AU2007234625A1 - Sugar and lipid metabolism regulators in plants II - Google Patents

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Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

DIVISIONAL

APPLICANT:

Invention Title: BASF PLANT SCIENCE GmbH SUGAR AND LIPID METABOLISM REGULATORS IN PLANTS II The following statement is a full description of this invention, including the best method of performing it known to me: WO 02/099076 PCT/US02/17937 Sugar and Lipid Metabolism Regulators in Plants II SBACKGROUND OF THE INVENTION

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z CROSS REFERENCE TO RELATED APPLICATIONS The present invention claims the priority benefit of U.S. Provisional Patent Application Serial No. 60/295,680 filed June 4, 2001, the entire contents of which are hereby I incorporated by reference.

SField of the Invention This invention relates generally to nucleic acid sequences encoding proteins that are 0related to the presence of seed storage compounds in plants. More specifically, the present invention relates to nucleic acid sequences encoding sugar and lipid metabolism regulator proteins and the use of these sequences in transgenic plants.

Background Art The study and genetic manipulation of plants has a long history that began even before the famed studies of Gregor Mendel. In perfecting this science, scientists have accomplished modification of particular traits in plants ranging from potato tubers having increased starch content to oilseed plants such as canola and sunflower having increased or altered fatty acid content. With the increased consumption and use of plant oils, the modification of seed oil content and seed oil levels has become increasingly widespread (e.g.

Tipfer et al. 1995, Science 268:681-686). Manipulation of biosynthetic pathways in transgenic plants provides a number of opportunities for molecular biologists and plant biochemists to affect plant metabolism giving rise to the production of specific higher-value products. The seed oil production or composition has been altered in numerous traditional oilseed plants such as soybean Patent No. 5,955,650), canola Patent No.

5,955,650), sunflower Patent No. 6,084,164) and rapeseed (T6pfer et al. 1995, Science 268:681-686), and non-traditional oil seed plants such as tobacco (Cahoon et al. 1992, Proc.

Natl. Acad. Sci. USA 89:11184-11188).

Plant seed oils comprise both neutral and polar lipids (see Table The neutral lipids contain primarily triacylglycerol, which is the main storage lipid that accumulates in oil bodies in seeds. The polar lipids are mainly found in the various membranes of the seed cells, e.g. the endoplasmic reticulum, microsomal membranes and the cell membrane. The neutral and polar lipids contain several common fatty acids (see Table 2) and a range of less common 1 WO 02/099076 PCT/US02/17937 fatty acids. The fatty acid composition of membrane lipids is highly regulated and only a select number of fatty acids are found in membrane lipids. On the other hand, a large number of unusual fatty acids can be incorporated into the neutral storage lipids in seeds of many plant species (Van de Loo F.J. et al. 1993, Unusual Fatty Acids in Lipid Metabolism in Plants pp. 91-126, editor TS Moore Jr. CRC Press; Millar et al. 2000, Trends Plant Sci. 5:95-101).

Table 1.

Plant Lipid Classes Neutral Lipids Triacylglycerol (TAG) Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar Lipids Monogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG) Phosphatidylglycerol (PG) Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylinositol (PI) Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol WO 02/099076 PCT/US02/17937 Table 2.

Common Plant Fatty Acids 16:0 Palmitic acid 16:1 Palmitoleic acid 16:3 Palmitolenic acid 18:0 Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 18:3 Linolenic acid y-18:3 Gamma-linolenic acid* 20:0 Arachidic acid 22:6 Docosahexanoic acid (DHA) 20:2 Eicosadienoic acid 20:4 Arachidonic acid (AA) 20:5 Eicosapentaenoic acid (EPA) 22:1 Erucic acid *These fatty acids do not normally occur in plant seed oils, but their production in transgenic plant seed oil is of importance in plant biotechnology.

