Patent Grant
7094953
The present invention provides plant retroelements and methods related to plant retroelements.
Eukaryotic retrotransposons are divided into two distinct classes of elements based on their structures: the long terminal repeat (LTR) retrotransposons and the LINE-like or non-LTR elements. See, Doolittle et al. (1989) Quart. Rev. Biol. 64:1–30; and Xiong and Eickbush (1990) EMBO J. 9:3353–3362. These element classes are related by the fact that each must undergo reverse transcription of an RNA intermediate to replicate, and each generally encodes its own reverse transcriptase. The LTR retrotransposons replicate by a mechanism resembling that of the retroviruses. See, Boeke and Sandmeyer (1991) Yeast transposable elements, in The Molecular and Cellular Biology of the Yeast Saccharomyces, ed. Broach, Jones, and Pringle, pp. 193–261 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). LTR retrotransposons typically use a specific tRNA to prime reverse transcription, and a linear cDNA is synthesized through a series of template transfers that require redundant LTR sequences at each end of the mRNA. This process occurs within a virus-like particle formed from proteins encoded by the retrotransposon mRNA. After reverse transcription, an integration complex is organized that directs the resulting cDNA to a new site in the genome of the host cell.
Phylogenetic analyses based on reverse transcriptase amino acid sequences have separated the LTR retrotransposons into two families: the Ty3/gypsy retrotransposons (Metaviridae), and the Ty1/copia elements (Pseudoviridae). See, Boeke et al. (1998) Metaviridae, in Virus Taxonomy: ICTV VIIth Report, ed. Murphy (Springer-Verlag, NY); and Boeke et al. (1998) Pseudoviridae, in Virus Taxonomy: ICTV VIIth Report, ed. Murphy (Springer Verlag, NY); Xiong and Eickbush supra. Although distinct, Ty3/gypsy elements are more closely related to retroviruses than to Ty1/copia elements. Ty3/gypsy elements share a similar genetic organization with the retroviruses, principally in the order of integrase and reverse transcriptase in their pol genes. Reverse transcriptase precedes integrase in the Ty3/gypsy elements, and this order is reversed for the Ty1/copia elements. In addition, some Ty3/gypsy elements have an extra open reading frame (ORF) encoding a polypeptide that is similar to retroviral envelope (env) proteins, which is required for viral infectivity. The Drosophila melanogaster gypsy retrotransposons encode an env-like ORF and can be transmitted between cells. See, Kim et al. (1994) Proc. Natl. Acad. Sci. USA 91:1285–1289; and Song et al. (1994) Genes Dev. 8:2046–2057. The retroviruses and the Ty3/gypsy retrotransposons that encode envelope-like proteins make up two distinct lineages of infectious LTR retroelements. The Ty3/gypsy elements have been further divided into two genera, the metaviruses and the errantiviruses, the latter including all elements with env-like genes. See, Boeke et al. Metaviridae (supra).
Retrotransposons have been extremely successful in plants. See, e.g., Bennetzen (1996) Trends Microbiol. 4:347–353; and Voytas (1996) Genetics 142:569–578. The enormous size of many plant genomes allows a great tolerance for repetitive DNA, a substantial proportion of which appears to be composed of retrotransposons. Because of their abundance, retrotransposons have undoubtedly influenced plant gene evolution. Retrotransposons can cause mutations in coding sequences (Grandbastien et al. (1989) Nature 337:376–380; Hirochika et al. (1996) Proc. Natl. Acad. Sci. USA 93:7783–7788; and Purugganan and Wessler (1994) Proc. Natl. Acad. Sci. USA 91:11674–11678), and the promoter regions of some plant genes contain relics of retrotransposon insertions that contribute transcriptional regulatory sequences. See, White et al. (1994) Proc. Natl. Acad. Sci. USA 91:11792–11796. Retrotransposons also can generate gene duplications, as repetitive retrotransposon sequences provide substrates for unequal crossing over. Such an event is thought to have caused a zein gene duplication in maize (White et al., supra). Cellular mRNAs occasionally are reverse transcribed, and the resultant cDNA recombines into the genome to give rise to new genes or, more frequently, cDNA pseudogenes. See, Maestre et al. (1995) EMBO J. 14:6333–6338. The transduction of gene sequences during reverse transcription, which produced the oncogenic retroviruses, also has been documented for a plant retrotransposon (Bureau et al. (1994) Cell 77:479–480; and Jin and Bennetzen (1994) Plant Cell 6:1177 1186). A maize Bs1 insertion in Adh1 carries part of an ATPase gene and is the only known example of a retrotransposon-mediated gene transduction event.
Plant genomes can encode representatives of the two major lineages of LTR retrotransposons that have been identified in other eukaryotes. Among these are numerous examples of Ty1/copia elements (see, e.g., Konieczny et al. (1991) Genetics 127:801–809; Voytas and Ausubel (1988) Nature 336:242–244; and Voytas et al. (1990) Genetics 126:713–721). Also prevalent are Ty3/gypsy elements that are member of the genus Metaviridae (Smyth et al. 1989 Proc. Natl. Acad. Sci. USA 86:5015–5019; Purugganan and Wessler (1994) Proc. Natl. Acad. Sci. USA 91:11674–11678; and Su and Brown (1997) Plasmid 38:148–157). As stated above, some metaviruses, including plant metaviruses, contain an envelope gene characteristic of the retroviruses. It has been suggested that the envelope gene may be required for cell-to-cell transmission of plant metaviruses (Bennetzen, supra). The uncertainty, however, has been described with respect to Cyclops, a retroelement identified from pea: “Since genes encoding ENV functions are very heterogeneous at the sequence level and difficult to identify by homology even between retroviruses, the possibility cannot be completely excluded at the present time that the 3′ ORF of Cyclops is, in fact, an env gene and, hence, Cyclops is a retrovirus or a descendant of one.” Chavanne et al. (1998) Plant Mol. Biol. 37:363–375.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
The present invention provides nucleic acids, as well as vectors, cells, and plants (including plant parts, seeds, and embryos) containing the nucleic acids. In particular, molecular tools are provided in the form of nucleic acids that are retroelements or that contain retroelement sequences. The invention also features methods for manipulating such nucleic acids. For example, the invention features methods to introduce nucleic acids containing retroelements or retroelement sequences into cells, especially retroelements carrying at least one agronomically significant characteristic. Specifically, the invention provides a method to transfer agronomically significant characteristics to plants, in which a helper cell line that expresses gag, pol, and env sequences is used to enable transfer of a secondary construct that carries an agronomically significant characteristic and has retroelement sequences that allow for replication and integration.
In one aspect, the invention features an isolated nucleic acid molecule containing a nucleotide sequence selected from the group consisting of: (a) a sequence having at least 60% identity to a nucleotide sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164; (b) a sequence encoding a polypeptide with an amino acid sequence having at least 60% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165; (c) a sequence that is an allelic variant of (a) or (b); and (d) a sequence that is fully complementary to a nucleotide sequence of (a), (b), or (c). The invention also features a stably transformed cell containing the isolated nucleic acid molecule. The cell can be a plant cell or a helper cell expressing gag, pol, and env genes. The invention also features a transgenic seed containing the nucleic acid molecule. The nucleic acid molecule can further contain a regulatory element operably linked to the nucleotide sequence.
In another aspect, the invention features an isolated nucleic acid molecule containing a nucleotide sequence that encodes a reverse transcriptase. The reverse transcriptase can contain any two of the following amino acid motifs: (a) Trp-Val-Ser within reverse transcriptase domain 1; (b) Thr-Val/Ile-Val/Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Asp/Glu-Leu-Val/Ile (SEQ ID NO:191) between reverse transcriptase domains 1 and 2; (c) Thr/Ser-Arg/Lys-Arg/Lys-Asp-His (SEQ ID NO:192) within reverse transcriptase domain 2; (d) Met-Leu-Asp/Glu-Arg-Leu (SEQ ID NO:193) spanning the boundary between reverse transcriptase domains 2 and 3; (e) Cys-Phe-Leu-Asp-Gly-Tyr-Ser (SEQ ID NO:194) within reverse transcriptase domain 3; (f) Phe-Thr-Cys-Pro (SEQ ID NO:195) within reverse transcriptase domain 3; (g) Phe-Gly-Leu-Cys-Asn-Ala-Pro (SEQ ID NO:196) within reverse transcriptase domain 4; (h) Phe-Met-Asp-Asp-Phe (SEQ ID NO:197) within reverse transcriptase domain 5; (i) Leu-Val/Ile-Leu-Asn-Trp-Glu-Lys-Cys-His-Phe-Met-Val/Ile (SEQ ID NO:198) spanning the boundary between reverse transcriptase domains 6 and 7; and (j) Gly-Leu/Ile-Val-Leu-Gly-His (SEQ ID NO:200) within reverse transcriptase domain 7. The invention also features a stably transformed cell containing the isolated nucleic acid molecule. The stably transformed cell can be a plant cell, a helper cell expressing gag, pol, and env genes, or a prokaryotic cell. The invention also features a transgenic seed containing the nucleic acid molecule. The isolated nucleic acid molecule can further contain a regulatory element operably linked to the nucleotide sequence. Furthermore, the invention features an isolated reverse transcriptase. The isolated reverse transcriptase can contain any two of the amino acid motifs described above.
In another aspect, the invention features an Athila retrotransposon containing an open reading frame that has no substantial sequence similarity to retrotransposon gag or pol open reading frames. The open reading frame can be positioned downstream from an Athila integrase open reading frame.
In another aspect, the invention provides isolated nucleic acid molecules encoding at least a portion of a plant retroelement and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence that is a plant retroelement primer binding site having at least 95% identity to SEQ ID NO:2;
(b) a sequence that is at least a portion of a plant retroelement envelope sequence having at least 50% identity to SEQ ID NO:5;
(c) a sequence that is at least a portion of a plant retroelement gag sequence having at least 50% identity to SEQ ID NO:7;
(d) a sequence that is at least a portion of a plant retroelement integrase sequence having at least 70% identity to SEQ ID NO:9;
(e) a sequence that is at least a portion of a plant retroelement reverse transcriptase sequence having at least 70% identity to SEQ ID NO:11;
(f) a sequence that is at least a portion of a plant retroelement protease sequence having at least 50% identity to SEQ ID NO:13;
(g) a sequence that is at least a portion of a plant retroelement RNaseH sequence having at least 70% identity to SEQ ID NO:15;
(h) a sequence that is at least a portion of a plant retroelement sequence having at least 50% identity to SEQ ID NO:17;
(i) a sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17;
(j) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement envelope amino acid sequence having at least 30% identity to SEQ ID NO:6;
(k) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement gag amino acid sequence having at least 30% identity to SEQ ID NO:8;
(l) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement integrase amino acid sequence having at least 75% identity to SEQ ID NO:10;
(m) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement reverse transcriptase amino acid sequence having at least 79% identity to SEQ ID NO:12;
(n) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement protease amino acid sequence having at least 55% identity to SEQ ID NO:14;
(o) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement RNaseH amino acid sequence having at least 90% identity to SEQ ID NO:16;
(p) a sequence that encodes an amino acid sequence that is at least a portion of a plant retroelement amino acid sequence having at least 40% identity to SEQ ID NO: 18;
(q) a sequence that encodes a polypeptide with an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18;
(r) a sequence that is an allelic variant of an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18; and
(s) a sequence fully complementary to a nucleotide sequence of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), or (r).
Seeds and plants containing the nucleic acids described above also are provided. Nucleic acid molecules as described above that further contain gag, pol, and env genes with adenine-thymidine-guanidine as the gag gene start codon also are provided. Furthermore, nucleic acids that contain gag, pol, and env genes with adenine-thymidine-guanidine as the gag gene start codon, and further contain SEQ ID NO:4 also are provided.
