTGA TAATGATATA GT - #TTGCAAAG4320- TCACAGTACT ATCGGTTACA TGCTGTGTCG ATGAATTCGT GTCGCCGTTT GG - #TGAAGTTG4380- TTATTACAGT TACGTTAGTT GTAGATGTTT GGGTAGATAT GGTGGAAATA GT - #TGAGGTCA4440- CGTCTGTGCC TTTTACAGAG CTTGCAGTGA ATCCTGTGGA TGTGTTGACG TT - #GCCATTGG4500- AGGATGTGAA CATAGTGGTA GACATTTCGG TGGTTTGTAA CGTAGATGTC AG - #TTGTGTAG4560- TAGATATTAA GCTTGTGGGT GTAATCGACG TGGAAGTATT GGCGATAGTG GT - #GTTGTTAC4620- ACTTGCTTTT CTGCAGAATC CAAAAAATAA TAAACATGCA TATTATTTGC GT - #ATATGATG4680# 4711 ATGT TGTGTGCGCA T- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 159 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Met Leu Arg Arg Leu His His - # Pro Ile Leu Asn Pro His ThrAsn# 15- Ile Leu Ser Val Arg Tyr Met Gln - # Leu Thr Ala Tyr Met Leu PheVal# 30- Val Cys Pro Leu Ala Val His Leu - # Leu Glu Leu Glu Asp Tyr AspLys# 45- Arg Cys Arg Cys Asn Asn Gln Ile - # Leu Leu Asn Thr Leu Pro IleGly# 60- Thr Glu Leu Leu Lys Pro Ile Ala - # Ala Ser Glu Ser Cys Asn ArgGln# 80- Glu Val Leu Ala Ile Leu Lys Asp - # Lys Gly Thr Lys Cys Leu AsnPro# 95- Asn Ala Gln Ala Val Arg Arg His - # Ile Asn Arg Leu Phe Phe ArgLeu# 110- Ile Leu Asp Glu Glu Gln Arg Ile - # Tyr Asp Val Val Ser Thr AsnIle# 125- Glu Phe Gly Ala Trp Pro Val Pro - # Thr Ala Tyr Lys Ala Phe LeuTrp# 140- Lys Tyr Ala Lys Lys Leu Asn Tyr - # His His Phe Arg Leu Arg Trp# 155- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 118 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Met Arg Leu Ile Phe Gly Ser Leu - # Ile Ser Leu Leu Met Ala PheMet# 15- Tyr Tyr His Gly Val His Ser Arg - # Glu Leu Arg Cys Pro Cys ThrHis# 30- Lys Ala Leu His His Pro Ile Gly - # Gly Leu Phe Trp Val Gly ArgAsp# 45- Pro Pro Asn Pro Pro Glu Cys Asp - # Lys Pro Gln His Tyr Leu LeuPro# 60- Pro Arg Gly Lys Pro Val Cys Leu - # Ala Pro Asp His His Leu SerLys# 80- Trp Leu Asp Gly Lys Lys Asp Asn - # Ser Trp His Arg Val Leu ValLys# 95- Val Lys Asp Ser Asn Gly Pro His - # Val Glu Glu Asn Ala Val ThrAsn# 110- Lys Arg Pro Arg Trp Lys 115- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 278 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Met Thr Arg Ala Pro Val Arg Ala - # Ile Ala Phe Thr Ser Thr AlaSer# 15- Thr Ile Thr Asn Ser Ser Asn Ser - # Val Thr Asp Ala Asn Ser ThrSer# 30- Ala Ile Ala Asn Gly Thr Thr His - # Lys Pro Ser Thr Ala Ser SerVal# 45- Ala Ser Ala Thr Thr Ser Thr Leu - # Ser Lys Ser Ser Ser Ser AlaThr# 60- Pro Thr Leu Thr Phe Ser Thr Ile - # His Ser Thr Thr Pro Trp LeuAsn# 80- Thr Ser Asn Ile Thr Cys Asn Gly - # Ser Leu Tyr Thr Val Tyr LysHis# 95- Ser Asn Leu Asn Tyr Glu Val Ile - # Asn Val Thr Gly Tyr Val GlyGly# 110- Tyr Val Thr Leu Lys Asn Cys Ser - # Arg Thr Asp Val Trp His AspIle# 125- Glu Trp Ile Lys Tyr Gly Pro Arg - # Ala His Gln Leu Cys Ser IleGly# 140- His Tyr Tyr Ser Thr Ser Pro Leu - # Asn Gly Met Cys Leu Asp CysAsn# 160- Lys Thr Ser Leu Thr Ile Tyr Asn - # Val Thr Thr Glu His Ala GlyLys# 175- Tyr Val Leu Gln Arg Tyr Ser Asp - # Gly Lys Lys Glu Asn Tyr TyrLeu# 190- Thr Val Leu Ser Gly Thr Ala Thr - # Ser Ser Pro Ile Pro Asp LysCys# 205- Lys Thr Lys Glu Glu Ser Asp Gln - # His Asn Ser Arg Thr Trp AspAsn# 220- Val Ile Lys Thr Val Lys Asn Thr - # Asn Ile Pro Leu Gly Ile HisAla# 240- Val Trp Ala Gly Ile Val Val Ser - # Val Ala Leu Ile Ala Leu TyrMet# 255- Gly Ser Arg Arg Val Pro Arg Arg - # Pro Arg Tyr Thr Lys Leu ProLys# 270- Tyr Asp Pro Asp Glu Phe 275- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 399 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Met Arg Thr Gln His Arg Arg Trp - # Asn Lys Ser Ser Tyr Thr GlnIle# 15- Ile Cys Met Phe Ile Ile Phe Trp - # Ile Leu Gln Lys Ser Lys CysAsn# 30- Asn Thr Thr Ile Ala Asn Thr Ser - # Thr Ser Ile Thr Pro Thr SerLeu# 45- Ile Ser Thr Thr Gln Leu Thr Ser - # Thr Leu Gln Thr Thr Glu MetSer# 60- Thr Thr Met Phe Thr Ser Ser Asn - # Gly Asn Val Asn Thr Ser ThrGly# 80- Phe Thr Ala Ser Ser Val Lys Gly - # Thr Asp Val Thr Ser Thr IleSer# 95- Thr Ile Ser Thr Gln Thr Ser Thr - # Thr Asn Val Thr Val Ile ThrThr# 110- Ser Pro Asn Gly Asp Thr Asn Ser - # Ser Thr Gln His Val Thr AspSer# 125- Thr Val Thr Leu Gln Thr Ile Ser - # Leu Ser Thr Asn Thr Thr ThrMet# 140- Ile Asn Ala Asn Glu Asn Val Thr - # Thr Pro Leu Pro Thr Cys SerSer# 160- Pro Asn Ser Thr Asn Asn Thr Ile - # Ser Lys Glu Ser Glu Thr LeuLeu# 175- Glu Ala Ala Gln Gly Asp Asn Ile - # Thr Ile Thr His Asn Leu ThrIle# 190- Thr Ser Cys Tyr Lys Thr Ala Trp - # Leu Arg His Phe Asn Ile SerThr# 205- His Gly Lys Tyr Thr His Pro Asn - # Ile Arg Asn Gly Lys Tyr HisAsn# 220- His Ser Leu Lys Ile Leu His Ser - # Arg Ile Leu Cys Glu Trp HisThr# 240- Asn Tyr Leu Lys His His Tyr Asp - # Leu Cys Phe Thr Cys Asp ArgAsn# 255- Leu Ser Leu Ser Leu Tyr Gly Leu - # Asn Phe Thr His Ser Gly LysTyr# 270- Ser Phe Arg Cys Tyr Lys Thr Gly - # His Pro Ser Glu Gln Asn GlnAsn# 285- Phe Asn Leu Gln Ile His Pro Arg - # Asn Asn Thr Asn Gly Thr HisVal# 300- Asn Pro Trp Val Cys Glu Glu Pro - # Lys His Glu Trp Asp Thr SerHis# 320- Lys Pro Thr Asn Tyr Glu Asp Asn - # Thr Ala Thr Ser Ser Ile AspHis# 335- Leu Tyr Arg Tyr Asn Asn His Ser - # Asn Thr Ser His Gly Arg ArgThr# 350- Thr Trp Thr Leu Ala Leu Ile Cys - # Val Ala Cys Ile Leu Leu PhePhe# 365- Val Arg Arg Ala Leu Asn Lys Lys - # Tyr His Pro Leu Ser Asp AspIle# 380- Ser Glu Ser Glu Phe Ile Val Arg - # Tyr Asn Pro Glu His Glu Asp# 395- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18318 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: Human CMV (B) STRAIN: Toledo- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 511..1281#/product = "UL133" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1401..2384#/product = "UL135" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2478..3197#/product = "UL136" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3283..3789#/product = "UL138" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4355..4759#/product = "UL139" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4944..5285#/product = "UL140" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 5558..6832#/product = "UL141" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 6908..7825#/product = "UL142" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 7813..8088#/product = "UL143" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 8468..8995#/product = "UL144" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9327..9626#/product = "UL145" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9910..10260#/product = "UL146" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 10328..10804#/product = "UL147" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 11106..12053#/product = "UL148" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 12133..12942#/product = "UL132" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 13569..14210#/product = "UL130" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 16216..16581#/product = "UL149" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1004..1528#/product = "UL134" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3063..3350#/product = "UL137" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 16337..18262#/product = "UL150" INFORMATION:- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 17752..18759#/product = "UL151" INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- CGCTGTAGGG ATAAATAGTG CGATGGCGTT TGTGGGAGAA