Lipids are synthesized from fatty acids and their synthesis may be divided into two parts: the prokaryotic pathway and the eukaryotic pathway (Browse et al. 1986, Biochemical J. 235:25-31; Ohlrogge Browse 1995, Plant Cell 7:957-970). The prokaryotic pathway is located in plastids that are the primary site of fatty acid biosynthesis. Fatty acid synthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase). Malonyl-CoA is converted to malonyl-acyl carrier protein (ACP) by the malonyl- CoA:ACP transacylase. The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes a condensation reaction in which the acyl group from acetyl-CoA is transferred to malonyl- ACP to form 3-ketobutyryl-ACP. In a subsequent series of condensation, reduction and dehydration reactions the nascent fatty acid chain on the ACP cofactor is elongated by the step-by-step addition (condensation) of two carbon atoms donated by malonyl-ACP until a 16- or 18-carbon saturated fatty acid chain is formed. The plastidial delta-9 acyl-ACP desaturase introduces the first unsaturated double bond into the fatty acid. Thioesterases cleave the fatty acids from the ACP cofactor and free fatty acids are exported to the WO 02/099076 PCT/US02/17937 cytoplasm where they participate as fatty acyl-CoA esters in the eukaryotic pathway. In this pathway the fatty acids are esterified by glycerol-3-phosphate acyltransferase and Slysophosphatidic acid acyltransferase to the sn-1 and sn-2 positions of glycerol-3-phosphate, respectively, to yield phosphatidic acid The PA is the precursor for other polar and

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Z neutral lipids, the latter being formed in the Kennedy pathway (Voelker 1996, Genetic Engineering ed.:Setlow 18:111-113; Shanklin Cahoon 1998, Annu. Rev. Plant Physiol.

Plant Mol. Biol. 49:611-641; Frentzen 1998, Lipids 100:161-166; Millar et al. 2000, Trends tn Plant Sci. 5:95-101).

SStorage lipids in seeds are synthesized from carbohydrate-derived precursors. Plants m have a complete glycolytic pathway in the cytosol (Plaxton 1996, Annu. Rev. Plant Physiol.

Plant Mol. Biol. 47:185-214) and it has been shown that a complete pathway also exists in the Splastids of rapeseeds (Kang Rawsthome 1994, Plant J. 6:795-805). Sucrose is the primary source of carbon and energy, transported from the leaves into the developing seeds. During the storage phase of seeds, sucrose is converted in the cytosol to provide the metabolic precursors glucose-6-phosphate and pyruvate. These are transported into the plastids and converted into acetyl-CoA that serves as the primary precursor for the synthesis of fatty acids. Acetyl-CoA in the plastids is the central precursor for lipid biosynthesis. Acetyl-CoA can be formed in the plastids by different reactions and the exact contribution of each reaction is still being debated (Ohlrogge Browse 1995, Plant Cell 7:957-970). It is however accepted that a large part of the acetyl-CoA is derived from glucose-6-phospate and pyruvate that are imported from the cytoplasm into the plastids. Sucrose is produced in the source organs (leaves, or anywhere that photosynthesis occurs) and is transported to the developing seeds that are also termed sink organs. In the developing seeds, the sucrose is the precursor for all the storage compounds, i.e. starch, lipids and partly the seed storage proteins.

Therefore, it is clear that carbohydrate metabolism in which sucrose plays a central role is very important to the accumulation of seed storage compounds.

Although lipid and fatty acid content of seed oil can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the seed oil content of a plant, and in some cases, has allowed for the alteration of seed oils in ways that could not be accomplished by breeding alone (see, e.g., Topfer et al. 1995, Science 268:681-686). For example, introduction of a Al 2 -hydroxylase nucleic acid sequence into transgenic tobacco resulted in the introduction of a novel fatty acid, ricinoleic acid, into the tobacco seed oil (Van de Loo et al. 1995, Proc. Natl. Acad. Sci USA 92:6743-6747). Tobacco plants have also been engineered to produce low levels of WO 02/099076 PCT/US02/17937 petroselinic acid by the introduction and expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992, Proc. Natl. Acad. Sci USA 89:11184-11188).

The modification of seed oil content in plants has significant medical, nutritional and economic ramifications. With regard to the medical ramifications, the long chain fatty acids Z (C18 and longer) found in many seed oils have been linked to reductions in "N hypercholesterolemia and other clinical disorders related to coronary heart disease (Brenner 1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a plant having increased i levels of these types of fatty acids may reduce the risk of heart disease. Enhanced levels of ND seed oil content also increase large-scale production of seed oils and thereby reduce the cost ¢C of these oils.