The invention also provides plant envelope nucleic acid sequences, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant envelope amino acid sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:5;
(b) the sequence of SEQ ID NO:5;
(c) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to SEQ ID NO:6;
(d) a sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:6;
(e) a sequence that is an allelic variant of SEQ ID NO:6; and
(f) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), (c), (d), or (e).
The invention also provides plant integrase nucleic acid sequences, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant integrase amino acid sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:9;
(b) the sequence of SEQ ID NO:9;
(c) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to SEQ ID NO:10;
(d) a sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:10;
(e) a sequence that is an allelic variant of SEQ ID NO:10; and
(f) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), (c), (d), or (e).
The invention also provides plant reverse transcriptase nucleic acid sequences, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant reverse transcriptase amino acid sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:11;
(b) the sequence of SEQ ID NO:11;
(c) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to SEQ ID NO:12;
(d) a sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:12;
(e) a sequence that is an allelic variant of SEQ ID NO:12; and
(f) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), (c), (d), or (e).
The invention also provides plant RNaseH nucleic acid sequences, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant RNaseH amino acid sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:15;
(b) the sequence of SEQ ID NO:15;
(c) a sequence encoding a polypeptide with an amino acid sequence having at least 95% identity to SEQ ID NO:16;
(d) a sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:16;
(e) a sequence that is an allelic variant of SEQ ID NO:16; and
(f) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), (c), (d), or (e).
The invention also provides plant retroelement nucleic acid sequences, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant retroelement amino acid sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 95% identity to a nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17;
(b) a sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17;
(c) a sequence encoding a polypeptide with an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18;
(d) a sequence encoding a polypeptide with an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18;
(e) a sequence that is an allelic variant of an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18; and
(f) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), (c), (d), or (e).
The invention also provides isolated nucleic acid molecules encoding at least a portion of a plant retroelement reverse transcriptase and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 85% identity to a nucleic acid sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 through SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a) or (b).
The invention also features nucleic acid molecules such as those described above, which further contain gag, pol, and env genes and have adenine-thymidine-guanidine as the gag gene start codon. In addition, the nucleic acid molecules described herein can further contain SEQ ID NO:5. In particular, the invention features isolated nucleic acid molecules encoding at least a portion of a plant envelope sequence and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:5;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to SEQ ID NO:6;
(c) a sequence that is an allelic variant of SEQ ID NO:5; and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), or (c).
The invention also features isolated nucleic acid molecules encoding at least a portion of a plant retroelement reverse transcriptase and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 95% identity to a nucleic acid sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 95% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 through SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a) or (b).
The nucleic acid molecules described above can further contain gag, pol, and env genes, with adenine-thymidine-guanidine as the gag gene start codon. In addition, the invention provides nucleic acid molecules that further contain SEQ ID NO:5.
The invention also features isolated nucleic acid molecules encoding at least a portion of a plant retroelement reverse transcriptase and containing a nucleotide sequence selected from the group consisting of:
(a) a sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 through SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a) or (b).
The invention also features plant retroelements containing the nucleic acid sequences described herein. Plant cells containing the plant retroelement nucleic acid molecules also are provided, as are plant retroelement proteins encoded by the nucleic acid molecules.
The invention also features plant retroelement nucleic acid sequences containing specialized signals, as well as constructs, cells, seeds, embryos, and plants containing such nucleic acid sequences. In particular, the invention provides isolated nucleic acid molecules containing a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 95% identity to SEQ ID NO:2;
(b) the sequence of SEQ ID NO:2;
(c) a sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:4; and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), or (c).
Seeds and plants containing the nucleic acid molecules described herein also are provided, as are nucleic acids that further contain gag, pol, and env genes and have adenine-thymidine-guanidine as the gag gene start codon. In addition, the nucleic acids provided herein can further contain SEQ ID NO:5. The invention also provides methods to impart agronomically significant characteristics to a plant cell. Such methods can involve contacting at least one plant cell with a nucleic acid molecule of the invention, under conditions sufficient to allow the nucleic acid molecule to enter the cell.
Nucleic acid molecules of the invention also can contain at least one nucleic acid sequence that imparts an agronomically significant characteristic. Agronomically significant characteristics can include, without limitation, those selected from the group consisting of: male sterility, self-incompatibility, foreign organism resistance, improved biosynthetic pathways, environmental tolerance, photosynthetic pathways, nutrient content, fruit ripening, oil biosynthesis, pigment biosynthesis, seed formation, starch metabolism, salt tolerance, cold/frost tolerance, drought tolerance, tolerance to anaerobic conditions, protein content, carbohydrate content (e.g., sugar and starch content), amino acid content, and fatty acid content.
In another aspect, the invention features seeds and plants containing the nucleic acid molecules provided herein. Suitable plants include, for example, soybean, maize, sugar cane, beet, tobacco, wheat, barley, poppy, rape, sunflower, alfalfa, sorghum, rose, carnation, gerbera, carrot, tomato, lettuce, chicory, pepper, melon, cabbage, oat, rye, cotton, flax, potato, pine, walnut, citrus fruits, hemp, oak, rice, petunia, orchids, Arabidopsis, broccoli, cauliflower, Brussels sprouts, onion, garlic, leek, squash, pumpkin, celery, peas, beans, strawberries, grapes, apples, pears, peaches, banana, palm, cocoa, cucumber, pineapple, apricot, plum, sugar beet, lawn grasses, maple, triticale, safflower, peanut, and olive.
In another aspect, the invention features methods for transferring a nucleic acid molecule into a plant cell. The methods can involve contacting a plant cell with a nucleic acid molecule of the present invention, under conditions sufficient to allow the nucleic acid molecule to enter the at least one plant cell. In particular, the invention provides methods to impart agronomically significant characteristics to at least one plant cell. Such methods can involve contacting at least one plant cell with a plant retroelement nucleic acid molecule encoding an agronomically significant characteristic such as those described above, under conditions sufficient to allow the nucleic acid molecule to enter the cell.
The nucleic acid molecules of the invention can be transmissible either to all plants or to a limited set of plants, such as a species. For example, plant viruses in general only infect a narrow host range or may infect a single species, and the present sequences can be genetically engineered to be similar. However, if a broad host range is desirable, those features that cause specificity can be removed, or can be overridden by the feature of broad transmissibility. The present invention is drawn to both of these embodiments, as well as to other variations.
By “agronomically significant” it is meant any trait of a plant that is recognized by members of the agricultural industry as desirable. Agronomically significant characteristics can include, for example, male sterility, self-incompatibility, foreign organism resistance, improved biosynthetic pathways, environmental tolerance, photosynthetic pathways, nutrient content, fruit ripening, oil biosynthesis, pigment biosynthesis, seed formation, starch metabolism, salt tolerance, cold/frost tolerance, drought tolerance, tolerance to anaerobic conditions, protein content, carbohydrate content, amino acid content, and fatty acid content.
“Fragment” is meant to refer to any subset of the referent nucleic acid molecule.
By “plant” it is meant one or more plant seed, plant embryo, plant part or whole plant. The plant may be an angiosperm (monocot or dicot), gymnosperm, man-made or naturally occurring.
By “proteins” it is meant any compounds that comprise amino acids, including peptides, polypeptides, fusion proteins, etc.
Moreover, for the purposes of the present invention, the term “a” or “an” entity refers to one or more of that entity; for example, “a protein” or “a nucleic acid molecule” refers to one or more of those compounds or at least one compound. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds.
According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. An “isolated nucleic acid” refers to a nucleic acid that is separated from the other nucleic acid molecules by which it is naturally flanked in the genome, including nucleic acids that normally flank one or both sides of a retroelement nucleic acid sequence in a plant genome. An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences, as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., any, retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not considered an isolated nucleic acid. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to that the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
Lastly, “more than” and “greater than” are interchangeable, and when used to modify a percent identity, i.e. “more than 90% identity”, mean any increment to 100%, so long as the increment were greater than the percentage specifically named. In the example of “more than 90% identity”, the term would include, among all other possibilities, 90.00001, 93.7, 98.04 and 99.0827 and 100%.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to that this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The following is a summary of the sequence listing, provided as a convenient reference.
The present invention provides nucleic acids, as well as vectors, cells, and plants (including plant parts, seeds, and embryos) containing the nucleic acids. In particular, molecular tools are provided in the form of nucleic acids that are retroelements or that contain retroelement sequences. The invention also features methods for manipulating such nucleic acids. For example, the invention features methods to introduce nucleic acids containing retroelements or retroelement sequences into cells, especially retroelements carrying at least one agronomically significant characteristic.
1. Nucleic Acid Molecules
The invention provides isolated nucleic acid molecules. The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid can be double-stranded or single-stranded. A single-stranded nucleic acid can be a sense strand or an antisense strand. In addition, a nucleic acid can be circular or linear.
The invention provides nucleic acid molecules having nucleotide sequences with a specified percent identity to the nucleotide sequences provided in the sequence listing. For example, a nucleic acid molecule of the invention can contain a nucleotide sequence that is at least 60% identical (e.g., 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical) to one or more of the nucleic acid sequences of SEQ ID NO:1 to SEQ ID NO:165. Furthermore, the isolated nucleic acid molecules provided herein typically are at least 15 nucleotides in length, and can be up to about 30 kilobases in length. An isolated nucleic acid molecule thus can be, for example, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2500, 5000, 10000, 20000, or about 30000 nucleotides in length.
The nucleic acid molecules provided herein can contain plant retroelement sequences. Such sequences can include, for example, a retroelement LTR, polypurine tract (PPT), primer binding site (PBS), gag gene, pol gene, and/or env gene, or portions of a pol gene (e.g., an integrase, RNaseH, or reverse transcriptase gene). An isolated nucleic acid molecule provided by the present invention can be, for example, a nucleic acid molecule encoding at least a portion of a plant retroelement reverse transcriptase. Such a nucleic acid molecule can contain a nucleotide sequence selected from the group consisting of:
(a) a sequence having at least 85% identity to a nucleic acid sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), or (c).
Such isolated nucleic acid molecules further can contain gag, pol, and env genes that have adenine-thymidine-guanidine as the gag gene start codon. In addition, such nucleic acid molecules also can contain SEQ ID NO:5 or sequences homologous to SEQ ID NO:5. The invention thus provides isolated nucleic acid molecules encoding at least a portion of a plant envelope sequence and containing a nucleic acid sequence selected from the group consisting of:
(a) a sequence having at least 90% identity to SEQ ID NO:5;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 85% identity to SEQ ID NO:6;
(c) a sequence that is an allelic variant of SEQ ID NO:5; and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b) or (c).
Isolated nucleic acid molecules of the invention also can encode at least a portion of a plant retroelement reverse transcriptase and contain a nucleic acid sequence selected from the group consisting of:
(a) a sequence having at least 95% identity to a nucleic acid sequence of one or more of the even-numbered SEQ IDNOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 95% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), or (c).
In some embodiments, isolated nucleic acid molecules provided herein can encode at least a portion of a plant retroelement reverse transcriptase and can contain a nucleic acid sequence selected from the group consisting of:
(a) a sequence having at least 60% identity to a nucleic acid sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence having at least 60% identity to an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a sequence that is fully complementary to a nucleic acid sequence of (a), (b), or (c).
Furthermore, an isolated nucleic acid molecule can encode at least a portion of a plant retroelement reverse transcriptase and can contain a nucleic acid sequence selected from the group consisting of:
(a) a sequence of one or more of the even-numbered SEQ ID NOs from SEQ ID NO:42 to SEQ ID NO:164;
(b) a sequence encoding a polypeptide with an amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 through SEQ ID NO:165;
(c) a sequence that is an allelic variant of a nucleic acid sequence of (a) or (b); and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence of (a), (b), or (c).