CGCAGTAGCG AT - #GGGTTGCG 60- ACGTGCACGA TCCTTCGTGG CAATGCCAAT GGGGCGTTCC CACGATTATC GT - #GGCCTGGA 120- TAACATGCGC GGCTTTAGGA ATTTGGTGTT TGGCGGGATC GTCGGCGGAT GT - #CTCTTCGG 180- GACCCGGCAT CGCAGCCGTA GTCGGCTGTT CTGTTTTCAT GATTTTCCTC TG - #CGCGTATC 240- TCATCCGTTA CCGGGAATTC TTCAAAGACT CCGTAATCGA CCTCCTTACC TG - #CCGATGGG 300- TTCGCTACTG CAGCTGCAGC TGTAAGTGCA GCTGCAAATG CATCTCGGGC CC - #CTGTAGCC 360- GCTGCTGTTC AGCGTGTTAC AAGGAGACGA TGATTTACGA CATGGTCCAA TA - #CGGTCATC 420- GACGGCGTCC CGGACACGGC GACGATCCCG ACAGGGTGAT CTGCGAGATA GT - #CGAGAGTC 480- CCCCGGTTTC GGCGCCGACG GTGTCCGTCC CCCCGCCGTC GGAGGAGTCC CA - #CCAGCCCG 540- TCATCCCACC GCAGCCGCCA GCACCGACAT CGGAACCCAA ACCGAAGAAA GG - #TAGGGCGA 600- AAGATAAACC GAAGGGTAGA CCGAAAGACA AACCTCCGTG CGAACCGACG GT - #GAGTTCAC 660- AACCACCGTC GCAGCCGACG GCAATGCCCG GCGGTCCGCC CGACGCGCCT CC - #CCCCGCCA 720- TGCCGCAGAT GCCACCCGGC GTGGCCGAGG CGGTACAAGC TGCCGTGCAG GC - #GGCCGTGG 780- CCGCGGCTCT ACAACAACAG CAGCAGCATC AGACCGGAAC GTAACCCGCC CC - #CGGTGCGA 840- TAAGGAATTT TCCGACTTGG CGCACATCTC CTTCCTCAAT GTTTGGACAA TA - #AACACATT 900- CCTTGCCAAA AAATGACGTT TCCAGAAATC CAAGGCATAA ATGTCCGTAC AC - #CGGCCCTT 960- CCCAACACGG AGTTTGAGAT TCCAAGCAGG AGAGAAGATC ATGGTGTGGA TA - #TGGCTCGG1020- CATCGGGCTC CTCGGCGGTA CCGGACTGGC TTCCCTGGTC CTGGCCATTT CC - #TTATTTAC1080- CCAGCGCCGA GGCCGCAAGC GATCCGACGA GACTTCGTCG CGAGGCCGGC TC - #CCGGGTGC1140- TGCTTCTGAT AAGCGTGGTG CCTGCGCGTG CTGCTATCGA AATCCGAAAG AA - #GACGTCGT1200- CGAGCCGCTG GATCTGGAAC TGGGGCTCAT GCGGGTGGAC ACCCACCCGC CG - #ACGCCGCA1260- GGTGCCGCGG TGTACGTCGC TCTACATAGG AGAGGATGGT CTGCCGATAG AT - #AAACCCGA1320- GTTTCCTCCG GCGCGGTTCG AGATCCCCGA CGTATCCACG CCGGGAACGC CG - #ACCAGCAT1380- CGGCCGATCT CCGTCGCATT GCTCCTCGTC GAGCTCTTTG TCGTCCTCGA CC - #AGCGTCGA1440- CACGGTGCTG TATCAGCCGC CGCCATCCTG GAAGCCACCT CCGCCGCCCG GG - #CGCAAGAA1500- GCGGCCGCCT ACGCCGCCGG TCCGGGCCCC CACCACGCGG CTGTCGTCGC AC - #AGACCCCC1560- GACGCCGATA CCCGCGCCGC GTAAGAACCT GAGCACGCCG CCCACCAAGA AA - #ACGCCGCC1620- GCCCACGAAA CCCAAGCCGG TCGGCTGGAC ACCGCCGGTG ACACCCAGGC CC - #TTCCCGAA1680- AACGCCGACG CCACAAAAGC CGCCGCGGAA TCCGAGACTA CCGCGCACCG TC - #GGTCTGGA1740- GAATCTCTCG AAGGTGGGAC TCTCGTGTCC CTGTCCCCGA CCCCGCACGC CG - #ACGGAGCC1800- GACCACGCTG CCTATCGTGT CGGTTTCCGA GCTAGCCCCG CCTCCTCGAT GG - #TCGGACAT1860- CGAGGAACTC TTGGAACAGG CGGTGCAGAG CGTCATGAAG GACGCCGAGT CG - #ATGCAGAT1920- GACCTGAGAC CGAAAGAGCG AGCGCGTCCG TTGTACAGTT GTATAGCAGC AC - #ACGCCTTC1980- CCTCTTTTTC ACCGCAGCTA AGAGAGAGAA AGAGAGTATG TCAGTCAAGG GC - #GTGGAGAT2040- GCCAGAAATG ACGTGGGACT TGGACGTTAG AAATAAATGG CGGCGTCGAA AG - #GCCCTGAG2100- TCGCATTCAC CGGTTCTGGG AATGTCGGCT ACGGGTGTGG TGGCTGAGTG AC - #GCCGGCGT2160- AAGAGAAACC GACCCACCGC GTCCCCGACG CCGCCCGACT TGGATGACCG CG - #GTGTTTCA2220- CGTTATCTGT GCCGTTTTGC TTACGCTTAT GATTATGGCC ATCGGCGCGC TC - #ATCGCGTA2280- CTTAAGATAT TACCACCAGG ACAGTTGGCG AGACATGCTC CACGATCTAT TT - #TGCGGCTG2340- TCATTATCCC GAGAAGTGCC GTCGGCACCA CGAGCGGCAG AGAAGGAGAC GG - #CAAGCCAT2400- GGATGTGCCC GACCCGGAAC TCGGCGACCC GGCCCGCCGG CCGTTGAACG GA - #GCTATGTA2460- CTACGGCAGC GGCTGTCGCT TCGACACGGT GGAAATGGTG GACGAGACGA GA - #CCCGCGCC2520- GCCGGCGCTG TCATCGCCCG AAACCGGCGA CGATAGCAAC GACGACGCGG TT - #GCCGGCGG2580- AGGTGCTGGC GGGGTAACAT CACCCGCGAC TCGTACGACG TCGCCGAACG CA - #CTGCTGCC2640- AGAATGGATG GATGCGGTGC ATGTGGCGGT CCAAGCCGCC GTTCAAGCGA CC - #GTGCAAGT2700- AAGTGGCCCG CGGGAGAACG CCGTATCTCC CGCTACGTAA GAGGGTTGAG GG - #GGCCGTTC2760- CCGCGCGAGT GCTGTACAAA AGAGAGAGAC TGGGACGTAG ATCCGGACAG AG - #GACGGTCA2820- CCATGGACGA TCTGCCGCTG AATGTCGGGT TACCCATCAT CGGCGTGATG CT - #CGTGCTGA2880- TCGTGGCCAT CCTCTGCTAT CTGGCTTACC ACTGGCACGA CACCTTCAAA CT - #GGTGCGCA2940- TGTTTCTGAG CTACCGCTGG CTGATCCGCT GTTGCGAGCT GTACGGGGAG TA - #CGAGCGCC3000- GGTTCGCGGA CCTGTCGTCT CTGGGCCTCG GCGCCGTACG GCGGGAGTCG GA - #CAGACGAT3060- ACCGTTTCTC CGAACGGCCC GACGAGATCT TGGTCCGTTG GGAGGAAGTG TC - #TTCCCAGT3120- GCAGCTACGC GTCGTCGCGG ATAACAGACC GCCGTGTGGG TTCATCGTCT TC - #GTCGTCGG3180- TCCACGTCGC TAGCCAGAGA AACAGCGTGC CTCCGCCGGA CATGGCGGTG AC - #GGCGCCGC3240- TGACCGACGT CGATCTGTTG AAACCCGTGA CGGGATCCGC GACGCAGTTC AC - #CACCGTAG3300- CCATGGTACA TTATCATCAA GAGTACACGT GAATGAGAAA AAGAAAAAAG AG - #GGGAGCGG3360- ATCGCGATAA TGTCGCTTTG ACATTCTCTG CTCGATCTAC TCAGCGTCTG CA - #CGAAACGG3420- CATCCGCACG GAGGCGAGCC CAAGCGTATC TGCAGCAAGC GGTTCTTTCC CT - #CGGTGATG3480- GTGGCAGCAT CGGTGGCGGG AGCTTGTTCG GACGATGGAC GGTGAGGAGT CC - #CTGGCGAT3540- CAGGCGGCTC CCGGGTGTGG AGTTCAACGG GTGGTAATGG TGGCGGTGAT CG - #GTGTTAGA3600- AAACGGTGGC CCTGGCAAAC ATATATCTAC TGTAAACCCT CTGCTCTGTT AA - #TAAAAAGC3660- ACACTTTTCA CATGAGTTCG TAATTTTATT GTGTAGTGGA AATTTTTACG TC - #ATTGGGAA3720- ACCCCAGAAT GAAAGAGTAT AATGTGCATA TCACCGGGGG TTCCCTGTCA GT - #ACGAATGT3780- ACACAACGCG GGTTACATTA CGATAAACTT TCCGGTAAAA CGATGCCGAT AC - #AGCGTGTA3840- TAACGCTGAT TGTTACGACA AACGAGTTGG TATATCCATT ATATAGTAAC GA - #ACATGCTG3900- TGGATATTAG TTTTATTTGC ACTCGCCGCA TCGGCGAGTG AAACCACTAC AG - #GTACCAGC3960- TCTAATTCCA GTCAATCTAC TAGTGCTACC GCCAACACGA CCGTATCGAC AT - #GTATTAAT4020- GCCTCTAACG GCAGTAGCTG GACAGTACCA CAGCTCGCGC TGCTTGCCGC TA - #GCGGCTGG4080- ACATTATCTG GACTCCTTCT CTTATTTACC TGCTGCTTTT GCTGCTTTTG GC - #TAGTACGT4140- AAAATCTGCA GCTGCTGCGG CAACTCCTCC GAGTCAGAGA GCAAAACAAC CC - #ACGCGTAC4200- ACCAATGCCG CATTCACTTC TTCCGACGCA ACGTTACCCA TGGGCACTAC AG - #GGTCGTAC4260- ACTCCCCCAC AGGACGGCTC ATTTCCACCT CCGCCTCGGT GACGTAGGCT AA - #ACCGAAAC4320- CCACGTTGAA CCTAACGCGG TTTCGGAAGG CCTGAGACGT CACTTTCACA AT - #GACGTCCG4380- TATACACGTT CATCATAAAA CACCGTAGAG GCTAAGGCTT CGGTAGGGAG AG - #ACCTCAAC4440- TGTTCCTGAT GAGCACCCGT GCTCTCATCT CTTCAGACTT GTCATGACCC CC - #GCTCAGAC4500- TAACGCGACT ACCACCGTGC ACCCGCACGA CGCAAAAAAC GGCAGCGGCG GT - #AGTGCCCT4560- GCCGACCCTC GTCGTTTTCG GCTTTATCGT TACGCTACTT TTCTTTCTCT TT - #ATGCTCTA4620- CTTTTGGAAC AACGACGTGT TCCGTAAGCT GCTCCGTGCG CTTGGATCCA GC - #GCTGTTGC4680- GACCGCTTCG ACGCGTGGCA AGACGAGGTC ATCTACCGTC GTCCATCACG TC - #GTTCCCAG4740- AGCGACGACG AGAGTCGTAC TAACAGCGTG TCATCGTACG TTCTTTTATC AC - #CCGCGTCC4800- GATGGCGGTT TTGACAACCC GGCACTGACA GAGGCCGTCG ACAGCGTGGA CG - #ACTGGGCG4860- ACCACCTCGG TTTTCTACGC CACGTCCGAC GAAACGGCGG ACGCCGAGCG CC - #GAGACTCG4920- CAGCAACTGC TCATCGAGCT TCCGCCGGAG CCGCTCCCGC CCGACGTGGT GG - #CGGCCATG4980- CAGAAAGCAG TGAAACGCGC TGTACAGAAC GCACTACGAC ACAGCCACGA CT - #CTTGGCAG5040- CTTCATCAGA CCCTGTGACG CCAGATGAAC GTTCCTTCTT AAACATCCGA GG - #TAGCAATG5100- AGACAGGTCG CGTACCGCCG GCGACGCGAG AGTTCCTGCG CGGTGCTGGT CC - #ACCACGTC5160- GGCCGCGACG GCGACGGCGA GGGGGAGGCA GCAAAAAAGA CCTGCAAAAA AA - #CCGGACGC5220- TCAGTTGCGG GCATCCCGGG CGAGAAGCTG CGTCGCACGG TGGTCACCAC CA - #CGCCGGCC5280- CGACGTTTGA GCGGCCGACA CACGGAGCAG GAGCAGGCGG GCATGCGTCT CT - #GTGAAAAA5340- GGGAAGAAAA GAATCATCAT GTGCCGCCGG GAGTCGCTCC GAACTCTGCC GT - #GGCTGTTC5400- TGGGTGCTGT TGAGCTGCCC GCGACTCCTC GAATATTCTT CCTCTTCGTT CC - #CCTTCGCC5460- ACCGCTGACA TTGCCGAAAA GATGTGGGCC GAGAATTATG AGACCACGTC GC - #CGGCGCCG5520- GTGTTGGTCG CCGAGGGAGA GCAAGTTACC ATCCCCTGCA CGGTCATGAC AC - #ACTCCTGG5580- CCCATGGTCT CCATTCGCGC ACGTTTCTGT CGTTCCCACG ACGGCAGCGA CG - #AGCTCATC5640- CTGGACGCCG TCAAAGGCCA TCGGCTGATG AACGGACTCC AGTACCGCCT GC - #CGTACGCC5700- ACTTGGAATT TCTCGCAATT GCATCTCGGC CAAATATTCT CGCTTACTTT TA - #ACGTATCG5760- ATGGACACAG CCGGCATGTA CGAATGCGTG CTACGCAACT ACAGCCACGG CC - #TCATCATG5820- CAACGCTTCG TAATTCTCAC GCAGCTGGAG ACGCTCAGCC GGCCCGACGA