In order to increase or alter the levels of compounds such as seed oils in plants, 0 nucleic acid sequences and proteins regulating lipid and fatty acid metabolism must be identified. As mentioned earlier, several desaturase nucleic acids such as the A 6 -desaturase nucleic acid, A 12 -desaturase nucleic acid and acyl-ACP desaturase nucleic acid have been cloned and demonstrated to encode enzymes required for fatty acid synthesis in various plant species. Oleosin nucleic acid sequences from such different species as Brassica, soybean, carrot, pine and Arabidopsis thaliana have also been cloned and determined to encode proteins associated with the phospholipid monolayer membrane of oil bodies in those plants.

It has also been determined that two phytohormones, gibberellic acid (GA) and absisic acid (ABA), are involved in overall regulatory processes in seed development (e.g.

Ritchie Gilroy 1998, Plant Physiol. 116:765-776; Arenas-Huertero et al. 2000, Genes Dev.

14:2085-2096). Both the GA and ABA pathways are affected by okadaic acid, a protein phosphatase inhibitor (Kuo et al. 1996, Plant Cell. 8:259-269). The regulation of protein phosphorylation by kinases and phosphatases is accepted as a universal mechanism of cellular control (Cohen 1992, Trends Biochem. Sci. 17:408-413. Likewise, the plant hormones ethylene Zhou et al. 1998, Proc. Natl. Acad. Sci. USA 95:10294-10299; Beaudoin et al. 2000, Plant Cell 2000:1103-1115) and auxin Colon-Carmona et al. 2000, Plant Physiol. 124:1728-1738) are involved in controlling plant development as well.

Although several compounds are known that generally affect plant and seed development, there is a clear need to specifically identify factors that are more specific for the developmental regulation of storage compound accumulation and to identify genes which have the capacity to confer altered or increased oil production to its host plant and to other plant species. This invention discloses a large number of nucleic acid sequences from Arabidopsis thaliana. These nucleic acid sequences can be used to alter or increase the levels of seed storage compounds such as proteins, sugars and oils, in plants, including transgenic WO 02/099076 PCT/US02/17937 plants, such asrapeseed, canola, linseed, soybean, sunflower maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut, which are oilseed Splants containing high amounts of lipid compounds.

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Z SUMMARY OF THE INVENTION The present invention provides novel isolated nucleic acid and amino acid sequences associated with the metabolism of seed storage compounds in plants.

SThe present invention also provides an isolated nucleic acid from Arabidopsis ND encoding a Lipid Metabolism Protein (LMP), or a portion thereof. These sequences may be c used to modify or increase lipids and fatty acids, cofactors and enzymes in microorganisms and plants.

SArabidopsis plants are known to produce considerable amounts of fatty acids like linoleic and linolenic acid (see, Table 2) and for their close similarity in many aspects (gene homology etc.) to the oil crop plant Brassica. Therefore nucleic acid molecules originating from a plant like Arabidopsis thaliana are especially suited to modify the lipid and fatty acid metabolism in a host, especially in microorganisms and plants. Furthermore, nucleic acids from the plant Arabidopsis thaliana can be used to identify those DNA sequences and enzymes in other species which are useful to modify the biosynthesis of precursor molecules of fatty acids in the respective organisms.

The present invention further provides an isolated nucleic acid comprising a fragment of at least 15 nucleotides of a nucleic acid from a plant (Arabidopsis thaliana) encoding a Lipid Metabolism Protein (LMP), or a portion thereof.

Also provided by the present invention are polypeptides encoded by the nucleic acids, and heterologous polypeptides comprising polypeptides encoded by the nucleic acids, and antibodies to those polypeptides.

Additionally, the present invention relates to and provides the use of LMP nucleic acids in the production of transgenic plants having a modified level of a seed storage compound. A method of producing a transgenic plant with a modified level of a seed storage compound includes the steps of transforming a plant cell with an expression vector comprising a LMP nucleic acid, and generating a plant with a modified level of the seed storage compound from the plant cell. In a preferred embodiment, the plant is an oil producing species selected from the group consisting of rapeseed, canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut, for example.