The invention provides isolated nucleic acid molecules that contain a nucleotide sequence having (1) a length, and (2) a percent identity to an identified nucleotide sequence over that length. The invention also provides isolated nucleic acid molecules that contain a nucleotide sequence encoding a polypeptide that contains an amino acid sequence having (1) a length, and (2) a percent identity to an identified amino acid sequence over that length. Typically, the identified nucleic acid or amino acid sequence is a sequence referenced by a particular sequence identification number, and the nucleic acid or amino acid sequence being compared to the identified sequence is referred to as the target sequence. For example, an identified sequence can be the sequence set forth in SEQ ID NO:42.
A length and percent identity over that length for any nucleic acid or amino acid sequence is determined as follows. First, a nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from the State University of New York—Old Westbury campus library as well as at Fish & Richardson's web site (World Wide Web at fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ.
Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1-r 2. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt j c:\seq2.txt -p blastp -o c:\output.txt. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences. Once aligned, a length is determined by counting the number of consecutive nucleotides or amino acid residues from the target sequence presented in alignment with sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide or amino acid residue is presented in both the target and identified sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acid residues. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides or amino acid residues are counted, not nucleotides or amino acid residues from the identified sequence.
The percent identity over a determined length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (1) a 1000 nucleotide target sequence is compared to the sequence set forth in SEQ ID NO:42, (2) the Bl2seq program presents 775 nucleotides from the target sequence aligned with a region of the sequence set forth in SEQ ID NO:42 where the first and last nucleotides of that 775 nucleotide region are matches, and (3) the number of matches over those 775 aligned nucleotides is 700, then the 1000 nucleotide target sequence contains a length of 775 and a percent identity over that length of 90.3 (i.e., 700÷775×100=90.3).
It will be appreciated that a single nucleic acid or amino acid target sequence that aligns with an identified sequence can have many different lengths with each length having its own percent identity.
It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also noted that the length value will always be an integer.
The nucleic acid molecules described herein can further contain at least one nucleic acid sequence encoding an agronomically significant characteristic. Useful agronomically significant characteristics can include, for example: male sterility, self-incompatibility, foreign organism resistance, improved biosynthetic pathways, environmental tolerance, photosynthetic pathways, nutrient content, fruit ripening, oil biosynthesis, pigment biosynthesis, seed formation, starch metabolism, salt tolerance, cold/frost tolerance, drought tolerance, tolerance to anaerobic conditions, protein content, carbohydrate content (e.g., sugar and starch content), amino acid content, and fatty acid content.
Recombinant molecules of the present invention also can contain secretory signals (e.g., signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein. Furthermore, recombinant nucleic acid molecules can contain fusion sequences that lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Recombinant molecules also can include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Included within the scope of the present invention, with particular regard to the nucleic acids above, are allelic variants, degenerate sequences and homologues. As used herein, the term “allelic variant” refers to a full-length gene or partial sequence of a full-length gene that has a nucleotide sequence that is similar but not identical to that of the reference gene. Such sequence differences can be due to natural variations caused by, for example, mutation or recombination. Allelic variants can encode polypeptides with amino acid sequences that vary from the amino acid sequence encoded by the reference gene. An allelic variant typically encodes a protein having an activity similar to that of the protein encoded by the gene to which it is being compared. Allelic variants also can contain alterations in the 5′ or 3′ untranslated regions of the gene (e.g., in regulatory control regions).
The present invention includes variants that arise during laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification or site directed mutagenesis. There is a substantial amount of redundancy in the various codons that code for specific amino acids. Therefore, this invention also is directed to those nucleic acid sequences containing alternative codons that code for the eventual translation of the identical amino acid. Also included within the scope of the invention are mutations either in the nucleic acid sequence or in the translated protein that do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagines for glutamine may not cause a change in functionality of the polypeptide.
The nucleic acid molecules provided herein typically encode plant retroelement polypeptides. For example, a nucleic acid molecule of the invention can encode a reverse transcriptase polypeptide having the amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. Alternatively, a nucleic acid molecule can encode a reverse transcriptase that is an allelic variant of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. Furthermore, a nucleic acid molecule can encode a reverse transcriptase polypeptide that contains two or more amino acid sequences that are conserved (i.e., “invariant”) among the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. The conserved nucleic acid sequences are contiguous and reside within or between one of the seven amino acid sequence domains that define reverse transcriptases (Xiong and Eickbush, supra). For example, a nucleic acid molecule provided herein can encode a reverse transcriptase containing the amino acid sequence Trp-Val-Ser within reverse transcriptase domain 1; the amino acid sequence Thr-Val/Ile-Val/Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Asp/Glu-Leu-Val/Ile (SEQ ID NO:191) between reverse transcriptase domains 1 and 2; the amino acid sequence Thr/Ser-Arg/Lys-Arg/Lys-Asp-His (SEQ ID NO:192) within reverse transcriptase domain 2; and/or the amino acid sequence Met-Leu-Asp/Glu-Arg-Leu (SEQ ID NO:193) spanning the boundary between reverse transcriptase domains 2 and 3. For example, the amino acid sequence Met-Leu-Asp/Glu can be present within reverse transcriptase domain 2, the Arg can be between domains 2 and 3, and the Leu can be within domain 3. Furthermore, a nucleic acid molecule provided herein can encode a reverse transcriptase containing the amino acid sequence the amino acid sequence Cys-Phe-Leu-Asp-Gly-Tyr-Ser (SEQ ID NO:194) within reverse transcriptase domain 3; the amino acid sequence Phe-Thr-Cys-Pro (SEQ ID NO:195) within reverse transcriptase domain 3; the amino acid sequence Phe-Gly-Leu-Cys-Asn-Ala-Pro (SEQ ID NO:196) within reverse transcriptase domain 4; the amino acid sequence Phe-Met-Asp-Asp-Phe (SEQ ID NO:197) within reverse transcriptase domain 5; the amino acid sequence Leu-Val/Ile-Leu-Asn-Trp-Glu-Lys-Cys-His-Phe-Met-Val/Ile (SEQ ID NO:198) spanning the boundary between reverse transcriptase domains 6 and 7 (e.g., with the amino acid sequence Leu-Val/Ile-Leu-Asn-Trp-Glu-Lys-Cys-His-Phe (SEQ ID NO:199) within domain 6, the Met between domains 6 and 7, and the Val/Ile within domain 7); and/or the amino acid sequence Gly-Leu/Ile-Val-Leu-Gly-His (SEQ ID NO:200) within reverse transcriptase domain 7.
Knowing the nucleic acid sequences of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain similar nucleic acid molecules from other species. Such nucleic acid molecules can be obtained in a variety to of ways including screening appropriate expression libraries of DNA; and PCR amplification of appropriate libraries of DNA using oligonucleotide primers of the present invention. Libraries to screen or from which to amplify nucleic acid molecules can include cDNA libraries as well as genomic DNA libraries. Similarly, DNA sources to screen or from which to amplify nucleic acid molecules can include adult cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press).
Recombination constructs can be made using the starting materials above or with additional materials, using methods well-known in the art. In general, the sequences can be manipulated to have ligase-compatible ends, and incubated with ligase to generate full constructs. For example, restriction enzymes can be chosen on the basis of their ability to cut at an acceptable site in both sequence to be ligated, or a linker can be added to adapt the sequence end(s) to be compatible. The methods for conducting these types of molecular manipulations are well known in the art, and are described in detail in Sambrook et al., supra; and Ausubel et al. (1993) Current Protocols in Molecular Biology (Greene Publishing Associates, Inc.). The methods described herein according to Tinland et al. (1994) Proc. Natl. Acad. Sci. USA 91:8000–8004 also can be used.
The invention also provides vectors containing the nucleic acid molecules described herein. Such vectors can be, without limitation, viral vectors, plasmids, phage, and cosmids. For example, vectors can be of viral origin (e.g., paramyxovirus vectors, SV40 vectors, molecular conjugate vectors, or vectors derived from adenovirus, adeno-associated virus, herpes virus, lentivirus, retrovirus, parvovirus, or Sindbis virus) or of non-viral origin (e.g., vectors from bacteria or yeast). An isolated nucleic acid encoding a plant retroelement polypeptide can be inserted into a vector such that the polypeptide is expressed. For example, a nucleic acid provided herein can be inserted into an expression vector. “Expression vectors” can contain one or more regulatory elements (e.g., a sequence that controls and regulates the transcription and/or translation of another sequence. Such regulatory elements can include, for example, promoter sequences, enhancer sequences, response elements, protein recognition sites, or inducible elements that modulate expression of a nucleic acid sequence.
Nucleic acid constructs of the invention can contain one or more regulatory elements operably linked to a nucleic acid coding sequence. As used herein, “operably linked” refers to positioning of a regulatory element in a construct relative to a nucleic acid coding sequence in such a way as to permit or facilitate expression of the encoded polypeptide. The choice of element(s) that can be included depends upon several factors, including, but not limited to, replication efficiency, selectability, inducibility, desired expression level, and cell or tissue specificity.
Expression vectors of the present invention can contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention can include transcription control sequences. Transcription control sequences are sequences that control the initiation, elongation and termination of transcription. Particularly important transcription control sequences are those that control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequences that can function in at least one of the recombinant cells of the present invention.
A variety of transcription control sequences are known to those skilled in the art. Useful transcription control sequences can include those that function in bacterial, yeast, insect and mammalian cells, such as, without limitation, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alphamating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxyirus, other poxyirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Suitable transcription control sequences also can include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with plants.
The following promoters can be particularly useful in early expression of the present sequences: Ogs4B (Tsuchiya et al. (1994) Plant Cell Physiol. 36:487; TA29 (Koltunow et al. (1990) Plant Cell 2:1201; and A3 and A9 (Paul et al. (1992) Plant Mol. Biol. 19:611). In order to then constitutively express the sequences described above, the construct optionally can contain, for example, a 35S promoter.
Other suitable regulatory elements include promoters that initiate transcription only, or predominantly, in certain cell types. For example, promoters specific to plant vegetative tissues such as ground meristem, vascular bundle, cambium, phloem, cortex, shoot apical meristem, lateral shoot meristem, root apical meristem, lateral root meristem, leaf primordium, leaf mesophyll, or leaf epidermis can be suitable regulatory elements. In other embodiments, a promoter specific to a reproductive tissue (e.g., fruit, ovule, seed, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo, zygote, endosperm, integument, seed coat or pollen) is used. A cell type or tissue-specific promoter can drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a cell type or tissue-specific promoter is one that drives expression preferentially in the target tissue, but also can lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano et al. (1989) Plant Cell 1:855–866; Bustos et al. (1989) Plant Cell 1:839–854; Green et al. (1988) EMBO J. 7:4035–4044; Meier et al. (1991) Plant Cell 3:309–316; and Zhang et al. (1996) Plant Physio. 110:1069–1079.