AC - #CTTGCTGC5880- ACACCGGCGT TAGGTCGCTA CTCGCTGGGA GACCAGATCT GGTCGCCGAC GC - #CCTGGCGT5940- CTACGGAATC ACGACTGCGG AACGTACCGC GGCTTTCAAC GCAACTACTT CT - #ATATCGGC6000- CGCGCCGACG CCGAGGATTG CTGGAAACCC GCATGTCCGG ACGAGGAACC CG - #ACCGCTGT6060- TGGACAGTGA TACAGCGTTA CCGGCTCCCC GGCGACTGCT ACCGTTCGCA GC - #CACACCCG6120- CCGAAATTTT TACCGGTGAC GCCAGCACCG CCGGCCGACA TAGACACCGG GA - #TGTCTCCC6180- TGGGCCACTC GGGGAATCGC GGCGTTTTTG GGGTTTTGGA GTATTTTTAC CG - #TATGTTTC6240- CTATGCTACC TGTGTTATCT GCAGTGTTGT GGACGCTGGT GTCCCACGCC GG - #GAAGGGGA6300- CGACGAGGCG GTGAGGGCTA TCGACGCCTA CCGACTTACG ATAGTTACCC CG - #GTGTTAGA6360- AAGATGAAGA GGTGAGAACA CGTATAAAAT AAAAAAATAA TATGTTAAAA AA - #TGCAGTGT6420- GTGAAGTGTG AATAGTGTGA TTAAAATATG CGGATTGAAT GGGTGTGGTG GT - #TATTCGGA6480- TACTTTGTGT CATCCGTTGG GAGCGAACGG TCATTATCCT ATCGTTACCA CT - #TGGAATCT6540- AATTCATCTA CCAACGTGGT TTGCAACGGA AACATTTCCG TGTTTGTAAA CG - #GCACCCTA6600- GGTGTGCGGT ATAACATTAC GGTAGGAATC AGTTCGTCTT TATTAATAGG AC - #ACCTTACT6660- ATACAAGTAT TGGAATCATG GTTCACACCC TGGGTCCAAA ATAAAAGTTA CA - #ACAAACAA6720- CCCCTAGGTG ACACTGAAAC GCTTTATAAT ATAGATAGCG AAAACATTCA TC - #GCGTATCT6780- CAATATTTTC ACACAAGATG GATAAAATCT CTGCAAGAGA ATCACACTTG CG - #ACCTCACA6840- AACAGTACAC CTACCTATAC ATATCAAGTA AACGTGAACA ACACGAATTA CC - #TAACACTA6900- ACATCCTCGG GATGGCAAGA CCGTCTAAAT TACACCGTCA TAAATAGTAC AC - #ACTTTAAC6960- CTCACAGAAT CGAACATAAC CAGCATTCAA AAATATCTCA ACACTACCTG CA - #TAGAAAGA7020- CTCCGTAACT ACACCTTGGA GTCCGTATAC ACCACAACTG TGCCTCAAAA CA - #TAACAACA7080- TCTCAACACG CAACAACCAC TATGCACACA ATACCTCCAA ATACAATAAC AA - #TTCAAAAT7140- ACAACTCAAA GCCATACTGT ACAGACGCCG TCTTTTAACG ACACACATAA CG - #TGACGAAA7200- CACACGTTAA ACATAAGCTA CGTTTTATCA CAAAAAACGA ATAACACAAC AT - #CACCGTGG7260- ATATATGCCA TACCTATGGG CGCTACAGCC ACAATAGGCG CCGGTTTATA TA - #TCGGGAAA7320- CACTTTACGC CGGTTAAGTT CGTATACGAG GTATGGCGCG GTCAGTAAAG AC - #GATTCGGA7380- TTCAACACAT ATACTCCCCA CGATCCTCGA ACACCTTACA GCATATGAGC AA - #AAAACAAG7440- AAAGTATAGC CACAATCACA TTTGGGCGAA TAACATGCTG TCATCCACTA GC - #GTCTATTA7500- ATCTAATGTT TAACGGGAGC TGTACTGTCA CCGTTAAAAT ATCCATGGGA AT - #CAACGGGT7560- CAACCAACGT CCATCAGCTT GTGATTGTGC TCCATCTGGG TAACCGCTGT CA - #GCCTTGGC7620- GACAGGTGTA ATCACAGCTG TCACATAACT CACGAAGCCT CCAATCACAG CA - #GCACACAT7680- AGTCCTAACG CCATTGGCGT GTATAAAAGT TCGGAAAACT TGACGGTTGT AC - #GGCACGAC7740- AAATCGATGT AGTGGTATGT TTTTCCAGCA GAGACCGTGT GCGGTCTCTT AG - #GTTCGCTA7800- TACTGTGGCT GGAAACTGGT TACCTGTGAA GATGGCTAAC TATCCTGTTC TG - #TCCTGGAA7860- AAACTTTTGG CGTCGTAGGT GGACTTTGCA GTATGCGGGT TAGTGAAGTT AT - #GTCATTTA7920- TTTACGTTTA CGATCTCGTA TTACAAACCG CGGAGAGGAT GATACCGTTC GG - #CCCCATGA7980- GTTATTTTTA TTCTTCCGGT AGGAGGCATG AAGCCTCTGA TAATGCTCAT CT - #GCTTTGCT8040- GTGATATTAT TGCAGCTTGG AGTGACTAAA GTGTGTCAGC ATAATGAAGT GC - #AACTGGGC8100- AATGAGTGCT GCCCTCCGTG TGGTTCGGGA CAAAGAGTTA CTAAAGTATG CA - #CGGATTAT8160- ACCAGTGTAA CGTGTACCCC TTGCCCCAAC GGCACGTATG TATCGGGACT TT - #ACAACTGT8220- ACCGATTGCA CTCAATGTAA CGTCACTCAG GTCATGATTC GTAACTGCAC TT - #CCACCAAT8280- AATACCGTAT GCGCACCTAA GAACCATACG TACTTTTCCA CTCCAGGCGT CC - #AACATCAC8340- AAACAACGAC AGCAAAATCA TACCGCACAT ATAACCGTCA AACAAGGAAA AA - #GCGGTCGT8400- CATACTCTAG CCTGGTTGTC TCTCTTTATC TTTCTTGTGG GTATCATACT TT - #TAATTCTC8460- TATCTTATAG CCGCCTATCG GAGTGAGAGA TGCCAACAGT GTTGCTCAAT CG - #GCAAAATT8520- TTCTACCGCA CCCTGTAAGC TTCCTGTTGT TGTTTTTACA TCACGGTACG AT - #GAAGTCAC8580- ACAGATAATT ACAGATGAGC TGTTCATATT TTTTATTATT TTTTCCAATT CC - #TGCACTAA8640- AAAAAGAAGC ACTTTACGGA ACCGTGTCTG AGTATCTGTG GGGAATTTAG GT - #ACTTTTTG8700- CCGACGTCAG GAAAAATAAG TGTCGCCTAC ATAAGAGCCC GGTGCTATCG TG - #CTGTCACT8760- CTTTCTTGTT GCCTTCGATG TACGGCGTCC TGGCTCATTA CTACTCCTTC AT - #CAGTAGCC8820- CCAGCGTTAT GGTTAATTTT AAGCATCATA ACGCCGTGCA GCTGTTATGT GC - #ACGGACCC8880- GAGACGCACT GCCGGATGGG AACGTTTAAC CCATCATGCG TCGTATCACG CG - #AACTACGG8940- GGCATACGCC GTGTTGATGG CTACATCGCA AAGAAAGTCC CTAGTGTTAC AT - #CGATACAG9000- TGCCGTGACA GCCGTGGCCC TGCAGCTCAT GCCTGTTGAG ATCGTCCGCA AG - #CTAGATCA9060- GTCGGACTGG GTGCGGGGTG CCTGGATCGT GTCAGAGACT TTTCCAACTA GC - #GACCCCAA9120- AGGAGTTTGG AGCGACGATG ACTCCTCGAT GGGTGGAAGT GATGATTGAT GA - #TGAGAACC9180- TGACAAGAAA GACGAGAGAG AAATTTAGAG CTGTCATTGT AGAATTAGTC TA - #GATTCCTG9240- ATAATAAACA GTATCGATTT TGAAACCTAA TTGACGTGTG ATCGATTTTT AA - #ACCTCTGT9300- GTTGTGTGAT TGATTGGTAT GTGGGGGGAT CCGATTTCAA AGGGGGGTAC TT - #ATCGGGAA9360- TTGATGTGTC ATGGACGCAG TTTTGAGCGA TTTTCCGGGA ATACCGGATA TT - #ACGAATTA9420- CTGGTAGTGA CGTAGATAAT AAAATTATAA TGCGATTAAT TTTTGGTGCG TT - #GATTATTT9480- TTTTAGCATA TGTGTATCAT TATGAGGTGA ATGGAACAGA ATTACGCTGC AG - #ATGTCTTC9540- ATAGAAAATG GCCGCCTAAT AAAATTATAT TGGGTAATTA TTGGCTTCAT CG - #CGATCCCA9600- GAGGGCCCGG ATGCGATAAA AATGAACATT TATTGTATCC AGACGGAAGG AA - #ACCGCCTG9660- GACCTGGAGT ATGTTTATCG CCCGATCACC TCTTCTCAAA ATGGTTAGAC AA - #ACACAACG9720- ATAATAGGTG GTATAATGTT AACATAACGA AATCACCAGG ACCGAGACGA AT - #AAATATAA9780- CCTTGATAGG TGTTAGAGGA TAATATTTAA TGTATGTTTT CAAACAGACA AG - #TTCGTTAA9840- AACAAAATAT TACAGTATGT GTTTAATATG GTGCTAACAT GGTTGCACCA TC - #CGGTTTCA9900- AACTCGCATA TCAATCTGTT ATCGGTACGA CACCTGTCAT TAATCGCATA TA - #TGTTACTT9960- ACCATATGTC CCCTAGCCGT CCATGTTTTA GAACTAGAAG ATTACGACAG GC - #GCTGCCGT10020- TGCAACAACC AAATTCTGTT GAATACCCTG CCGGTCGGAA CCGAATTGCT TA - #AGCCAATC10080- GCAGCGAGCG AAAGCTGCAA TCGTCAGGAA GTGCTGGCTA TTTTAAAGGA CA - #AGGGAACC10140- AAGTGTCTCA ATCCTAACGC GCAAGCCGTG CGTCGTCACA TCAACCGGCT AT - #TTTTTCGG10200- TTAATCTTAG ACGAGGAACA ACGCATTTAC GACGTAGTGT CTACCAATAT TG - #AGTTCGGT10260- GCCTGGCCAG TCCCTACGGC CTACAAAGCC TTTCTTTGGA AATACGCCAA GA - #GACTGAAC10320- TACCACCACT TCAGACTGCG CTGGTGATCA TGTCCCTATT TTACCGTGCG GT - #AGCTCTGG10380- GCACGCTAAG CGCTTTGGTG TGGTACAGCA CTAGCATCCT CGCAGAGATT AA - #CGAAAATT10440- CCTGCTCCTC ATCTTCTGCG GATCACGAAG ACTGCGAGGA ACCGGACGAG AT - #CGTTCGCG10500- AAGAGCAAGA CTATCGGGCT CTGCTGGCCT TTTCCCTAGT GATTTGCGGT AC - #GCTCCTCG10560- TCACTTGTGT GATCTGAGAC GTCATGCTGG TAGCGTTTAT GAGTCGGGCG GT - #GGCCGACA10620- CGCCGCATTT CCTAACCCGC GCAGCATGTT GCGCTTGCTG TTCACGCTCG TC - #CTGCTGGC10680- CCTCCACGGG CAGTCTGTCG GCGCTAGCCG CGACTATGTG CATGTTCGGC TA - #CTGAGCTA10740- CCGAGGCGAC CCCCTGGTCT TCAAGCACAC TTTCTCGGGT GTGCGTCGAC CC - #TTCACCGA10800- GCTAGGCTGG GCTGCGTGTC GCGACTGGGA CAGTATGCAT TGCACACCCT TC - #TGGTCTAC10860- CGATCTGGAG CAGATGACCG ACTCGGTGCG GCGTTACAGC ACGGTGAGCC CC - #GGCAAGGA10920- AGTGACGCTT CAGCTTCACG GGAACCAAAC CGTACAGCCG TCGTTTCTAA GC - #TTTACGTG10980- CCGCCTGCAG CTAGAACCCG TGGTGGAAAA TGTTGGCCTC TACGTGGCCT AC - #GTGGTCAA11040- CGACGGCGAA CGCCCACAAC AGTTTTTTAC ACCGCAGGTA GACGTGGTAC GC - #TTTGCTCT11100- ATATCTAGAA ACACTCTCCC GGATCGTGGA ACCGTTAGAA TCAGGTCGCC TG - #GCAGTGGA11160- ATTTGATACG CCTGACCTAG CTCTGGCGCC CGATTTAGTA AGCAGCCTCT TC - #GTGGCCGG11220- ACACGGCGAG ACCGACTTTT ACATGAACTG GACGCTGCGT CGCAGTCAGA CC - #CACTACCT11280- GGAGGAGATG GCCTTACAGG TGGAGATTCT AAAACCCCGC GGCGTACGTC AC - #CGCGCTAT11340- TATCCACCAT CCGAAGCTAC AGCCGGGCGT TGGCCTGTGG ATAGATTTCT GC - #GTGTACCG11400- CTACAACGCG CGCCTGACCC GCGGCTACGT ACGATACACC CTGTCACCGA AA - #GCGCGCTT11460- GCCCGCAAAA GCAGAGGGTT GGCTGGTGTC ACTAGACAGA TTCATCGTGC AG - #TACCTCAA11520- CACATTGCTG ATTACAATGA TGGCGGCGAT ATGGGCTCGC GTTTTGATAA CC - #TACCTGGT11580- GTCGCGGCGT CGGTAGAGGC TTGCGGAAAC CACGTCCTCG TCACACGTCG TT - #CGCGGACA11640- TAGCAAGAAA TCCACGTCGC CACATCTCGA