According to the present invention, the compositions and methods described herein r can be used to increase or decrease the level of a LMP in a transgenic plant comprising Sincreasing or decreasing the expression of a LMP nucleic acid in the plant. Increased or decreased expression of the LMP nucleic acid can be achieved through in vivo mutagenesis 0 Z of the LMP nucleic acid. The present invention can also be used to increase or decrease the N, level of a lipid in a seed oil, to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch in a seed or plant.

t Also included herein is a seed produced by a transgenic plant transformed by a LMP O DNA sequence, wherein the seed contains the LMP DNA sequence and wherein the plant is Cc true breeding for a modified level of a seed storage compound. The present invention r additionally includes a seed oil produced by the aforementioned seed.

Further provided by the present invention are vectors comprising the nucleic acids, host cells containing the vectors, and descendent plant materials produced by transforming a plant cell with the nucleic acids and/or vectors.

According to the present invention, the compounds, compositions, and methods described herein can be used to increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch or other carbohydrate in a seed or plant. A method of producing a higher or lower than normal or typical level of storage compound in a transgenic plant, comprises expressing a LMP nucleic acid from Arabidopsis thaliana in the transgenic plant, wherein the transgenic plant is Arabidopsis thaliana or a species different from Arabidopsis thaliana. Also included herein are compositions and methods of the modification of the efficiency of production of a seed storage compound.

Accordingly, the present invention provides novel isolated LMP nucleic acids and isolated LMP amino acid sequences from Arabidopsis thaliana, as well as active fragments, analogs and orthologs thereof.

The present invention also provides transgenic plants having modified levels of seed storage compounds, and in particular, modified levels of a lipid, a fatty acid or a sugar.

The invention further provides methods for producing such aforementioned transgenic plants.

In yet another aspect, the present invention provides seeds and seed oils from such aforementioned transgenic plants.

AO 752756.1 "7 These and other aspects, features and advantages of the present invention will become O apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

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CN BRIEF DESCRIPTION OF THE FIGURES

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Figures 1A-B: Figure 1A shows the polynucleotide sequences of the open reading lt frame of Clone ID NO: pk002b from Arabidopsis thaliana (SEQ ID NO:1) of the present \invention. The polynucleotide sequence contains 2253 nucleotides. Figure 1B shows the deduced amino acid sequence of SEQ ID NO:1 (SEQ ID NO:2) (Clone ID NO: pk002b) of the present invention. The polypeptide sequence contains 750 amino acids. The standard one- 0letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figure 2A-B: Figure 2A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk020b from Arabidopsis thaliana (SEQ ID NO:3) of the present invention. The polynucleotide sequence contains 681 nucleotides. Figure 2B shows the deduced amino acid sequence of SEQ ID NO:3 (SEQ ID NO:4) (Clone ID NO: pk020b) of the present invention. The polypeptide sequence contains 226 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 3A-B: Figure 3A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk033 from Arabidopsis thaliana (SEQ ID NO:5) of the present invention. The polynucleotide sequence contains 1815 nucleotides. Figure 3B shows the deduced amino acid sequence of SEQ ID NO:5 (SEQ ID NO:6) (Clone ID NO: pk033) of the present invention. The polypeptide sequence contains 604 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 4A-B: Figure 4A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk037 from Arabidopsis thaliana (SEQ ID NO:7) of the present invention. The polynucleotide sequence contains 1773 nucleotides. Figure 4B shows the deduced amino acid sequence of SEQ ID NO:7 (SEQ ID NO:8) (Clone ID NO: pk037) of the present invention. The polypeptide sequence contains 590 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 5A-B: Figure 5A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk038 from Arabidopsis thaliana (SEQ ID NO:9) of the present invention. The polynucleotide sequence contains 1035 nucleotides. Figure 5B shows the deduced amino acid sequence of SEQ ID NO:9 (SEQ ID NO:10) (Clone ID NO: pk038) of AO 752756.1 o WO 02/099076 PCT/U S02/17937 Figures 6A-B: Figure 6A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk042a from Arabidopsis thaliana (SEQ ID NO:11) of the present Sinvention. The polynucleotide sequence contains 804 nucleotides. Figure 6B shows the deduced amino acid sequence of SEQ ID NO:11 (SEQ ID NO:12) (Clone ID NO: pk042a) of