Reproductive tissue promoters include, for example, those derived from the following seed-genes: zygote and embryo LECI (Lotan (1998) Cell 93:1195–1205); suspensor G564 (Weterings (2001) Plant Cell 13:2409–2425); maize MAC1 (Sheridan (1996) Genetics 142:1009–1020); maize Cat3 (GenBank No. L05934; Abler (1993) Plant Mol. Biol. 22:10131–1038); Arabidopsis viviparous-l (GenBank No. U93215); Arabidopsis atmycl (Urao (1996) Plant Mol. Biol. 32:571–57; Conceicao (1994) Plant 5:493–505); and Brassica napus napin gene family, including napA (GenBank No. J02798; Josefsson (1987) JBL 26:12196–1301; and Sjodahl (1995) Planta 197:264–271). Other examples of reproductive tissue-specific promoters include those derived from the pollen genes described in, for example: Guerrero (1990) Mol. Gen. Genet. 224:161–168; Wakeley (1998) Plant Mol. Biol. 37:187–192; Ficker (1998) Mol. Gen. Genet. 257:132–142; Kulikauskas (1997) Plant Mol. Biol. 34:809–814; and Treacy (1997) Plant Mol. Biol. 34:603–611. Still other suitable reproductive tissue promoters include those derived from the following embryo genes: Brassica napus 2s storage protein (Dasgupta (1993) Gene 133:301–302); Arabidopsis 2s storage protein (GenBank No. AL161566); soybean β-conglycinin (GenBank No. S44893); Brassica napus oleosin 20 kD gene (GenBank No. M63985); soybean oleosin A (GenBank No. U09118); soybean oleosin B (GenBank No. U09119); soybean lectin1 (GenBank K00821); soybean Kunitz trypsin inhibitor 3 (GenBank No. AF233296); soybean glycinin1 (GenBank No. X15121); Arabidopsis oleosin (GenBank No. Z17657); maize oleosin 18 kD (GenBank No. J05212; Lee (1994) Plant Mol. Biol. 26:1981–1987); and the gene encoding low molecular weight sulfur rich protein from soybean (Choi (1995) Mol. Gen. Genet. 246:266–268). Other examples of reproductive tissue promoters include those derived from the following endosperm genes: Arabidopsis Fie (GenBank No. AF129516); Arabidopsis Mea; Arabidopsis Fis2 (GenBank No. AF096096); rice Glu1 (GenBank No. M28156); and rice 26 kDa globulin (GenBank No. D50643). Further examples of reproductive tissue promoters include those derived from the following genes: ovule BEL1 (Reiser (1995) Cell 83:735–742; Ray (1994) Proc. Natl. Acad. Sci. USA 91:5761–5765; and GenBank No. U39944); central cell FIE (GenBank No. AF129516); flower primordia Arabidopsis APETALA1 (a.k.a. AP1; Gustafson-Brown (1994) Cell 76:131–143; Mandrel (1992) Nature 360:273–277); flower Arabidopsis AP2 (Jofuku (1994) Plant Cell 6:1211–1225); Arabidopsis flower ufo, expressed at the junction between sepal and petal primordia (Bossinger (1996) Development 122:1093–1102); fruit-specific tomato E8; a tomato gene expressed during fruit ripening, senescence and abscission of leaves and flowers (Blume (1997) Plant J. 12:731–746); pistil-specific potato SK2 (Ficker (1997) Plant Mol. Biol. 35:425–431); Arabidopsis DMC1 (GenBank No. U76670); and Arabidopsis DMT1 (Choi (2002) Cell 110:33–42).
Suitable vegetative tissue promoters include, for example, those derived from the following genes: pea Blec4, active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa; potato storage protein patatin gene (Kim (1994) Plant Mol. Biol. 26:603–615; Martin (1997) Plant J., 11:53–62); root Agrobacterium rhizogenes ORF13 (Hansen (1997) Mol. Gen. Genet. 254:337–343); genes active during taro corm development (Bezerra (1995) Plant Mol. Biol. 28:137–144); de Castro (1992) Plant Cell 4:1549–1559); root meristem and immature central cylinder tobacco gene TobRB7 (Yamamoto (1991) Plant Cell 3:371–382); ribulose biphosphate carboxylase genes RBCS1, RBCS2, and RBCS3A expressed in tomato leaves (Meier (1997) FEBS Lett. 415:91–95); ribulose biphosphate carboxylase genes expressed in leaf blade and leaf sheath mesophyll cells (Matsuoka (1994) Plant J. 6:311–319); leaf chlorophyll a/b binding protein (Shiina (1997) Plant Physiol. 115:477–483; Casal (1998) Plant Physiol. 116:1533–1538); Arabidopsis Atmyb5, expressed in developing leaf trichomes, stipules, in epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage (Li (1996) FEBS Lett. 379:117–121); a maize leaf-specific gene described by Busk (1997) Plant J. 11:1285–1295; “SHOOTMERISTEMLESS” and “SCARECROW” genes active in developing shoot or root apical meristems (Di Laurenzio (1996) Cell 86:423–433; Long (1996) Nature 379:66–69); 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2, expressed in meristematic tissue, and floral reductase HMG2, expressed in meristematic and floral (e.g., secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (Enjuto (1995) Plant Cell 7:517–527); meristem kn1-related genes from maize and other species (Granger (1996) Plant Mol. Biol. 31:373–378; Kerstetter (1994) Plant Cell 6:1877–1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45–51; Lincoln (1994) Plant Cell 6:1859–1876); and constitutive Cauliflower mosaic virus 35S.
Other examples of suitable plant promoters include those described in U.S. Pat. Nos. 6,184,443, 6,054,635, 6,005,092, 5,866,793, and 5,792,932, for example.
Cell type or tissue-specific promoters derived from viruses also can be suitable regulatory elements. Such viral promoters include, for example, the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679–1683); the phloem-specific tungro bacilliform virus (RTBV) promoter; the cassaya vein mosaic virus (CVMV) promoter, expressed most strongly in vascular elements, leaf mesophyll cells, and root tips (Verdaguer (1996) Plant. Mol. Biol. 31:1129–1139).
Vectors can be obtained from various commercial sources, including Clontech Laboratories, Inc. (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Mass.) and Promega (Madison, Wis.), for example. Vectors that can be used to transfer the sequences disclosed herein into plant cells or plant parts can be particularly useful.
The present invention includes nucleic acid molecules that are oligonucleotides, as well as nucleic acid molecules that contain entire retroelement nucleotide sequences. Thus, nucleic acid molecules of the invention can range in length from about 10 nucleotides to more than 10,000 nucleotides. Oligonucleotides, for example, can be between about 10 and about 30 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28, or 30 nucleotides in length). Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides typically is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence within a nucleic acid molecule of the present invention, under stringent hybridization conditions. The present invention includes oligonucleotides that can be used as probes to identify nucleic acid molecules, primers to produce nucleic acid molecules or therapeutic reagents, for example. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, e.g., Sambrook et al., supra, and Meinkoth et al. (1984) Anal. Biochem. 138:267–284.
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with that those nucleic acid molecules are transcribed, the efficiency with that those nucleic acid molecules are transcribed, the efficiency with that the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgamo sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
Nucleic acid molecules within the scope of the invention can be obtained using any method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to construct nucleic acid molecules that contain plant retroelement sequences. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein.
The invention also provides isolated plant retroelements containing the nucleic acid molecules described herein, as well as plant cells containing the nucleic acid molecules described herein. The invention features both plants and plant parts (e.g., seeds) containing the nucleic acid molecules and polypeptides provided herein. Suitable plants can include those selected from the group consisting of: soybean, maize, sugar cane, beet, tobacco, wheat, barley, poppy, rape, sunflower, alfalfa, sorghum, rose, carnation, gerbera, carrot, tomato, lettuce, chicory, pepper, melon, cabbage, oat, rye, cotton, flax, potato, pine, walnut, citrus fruit (e.g., orange and grapefruit), hemp, oak, rice, petunia, orchids, Arabidopsis, broccoli, cauliflower, Brussels sprouts, onion, garlic, leek, squash, pumpkin, celery, pea, bean, strawberries, grapes, apples, pears, peaches, banana, palm, cocoa, cucumber, pineapple, apricot, plum, sugar beet, lawn grasses, maple, triticale, safflower, peanut, and olive.
Nucleic acids of the present invention can be transferred to cells according to the methods of the present invention, as well as using any suitable means known in the art. The transformed cells can be induced to form transformed plants via organogenesis or embryogenesis, according to the procedures of Dixon (1987) Plant Cell Culture: A Practical Approach (IRL Press, Oxford).
Any seed, embryo, plant or plant part is amenable to the methods provided herein. Agronomically significant seeds, embryos, plants or plant parts are particularly useful. For example, soybean, maize, sugar cane, beet, tobacco, wheat, barley, poppy, rape, sunflower, alfalfa, sorghum, rose, carnation, gerbera, carrot, tomato, lettuce, chicory, pepper, melon, cabbage, oat, rye, cotton, flax, potato, pine, walnut, citrus (including oranges, grapefruit etc.), hemp, oak, rice, petunia, orchids, Arabidopsis, broccoli, cauliflower, Brussels sprouts, onion, garlic, leek, squash, pumpkin, celery, pea, bean (including various legumes), strawberries, grapes, apples, pears, peaches, banana, palm, cocoa, cucumber, pineapple, apricot, plum, sugar beet, lawn grasses, maple, triticale, safflower, peanut, and olive are among the useful seeds, embryos plants or plant parts. Particularly useful are: soybean, tobacco and maize seeds, embryos, plants or plant parts. However, Arabidopsis seeds, embryos, plants or plant parts are also useful, since Arabidopsis is an excellent system for study of plant genetics.
2. Polypeptides
The invention provides polypeptides encoded by the nucleic acid molecules described herein. As used herein, a “polypeptide” refers to a chain of amino acid residues, regardless of post-translational modification (e.g., phosphorylation or glycosylation). Polypeptides of the invention can be plant retroelement polypeptides. Polypeptides of the invention typically have amino acid sequences that are at least 60% (e.g., 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) identical to the sequences set forth in one or more of the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:165 (e.g., the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165).
Plant retroelement polypeptides of the invention can contain at least one amino acid substitution relative to the corresponding wild type polypeptides (e.g., the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165). Such amino acid substitutions typically are located at positions that are not required for or are minimally involved in reverse transcriptase function. Amino acid substitutions can be conservative or non-conservative. Conservative amino acid substitutions replace an amino acid with an amino acid of the same class, whereas non-conservative amino acid substitutions replace an amino acid with an amino acid of a different class. Examples of conservative substitutions include amino acid substitutions within the following groups: (1) glycine and alanine; (2) valine, isoleucine, and leucine; (3) aspartic acid and glutamic acid; (4) asparagine, glutamine, serine, and threonine; (5) lysine, histidine, and arginine; and (6) phenylalanine and tyrosine.
Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions also can make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid or a polar amino acid for an acidic amino acid.
Polypeptides of the invention can be any plant retroelement polypeptide. A polypeptide can be, for example, a gag, pol, env, RNaseH, invertase, or reverse transcriptase polypeptide. Such polypeptides can contain a portion or all of one or more of the amino acid sequences provided herein. For example, a plant retroelement polypeptide can be a reverse transcriptase polypeptide having the amino acid sequence of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. Alternatively, a reverse transcriptase polypeptide can be an allelic variant of one or more of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. Furthermore, a reverse transcriptase polypeptide can contain two or more particular sequences that are conserved (i.e., “invariant”) among the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165. The conserved amino acid sequences are contiguous and reside within or between one of the seven amino acid sequence domains that define reverse transcriptases (Xiong and Eickbush, supra). For example, a reverse transcriptase can contain the amino acid sequence Tip-Val-Ser within reverse transcriptase domain 1; the amino acid sequence Trp-Val-Ser within reverse transcriptase domain 1; the amino acid sequence Thr-Val/Ile-Val/Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Asp/Glu-Leu-Val/Ile (SEQ ID NO:191) between reverse transcriptase domains 1 and 2; the amino acid sequence Thr/Ser-Arg/Lys-Arg/Lys-Asp-His (SEQ ID NO:192) within reverse transcriptase domain 2; and/or the amino acid sequence Met-Leu-Asp/Glu-Arg-Leu (SEQ ID NO:193) spanning the boundary between reverse transcriptase domains 2 and 3. For example, the amino acid sequence Met-Leu-Asp/Glu can be present within reverse transcriptase domain 2, the Arg can be between domains 2 and 3, and the Leu can be within domain 3. Furthermore, a nucleic acid molecule provided herein can encode a reverse transcriptase containing the amino acid sequence the amino acid sequence Cys-Phe-Leu-Asp-Gly-Tyr-Ser (SEQ ID NO:194) within reverse transcriptase domain 3; the amino acid sequence Phe-Thr-Cys-Pro (SEQ ID NO:195) within reverse transcriptase domain 3; the amino acid sequence Phe-Gly-Leu-Cys-Asn-Ala-Pro (SEQ ID NO:196) within reverse transcriptase domain 4; the amino acid sequence Phe-Met-Asp-Asp-Phe (SEQ ID NO:197) within reverse transcriptase domain 5; the amino acid sequence Leu-Val/Ile-Leu-Asn-Trp-Glu-Lys-Cys-His-Phe-Met-Val/Ile (SEQ ID NO:198) spanning the boundary between reverse transcriptase domains 6 and 7 (e.g., with the amino acid sequence Leu-Val/Ile-Leu-Asn-Trp-Glu-Lys-Cys-His-Phe (SEQ ID NO:199) within domain 6, the Met between domains 6 and 7, and the Val/Ile within domain 7); and/or the amino acid sequence Gly-Leu/Ile-Val-Leu-Gly-His (SEQ ID NO:200) within reverse transcriptase domain 7.