GAATGCCGGC CTTGCGGGGT CC - #CCTTCGCG11700- CAACATTCCT GGCCCTGGTC GCGTTCGGGT TGCTGCTTCA GATAGACCTC AG - #CGACGCTA11760- CGAATGTGAC CAGCAGCACA AAAGTCCCTA CTAGCACCAG CAACAGAAAT AA - #CGTCGACA11820- ACGCCACGAG TAGCGGACCC ACAACCGGGA TCAACATGAC CACCACCCAC GA - #GTCTTCCG11880- TTCACAACGT GCGCAATAAC GAGATCATGA AAGTGCTGGC TATCCTCTTC TA - #CATCGTGA11940- CAGGCACCTC CATTTTCAGC TTCATAGCGG TACTGATCGC GGTAGTTTAC TC - #CTCGTGTT12000- GCAAGCACCC GGGCCGCTTT CGTTTCGCCG ACGAAGAGGC CGTCAACCTG TT - #GGACGACA12060- CGGACGACAG TGGCGGCAGC AGCCCGTTTG GCAGCGGTTC CCGACGAGGT TC - #TCAGATCC12120- CCGCCGGATT TTGTTCCTCG AGCCCTTATC AGCGGTTGGA AACTCGGGAC TG - #GGACGAGG12180- AGGAGGAGGC GTCCGCGGCC CGCGAGCGCA TGAAACATGA TCCTGAGAAC GT - #CATCTATT12240- TCAGAAAGGA TGGCAACTTG GACACGTCGT TCGTGAATCC CAATTATGGG AG - #AGGCTCGC12300- CTTTGACCAT CGAATCTCAC CTCTCGGACA ATGAGGAGGA CCCCATCAGG TA - #CTACGTTT12360- CGGTGTACGA TGAACTGACC GCCTCGGAAA TGGAAGAACC TTCGAACAGC AC - #CAGCTGGC12420- AGATTCCCAA ACTAATGAAA GTTGCCATGC AACCCGTCTC GCTCAGAGAT CC - #CGAGTACG12480- ACTAGGCTTT TTTTTTTGTC TTTCGGTTCC AACTCTTTCC CCGCCCCATC AC - #CTCGCCTG12540- TACTATGTGT ATGATGTCTC ATAATAAAGC TTTCTTTCTC AGTCTGCAAC AT - #GCAGCTGT12600- GTCGGGTGTG GCTGTCTGTT TGTCTGTGCG CCGTGGTGCT GGGTCAGTGC CA - #GCGGGAAA12660- CCGCGGAAAA AAACGATTAT TACCGAGTAC CGCATTACTG GGACGCGTGC TC - #TCGCGCGC12720- TGCCCGACCA AACCCGTTAC AAGTATGTGG AACAGCTCGT GGACCTCACG TT - #GAACTACC12780- ACTACGATGC GAGCCACGGC TTGGACAACT TTGACGTGCT CAAGAGGTGA GG - #GTACGCGC12840- TAAAGGTGCA TGACAACGGG AAGGTAAGGG CGAACGGGTA ACGGCTAAGT AA - #CCGCATGG12900- GGTATGAAAT GACGTTTGGA ACCTGTGCTT GCAGAATCAA CGTGACCGAG GT - #GTCGTTGC12960- TCATCAGCGA CTTTAGACGT CAGAACCGTC GCGGCGGCAC CAACAAAAGG AC - #CACGTTCA13020- ACGCCGCCGG TTCGCTGGCG CCACACGCCC GGAGCCTCGA GTTCAGCGTG CG - #GCTCTTTG13080- CCAACTAGCC TGCGTCACGG GAAATAATAT GCTGCGGCTT CTGCTTCGTC AC - #CACTTTCA13140- CTGCCTGCTT CTGTGCGCGG TTTGGGCAAC GCCCTGTCTG GCGTCTCCGT GG - #TCGACGCT13200- AACGGCAAAC CAGAATCCGT CCCCGCCATG GTCTAAACTG ACGTATTCCA AA - #CCGCATGA13260- CGCGGCGACG TTTTACTGTC CTTTTCTCTA TCCCTCGCCC CCACGGTCCC CC - #TTGCAATT13320- CTCGGGGTTC CAGCAGGTAT CAACGGGTCC CGAGTGTCGC AACGAGACCC TG - #TATCTGCT13380- GTACAACCGG GAAGGCCAGA CCTTGGTGGA GAGAAGCTCC ACCTGGGTGA AA - #AAGGTGAT13440- CTGGTATCTG AGCGGTCGCA ACCAGACCAT CCTCCAACGG ATGCCCCAAA CG - #GCTTCGAA13500- ACCGAGCGAC GGAAACGTGC AGATCAGCGT GGAAGACGCC AAGATTTTTG GA - #GCGCACAT13560- GGTGCCCAAG CAGACCAAGC TGCTACGCTT CGTCGTCAAC GATGGCACGC GT - #TATCAGAT13620- GTGTGTGATG AAGCTGGAGA GCTGGGCCCA CGTCTTCCGG GACTACAGCG TG - #TCTTTTCA13680- GGTGCGATTG ACGTTCACCG AGGCCAATAA CCAGACTTAC ACCTTCTGTA CC - #CATCCCAA13740- TCTCATCATT TGAGCCCGTC GCGCGCGCAG GGAATTTTGA AAACCGCGCG TC - #ATGAGTCC13800- CAAAGACCTG ACGCCGTTCT TGACGACGTT GTGGCTGCTA TTGGGTCACA GC - #CGCGTGCC13860- GCGGGTGCGC GCAGAAGAAT GTTGCGAATT CATAAACGTC AACCACCCGC CG - #GAACGCTG13920- TTACGATTTC AAAATGTGCA ATCGCTTCAC CGTCGCGTAC GTATTTTCAT GA - #TTGTCTGC13980- GTTCTGTGGT GCGTCTGGAT TTGTCTCTCG ACGTTTCTGA TAGCCATGTT CC - #ATCGACGA14040- TCCTCGGGAA TGCCAGAGTA GATTTTCATG AATCCACAGG CTGCGGTGTC CG - #GACGGCGA14100- AGTCTGCTAC AGTCCCGAGA AAACGGCTGA GATTCGCGGG ATCGTCACCA CC - #ATGACCCA14160- TTCATTGACA CGCCAGGTCG TACACAACAA ACTGACGAGC TGCAACTACA AT - #CCGTAAGT14220- CTCTTCCTCG AGGGCCTTAC AGCCTATGGG AGAGTAAGAC AGAGAGGGAC AA - #AACATCAT14280- TAAAAAAAAA AGTCTAATTT CACGTTTTGT ACCCCCCTTC CCCTCCGTGT TG - #TAGCCCAT14340- CGGCCGCGGC GATCTCCTAG TAACACTCGT CCGACACTTC CACCATCTCC AG - #CTCGGCCG14400- GCGGTTCGGC ATCCTCTACC AGCGGCGTCG TCTCATCTTT GCCGCAGCAG CG - #GACGCACA14460- CCTTCTCCAG GCAGAACGCC ACCAGCTGCC GCCGAACGTA CCACAGGTAC AC - #GTGCAGAC14520- CTGCGAACAG GACTACGGAG GTCATGACCA CCACGACGCA CACGGGAATC CA - #GGGATCGA14580- GATTGTTGCT GGAACTCATG GCTATCGCCA CCGACGTGCC CGCGTCTGTC TC - #ACCGCCGC14640- TCGCCCGATG TCGCGCGGCT TGTTATACGC TAGCCCGTCG CCGCCTCGGG GC - #ACGGTGCC14700- CTCCTACCCA CGTAACTTCC TCCGTGACTT AAAGTCGCGT GTGGTAGATC TC - #CTGCTCCG14760- TGGACGAACC GTCCGGCAGG ATAGCGGTTA AGGATTCGGT GCTAAGGCCG TG - #TCGCCAAC14820- GTCGAATGCT ACGTTGCAAC AGCTTCGACG GACGGCCATC CCCTCTCTCA TC - #GCAATAAT14880- AAAACACCAG CAGCGCGCAC GACGCGATCA CGGTGACACC CATGATTAGA CC - #CACGCAGA14940- TAGCCAGCCC CGCTAGCGTA TCTAGCGCCA TCCCGTTCGC TCCCGTTGTC TC - #CTGAGCGA15000- AGCAACTTCT CGGTCCCCGT TTTCAACAGT TTTTGTTTCC TTCTCCGCGA CT - #AGATGTTA15060- ACGCCCGCGG TCTTTCCGGC CGTGCTCTAC CTCCTGGCGC TTGTCGTCTG GG - #TTGAGATG15120- TTCTGCCTCG TCGCCGTAGC CGTCGTCGAG CGCGAGATCG CCTGGGCGCT GC - #TGCTGCGG15180- ATGCTGGTCG TTGGCCTGAT GGTGGAAGTC GGCGCCGCCG CCGCTTGGAC CT - #TCGTGCGT15240- TGTCTTGCCT ATCAGCGCTC CTTCCCCGTG CTTACGGCCT TCCCCTGAAA CC - #CACGTTAA15300- CCGACCGTCC CAAAAACGCC GGTGTTAACA CAGGAAAAAA AGAAACCACG CA - #GGAACCGC15360- GCAGGAACCA CGCGGAACAT GGGACACTAT CTGGAAATCC TGTTCAACGT CA - #TCGTCTTC15420- ACTCTGCTGC TCGGCGTCAT GGTCAGTATC GTCGCTTGGT ACTTCACGTG AA - #CCACCGTC15480- GTCCCGGTTT AAAAACCATC ATCGACGGCC GTTATAAAGC CACCCGGACA CG - #CGCCGCGG15540- CACTTGCCTA CGGCGCTGCT TCAGGGAAAC TCCTCTTCCT TCTGCTCTTC CT - #CCTTCACC15600- GCAGGGATCG TTTCCCTCGA CCAGGGACTC GCCGAAGCAA CCGCCGGAGC AA - #CCTGGAGG15660- AGTCGCGGCA TGACGGCGCC CAAGTGTGTC ACCACCAGTA CTTATCTGGT CA - #AGACCAAG15720- GAACAGCCCT GGTGGCCCGA CAACGCCATC AGGAGATGGT GGATCAGTGT TG - #CTATCGTC15780- ATCTTCATCG GAGTCTGTCT GGTGGCCCTG ATGTACTTTA CGCAGCAGCA GG - #CACGCAGC15840- GGGAGCAGCA GCGGCTAGAC AAGTCTCTGG CGGCTACAGC TCCAAGCGCC GT - #AGCCGGGC15900- CGCCTGCCGA TCGCGACGTC GTGGACCATC GAACAGAGAC TCACGCGTAC GA - #GACCCCGA15960- GGTACGCCAC GCGGTGCCTA ACGCGGTATA CCACACCCGT ACGGTCTGCA GT - #GCGGCGTA16020- CAACGTGTGG AAAACGCGTT GCGTCGCAGA GTCCGCCACG TTCCTGTCTT GT - #CGCTCCCC16080- AATCGTCTCC CGCACACCCC CCGCGACACC CAGAGGGCGG GTGAGCCAAG TA - #TTCTTAAG16140- GCCGTTCTTT GTTCCATAGC CCATAAATTG TTGATTCCGG AGCTCGTTGG CG - #CGGAAATA16200- GCCGGATAAG GGGAGCAACA ACCGTTGGCG AAAGCCGTCC CGCTCATTCA GT - #CCGGGTTT16260- CGCGTCCAGT CGGACGTGTG ACCGTTGGGC AACGGAACGG CGTTTCACTG CC - #AAAATCGT16320- ATCGGGTAGT GTACGAGACG TCGGCGGTGC AGAATGCGAC TCGCGGCGTA GC - #TCGCCGTC16380- GCTATGCGGC TCGTCGCCGT GTGGCGCGGC CTGGCCGGCT GTCTGCGTCC AG - #ATCTGTTG16440- GCCTTTTGGT TCCTCTGGCT GCTGCTGCGT GTGTGCTTTG GTAGACGCGG TG - #GCAGTTTG16500- CGGTCTGCGG TAAGTGAGGA TGTCGCCGAG CAAACGCACT TGCGGCGCGT GG - #GCGGCACG16560- CGTGTCATTG TAGGTTCGTT GCCAGATGGC AAGTGCTGTC AACAGCAGGC GT - #TGTGGGCG16620- GTCGGTGTAT TTTTGTGGGT TGCGGTGAGA GTCGGCACTC GGTGTTTTGT GA - #GTCATCTC16680- AACTATCTGT GTTGCTTTGA GCAGCGTCCA GAACAGCGAC GCGACTTTGG GG - #ATGGCCTC16740- GTGCTCACCT CCGCGGAGAG CGCCGCCGGA CCTGCTCGTC AGCAGCGAGC TA - #CGCAGACG16800- GAATATCTGG AGGAGAGTTA CGTGTGTCAC AGGAGAGCGC GGGTCTCCGG CG - #GTAACGAC16860- GGCGGTGTCG TCGACACGTG TGCGGCCTGT TGTGCTCTGC GGAAAAGTGC CG - #GTCTCGGA16920- GACCGTGGAC GAAAAAGAGA ACGCAGCAGC TACCGCTGGC GGCGGCGGCG TT - #AATGCAGC16980- CGTTGATGTT CGACGTTGTG AGCACTCGGA AACAGCGGTG AGGCAGAAGG TC - #GATTCTCC17040- AGGGAACGAC AGTCGATGCG TGGTAGCCGC AGCAGGTGAG GTTGGGGCGG AC - #AACGTGTT17100- GCGGATTGTG GCGAGAACGT CGTCCTCCCC TTCTTCACCG CCCCACCCAC CC - #TCGGTTGG17160- TGTTTCTTTT TTCTTGTGTC CTGCAGATAG TTCCACGGAC AGCGACGGCA AG - #TCCATAAT17220- CAGCGGTGTG CAAGTGGTGG AACACGACGA AGATATCATC GCGCCGCAGA GT - #TTGTGGTG17280- CACGGCGTTC AAGGAAGCCC TCTGGGATGT GGCTCTGTTG GAAGTGCCGC GT - #TGGGCGTG17340- GCAGGGCTGG AAGAGGTGGC GCAACAGCGA GGCCGGGCGT CGATGGAGTG CT - #GGGTCTGC17400- GTCGGCTTCC AGCTTGTCTG