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Z the present invention. The polypeptide sequence contains 267 amino acids. The standard one- C1 letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 7A-B: Figure 7A shows the polynucleotide sequences of the open reading t frame of Clone ID NO: pk053b from Arabidopsis thaliana (SEQ ID NO:13) of the present D0 invention. The polynucleotide sequence contains 272 nucleotides. Figure 7B shows the m deduced amino acid sequence of SEQ ID NO: 13 (SEQ ID NO: 14) (Clone ID NO: pk053b) of the present invention. The polypeptide sequence contains 90 amino acids. The standard one- O letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 8A-B: Figure 8A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk060 from Arabidopsis thaliana (SEQ ID NO:15) of the present invention. The polynucleotide sequence contains 603 nucleotides. Figure 8B shows the deduced amino acid sequence of SEQ ID NO:15 (SEQ ID NO:16) (Clone ID NO: pk060) of the present invention. The polypeptide sequence contains 200 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 9A-B: Figure 9A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk063b from Arabidopsis thaliana (SEQ ID NO:17) of the present invention. The polynucleotide sequence contains 1746 nucleotides. Figure 9B shows the deduced amino acid sequence of SEQ ID NO:17 (SEQ ID NO:18) (Clone ID NO: pk063b) of the present invention. The polypeptide sequence contains 581 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 10A-B: Figure 10A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk064 from Arabidopsis thaliana (SEQ ID NO:19) of the present invention. The polynucleotide sequence contains 1347 nucleotides. Figure 10B shows the deduced amino acid sequence of SEQ ID NO:19 (SEQ ID NO:20) (Clone ID NO: pk064) of the present invention. The polypeptide sequence contains 448 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 11A-B. Figure 11A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk073 from Arabidopsis thaliana (SEQ ID NO:21) of the present invention. The polynucleotide sequence contains 564 nucleotides. Figure 11B shows the deduced amino acid sequence of SEQ ID NO:21 (SEQ ID NO:22) (Clone ID NO: pk073) of WO 02/099076 PCT/US02/17937 the present invention. The polypeptide sequence contains 187 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

SFigures 12A-B. Figure 12A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk082 from Arabidopsis thaliana (SEQ ID NO:23) of the present Z invention. The polynucleotide sequence contains 753 nucleotides. Figure 12B shows the deduced amino acid sequence of SEQ ID NO:23 (SEQ ID NO:24) (Clone ID NO: pk082) of the present invention. The polypeptide sequence contains 250 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

SFigures 13A-B. Figure 13A shows the polynucleotide sequences of the open reading S frame of Clone ID NO: pk084 from Arabidopsis thaliana (SEQ ID NO:25) of the present invention. The polynucleotide sequence contains 1218 nucleotides. Figure 13B shows the Sdeduced amino acid sequence of SEQ ID NO:25 (SEQ ID NO:26) (Clone ID NO: pk084) of the present invention. The polypeptide sequence contains 405 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 14A-B. Figure 14A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk086 from Arabidopsis thaliana (SEQ ID NO:27) of the present invention. The polynucleotide sequence contains 420 nucleotides. Figure 14B shows the deduced amino acid sequence of SEQ ID NO:27 (SEQ ID NO:28) (Clone ID NO: pk086) of the present invention. The polypeptide sequence contains 139 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 15A-B. Figure 15A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk088 from Arabidopsis thaliana (SEQ ID NO:29) of the present invention. The polynucleotide sequence contains 549 nucleotides. Figure 15B shows the deduced amino acid sequence of SEQ ID NO:29 (SEQ ID NO:30) (Clone ID NO: pk088) of the present invention. The polypeptide sequence contains 182 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 16A-B. Figure 16A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk095 from Arabidopsis thaliana (SEQ ID NO:31) of the present invention. The polynucleotide sequence contains 987 nucleotides. Figure 16B shows the deduced amino acid sequence of SEQ ID NO:31 (SEQ ID NO:32) (Clone ID NO: pk095) of the present invention. The polypeptide sequence contains 328 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 17A-B. Figure 17A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk096 from Arabidopsis thaliana (SEQ ID NO:33) of the present invention. The polynucleotide sequence contains 2322 nucleotides. Figure 17B shows the WO 02/099076 PCT/US02/17937 deduced amino acid sequence of SEQ ID NO:33 (SEQ ID NO:34) (Clone ID NO: pk096) of the present invention. The polypeptide sequence contains 773 amino acids. The standard one- O letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 18A-D. Figure 18A shows the polynucleotide sequences of the open reading Z frame of Clone ID NO: pk097a from Arabidopsis thaliana (SEQ ID NO:35) of the present C1 invention. The polynucleotide sequence contains 2217 nucleotides. Figure 1SB shows the deduced amino acid sequence of SEQ ID NO:35 (SEQ ID NO:36) (Clone ID NO: pk097a) of I the present invention. The polypeptide sequence contains 738 amino acids. The standard one- 0 letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