As used herein, “reverse transcriptase” refers to a polypeptide having enzymatic activity that catalyzes the incorporation of deoxynucleotides into a nucleic acid molecule, using RNA as a template. For example, a reverse transcriptase can catalyze the incorporation of deoxynucleotides into a cDNA molecule, using mRNA as a template and oligo(dT) as a primer. A reverse transcriptase polypeptide can be identified by measuring its enzymatic activity. Typically, one “unit” of a reverse transcriptase can catalyze the incorporation of 1 mmol dNTP into acid- (e.g., trichloroacetic acid-) precipitatable material in 10 minutes. As such, reverse transcriptase polypeptides such as those containing the sequences specified above can be used to prepare double-stranded nucleic acid molecules from RNA molecules. Reverse transcriptase polypeptides are further defined as amino acid sequences having the seven conserved amino acid sequence domains that characterize all known reverse transcriptases (Xiong and Eickbush, supra). These seven amino acid sequence domains can be identified using a standard amino acid sequence alignment program (e.g. ClustalX; see, Thompson et al. (1997) Nucl. Acids Res. 24:4876–4882). Input data for alignments can include amino acid sequences for reverse transcriptases from characterized retrotransposons, as well as retroelement and reverse transcriptase amino acid sequences such as those described above. Alignment can be performed using default parameters, and the seven conserved domains can be identified in by comparison to the domains identified in the alignment of Xiong and Eickbush, supra.
Plant retroelement polypeptides can be produced by a number of different methods. For example, polypeptides can be obtained by extraction from viruses, isolated cells, tissues, and plant fluids. Polypeptides also can be produced by chemical synthesis. Alternatively, polypeptides of the invention can be produced by standard recombinant technology using heterologous expression vectors encoding retroelement polypeptides. Expression vectors can be introduced into host cells (e.g., by transformation or transfection) for expression of the encoded polypeptide, which then can be purified. Expression systems that can be used for small or large scale production of plant retroelement polypeptides include, without limitation, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention, and yeast (e.g., S. cerevisiae) transformed with recombinant yeast expression vectors containing the nucleic acid molecules of the invention. Useful expression systems also include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the nucleic acid molecules of the invention, and plant cell systems infected with recombinant virus expression vectors (e.g., tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the nucleic acid molecules of the invention. Plant retroelement polypeptides also can be produced using mammalian expression systems, which include cells (e.g., primary cells or immortalized cell lines such as COS cells, Chinese hamster ovary cells, HeLa cells, human embryonic kidney 293 cells, and 3T3 L1 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter and the cytomegalovirus promoter), along with the nucleic acids of the invention.
3. Host Cells—Eukaryotic Organisms
The term “host” or “host cell” includes not only prokaryotes, such as E. coli, but also eukaryotic cells such as fungus, insect, plant, and animal cells. Animal cells include, for example, COS cells and HeLa cells. Fungal cells include yeast cells, such as Saccharomyces cereviseae cells. Host cells containing a nucleic acid (e.g., a vector) of the present invention can be used for such purposes as propagating a vector, producing a nucleic acid (e.g., DNA, RNA, or antisense RNA), or expressing a polypeptide or a fragment thereof.
Host cells can be transformed with a DNA molecule (e.g., a vector) using techniques known to those of ordinary skill in the art, such as, without limitation, calcium phosphate or lithium acetate precipitation, electroporation, lipofection and particle bombardment. Other useful methods include, for example, infective, vector-containing bacterial strains (such as Agrobacterium rhizogenes and Agrobacterium tumefaciens) according to Zambryski (1992) Ann. Rev. Plant Mol. Biol. 43:465; pollen-tube transformation (Zhon-xun et al. (1989) Plant Cell 1:133); polyethylene glycol or electroporation transformation (Christou et al. (1987) Proc. Nat. Acad. Sci. USA 84:3662); and bilistic processes (Yang and Christou (1994) Particle Bombardment Technology for Gene Transfer).
As used herein, the term “transformed” is meant to encompass any suitable method for introducing a nucleic acid molecule into a host cell (e.g., transformation, transfection, transduction, or infection). A host cell can be transformed using any of the methods disclosed herein, for example. Furthermore, host cells that integrate an introduced nucleic acid sequence into their genome are called stably transformed cells. Stably transformed cells typically retain the introduced nucleic acid sequence with each cell division. Cells that contain introduced nucleic acid sequences that are not integrated into the genome are called transiently transformed cells. Transiently transformed cells typically lose at least a portion of the introduced nucleic acid sequence with each cell division. Thus, transformed cells can be either transiently and/or stably transformed.
Plants: Among the eukaryotic organisms featured in the invention are plants containing an exogenous nucleic acid that encodes a polypeptide of the invention.
Accordingly, a method according to the invention comprises making a plant having a nucleic acid construct described herein. Techniques for introducing exogenous nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Pat. Nos. 5,204,253 and 6,013,863. If a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures by techniques known to those skilled in the art. Transgenic plants can be entered into a breeding program, e.g., to introduce a nucleic acid encoding a polypeptide into other lines, to transfer the nucleic acid to other species or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants. Seeds (i.e., transgenic seeds) produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid encoding a novel polypeptide.
Polypeptides of the invention can be expressed in plants in a cell- or tissue-specific manner according to the regulatory elements included within a particular nucleic acid construct present in the plant. Suitable cells, tissues and organs in which to express a polypeptide of the invention include, without limitation, egg cell, central cell, synergid cell, zygote, ovule primordia, nucellus, integuments, endothelium, female gametophyte cells, embryo, axis, cotyledons, suspensor, endosperm, seed coat, ground meristem, vascular bundle, cambium, phloem, cortex, shoot or root apical meristems, lateral shoot or root meristems, floral meristem, leaf primordia, leaf mesophyll cells, and leaf epidermal cells, e.g., epidermal cells involved in forming the cuticular layer. Also suitable are cells and tissues grown in liquid media or on semi-solid media.
Fungi: Other eukaryotic organisms featured in the invention are fungi containing an exogenous nucleic acid that encodes a chimeric polypeptide of the invention.
Accordingly, a method according to the invention comprises introducing a nucleic acid construct as described herein into a fungus. Techniques for introducing exogenous nucleic acids into many fungi are known in the art, e.g., U.S. Pat. Nos. 5,252,726 and 5,070,020. Transformed fungi can be cultured by techniques known to those skilled in the art. Such fungi can be used to introduce a nucleic acid encoding a polypeptide into other fungal strains, to transfer the nucleic acid to other species or for further selection of other desirable traits.
A suitable group of fungi with which to practice the invention include fission yeast and budding yeast, such as Saccharomyces cereviseae, S. pombe, S. carlsbergeris and Candida albicans. Filamentous fungi such as Aspergillus spp. and Penicillium spp. are also useful.
Animals: Other eukaryotic host cells featured in the invention are animals cells (e.g., cells from insects such mosquitoes and flies, fish, and mammals such as rodents, bovines and porcines, including cell lines such as Chinese hamster ovary cells, HeLa cells, and COS cells) that contain an exogenous nucleic acid that encodes a polypeptide of the invention (e.g., a nucleic acid molecule encoding a polypeptide having the amino acid sequence of one or more one of the odd-numbered SEQ ID NOs from SEQ ID NO:43 to SEQ ID NO:165). In some embodiments, the animal cells are within transgenic animals. A variety of techniques known in the art can be used to generate such transgenic animals. Such techniques typically involve generating a plurality of animals whose genomes can be screened for the presence or absence of the transgene. For example, a transgene can be introduced into a non-human mammal by pronuclear microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82:6148), gene targeting into embryonic stem cells (Thompson et al. (1989) Cell 56:313), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3:1803), and transformation of somatic cells in vitro followed by nuclear transplantation (Wilmut et al. (1997) Nature 385:810–813; and Wakayama et al. (1998) Nature 394:369–374). When using mice to make a transgenic animal, suitable genetic backgrounds for use in making founder lines include, without limitation, C57B6, SJL/J, FVB/N, 129SV, BALB/C, C3H, and hybrids thereof.
The invention also provides methods to transfer nucleic acid molecules into host cells (e.g., prokaryotic, fungal, plant, and animal cells). Such methods can involve contacting a host cell with a nucleic acid molecule of the present invention, under conditions sufficient to allow the nucleic acid molecule to enter the cell. In particular, the invention provides methods to impart agronomically significant characteristics to plant cells, by contacting a plant cell with a nucleic acid molecule encoding an agronomically significant characteristic, under conditions sufficient to allow the nucleic acid molecule to enter the cell. Useful agronomically significant characteristics include, for example, those selected from the group consisting of: male sterility, self-incompatibility, foreign organism resistance, improved biosynthetic pathways, environmental tolerance, photosynthetic pathways, nutrient content, fruit ripening, oil biosynthesis, pigment biosynthesis, seed formation, starch metabolism, salt tolerance, cold/frost tolerance, drought tolerance, tolerance to anaerobic conditions, protein content, carbohydrate content, amino acid content, and fatty acid content.
Cultured cell lines: In some embodiments, host cells can be cultured cell lines. Cultured helper cell lines that express gag, pol, and env genes can be particularly useful. In some embodiments, a cell line can be transformed with a retroelement vector containing LTR sequences flanking a PPT, a PBS, and a nucleotide sequence of interest (e.g., a nucleotide sequence encoding an agronomically significant characteristic).
The following paragraph is designed to elaborate on the best mode and is not indicative of the sole means for making and carrying out the present invention. This paragraph is not intended to be limiting. The best way to make the present nucleic acids is to clone the nucleic acids from the respective organisms or amplify them from genomic cDNA by the polymerase chain reaction using appropriate primers. The best way to make the present retroelements is to assemble the nucleic acids using standard cloning procedures. Transcriptional controls can be manipulated by inserting enhancers in or near the 5′ LTR. Marker genes or genes of interest can be inserted within the retroelement.
In these new aspects of the invention, it is understood that the materials and methods described previously are useful in obtaining the present materials. Moreover, the discussion as to scope and usefulness of the invention, including the percent identities, retroelement uses and constructs, plants transfected, methods for improving crops, etc. are applicable for the present new aspects as well. For instance, combination of the previously disclosed materials with the present materials are certainly within the scope of the present disclosure.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Plant material and Southern hybridizations: The Arabidopsis Information Service supplied the following seed stocks (Kranz and Kirchheim (1987) Arabidopsis Inform. Serv. 24): Col-0, La-0, Kas-1, Co-4, Sei-0, Mv-0, L1-0, Cvi-0, Fi-3, Ba-1, Hau-0, Aa-0, Ms-0, Ag-0, Ge-0, No-0 and Mh-0. Genomic DNA was extracted using Qiagen genomic tips and protocols supplied by Qiagen (Valencia, Calif.). For Southern hybridizations, the resulting DNA was digested with EcoRI, electrophoresed on 0.8% agarose and transferred to Gene Screen Plus membranes using the manufacturer's alkaline transfer protocol (Perkin Elmer™ Life Sciences, Boston, Mass.). All hybridizations were performed as described by Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991–1995.