ACTTGGCGGG CGAGGCCGTT GGAGAATTGG TG - #GGATCGGT17460- CGTCGCGTAC GTGATCCTTG AACGTCTGTG GTTGGCAGCC AGAGGTTGGG TG - #TGCGAAAC17520- AGGTGTGGAA GCCGAGGAGG CCATGTCGCG GCGGCGACAG CGCATGCTGT GG - #CGTATTGT17580- TCTCTCGTGG AGGCGACGGC GAATGCAGCA GACGGTGTTC GATGGAGATG GC - #GTGCGGGG17640- AAGAAAGCGC CGTGTTGTGA GCAGACGACG TAGGATGCGG GACGTCGGAG CA - #CATGGGCC17700- ATGTGTGGTG GCAGATGGCG GTGTCCGCTG GTGTCTGCTG CGGCAGTGCA TA - #GACGAAGC17760- AACATGTCGC TGTGAAGAGA TAGAGTGTGA GCATAGCTGC ATGCAGCGTT GC - #GTGTATAA17820- GCGGGGGGGA TTAAGACGTT AATAAAGAAT AGCGGCGGTT CTGATAGGGC GA - #CCGCTGAA17880- GTGAGCTGCG TGTGCGTGTG GTTTGTGGAG TCCCCGCCGC CCCCGGTCCC GT - #GTCCGCCG17940- GCAAAGCCCC CCGGNTCCGC ACACTCCTGG CCGCGCAACC CTCGTCGCTG CA - #AAAGCCCC18000- CCGTCCCCGC ACACCCCCGC GACCGCCGGT CCCGCGAGTC CCCGTCCCCG CC - #GCAAAAGG18060- CCCCCGTCCT CGCCGCAAAC ACCCCCGTCA CCCCCGTCCC TCAGNCCGGG TC - #CGCGAGTC18120- CCCGTTCCCA GCGTAATCCC CGTACCCGCA ACGNCCCGGN CCCACCGTCG TC - #CCGCACAC18180- CCCCCGTCCC CCAGCCCGGT GCCCAGCGTG CGAAAAAAGC TCCGTCCCTC AC - #ACCCGCAG18240- AAAGATCCCT CAGCGCGGTG AAACCCCGTC CCCAGCGCCG TGCCGCTGAC AA - #AGACCATG18300# 183 - #18- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 257 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.01- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..257#/label= UL133OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Met Gly Cys Asp Val His Asp Pro - # Ser Trp Gln Cys Gln Trp GlyVal# 15- Pro Thr Ile Ile Val Ala Trp Ile - # Thr Cys Ala Ala Leu Gly IleTrp# 30- Cys Leu Ala Gly Ser Ser Ala Asp - # Val Ser Ser Gly Pro Gly IleAla# 45- Ala Val Val Gly Cys Ser Val Phe - # Met Ile Phe Leu Cys Ala TyrLeu# 60- Ile Arg Tyr Arg Glu Phe Phe Lys - # Asp Ser Val Ile Asp Leu LeuThr# 80- Cys Arg Trp Val Arg Tyr Cys Ser - # Cys Ser Cys Lys Cys Ser CysLys# 95- Cys Ile Ser Gly Pro Cys Ser Arg - # Cys Cys Ser Ala Cys Tyr LysGlu# 110- Thr Met Ile Tyr Asp Met Val Gln - # Tyr Gly His Arg Arg Arg ProGly# 125- His Gly Asp Asp Pro Asp Arg Val - # Ile Cys Glu Ile Val Glu SerPro# 140- Pro Val Ser Ala Pro Thr Val Ser - # Val Pro Pro Pro Ser Glu GluSer# 160- His Gln Pro Val Ile Pro Pro Gln - # Pro Pro Ala Pro Thr Ser GluPro# 175- Lys Pro Lys Lys Gly Arg Ala Lys - # Asp Lys Pro Lys Gly Arg ProLys# 190- Asp Lys Pro Pro Cys Glu Pro Thr - # Val Ser Ser Gln Pro Pro SerGln# 205- Pro Thr Ala Met Pro Gly Gly Pro - # Pro Asp Ala Pro Pro Pro AlaMet# 220- Pro Gln Met Pro Pro Gly Val Ala - # Glu Ala Val Gln Ala Ala ValGln# 240- Ala Ala Val Ala Ala Ala Leu Gln - # Gln Gln Gln Gln His Gln ThrGly# 255- Thr- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 175 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.02- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..175#/label= UL134OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Met Ala Arg Thr Arg Glu Ala Ser - # Pro Val Pro Pro Arg Ser ProMet# 15- Pro Ser His Ile His Thr Met Ile - # Phe Ser Pro Ala Trp Asn LeuLys# 30- Leu Arg Val Gly Lys Gly Arg Cys - # Thr Asp Ile Tyr Ala Leu AspPhe# 45- Trp Lys Arg His Phe Leu Ala Arg - # Asn Val Phe Ile Val Gln ThrLeu# 60- Arg Lys Glu Met Cys Ala Lys Ser - # Glu Asn Ser Leu Ser His ArgGly# 80- Arg Val Thr Phe Arg Ser Asp Ala - # Ala Ala Val Val Val Glu ProArg# 95- Pro Arg Pro Pro Ala Arg Gln Leu - # Val Pro Pro Arg Pro Arg ArgVal# 110- Ala Ser Ala Ala Trp Arg Gly Glu - # Ala Arg Arg Ala Asp Arg ArgAla# 125- Leu Pro Ser Ala Ala Thr Val Val - # Val Asn Ser Pro Ser Val ArgThr# 140- Glu Val Cys Leu Ser Val Tyr Pro - # Ser Val Tyr Leu Ser Pro TyrLeu# 160- Ser Ser Val Trp Val Pro Met Ser - # Val Leu Ala Ala Ala Val Gly# 175- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 328 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.03- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..328#/label= UL135OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Met Ser Val His Arg Pro Phe Pro - # Thr Arg Ser Leu Arg Phe GlnAla# 15- Gly Glu Lys Ile Met Val Trp Ile - # Trp Leu Gly Ile Gly Leu LeuGly# 30- Gly Thr Gly Leu Ala Ser Leu Val - # Leu Ala Ile Ser Leu Phe ThrGln# 45- Arg Arg Gly Arg Lys Arg Ser Asp - # Glu Thr Ser Ser Arg Gly ArgLeu# 60- Pro Gly Ala Ala Ser Asp Lys Arg - # Gly Ala Cys Ala Cys Cys TyrArg# 80- Asn Pro Lys Glu Asp Val Val Glu - # Pro Leu Asp Leu Glu Leu GlyLeu# 95- Met Arg Val Asp Thr His Pro Pro - # Thr Pro Gln Val Pro Arg CysThr# 110- Ser Leu Tyr Ile Gly Glu Asp Gly - # Leu Pro Ile Asp Lys Pro GluPhe# 125- Pro Pro Ala Arg Phe Glu Ile Pro - # Asp Val Ser Thr Pro Gly ThrPro# 140- Thr Ser Ile Gly Arg Ser Pro Ser - # His Cys Ser Ser Ser Ser SerLeu# 160- Ser Ser Ser Thr Ser Val Asp Thr - # Val Leu Tyr Gln Pro Pro ProSer# 175- Trp Lys Pro Pro Pro Pro Pro Gly - # Arg Lys Lys Arg Pro Pro ThrPro# 190- Pro Val Arg Ala Pro Thr Thr Arg - # Leu Ser Ser His Arg Pro ProThr# 205- Pro Ile Pro Ala Pro Arg Lys Asn - # Leu Ser Thr Pro Pro Thr LysLys# 220- Thr Pro Pro Pro Thr Lys Pro Lys - # Pro Val Gly Trp Thr Pro ProVal# 240- Thr Pro Arg Pro Phe Pro Lys Thr - # Pro Thr Pro Gln Lys Pro ProArg# 255- Asn Pro Arg Leu Pro Arg Thr Val - # Gly Leu Glu Asn Leu Ser LysVal# 270- Gly Leu Ser Cys Pro Cys Pro Arg - # Pro Arg Thr Pro Thr Glu ProThr# 285- Thr Leu Pro Ile Val Ser Val Ser - # Glu Leu Ala Pro Pro Pro ArgTrp# 300- Ser Asp Ile Glu Glu Leu Leu Glu - # Gln Ala Val Gln Ser Val MetLys# 320- Asp Ala Glu Ser Met Gln Met Thr# 325- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 240 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.04- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..240#/label= UL136OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Met Ser Val Lys Gly Val Glu Met - # Pro Glu Met Thr Trp Asp LeuAsp# 15- Val Arg Asn Lys Trp Arg Arg Arg - # Lys Ala Leu Ser Arg Ile HisArg# 30- Phe Trp Glu Cys Arg Leu Arg Val - # Trp Trp Leu Ser Asp Ala GlyVal# 45- Arg Glu Thr Asp Pro Pro Arg Pro - # Arg Arg Arg Pro Thr Trp MetThr# 60- Ala Val Phe His Val Ile Cys Ala - # Val Leu Leu Thr Leu Met IleMet# 80- Ala Ile Gly Ala Leu Ile Ala Tyr - # Leu Arg Tyr Tyr His Gln AspSer# 95- Trp Arg Asp Met Leu His Asp Leu - # Phe Cys Gly Cys His Tyr ProGlu# 110- Lys Cys Arg Arg His His Glu Arg - # Gln Arg Arg Arg Arg Gln AlaMet# 125- Asp Val Pro Asp Pro Glu Leu Gly - # Asp Pro Ala Arg Arg Pro LeuAsn# 140- Gly Ala Met Tyr Tyr Gly Ser Gly - # Cys Arg Phe Asp Thr Val GluMet# 160- Val Asp Glu Thr Arg Pro Ala Pro - # Pro Ala Leu Ser Ser Pro GluThr# 175- Gly Asp Asp Ser Asn Asp Asp Ala - # Val Ala Gly Gly Gly Ala GlyGly# 190- Val Thr Ser Pro Ala Thr Arg Thr - # Thr Ser Pro Asn Ala Leu LeuPro# 205- Glu Trp Met Asp Ala Val His Val - # Ala Val Gln Ala Ala Val GlnAla# 220- Thr Val Gln Val Ser Gly Pro Arg - # Glu Asn Ala Val Ser Pro AlaThr# 240- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 96 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.05- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..96#/label= UL137OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Met Ala Thr Ile Ser Thr Ser Ile - # Thr Pro Met Met Gly Asn ProThr# 15- Phe Ser Gly Arg Ser Ser Met Val - # Thr Val Leu Cys Pro Asp LeuArg# 30- Pro Ser Leu Ser Leu Leu Tyr Ser - # Thr Arg Ala Gly Thr Ala ProSer# 45- Thr Leu Leu Arg Ser Gly Arg Tyr - # Gly Val Leu Pro Arg Ala ThrTyr# 60- Leu His Gly Arg Leu Asn Gly Gly - # Leu Asp Arg His Met His ArgIle# 80- His Pro Phe Trp Gln Gln Cys Val - # Arg Arg Arg Arg