mc Figure 18C shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk097b from Arabidopsis thaliana (SEQ ID NO:37) of the present invention. The O polynucleotide sequence contains 2154 nucleotides. Figure 18D shows the deduced amino acid sequence of SEQ ID NO:37 (SEQ ID NO:38) (Clone ID NO: pk097b) of the present invention. The polypeptide sequence contains 717 amino acids. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 19A-B. Figure 19A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk098 from Arabidopsis thaliana (SEQ ID NO:39) of the present invention. The polynucleotide sequence contains 690 nucleotides. Figure 19B shows the deduced amino acid sequence of SEQ ID NO:39 (SEQ ID NO:40) (Clone ID NO: pk098) of the present invention. The polypeptide sequence contains 229 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 20A-B. Figure 20A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk099 from Arabidopsis thaliana (SEQ ID NO:41) of the present invention. The polynucleotide sequence contains 732 nucleotides. Figure 20B shows the deduced amino acid sequence of SEQ ID NO:41 (SEQ ID NO:42) (Clone ID NO: pk099) of the present invention. The polypeptide sequence contains 243 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 21A-B. Figure 21A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pkl00 from Arabidopsis thaliana (SEQ ID NO:43) of the present invention. The polynucleotide sequence contains 2148 nucleotides. Figure 21B shows the deduced amino acid sequence of SEQ ID NO:43 (SEQ ID NO:44) (Clone ID NO: pkl00) of the present invention. The polypeptide sequence contains 715 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

WO 02/099076 PCT/US02/17937 Figures 22A-B. Figure 22A shows the polynucleotide sequences of the open reading frame of Clone ID NO: pk105 from Arabidopsis thaliana (SEQ ID NO:45) of the present invention.

SThe polynucleotide sequence contains 1305 nucleotides. Figure 22B shows the deduced amino acid sequence of SEQ ID NO:45 (SEQ ID NO:46) (Clone ID NO: pkl05) of the Z present invention. The polypeptide sequence contains 434 amino acids. The standard oneletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

Figures 23A-B. Figure 23A shows the polynucleotide sequences of the open reading In frame of Clone ID NO: pkl07 from Arabidopsis thaliana (SEQ ID NO:47) of the present D invention. The polynucleotide sequence contains 2466 nucleotides. Figure 23B shows the c deduced amino acid sequence of SEQ ID NO:47 (SEQ ID NO:48) (Clone ID NO: pkl07) of the present invention. The polypeptide sequence contains 821 amino acids. The standard one- Sletter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein.

Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.

In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, provides an isolated nucleic acid from a plant (Arabidopsis thaliana) encoding a Lipid Metabolism Protein (LMP), or a portion thereof.

One aspect of the invention pertains to isolated nucleic acid molecules that encode LMP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of an LMP-encoding nucleic acid LMP DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules cDNA or genomic DNA) and RNA molecules mRNA) and analogs of the DNA or RNA generated using nucleotide 12 WO 02/099076 PCT/US02/17937 analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of a gene: at least about 1000 nucleotides of sequence upstream from the 0 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. The nucleic acid molecule can be single-stranded

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z or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid C molecule is one which is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is In substantially free of sequences which naturally flank the nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated LMP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of O nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived a Arabidopsis thaliana cell). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, a nucleic acid molecule having a nucleotide sequence of the Figures, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, an Arabidopsis thaliana LMP cDNA can be isolated from an Arabidopsis thaliana library using all or portion of one of the sequences of the Figures as a hybridization probe and standard hybridization techniques as described in Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratoiy, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of the Figures can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence a nucleic acid molecule encompassing all or a portion of one of the sequences of the Figures can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence the Figures). For example, mRNA can be isolated from plant cells by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.