Library screening, probe preparation and PCR: Tat1 clones were obtained by screening a Landsberg erecta (La-0) 1 phage library (Voytas et al. (1990) supra), using a probe derived by PCR amplification of La-0 DNA. The primers for probe amplification were based on the three published Tat1 sequences (DVO158, 5′-GGGATCCGCAATTAGAATCT-3′ (SEQ ID NO:170); DVO159, 5′-CGAATTCGGTCCACTTCGGA-3′ (SEQ ID NO:171)). See, Peleman et al. (1991) Proc. Natl. Acad. Sci. USA 88:3618–3622. Subsequent probes were restriction fragments of cloned Tat1 elements, and all probes were radiolabeled by random priming (Promega). Long PCR was performed using the Expand Long Template PCR System (Boehringer Mannheim) with LTR-specific primers DVO354 (5′-CCACAAGATTCTAATTGCGGATTC-3′; SEQ ID NO:172) and DVO355 (5′-CCGAAATGGACCGAACCCGACATC-3′; SEQ ID NO:173). The protocol used was for PCR amplification of DNA up to 15 kb in length. The following PCR primers were used to confirm the structure of Tat1–3: DVO405 (5′-TTTCCAGGCTCTTGACGAGATTTG-3′; SEQ ID NO:174) for the 3′ non-coding region, DVO385 (5′-CGACTCGAGCTCCATAGCGATG-3′; SEQ ID NO:175) for the second ORF of Tat1–3 (note that the seventh base was changed from an A to a G to generate XhoI and SalI restriction sites) and DVO371 (5′-CGGATTGGGCCGAAATGGACCGAA-3′; SEQ ID NO:176) for the 3′ LTR.
DNA sequencing: Clones were sequenced either by the DNA sequencing facility at Iowa State University or with the fmol sequencing kit (Promega). DNA from the λ phage clones was initially subcloned into the vector pBluescript II KS− and transformed into the E. coli host strain XL1 Blue (Stratagene). Ausubel et al, supra. Subclones in the vector pMOB were used for transposon mutagenesis with the TN 1000 sequencing kit (Gold Biotechnologies). Transposon-specific primers were used for DNA sequencing reactions.
Sequence analysis: Sequence analysis was performed using the GCG software package (Devereux et al. (1984) Nucl. Acids Res. 12:387–395), DNA Strider 1.2 (Marck (1991) DNA Strider 1.2, Gif-sur-Yvette, France), the BLAST search tool (Altschul et al. (1990) J. Mol. Biol. 215:403–410) and the tRNAscan-SE 1.1 program (Lowe and Eddy (1997) Nucl. Acids Res. 25:955–964). Phylogenetic relationships were determined by the neighbor-joining distance algorithm using Phylip (Felsenstein (1993) PHYLIP (Phylogeny Inference Package), Department of Genetics, University of Washington, Seattle; Saitou and Nei (1987) Mol. Biol. Evol. 4:406–425) and were based on reverse transcriptase amino acid sequence that had been aligned with ClustalW1.7. Thompson et al. (1994) Nucl. Acids Res. 22:4673–4680. Transmembrane helices were identified using the PHDhtm program. Rost et al. (1995) Port. Science 4:521–533. All DNA sequences have been submitted to the DDBS/EMBL/GenBank databases under the accession numbers X12345, X23456, X34567 and X45678.
Tat1 is a retrotransposon: Tat1 insertions share features with retrotransposon solo LTRs. We reasoned that if Tat1 is a retrotransposon, then there should be full-length elements in the genome consisting of two Tat1 sequences flanking an internal retrotransposon coding region. To test this hypothesis, additional Tat1 elements were isolated by screening a Landsberg (La-0) genomic DNA library with a Tat1 probe. Twenty-one 1 phage clones were isolated and Southern analysis revealed two clones (pDW42 and pDW99) each with two copies of Tat1 (data not shown). The two Tat1 elements in each clone were sequenced, along with the intervening DNA. All Tat1 sequences shared >89% nucleotide identity to the previously characterized Tat1a–Tat1c elements. Peleman et al., supra. In clone pDW99, the 5′ and 3′ Tat1 sequences were 433 bases in length and only differed at two base positions. These Tat1 sequences also had conserved features of LTRs, including the dinucleotide end-sequences (5′ TG-CA 3′) that were part of 12 base inverted terminal repeats. If the two Tat1 elements in clone pDW99 were retrotransposon LTRs, then both, along with the intervening DNA, should be flanked by a target site duplication. A putative five base target site duplication (TATGT) was present immediately adjacent to the 5′ and 3′ Tat1 elements, supporting the hypothesis that they and the intervening DNA inserted as a single unit. In clone pDW42, the 5′ Tat1 was 432 bases in length and shared 98% nucleotide sequence identity to the 3′ Tat1. The last ˜74 bases of the 3′ Tat1 was truncated during library construction and lies adjacent to one phage arm. A target site duplication, therefore, could not be identified in this clone.
DNA sequences were analyzed for potential coding information between the 5′ and 3′ Tat1 elements. Nearly identical ORFs of 424 and 405 amino acids were found encoded between the Tat1 sequences in PDW42 and pDW99, respectively. The derived amino acid sequences of these ORFs were used to search the DNA sequence database with the BLAST search tool, and significant similarity was found to the Zea mays retrotransposable element Zeon-1 (p=4.4×10−8). Hu et al. (1995) Mol. Gen. Genet. 248:471–480. The ORFs have ˜44% similarity across their entirety to the 628 amino acid ORF encoded by Zeon-1 (see below). The Zeon-1 ORF includes a zinc finger motif characteristic of retrotransposon gag protein RNA binding domains. Although the Tat1 ORFs do not include the zinc finger motif, the degree of similarity suggests that they are part of a related gag protein.
If the Tat1 sequences in pDW42 and pDW99 defined retrotransposon insertions, a PBS would be predicted to lie adjacent to the 5′ Tat1 elements in both clones. The putative Tat1 PBS shares similarity with the PBSs of Zeon-1 and another maize retrotransposon called Cinful (see below), but it is not complementary to an initiator methionine tRNA as is the case for most plant retrotransposons. Additionally, a possible polypurine tract (PPT), the primer for second strand cDNA synthesis, was observed one base upstream of the 3′ Tat1 sequence in both phage clones (5′-GAGGACTTGGGGGGCAAA-3′; SEQ ID NO:177). We concluded from the available evidence that Tat1 is a retrotransposon, and we have designated the 3960 base insertion in pDW42 as Tat1-1 and the 3879 base insertion in pDW99 as Tat1-2. It is apparent that both Tat1-2 and Tat1-2 are non-funtional. Their ORFs are truncated with respect to the coding information found in transposition-competent retrotransposons, and they lack obvious pol motifs.
In light of our findings, the previously reported Tat1 sequences can be reinterpreted. Tat1 a and Tat1b, which are flanked by putative target site duplications, are solo LTRs. Tat1c, the only element without a target site duplication, is actually the 5′ LTR and part of the coding sequence for a larger Tat1 element.
Copy number of Tat1 among A. thaliana ecotypes: To estimate Tat1 copy number, the 5′ LTR, gag and the 3′ non-coding region were used as separate probes in Southern hybridizations. The Southern filters contained genomic DNA from 17 ecotypes representing wild populations of A. thaliana from around the world. This collection of ecotypes had previously been used to evaluate retrotransposon population dynamics. Konieczny et al., supra; Voytas et al. (1990) supra; Wright et al. (1996) Genetics 142:569–578. Based on the hybridization with the gag probe, element copy number ranges from two to approximately ten copies per ecotype. The copy number of the LTRs is higher, likely due to the presence of two LTRs flanking full-length elements or solo LTRs scattered throughout the genome. The Tat1 copy number contrasts with the copy numbers (typically less than three per ecotype) observed for 28 other A. thaliana retrotransposon families. In addition, the Tat1-hybridizing restriction fragments are highly polymorphic among strains. This degree of polymorphism, coupled with the high copy number, suggested that Tat1 has been active in transposition since the separation of the ecotypes. The Tat1 3′ non-coding region contains DNA sequences from elsewhere in the genome: In an attempt to identify a complete and functional Tat1 element, LTR-specific primers were used in PCR reactions optimized for amplification of large DNA fragments. Most full-length retrotransposable elements are between five and six kb in length. DNAs from al 17 ecotypes were used as templates, and each gave amplification products of ˜3.2 kb, the size predicted for Tat1-1 and Tat1-2 (data not shown). In La-0, however, a 3.8 kb PCR product was also recovered. This PCR product was cloned, sequenced and called Tat1-3. This insertion is expected to be about 4.6 Kb in total length if the LTR sequences are included.
Tat1-3 differed from Tat1-1 and Tat1-2 in that it had two ORFs separated by stop codons and a 477 base insertion in the 3′ non-coding region. The first ORF (365 amino acids) was similar to but shorter than the ORFs of the other Tat1 elements. The sequences constituting the second ORF (188 amino acids) were not present in the other Tat1 insertions and were not related to other sequences in the DNA databases. Database searches with the 477 base insertion in the 3′ non-coding region, however, revealed three regions of similarity to other genomic sequences. A region of 113 bases matched a region of 26 bp repeats in the 5′ untranslated sequence of the AT-P5C1 mRNA, which encodes pyrroline-5-carboxylate reductase (p=2.1e-19). Verbruggen et al. (1993) Plant Physiol. 103: 771–781. In addition, 50 bases appear to be a remnant of another retrotransposon related to Tat1. These 50 bases are 71% identical to the 3′ end of the Tat1-3 LTR and the putative primer binding site. The putative primer binding site, however, is more closely related to those of other plant retrotransposons such as Huck-2 (Sanmiguel et al. (1996) Science 274: 765–768). Finally, sequences in the remainder of the insertion showed significant similarity to a region on chromosome 5. To confirm that Tat1-3 was not a PCR artifact, two additional primer pairs were used in separate amplifications. Both amplifications gave PCR products of the predicted sizes, which were cloned and confirmed to be Tat1-3 by DNA sequencing.
PCR amplification with the additional primer pairs also yielded a product 0.8 kb longer than that expected for Tat1-3. This product was cloned, sequenced and found to be another Tat1 element, designated Tat 1-4. This element has sequences similar to a Tat1 LTR, polypurine tract and the second ORF of Tat1-3. In Tat1-4, 1182 bases of DNA are found in the 3′ non-coding region at the position corresponding to the 477 base insertion in Tat1-3. This region does not match any sequences in the DNA databases.
Other Tat1-like elements in A. thaliana: A BLAST search of DNA sequences generated by the A. thaliana genome project identified two more solo LTRs similar to Tat1. All share similarities throughout, but most strikingly, they are very well conserved at the 5′ and 3′ ends where it is expected integrase would bind. Braiterman and Boeke (1994) Mol. Cell. Biol. 14:5731–5740. These conserved end-sequences suggests that the integrases encoded by full-length elements are also related, and that the LTRs have evolved under functional constraints; that is, they are not simply degenerate Tat1 LTRs. The two new LTRs are designated as Tat2-1 and Tat3-1. Tat2-1 is 418 bases long, is flanked by a five base target site duplication (CTATT) and is ˜63% identical to the Tat1-2 5′LTR. Tat3-1 is 463 bases long and is also flanked by a target site duplication (ATATT). Tat3-1 is ˜53% identical to the Tat1-2 5′ LTR.