Thr Ser ArgGly# 95- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 169 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.06- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..169#/label= UL138OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Met Asp Asp Leu Pro Leu Asn Val - # Gly Leu Pro Ile Ile Gly ValMet# 15- Leu Val Leu Ile Val Ala Ile Leu - # Cys Tyr Leu Ala Tyr His TrpHis# 30- Asp Thr Phe Lys Leu Val Arg Met - # Phe Leu Ser Tyr Arg Trp LeuIle# 45- Arg Cys Cys Glu Leu Tyr Gly Glu - # Tyr Glu Arg Arg Phe Ala AspLeu# 60- Ser Ser Leu Gly Leu Gly Ala Val - # Arg Arg Glu Ser Asp Arg ArgTyr# 80- Arg Phe Ser Glu Arg Pro Asp Glu - # Ile Leu Val Arg Trp Glu GluVal# 95- Ser Ser Gln Cys Ser Tyr Ala Ser - # Ser Arg Ile Thr Asp Arg ArgVal# 110- Gly Ser Ser Ser Ser Ser Ser Val - # His Val Ala Ser Gln Arg AsnSer# 125- Val Pro Pro Pro Asp Met Ala Val - # Thr Ala Pro Leu Thr Asp ValAsp# 140- Leu Leu Lys Pro Val Thr Gly Ser - # Ala Thr Gln Phe Thr Thr ValAla# 160- Met Val His Tyr His Gln Glu Tyr - # Thr# 165- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 135 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.07- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..135#/label= UL139OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- Met Leu Trp Ile Leu Val Leu Phe - # Ala Leu Ala Ala Ser Ala SerGlu# 15- Thr Thr Thr Gly Thr Ser Ser Asn - # Ser Ser Gln Ser Thr Ser AlaThr# 30- Ala Asn Thr Thr Val Ser Thr Cys - # Ile Asn Ala Ser Asn Gly SerSer# 45- Trp Thr Val Pro Gln Leu Ala Leu - # Leu Ala Ala Ser Gly Trp ThrLeu# 60- Ser Gly Leu Leu Leu Leu Phe Thr - # Cys Cys Phe Cys Cys Phe TrpLeu# 80- Val Arg Lys Ile Cys Ser Cys Cys - # Gly Asn Ser Ser Glu Ser GluSer# 95- Lys Thr Thr His Ala Tyr Thr Asn - # Ala Ala Phe Thr Ser Ser AspAla# 110- Thr Leu Pro Met Gly Thr Thr Gly - # Ser Tyr Thr Pro Pro Gln AspGly# 125- Ser Phe Pro Pro Pro Pro Arg# 135- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 114 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.08- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..114#/label= UL140OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:- Met Thr Pro Ala Gln Thr Asn Ala - # Thr Thr Thr Val His Pro HisAsp# 15- Ala Lys Asn Gly Ser Gly Gly Ser - # Ala Leu Pro Thr Leu Val ValPhe# 30- Gly Phe Ile Val Thr Leu Leu Phe - # Phe Leu Phe Met Leu Tyr PheTrp# 45- Asn Asn Asp Val Phe Arg Lys Leu - # Leu Arg Ala Leu Gly Ser SerAla# 60- Val Ala Thr Ala Ser Thr Arg Gly - # Lys Thr Arg Ser Ser Thr ValVal# 80- His His Val Val Pro Arg Ala Thr - # Thr Arg Val Val Leu Thr AlaCys# 95- His Arg Thr Phe Phe Tyr His Pro - # Arg Pro Met Ala Val Leu ThrThr# 110- Arg His- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 425 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.09- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..425#/label= UL141OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- Met Arg Gln Val Ala Tyr Arg Arg - # Arg Arg Glu Ser Ser Cys AlaVal# 15- Leu Val His His Val Gly Arg Asp - # Gly Asp Gly Glu Gly Glu AlaAla# 30- Lys Lys Thr Cys Lys Lys Thr Gly - # Arg Ser Val Ala Gly Ile ProGly# 45- Glu Lys Leu Arg Arg Thr Val Val - # Thr Thr Thr Pro Ala Arg ArgLeu# 60- Ser Gly Arg His Thr Glu Gln Glu - # Gln Ala Gly Met Arg Leu CysGlu# 80- Lys Gly Lys Lys Arg Ile Ile Met - # Cys Arg Arg Glu Ser Leu ArgThr# 95- Leu Pro Trp Leu Phe Trp Val Leu - # Leu Ser Cys Pro Arg Leu LeuGlu# 110- Tyr Ser Ser Ser Ser Phe Pro Phe - # Ala Thr Ala Asp Ile Ala GluLys# 125- Met Trp Ala Glu Asn Tyr Glu Thr - # Thr Ser Pro Ala Pro Val LeuVal# 140- Ala Glu Gly Glu Gln Val Thr Ile - # Pro Cys Thr Val Met Thr HisSer# 160- Trp Pro Met Val Ser Ile Arg Ala - # Arg Phe Cys Arg Ser His AspGly# 175- Ser Asp Glu Leu Ile Leu Asp Ala - # Val Lys Gly His Arg Leu MetAsn# 190- Gly Leu Gln Tyr Arg Leu Pro Tyr - # Ala Thr Trp Asn Phe Ser GlnLeu# 205- His Leu Gly Gln Ile Phe Ser Leu - # Thr Phe Asn Val Ser Met AspThr# 220- Ala Gly Met Tyr Glu Cys Val Leu - # Arg Asn Tyr Ser His Gly LeuIle# 240- Met Gln Arg Phe Val Ile Leu Thr - # Gln Leu Glu Thr Leu Ser ArgPro# 255- Asp Glu Pro Cys Cys Thr Pro Ala - # Leu Gly Arg Tyr Ser Leu GlyAsp# 270- Gln Ile Trp Ser Pro Thr Pro Trp - # Arg Leu Arg Asn His Asp CysGly# 285- Thr Tyr Arg Gly Phe Gln Arg Asn - # Tyr Phe Tyr Ile Gly Arg AlaAsp# 300- Ala Glu Asp Cys Trp Lys Pro Ala - # Cys Pro Asp Glu Glu Pro AspArg# 320- Cys Trp Thr Val Ile Gln Arg Tyr - # Arg Leu Pro Gly Asp Cys TyrArg# 335- Ser Gln Pro His Pro Pro Lys Phe - # Leu Pro Val Thr Pro Ala ProPro# 350- Ala Asp Ile Asp Thr Gly Met Ser - # Pro Trp Ala Thr Arg Gly IleAla# 365- Ala Phe Leu Gly Phe Trp Ser Ile - # Phe Thr Val Cys Phe Leu CysTyr# 380- Leu Cys Tyr Leu Gln Cys Cys Gly - # Arg Trp Cys Pro Thr Pro GlyArg# 400- Gly Arg Arg Gly Gly Glu Gly Tyr - # Arg Arg Leu Pro Thr Tyr AspSer# 415- Tyr Pro Gly Val Arg Lys Met Lys - # Arg# 425- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 306 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.10- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..306#/label= UL142OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:- Met Arg Ile Glu Trp Val Trp Trp - # Leu Phe Gly Tyr Phe Val SerSer# 15- Val Gly Ser Glu Arg Ser Leu Ser - # Tyr Arg Tyr His Leu Glu SerAsn# 30- Ser Ser Thr Asn Val Val Cys Asn - # Gly Asn Ile Ser Val Phe ValAsn# 45- Gly Thr Leu Gly Val Arg Tyr Asn - # Ile Thr Val Gly Ile Ser SerSer# 60- Leu Leu Ile Gly His Leu Thr Ile - # Gln Val Leu Glu Ser Trp PheThr# 80- Pro Trp Val Gln Asn Lys Ser Tyr - # Asn Lys Gln Pro Leu Gly AspThr# 95- Glu Thr Leu Tyr Asn Ile Asp Ser - # Glu Asn Ile His Arg Val SerGln# 110- Tyr Phe His Thr Arg Trp Ile Lys - # Ser Leu Gln Glu Asn His ThrCys# 125- Asp Leu Thr Asn Ser Thr Pro Thr - # Tyr Thr Tyr Gln Val Asn ValAsn# 140- Asn Thr Asn Tyr Leu Thr Leu Thr - # Ser Ser Gly Trp Gln Asp ArgLeu# 160- Asn Tyr Thr Val Ile Asn Ser Thr - # His Phe Asn Leu Thr Glu SerAsn# 175- Ile Thr Ser Ile Gln Lys Tyr Leu - # Asn Thr Thr Cys Ile Glu ArgLeu# 190- Arg Asn Tyr Thr Leu Glu Ser Val - # Tyr Thr Thr Thr Val Pro GlnAsn# 205- Ile Thr Thr Ser Gln His Ala Thr - # Thr Thr Met His Thr Ile ProPro# 220- Asn Thr Ile Thr Ile Gln Asn Thr - # Thr Gln Ser His Thr Val GlnThr# 240- Pro Ser Phe Asn Asp Thr His Asn - # Val Thr Lys His Thr Leu AsnIle# 255- Ser Tyr Val Leu Ser Gln Lys Thr - # Asn Asn Thr Thr Ser Pro TrpIle# 270- Tyr Ala Ile Pro Met Gly Ala Thr - # Ala Thr Ile Gly Ala Gly LeuTyr# 285- Ile Gly Lys His Phe Thr Pro Val - # Lys Phe Val Tyr Glu Val TrpArg# 300- Gly Gln 305- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 92 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.11- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..92#/label= UL143OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- Met Ala Arg Ser Val Lys Thr Ile - # Arg Ile Gln His Ile Tyr SerPro# 15- Arg Ser Ser Asn Thr Leu Gln His - # Met Ser Lys Lys Gln Glu SerIle# 30- Ala Thr Ile Thr Phe Gly Arg Ile - # Thr Cys Cys His Pro Leu AlaSer# 45- Ile Asn Leu Met Phe Asn Gly Ser - # Cys Thr Val Thr Val Lys IleSer# 60- Met Gly Ile Asn Gly Ser Thr Asn - # Val His Gln Leu Val Ile ValLeu# 80- His Leu Gly Asn Arg Cys Gln Pro - # Trp Arg Gln Val# 90- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 176 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.12- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..176#/label= UL144OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:- Met