1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL).

Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Figures. A nucleic acid of 13 WO 02/099076 PCT/US02/17937 the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.

O The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a LMP nucleotide Z sequence can be prepared by standard synthetic techniques, using an automated DNA N synthesizer.

In a preferred embodiment, an isolated nucleic acid of the invention comprises one of It the polynucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID \0 NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ mc ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, r- SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID SNO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47. These polynucleotide correspond to the Arabidopsis thaliana LMP cDNAs of the invention. These cDNAs comprise sequences encoding LMPs the "coding region"), as well as 5' untranslated sequences and 3' untranslated sequences. Alternatively, the nucleic acid molecules can comprise only the coding region of any of the polynucleotide sequences described herein.

Examples ofpolynucleotides comprising only the coding region or open reading frame (ORF) are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

For the purposes of this application, it will be understood that each of the polynucleotide sequences set forth in the Figures has an identifying entry number pk002b). Each of these sequences may generally comprise three parts: a 5' upstream region, a coding region, and a downstream region. The particular sequences shown in the figures represent the open reading frames. The putative functions of these proteins are indicated in Table 4.

In another preferred embodiment, an isolated nucleic acid molecule of the present invention encodes a polypeptide that is able to participate in the metabolism of seed storage compounds such as lipids, starch and seed storage proteins and that contains a DNA-binding (or transcription factor) domain, a protein kinase domain, a signal transduction domain, a protease domain, or a carbohydarte metabolism domain. Examples of isolated LMPs that contain such domains can be found in Table 5. LMPs containing a DNA-binding domain include those shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:24, SEQ 14 WO 02/099076 PCT/US02/17937 ID NO:26, SEQ ID NO:28, SEQ ID NO:32, or SEQ ID NO:44. LMPs containing a protein kinase domain include those shown in SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:48, SEQ 0 ID NO:22 or SEQ ID NO:16. LMPs containing a signal transduction domain include those shown in SEQ ID NO:2, SEQ ID NO: 14, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:42 or

O

z SEQ ID NO:46. LMPs containing a protease domain include those shown in SEQ ID NO:6 C, or SEQ ID NO:12. LMPs containing a carbohydrate metabolism domain include those shown in SEQ ID NO:10; SEQ ID NO:18 or SEQ ID I In another preferred embodiment, an isolated nucleic acid molecule of the invention I\D comprises a nucleic acid molecule which is a complement of any of the nucleic acid sequences disclosed herein, including one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID O NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a portion thereof. As used herein, the term "complementary" refers to a nucleotide sequence that can hybridize to one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, thereby forming a stable duplex.

In another preferred embodiment, an isolated nucleic acid of the invention comprises a polynucleotide sequence encoding a polypeptide selected from the group consisting of: SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown in the Figures, or a portion thereof. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, hybridizes under stringent conditions, to one of the nucleotide sequences WO 02/099076 PCT/US02/17937 shown in the Figures, or a portion thereof. These hybridization conditions include washing with a solution having a salt concentration of about 0.02 molar at pH 7 at about 60 0

C.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the sequences in the Figures, for example a fragment which can Z be used as a probe or primer or a fragment encoding a biologically active portion of a LMP.

C, The nucleotide sequences determined from the cloning of the LMP genes from Arabidopsis thaliana allows for the generation of probes and primers designed for use in identifying and/or cloning LMP homologues in other cell types and organisms, as well as LMP ND homologues from other plants or related species. Therefore this invention also provides Scompounds comprising the nucleic acids disclosed herein, or fragments thereof. These compounds include the nucleic acids attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in the Figures, an anti-sense sequence of one of the sequences set forth in the Figures, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the Figures can be used in PCR reactions to clone LMP homologues.

Probes based on the LMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a LMP, such as by measuring a level of a LMP-encoding nucleic acid in a sample of cells, detecting LMP mRNA levels or determining whether a genomic LMP gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid encoded by a sequence of the Figures such that the protein or portion thereof maintains the same or a similar function as the wild-type protein. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent an amino acid residue which has a similar side chain as an amino acid residue in one of the ORFs of a sequence of the Figures) amino acid residues to an amino acid sequence such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the WO 02/099076 PCT/US02/17937 production of seed storage compounds in plants, construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes.