Tat1 and Athila are related to Ty3/gypsy retrotransposons: Further analysis of data from the A. thaliana genome project revealed two slightly degenerate retrotransposons with similarity to the Tat1 ORF. These elements were identified within the sequence of the P1 phage clones MXA21 (Accession AB005247; bases 54,977–66,874) and MX110 (Accession AB005248; bases 24,125–35,848). Each has two LTRs, a putative PBS, and long ORFs between their LTRs. The genetic organization of these elements is depicted in
Because the characterized Tat1 insertions do not encode pol genes, this element family could not be classified. However, the amino acid sequence of the Tat1-2 ORF is 51% similar to the gag region of the MXA21 retrotransposon. Since plant retrotransposons within the Ty1/copia or Ty3/gypsy families, even those with highly similar pol genes, share little amino acid sequence similarity in their gag regions, Tat1 is likely a Ty3/gypsy element. This conclusion is further supported by the report that the Tat-like Zcon-1 retrotransposon is very similar to a Z. mays Ty3/gypsy element called cinful (Bennetzen, supra); however, only the 5′ LTR and putative primer binding site (PBS) sequences are available in the sequence database for analysis (Accession No. U68402). Because of the extent of similarity to Tat1, we have named the MXA21 insertion Tat4-1.
The gag region of the MX110 element is 62% similar (p=1.1×10−193) to the first ORF of Athila, which has previously been unclassified (Pelissier et al. (1995) Plant Mol. Biol. 29:441–452). This implies that Athila is also a Ty3/gypsy element, and we have designated the MX110 insertion as Athila1-1. Our classification of Athila as a Ty3/gypsy element is further supported by the observation that the Athila gag amino acid sequence shares significant similarity to the gag protein encoded by the Cyclops-2 Ty3/gypsy retrotransposon of pea (Accession AJ000640; p=1.1×10−46; data not shown). Further analysis of the available A. thaliana genome sequences identified three additional Athila homologs. They include an additional Athila1 element, designated Athila1-2, and two more distantly related Athila-like elements, designated Athila2-1 and Athila3-1.
In addition to similarities among their gag amino acid sequences, the Tat elements have short LTRs (<550 bp) and long 3′ non-coding regions (>2 kb). In contrast, the Athila-like elements have long LTRs (>1.2 kb) and are very large retrotransposons (>11 kb). One additional feature to note about both the Athila-like and Tat-like elements is the high degree of sequence degeneracy of their internal coding regions. This contrasts with the near sequence identity of their 5′ and 3′ LTRs, which is typically greater than 95%. Because a single template is used in the synthesis of both LTRs, LTR sequences are usually identical at the time of integration. The degree of sequence similarity between the LTRs suggests that most elements integrated relatively recently. The polymorphisms observed in the internal domains of these insertions, therefore, may have been present in their progenitors, and these elements may have been replicated in trans.
A novel, conserved coding region in Athila elements: A surprising feature of Athila1-1 is the presence of an additional ORF after integrase. Like gag, this ORF shares significant similarity across its entirety (p=3.8×10−8) to the second ORF of Athila. This ORF is also encoded by the Athila2-1 and Athila3-1 elements, although it is somewhat more degenerate. The presence of this coding sequence among these divergent retrotransposons suggests that it plays a functional role in the element replication cycle. However, the ORF shows no similarity to retrotransposon gag or pol genes. The retroviruses and some Ty3/gypsy retrotransposons encode an env gene after integrase. Although not well-conserved in primary sequence, both viral and retrotransposon envelope proteins share some structural similarities. They are typically translated from spliced mRNAs and the primary translation product encodes a signal peptide and a transmembrane domain near the C-terminus. All four families of Athila elements encode a domain near the center of the ORF that is strongly predicted to be a transmembrane region (70%–90% confidence, depending on the element analyzed). See, Rost et al., supra. Two retrotransposons, Athila and Athila2-1, also have a hydrophobic transmembrane domain near the 5′ end of their env-like ORFs, which may serve as a secretory signal sequence. Von Heijne (1986) Nucl. Acids Res. 14:4683–4690.
Two lineages of plant Ty3/gypsy retrotransposons: Relationships among Ty3/gypsy retrotransposons from A. thaliana and other organisms were assessed by constructing a neighbor-joining tree of their reverse transcriptase amino acid sequences. Including in the analysis were reverse transcriptases from two additional families of A. thaliana Ty3/gypsy elements that we identified from the unannotated genome sequence data (designated Tma elements; Tma1-1 and Tma3-1); two other Tma element families were identified in the genome sequence that did not encode complete reverse transcriptases (Tma2-1 and Tma4-1; Table 1). Also included in the phylogenetic analyses were reverse transcriptases from a faba bean retrotransposon and the cyclops-2 element from pea. The plant Ty3/gypsy group retrotransposons resolved into two lineages: One was made up of dell from lily, the IFG7 retrotransposon from pine, reina from Z. mays, and Tmal-1 and Tma3-1. This group of elements formed a single branch closely related to numerous fungal retrotransposons (branch 1). The second branch (branch 2) was well-separated from all other known Ty3/gypsy group elements, and was further resolved into two lineages: Athila1-1, cyclops-2 and the faba bean reverse transcriptase formed one lineage (the Athila branch), and Tat4-1 and Grande1-4 from Zea diploperennis formed a separate, distinct branch (the Tat branch).
Primer binding sites: Most plant Ty1/copia retrotransposons as well as the branch 1 Ty3/gypsy elements have PBSs complementary to the 3′-end of an initiator methionine tRNA. This is not the case for any of the branch 2 Ty3/gypsy elements. We compared the putative PBSs of Tat-branch and Athila-branch elements to known plant tRNA genes as well as to the 11 tRNA genes that had been identified to date in sequences generated by the A. thaliana genome project. In addition, we searched the unannotated A. thaliana genome sequences and identified 30 more A. thaliana tRNA genes using the program tRNAscan-SE (Lowe and Eddy (1997) Nucl. Acids Res. 25:955–964). The PBS of Tat1 is complementary to 10 bases at the 3′ end of the asparagines tRNA for the AAC codon; these 10 bases are followed by a two base mismatch and six additional bases of perfect complementarity. The Tat4-1 PBS is complementary to 20 bases at the 3′ end of the arginine tRNA for the AGG codon with one mismatch 10 bases from the 3′ end; Huck-2, Grande-zm1, Grande1-4, and the retrotransposon-like insertion in the 3′ non-coding region of Tat1-3 all have 20-base perfect complementarity to this tRNA. The PBS of Athila1-1 is perfectly complementary to 15 bases at the 3′ end of the aspartic acid tRNA for the GAC codon, and Athila and Athila2-1 have 13 bases of complementarity to this tRNA. At this time there is no known plant tRNA complementary to the PBS of Zeon-1, which has the same PBS as the maize retrotransposon cinful. As more tRNA sequences become available, a candidate primer may be identified for these elements.
After identifying the retroelement elements in A. thaliana, the element called Cyclops2 from Pisum sativum (Chavanne et al., supra) was examined. Comparison of this element to the Athila-like elements both in size and amino acid and nucleotide sequence composition was made. Cyclops2 also encodes an open reading frame (ORF) in the position corresponding to the env-like gene of the Athila elements. This Cyclops2 ORF was examined using the same methods used to characterize the Athila group env-like genes (see Example 1). The Cyclops2 ORF was found to have a potential splice site at its N-terminus and transmembrane domains at the N-terminus, the central region and the C-terminus. Based on the presence of these features, it was concluded that Cyclops2 is a retrovirus-like retroelement that encodes on env-like gene.
Library Screening and Southern Hybridization: A soybean genomic lambda phage library (line L85–3044) was initially screened with a reverse transcriptase probe under low stringency conditions (50° C. with a 1% SDS wash; Church and Gilbert supra). The library was previously described (Chen et al. (1998) Soybean Genetics Newsletter 25:132–134). The probe was obtained by PCR amplification of genomic P. sativum DNA using primers based on the reverse transcriptase of Cyclops2 (DVO701 and DVO702). All probes were radio-labeled using random primers and protocols supplied by Promega. For Southern hybridizations, DNA was digested, electrophoresed on 0.8% agarose gels, and transferred to Gene Screen Plus membranes using the manufacturer's alkaline transfer protocol (Perkin Elmer™ Life Sciences). All high stringency hybridizations were as described by Church and Gilbert, supra.
DNA sequencing: Lambda phage clones were subcloned into the vector pBluescript KSII—and transformed into the E. coli host strain XL1 Blue (Stratagene) as described by Ausubel et al., supra. Subclones were sequenced by primer walking at the Iowa State University DNA sequencing facility.
Sequence Analysis: DNA Sequence analysis was performed using the GCG software package (Devereux et al., supra), DNA Strider 1.2 (Marck (1991) DNA Strider 1.2, Gif-sur-Yvette, France) and the BLAST search tool (Altschul et al., supra). Phylogenetic relationships were determined by the neighbor-joining distance algorithm (Saitou and Nei, supra) using PAUP v4.0 beta 1 (Swofford (1993) Illinois Natural History Survey, Champaign, IL.) and were based on reverse transcriptase amino acid sequences that had been aligned with ClustalX v1.63 b (Thompson et al., supra). Transmembrane helices were identified using the PHDhtm program and TMPred (Rost et al., supra; and Hofmann and Stoffel (1993) Biol. Chem. 374:166).
Retrovirus-like elements in Glycine max. Soybean retrovirus-like elements were identified by a low stringency (50° C.) screen of a soybean lambda library using a reverse transcriptase probe. The probe was based on a sequence from Cyclops2 (Chavanne et al., supra). The screen produced 63 λ clones that appeared to contain a retrovirus-like reverse transcriptase based on hybridization to the probe. Thirty-five of these putative elements were sequenced to varying degrees and 24 encoded readily identifiable retrovirus-like sequences. Most of the elements were distantly related and had premature stop codons, frame shifts, deletions or insertions. A related group of three elements and another related pair were completely sequenced and analyzed. The three elements in the first group are referred to as Calypso1-1, Calypso1-2, and Calypso1-3. The elements in the second pair are referred to as Calypso2-1 and Calypso2-2. The remaining soybean retrovirus-like elements will be given the Calypso name and a sequential designator number based on their family grouping.
The Calypso retrovirus-like elements had the same overall structure and sequence homology as the previously described Athila and Cyclops elements. The elements were about 12 kb in length; they had a 5′ LTR, a Primer Binding Site (PBS), a gag protein, a pol protein, a spacer, an env-like protein, another spacer region, a Polypurine Tract (PPT) and a 3′ LTR. The LTRs varied from about 1.3 to about 1.5 kb in length, and characteristically began with TG and ended with CA. The PBS was similar to that used by the Athila and Cyclops elements; it is 4 to 6 bases past the 5′ LTR and matches the 3′ end of a soybean aspartic acid tRNA for 18 to 19 bases with 1 mismatch. The fact that the sequences of the Calypso primer binding sites are shared with the A. thaliana and P. sativum retrovirus-like elements, indicates that this sequence is a unique marker for envelope-encoding retroelements. The gag protein extends ˜850 amino acids and encodes a zinc finger domain (characterized by the amino acid motif CxxCxxxHxxxxC; SEQ ID NO:178) and a protease domain (characterized by the amino acid motif LIDLGA (SEQ ID NO:179)). These domains are located at approximately the same positions within gag as in other retroelements. The approximately 600 amino acid reverse transcriptase region follows gag and has the conserved plant retrovirus-like motifs that approximate the following amino acids: KTAF (SEQ ID NO:180), MP/SFGLCNA (SEQ ID NO:181), V/I/MEVFMDDFS/WV/I (SEQ ID NO:182), FELMCDASDYAI/VGAVLGQR (SEQ ID NO:183), and YATT/IEKEL/MLAIVF/YAL/FEKFR/KSYLI/VGSR/KV (SEQ ID NO:184), respectively. The approximately 450 amino acid integrase domain has the plant retrovirus-like integrase motifs that approximate HCHxSxxGGH30xCDxCQR (SEQ ID NO:185) for the Zn finger as well as two other motifs that approximate WGIDFI/V/MGP (SEQ ID NO:186), and PYHPQTxGQA/VE (SEQ ID NO:187). After integrase, there is a ˜0.7 kb spacer then a ˜450 amino acid env-like protein coding region. The env-like protein of the Calypso elements is well conserved through most of the ORF but conservation decreases toward the C-terminus. The conservation includes 2 or 3 presumed transmembrane domains and a putative RNA splice site acceptor. The coding sequence for the env-like protein is followed by a ˜2 kb spacer and then a polypurine tract with the approximate sequence ATTTGGGGG/AANNT (SEQ ID NO:188). The 3′ LTR starts immediately after the final T of the PPT.