Lys Pro Leu Ile Met Leu Ile - # Cys Phe Ala Val Ile Leu LeuGln# 15- Leu Gly Val Thr Lys Val Cys Gln - # His Asn Glu Val Gln Leu GlyAsn# 30- Glu Cys Cys Pro Pro Cys Gly Ser - # Gly Gln Arg Val Thr Lys ValCys# 45- Thr Asp Tyr Thr Ser Val Thr Cys - # Thr Pro Cys Pro Asn Gly ThrTyr# 60- Val Ser Gly Leu Tyr Asn Cys Thr - # Asp Cys Thr Gln Cys Asn ValThr# 80- Gln Val Met Ile Arg Asn Cys Thr - # Ser Thr Asn Asn Thr Val CysAla# 95- Pro Lys Asn His Thr Tyr Phe Ser - # Thr Pro Gly Val Gln His HisLys# 110- Gln Arg Gln Gln Asn His Thr Ala - # His Ile Thr Val Lys Gln GlyLys# 125- Ser Gly Arg His Thr Leu Ala Trp - # Leu Ser Leu Phe Ile Phe LeuVal# 140- Gly Ile Ile Leu Leu Ile Leu Tyr - # Leu Ile Ala Ala Tyr Arg SerGlu# 160- Arg Cys Gln Gln Cys Cys Ser Ile - # Gly Lys Ile Phe Tyr Arg ThrLeu# 175- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 100 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.13- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..100#/label= UL145OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Met Cys Thr Asp Pro Arg Arg Thr - # Ala Gly Trp Glu Arg Leu ThrHis# 15- His Ala Ser Tyr His Ala Asn Tyr - # Gly Ala Tyr Ala Val Leu MetAla# 30- Thr Ser Gln Arg Lys Ser Leu Val - # Leu His Arg Tyr Ser Ala ValThr# 45- Ala Val Ala Leu Gln Leu Met Pro - # Val Glu Ile Val Arg Lys LeuAsp# 60- Gln Ser Asp Trp Val Arg Gly Ala - # Trp Ile Val Ser Glu Thr PhePro# 80- Thr Ser Asp Pro Lys Gly Val Trp - # Ser Asp Asp Asp Ser Ser MetGly# 95- Gly Ser Asp Asp 100- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 117 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.14- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..117#/label= UL146OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:- Met Arg Leu Ile Phe Gly Ala Leu - # Ile Ile Phe Leu Ala Tyr ValTyr# 15- His Tyr Glu Val Asn Gly Thr Glu - # Leu Arg Cys Arg Cys Leu HisArg# 30- Lys Trp Pro Pro Asn Lys Ile Ile - # Leu Gly Asn Tyr Trp Leu HisArg# 45- Asp Pro Arg Gly Pro Gly Cys Asp - # Lys Asn Glu His Leu Leu TyrPro# 60- Asp Gly Arg Lys Pro Pro Gly Pro - # Gly Val Cys Leu Ser Pro AspHis# 80- Leu Phe Ser Lys Trp Leu Asp Lys - # His Asn Asp Asn Arg Trp TyrAsn# 95- Val Asn Ile Thr Lys Ser Pro Gly - # Pro Arg Arg Ile Asn Ile ThrLeu# 110- Ile Gly Val Arg Gly 115- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 159 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.15- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..159#/label= UL147OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:- Met Val Leu Thr Trp Leu His His - # Pro Val Ser Asn Ser His IleAsn# 15- Leu Leu Ser Val Arg His Leu Ser - # Leu Ile Ala Tyr Met Leu LeuThr# 30- Ile Cys Pro Leu Ala Val His Val - # Leu Glu Leu Glu Asp Tyr AspArg# 45- Arg Cys Arg Cys Asn Asn Gln Ile - # Leu Leu Asn Thr Leu Pro ValGly# 60- Thr Glu Leu Leu Lys Pro Ile Ala - # Ala Ser Glu Ser Cys Asn ArgGln# 80- Glu Val Leu Ala Ile Leu Lys Asp - # Lys Gly Thr Lys Cys Leu AsnPro# 95- Asn Ala Gln Ala Val Arg Arg His - # Ile Asn Arg Leu Phe Phe ArgLeu# 110- Ile Leu Asp Glu Glu Gln Arg Ile - # Tyr Asp Val Val Ser Thr AsnIle# 125- Glu Phe Gly Ala Trp Pro Val Pro - # Thr Ala Tyr Lys Ala Phe LeuTrp# 140- Lys Tyr Ala Lys Arg Leu Asn Tyr - # His His Phe Arg Leu Arg Trp# 155- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 316 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.16- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..316#/label= UL148OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:- Met Leu Arg Leu Leu Phe Thr Leu - # Val Leu Leu Ala Leu His GlyGln# 15- Ser Val Gly Ala Ser Arg Asp Tyr - # Val His Val Arg Leu Leu SerTyr# 30- Arg Gly Asp Pro Leu Val Phe Lys - # His Thr Phe Ser Gly Val ArgArg# 45- Pro Phe Thr Glu Leu Gly Trp Ala - # Ala Cys Arg Asp Trp Asp SerMet# 60- His Cys Thr Pro Phe Trp Ser Thr - # Asp Leu Glu Gln Met Thr AspSer# 80- Val Arg Arg Tyr Ser Thr Val Ser - # Pro Gly Lys Glu Val Thr LeuGln# 95- Leu His Gly Asn Gln Thr Val Gln - # Pro Ser Phe Leu Ser Phe ThrCys# 110- Arg Leu Gln Leu Glu Pro Val Val - # Glu Asn Val Gly Leu Tyr ValAla# 125- Tyr Val Val Asn Asp Gly Glu Arg - # Pro Gln Gln Phe Phe Thr ProGln# 140- Val Asp Val Val Arg Phe Ala Leu - # Tyr Leu Glu Thr Leu Ser ArgIle# 160- Val Glu Pro Leu Glu Ser Gly Arg - # Leu Ala Val Glu Phe Asp ThrPro# 175- Asp Leu Ala Leu Ala Pro Asp Leu - # Val Ser Ser Leu Phe Val AlaGly# 190- His Gly Glu Thr Asp Phe Tyr Met - # Asn Trp Thr Leu Arg Arg SerGln# 205- Thr His Tyr Leu Glu Glu Met Ala - # Leu Gln Val Glu Ile Leu LysPro# 220- Arg Gly Val Arg His Arg Ala Ile - # Ile His His Pro Lys Leu GlnPro# 240- Gly Val Gly Leu Trp Ile Asp Phe - # Cys Val Tyr Arg Tyr Asn AlaArg# 255- Leu Thr Arg Gly Tyr Val Arg Tyr - # Thr Leu Ser Pro Lys Ala ArgLeu# 270- Pro Ala Lys Ala Glu Gly Trp Leu - # Val Ser Leu Asp Arg Phe IleVal# 285- Gln Tyr Leu Asn Thr Leu Leu Ile - # Thr Met Met Ala Ala Ile TrpAla# 300- Arg Val Leu Ile Thr Tyr Leu Val - # Ser Arg Arg Arg# 315- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 214 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.19- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..214#/label= UL130OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:- Met Leu Arg Leu Leu Leu Arg His - # His Phe His Cys Leu Leu LeuCys# 15- Ala Val Trp Ala Thr Pro Cys Leu - # Ala Ser Pro Trp Ser Thr LeuThr# 30- Ala Asn Gln Asn Pro Ser Pro Pro - # Trp Ser Lys Leu Thr Tyr SerLys# 45- Pro His Asp Ala Ala Thr Phe Tyr - # Cys Pro Phe Leu Tyr Pro SerPro# 60- Pro Arg Ser Pro Leu Gln Phe Ser - # Gly Phe Gln Gln Val Ser ThrGly# 80- Pro Glu Cys Arg Asn Glu Thr Leu - # Tyr Leu Leu Tyr Asn Arg GluGly# 95- Gln Thr Leu Val Glu Arg Ser Ser - # Thr Trp Val Lys Lys Val IleTrp# 110- Tyr Leu Ser Gly Arg Asn Gln Thr - # Ile Leu Gln Arg Met Pro GlnThr# 125- Ala Ser Lys Pro Ser Asp Gly Asn - # Val Gln Ile Ser Val Glu AspAla# 140- Lys Ile Phe Gly Ala His Met Val - # Pro Lys Gln Thr Lys Leu LeuArg# 160- Phe Val Val Asn Asp Gly Thr Arg - # Tyr Gln Met Cys Val Met LysLeu# 175- Glu Ser Trp Ala His Val Phe Arg - # Asp Tyr Ser Val Ser Phe GlnVal# 190- Arg Leu Thr Phe Thr Glu Ala Asn - # Asn Gln Thr Tyr Thr Phe CysThr# 205- His Pro Asn Leu Ile Ile 210- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 122 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.20- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..122#/label= UL149OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:- Met Val Asp Gln Cys Cys Tyr Arg - # His Leu His Arg Ser Leu SerGly# 15- Gly Pro Asp Val Leu Tyr Ala Ala - # Ala Gly Thr Gln Arg Glu GlnGln# 30- Arg Leu Asp Lys Ser Leu Ala Ala - # Thr Ala Pro Ser Ala Val AlaGly# 45- Pro Pro Ala Asp Arg Asp Val Val - # Asp His Arg Thr Glu Thr HisAla# 60- Tyr Glu Thr Pro Arg Tyr Ala Thr - # Arg Cys Leu Thr Arg Tyr ThrThr# 80- Pro Val Arg Ser Ala Val Arg Arg - # Thr Thr Cys Gly Lys Arg ValAla# 95- Ser Gln Ser Pro Pro Arg Ser Cys - # Leu Val Ala Pro Gln Ser SerPro# 110- Ala His Pro Pro Arg His Pro Glu - # Gly Gly# 120- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 642 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.21- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..642#/label= UL150OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:- Met Gln Leu Cys Ser His Ser Ile - # Ser