Calypso elements are abundant and heterogeneous: The Calypso elements appear to be abundant in the soybean genome. High stringency Southern blots of soybean DNA probed with reverse transcriptase, gag or env-like sequences produced smeared hybridization patterns, suggesting that the elements are abundant and heterogeneous. Their heterogeneity was also supported by DNA sequence analysis, which revealed a maximum of 93% nucleotide identity among elements, and most elements averaged ˜88% nucleotide identify. This identity can be region-specific or dispersed over the element's entirety. For example, reverse transcriptase, integrase and envelope-like coding regions may be well conserved, whereas the LTR, gag and spacer regions may have very little sequence conservation.
Phylogenetic analysis of Calypso reverse transcriptase: The reverse transcriptase of retroelements is the preferred protein for assessment of phylogenetic relationships (Xiong and Eickbush, supra). This is due to the high degree of amino acid sequence conservation found in reverse transcriptase proteins from many sources. The Calypso retrovirus-like elements were compared to previously described Ty3/gypsy and retrovirus-like elements from plants, fungi and invertebrate animals. The Calypso elements formed a distinct group with other plant retrovirus-like elements from A. thaliana and P. sativum and Faba bean. This group did not include plant Ty3/gypsy elements that are members of the metavirus genus. This indicates that the plant retrovirus-like elements from these four plant species are closely related and form a new element group that may be present in all or most plant species.
The Calypso reverse transcriptase and integrase are well-conserved: Frame shifts in the retrovirus-like elements were repaired through sequence comparison between the retrovirus-like elements from A. thaliana, P. sativum and G. max. Restoration typically involved an insertion or deletion of a single nucleotide or a single nucleotide substitution. When the edited ORFs of seven plant retrovirus-like elements from three species were compared, it was found that the gag domain had very little conservation. The amino acid sequence around the protease domain was reasonably conserved (˜50%) but the reverse transcriptase and integrase domains were highly conserved (˜70%).
The env-like ORF of Calypso is well-conserved: Animal retrovirus env proteins share little in common. They are however cleaved into two functional units that consist of the surface (SU) and transmembrane (TM) peptides. The SU peptide contains a transmembrane secretory signal at the N-terminus. The TM peptide has two transmembrane domains, one at the N-terminus, which functions in membrane fusion, and another near the C-terminus, which acts as an anchor site. The retrovirus env protein is expressed from an RNA that is spliced near the beginning of the env ORF. There are currently nine Athila group elements from A. thaliana that have an identifiable env-like ORF. Alignment of the env-like amino acid sequence shows that there are five subgroups of env-like proteins in the Athila family. Three are distinct, four are closely related and another pair is closely related. As a whole, these env-like sequences share limited homology over the entire length of the ORF, but within subgroups, they share high homology (data not shown). Some of the Athila env-like proteins have an apparent secretory peptide and a central transmembrane domain, suggesting that they may have an env-like function.
Among the Calypso elements, seven have been characterized that encode env-like ORFs. These env-like ORFs form four families that have a high degree of overall sequence similarity beginning at the first methionine and continuing for three quarters of the ORF; sequence similarity falls off dramatically near the C-terminus. The amino acid sequence at the first methionine has the consensus sequence QMASR/KKRR/KA (SEQ ID NO:189), which appears to be a nuclear targeting signal, however, the program PSORT only predicts a 0.300 confidence level for this targeting role (Nakai and Horton (1999) Trends Biochem. Sci. 24:34–36). A similar sequence (ASKKRK; SEQ ID NO:190) is found at the same position in the env-like ORF of Cyclops2, suggesting that it serves a similar purpose. No other potential targeting peptide stands out from the sequence that has been analyzed so far. There is a conserved region that is predicted to be a transmembrane domain near the center of the Calypso env-like protein and a second transmembrane domain located at variable positions near the C-terminus. These may be the fusion and anchor functions of a TM peptide. It should also be noted that five of the seven ORFs are predicted to have a transmembrane domain that is just before and includes the first methionine. This N-terminal transmembrane domain may be a secretory signal of an SU peptide. The program Tmpred estimates these transmembrane domains to be significant based on a score >500 (Hofmann and Stoffel, supra). These three transmembrane domains are found in the Cyclops2 env-like protein at similar locations but at a reduced significance score. Another feature of the Calypso env-like ORF is the conserved splice site that is predicted to be at the first methionine by the program NetGene2 v. 2.4 with a confidence level of 1.00 (Hebsgaard et al. (1996) Nucl. Acids Res. 24:3439–3452); and Brunak et al. (1991) J. Mol. Biol. 220:49–65). There are other less preferred putative splice sites in the region, but only the splice site near the methionine is optimally placed and conserved in all seven env-like ORFs.
ClustalX v1.63b (Thompson et al., supra) was used to align nucleotide sequences of Calypso1-1, Calypso1-2 and Calypso1-3. A consensus sequence was generated from the ClustalX output. The consensus sequence file was then translated and compared using ClustalX to amino acid sequences of retrovirus-like elements from soybean, pea (Cyclops2) and A. thaliana (Athila-like elements) using the GCG computer software package (Devereux et al., supra). For coding regions encompassing protease, reverse transcriptase and integrase, a new consensus sequence was generated that best matched the coding information in all elements. This second consensus sequence forms the protease, reverse transcriptase and integrase genes of the generic element. The gag gene of the generic element is a consensus sequence generated by editing elements between Calypso1-1 and Calypso2-2. The env gene is a consensus sequence based on env gene sequence alignments of all Calypso elements. All non-coding regions for the generic element were obtained from Calypso1-2, with the exception of the LTRs, which were taken from Calypso1-1.
A generic retroelement will be constructed by first generating a DNA sequence that approximates the sequence of the generic element. An element that closely matches the consensus—for example, Calypso1-1—will be modified by PCR-based site-directed mutagenesis (Ausubel et al., supra). Modifications will be sequentially introduced into the starting element until it conforms to the sequence of the generic element.
The generic element will be modified so that it will be expressed at high levels in plant cells. This will be accomplished by inserting an enhancer—such as the cauliflower mosaic virus 35S enhancer—into the 5′ LTR. To monitor replication, a maker gene will be inserted into the virus between the end of the coding region for the env gene and the polypurine tract. The marker gene may encode resistance to an herbicide or antibiotic. The modified generic element will then be introduced into plant cells by standard means of plant transformation.
The degenerate oligos DVO1197 (5′ GTG-CGN-AAR-GAR-GTN-NTN-AAR-YT 3′ (SEQ ID NO:166) for the N terminal amino acid sequence VRKEVLKL (SEQ ID NO:167)) and DVO1198 (5′ AAC-YTT-NGW-RAA-RTC-YTT-DAT-RAA 3′ (SEQ ID NO:168) for the C terminal amino acid sequence VKSFDKIF (SEQ ID NO:169)) were used to amplify the Xiong/Eickbush plant retroelement reverse transcriptase domain from genomic DNA of the following plants: New sequences were obtained from Nicotiana tabacum (Tobacco), Platanus occidentalis (sycamore), Gossypium hirsutum (cotton), Lycopersicon esculentum (tomato), Solanum tuberosum (potato), Oryza sativa (rice), Triticum aestivum (wheat), Hordeum vulgare (barley), Sorghum bicolor (sorghum), Avena sativa (oat), Secale cereale (rye). No sequence was obtained for Pinus coulteri (big-cone pine), Zea mays (corn), Zea mays subspecies Parviglumis (teosinte), and a Tripsacum species. A positive control for PCR was used to obtain previously known sequences from: Arabidopsis thaliana, Pisum sativum (pea) and three varieties (Hard 89, L85 and Williams) of Glycine max (soybean).
The conditions for PCR were as follows: 50 μL reactions were set up with 5 μL of Promega Taq enzyme buffer, 1 μL of Taq enzyme, 5 μL of Promega 25 mM magnesium chloride, 100 ng genomic DNA, 5 μL of 2.5 mM Promega dNTP (deoxynucleotide mixture) and 7.5 μL of each oligo from a 20 pmol/μL solution. The reaction volume was brought to 50 μL with deionized water. PCR was done with a 92° C. melting temperature for 2 minutes for the first cycle and 20 seconds for each cycle thereafter, 50° C. annealing temperature for 30 seconds and 72° C. extension for 1 minute 30 seconds. There was a total of thirty cycles. Based on known sequence data, a 762 base pair band was expected for each PCR reaction.
The PCR reactions were run out on a 0.8% agarose gel, the approximately sized 762 based pair band was excised for each species and ligated to a T-vector pBLUESCRIPT II KS-. The ligations were transformed into the E. coli strain XL1 BLUE, selected and sequenced. The results are in the Sequence Listing, at SEQ ID NOS:42 through 165, with the even-numbered sequences in that range being the DNA sequences identified, and the odd-numbered 3sequences being the amino acid sequences deduced from the DNA sequences.
The amino acid sequences obtained through the PCR survey (Example 5) were analyzed to determine whether they contain conserved amino acid signatures that distinguish them from previously characterized reverse transcriptases. To accomplish this, reverse transcriptase amino acid sequences were obtained from characterized retrotransposons and retroviruses. These include Tat4-1, f26h6, Rire2, Grand1-4, Vulgar, Cinful, Mag, SURL, Mdg1, 412, Cft-1, boty, Skippy, Maggy, Grasshopper, Tf1, Tma1-1, Tma3-1, Del1, Reina, IFG7, Ty3, Tom, 297, 17.6, TED, Gypsy, Yoyo, Ulysses, Woot, Cer1, HIV1, RSV, and MuMLV. The boundaries of these reverse transcriptase amino acid sequences were as defined by Xiong and Eickbush, supra. The characterized reverse transcriptases then were compared to the derived amino acid sequences of the PCR products obtained from the survey of plant genomes (portions of the odd-numbered sequences from SEQ ID NO:43 to SEQ ID NO:165 within the boundaries defined by Xiong and Eickbush; SEQ ID NOS are as indicated in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation in part of U.S. patent application Ser. No. 09/586,106 filed on Jun. 2, 2000, now U.S. Pat. No. 6,720,479, which is a continuation in part of U.S. patent application Ser. No. 09/322,478 filed on May 28, 1999, now U.S. Pat. No. 6,331,662, which claims priority to U.S. Provisional Patent Application Ser. No. 60/087,125 filed on May 29, 1998. These are hereby incorporated by reference in their entirety for all purposes.
The present invention was funded, in part, by the United States Department of Agriculture, Contract Number 98-CRHF-0-6019 (USDA/CSREES Project number IOW03120). The United States Government may have certain rights in the invention.
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| Number | Date | Country | |
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| Parent | 09586106 | Jun 2000 | US |
| Child | 10395607 | US | |
| Parent | 09322478 | May 1999 | US |
| Child | 09586106 | US |