Ser Gln Arg His Val AlaSer# 15- Ser Met His Cys Arg Ser Arg His - # Gln Arg Thr Pro Pro Ser AlaThr# 30- Thr His Gly Pro Cys Ala Pro Thr - # Ser Arg Ile Leu Arg Arg LeuLeu# 45- Thr Thr Arg Arg Phe Leu Pro Arg - # Thr Pro Ser Pro Ser Asn ThrVal# 60- Cys Cys Ile Arg Arg Arg Leu His - # Glu Arg Thr Ile Arg His SerMet# 80- Arg Cys Arg Arg Arg Asp Met Ala - # Ser Ser Ala Ser Thr Pro ValSer# 95- His Thr Gln Pro Leu Ala Ala Asn - # His Arg Arg Ser Arg Ile ThrTyr# 110- Ala Thr Thr Asp Pro Thr Asn Ser - # Pro Thr Ala Ser Pro Ala LysSer# 125- Asp Lys Leu Glu Ala Asp Ala Asp - # Pro Ala Leu His Arg Arg ProAla# 140- Ser Leu Leu Arg His Leu Phe Gln - # Pro Cys His Ala Gln Arg GlyThr# 160- Ser Asn Arg Ala Thr Ser Gln Arg - # Ala Ser Leu Asn Ala Val HisHis# 175- Lys Leu Cys Gly Ala Met Ile Ser - # Ser Ser Cys Ser Thr Thr CysThr# 190- Pro Leu Ile Met Asp Leu Pro Ser - # Leu Ser Val Glu Leu Ser AlaGly# 205- His Lys Lys Lys Glu Thr Pro Thr - # Glu Gly Gly Trp Gly Gly GluGlu# 220- Gly Glu Asp Asp Val Leu Ala Thr - # Ile Arg Asn Thr Leu Ser AlaPro# 240- Thr Ser Pro Ala Ala Ala Thr Thr - # His Arg Leu Ser Phe Pro GlyGlu# 255- Ser Thr Phe Cys Leu Thr Ala Val - # Ser Glu Cys Ser Gln Arg ArgThr# 270- Ser Thr Ala Ala Leu Thr Pro Pro - # Pro Pro Ala Val Ala Ala AlaPhe# 285- Ser Phe Ser Ser Thr Val Ser Glu - # Thr Gly Thr Phe Pro Gln SerThr# 300- Thr Gly Arg Thr Arg Val Asp Asp - # Thr Ala Val Val Thr Ala GlyAsp# 320- Pro Arg Ser Pro Val Thr His Val - # Thr Leu Leu Gln Ile Phe ArgLeu# 335- Arg Ser Ser Leu Leu Thr Ser Arg - # Ser Gly Gly Ala Leu Arg GlyGly# 350- Glu His Glu Ala Ile Pro Lys Val - # Ala Ser Leu Phe Trp Thr LeuLeu# 365- Lys Ala Thr Gln Ile Val Glu Met - # Thr His Lys Thr Pro Ser AlaAsp# 380- Ser His Arg Asn Pro Gln Lys Tyr - # Thr Asp Arg Pro Gln Arg LeuLeu# 400- Leu Thr Ala Leu Ala Ile Trp Gln - # Arg Thr Tyr Asn Asp Thr ArgAla# 415- Ala His Ala Pro Gln Val Arg Leu - # Leu Gly Asp Ile Leu Thr TyrArg# 430- Arg Pro Gln Thr Ala Thr Ala Ser - # Thr Lys Ala His Thr Gln GlnGln# 445- Pro Glu Glu Pro Lys Gly Gln Gln - # Ile Trp Thr Gln Thr Ala GlyGln# 460- Ala Ala Pro His Gly Asp Glu Pro - # His Ser Asp Gly Glu Leu ArgArg# 480- Glu Ser His Ser Ala Pro Pro Thr - # Ser Arg Thr Leu Pro Asp ThrIle# 495- Leu Ala Val Lys Arg Arg Ser Val - # Ala Gln Arg Ser His Val ArgLeu# 510- Asp Ala Lys Pro Gly Leu Asn Glu - # Arg Asp Gly Phe Arg Gln ArgLeu# 525- Leu Leu Pro Leu Ser Gly Tyr Phe - # Arg Ala Asn Glu Leu Arg AsnGln# 540- Gln Phe Met Gly Tyr Gly Thr Lys - # Asn Gly Leu Lys Asn Thr TrpLeu# 560- Thr Arg Pro Leu Gly Val Ala Gly - # Gly Val Arg Glu Thr Ile GlyGlu# 575- Arg Gln Asp Arg Asn Val Ala Asp - # Ser Ala Thr Gln Arg Val PheHis# 590- Thr Leu Tyr Ala Ala Leu Gln Thr - # Val Arg Val Trp Tyr Thr AlaLeu# 605- Gly Thr Ala Trp Arg Thr Ser Gly - # Ser Arg Thr Arg Glu Ser LeuPhe# 620- Asp Gly Pro Arg Arg Arg Asp Arg - # Gln Ala Ala Arg Leu Arg ArgLeu# 640- Glu Leu- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 336 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.22- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..336#/label= UL151OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:- Met Val Phe Val Ser Gly Thr Ala - # Leu Gly Thr Gly Phe His ArgAla# 15- Glu Gly Ser Phe Cys Gly Cys Glu - # Gly Arg Ser Phe Phe Arg ThrLeu# 30- Gly Thr Gly Leu Gly Asp Gly Gly - # Cys Ala Gly Arg Arg Trp XaaArg# 45- Xaa Val Ala Gly Thr Gly Ile Thr - # Leu Gly Thr Gly Thr Arg GlyPro# 60- Gly Leu Arg Asp Gly Gly Asp Gly - # Gly Val Cys Gly Glu Asp GlyGly# 80- Leu Leu Arg Arg Gly Arg Gly Leu - # Ala Gly Pro Ala Val Ala GlyVal# 95- Cys Gly Asp Gly Gly Leu Leu Gln - # Arg Arg Gly Leu Arg Gly GlnGlu# 110- Cys Ala Xaa Pro Gly Gly Phe Ala - # Gly Gly His Gly Thr Gly GlyGly# 125- Gly Asp Ser Thr Asn His Thr His - # Thr Gln Leu Thr Ser Ala ValAla# 140- Leu Ser Glu Pro Pro Leu Phe Phe - # Ile Asn Val Leu Ile Pro ProAla# 160- Tyr Thr Arg Asn Ala Ala Cys Ser - # Tyr Ala His Thr Leu Ser LeuHis# 175- Ser Asp Met Leu Leu Arg Leu Cys - # Thr Ala Ala Ala Asp Thr SerGly# 190- His Arg His Leu Pro Pro His Met - # Ala His Val Leu Arg Arg ProAla# 205- Ser Tyr Val Val Cys Ser Gln His - # Gly Ala Phe Phe Pro Ala ArgHis# 220- Leu His Arg Thr Pro Ser Ala Ala - # Phe Ala Val Ala Ser Thr ArgGlu# 240- Gln Tyr Ala Thr Ala Cys Ala Val - # Ala Ala Ala Thr Trp Pro ProArg# 255- Leu Pro His Leu Phe Arg Thr Pro - # Asn Leu Trp Leu Pro Thr ThrAsp# 270- Val Gln Gly Ser Arg Thr Arg Arg - # Pro Ile Pro Pro Ile Leu GlnArg# 285- Pro Arg Pro Pro Ser Gln Thr Ser - # Trp Lys Pro Thr Gln Thr GlnHis# 300- Ser Ile Asp Ala Arg Pro Arg Cys - # Cys Ala Thr Ser Ser Ser ProAla# 320- Thr Pro Asn Ala Ala Leu Pro Thr - # Glu Pro His Pro Arg Gly LeuPro# 335- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 270 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (vii) IMMEDIATE SOURCE: (B) CLONE: tol.23- (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..270#/label= UL132OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:- Met Pro Ala Leu Arg Gly Pro Leu - # Arg Ala Thr Phe Leu Ala LeuVal# 15- Ala Phe Gly Leu Leu Leu Gln Ile - # Asp Leu Ser Asp Ala Thr AsnVal# 30- Thr Ser Ser Thr Lys Val Pro Thr - # Ser Thr Ser Asn Arg Asn AsnVal# 45- Asp Asn Ala Thr Ser Ser Gly Pro - # Thr Thr Gly Ile Asn Met ThrThr# 60- Thr His Glu Ser Ser Val His Asn - # Val Arg Asn Asn Glu Ile MetLys# 80- Val Leu Ala Ile Leu Phe Tyr Ile - # Val Thr Gly Thr Ser Ile PheSer# 95- Phe Ile Ala Val Leu Ile Ala Val - # Val Tyr Ser Ser Cys Cys LysHis# 110- Pro Gly Arg Phe Arg Phe Ala Asp - # Glu Glu Ala Val Asn Leu LeuAsp# 125- Asp Thr Asp Asp Ser Gly Gly Ser - # Ser Pro Phe Gly Ser Gly SerArg# 140- Arg Gly Ser Gln Ile Pro Ala Gly - # Phe Cys Ser Ser Ser Pro TyrGln# 160- Arg Leu Glu Thr Arg Asp Trp Asp - # Glu Glu Glu Glu Ala Ser AlaAla# 175- Arg Glu Arg Met Lys His Asp Pro - # Glu Asn Val Ile Tyr Phe ArgLys# 190- Asp Gly Asn Leu Asp Thr Ser Phe - # Val Asn Pro Asn Tyr Gly ArgGly# 205- Ser Pro Leu Thr Ile Glu Ser His - # Leu Ser Asp Asn Glu Glu AspPro# 220- Ile Arg Tyr Tyr Val Ser Val Tyr - # Asp Glu Leu Thr Ala Ser GluMet# 240- Glu Glu Pro Ser Asn Ser Thr Ser - # Trp Gln Ile Pro Lys Leu MetLys# 255- Val Ala Met Gln Pro Val Ser Leu - # Arg Asp Pro Glu Tyr Asp# 270__________________________________________________________________________

Number	Name	Date	Kind
5075213	Pande et al.	Dec 1991
5194256	Rasmussen et al.	Mar 1993

Human cytomegalovirus DNA sequences

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US Referenced Citations (2)

Non-Patent Literature Citations (23)

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Entry
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Zaia et al., Comparative Analysis of Human Cytomegalovirus alpha-Sequence in Multiple Clinical Isolates by Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assays, Journal of Clinical Microbiology, vol. 28, No. 12, pp. 2602-2 Dec. 1990.
Pande et al., Human Cytomegalovirus Strain Towne pp65 Gene: Nucleotide Sequence and Expression in Escherichia coli, Virology, vol. 182, pp. 220-228, see Abstract, Jan. 1991.
Pande et al., Human Cytomegalovirus Strain Towne pp28 Gene: Sequence Comparison to pp28 of HCMV AD169 and Stable Expression in Chinese Hamster Ovary Cells, Virology, vol. 184, pp. 762-767, see Abtract and Fig. 3, Jul. 1991.
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