C Regulatory proteins, such as DNA binding proteins, transcription factors, kinases, phosphatases, or protein members of metabolic pathways such as the lipid, starch and protein

O

z biosynthetic pathways, or membrane transport systems, may play a role in the biosynthesis of seed storage compounds. Examples of such activities are described herein (see putative annotations in Table Examples of LMP-encoding nucleic acid sequences are set forth in Ithe Figures.

SAs altered or increased sugar and/or fatty acid production is a general trait wished to Cc be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, 0 soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower and tagetes, 0solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut) and perennial grasses and forage crops, these crop plants are also preferred target plants for genetic engineering as one further embodiment of the present invention.

Portions of proteins encoded by the LMP nucleic acid molecules of the invention are preferably biologically active portions of one of the LMPs. As used herein, the term "biologically active portion of a LMP" is intended to include a portion, a domain/motif, of a LMP that participates in the metabolism of compounds necessary for the biosynthesis of seed storage lipids, or the construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes, or has an activity as set forth in Table 3. To determine whether a LMP or a biologically active portion thereof can participate in the metabolism of compounds necessary for the production of seed storage compounds and cellular membranes, an assay of enzymatic activity may be performed. Such assay methods are well known to those skilled in the art, and as described in Example 14 of the Exemplification.

Biologically active portions of a LMP include peptides comprising amino acid sequences derived from the amino acid sequence of a LMP an amino acid sequence of SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48 or the amino acid sequence of a protein homologous to a LMP, which include fewer amino acids than a full length LMP or the full length protein which is homologous to a LMP) and exhibit at least one activity of a LMP. Typically, 17

I

WO 02/099076 PCT/US02/17937 biologically active portions (peptides, peptides which are, for example, 5, 10, 15, 20, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif Swith at least one activity of a LMP. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and

O

z evaluated for one or more of the activities described herein. Preferably, the biologically N active portions of a LMP include one or more selected domains/motifs or portions thereof having biological activity.

tI Additional nucleic acid fragments encoding biologically active portions of a LMP can \O be prepared by isolating a portion of one of the sequences, expressing the encoded portion of Sthe LMP or peptide by recombinant expression in vitro) and assessing the activity of the encoded portion of the LMP or peptide.

O The invention further encompasses nucleic acid molecules that differ from one of the polynucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 (and portions thereof) due to degeneracy of the genetic code and thus encode the same LMP as that encoded by the nucleotide sequences shown in the Figures. In a further embodiment, the nucleic acid molecule of the invention encodes a full length protein which is substantially homologous to an amino acid sequence of a polypeptide encoded by an open reading frame shown in the Figures. In one embodiment, the full-length nucleic acid or protein or fragment of the nucleic acid or protein is from Arabidopsis thaliana.

In addition to the Arabidopsis thaliana LMP nucleotide sequences described herein, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of LMPs may exist within a population the Arabidopsis thaliana population). Such genetic polymorphism in the LMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a LMP, preferably a Arabidopsis thaliana LMP. Such natural variations can typically result in 1-40% variance in the nucleotide sequence of the LMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in LMP that are the result of natural variation and that do not alter the functional activity of LMPs are intended to be within the scope of the invention.

WO 02/099076 PCT/US02/17937 Nucleic acid molecules corresponding to natural variants and non-Arabidopsis thaliana orthologs of the Arabidopsis thaliana LMP cDNA of the invention can be isolated 0 based on their homology to Arabidopsis thaliana LMP nucleic acid disclosed herein using the Arabidopsis thaliana cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. As used herein, 0 the term "orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having ^I the same or similar functions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of the Figures. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length.

As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley Sons, N.Y.

(1989) 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 0 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a polynucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature encodes a natural protein). In one embodiment, the nucleic acid encodes a natural Arabidopsis thaliana LMP.

In addition to naturally-occurring variants of the LMP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the Figures, thereby leading to changes in the amino acid sequence of the encoded LMP, without altering the functional ability of the LMP. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino 19 WO 02/099076 PCT/US02/17937 acid residues can be made in a a LMP polynucleotide sequence. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the LMPs

O

0 provided herein without altering the activity of said LMP, whereas an "essential" amino acid Sresidue is required for LMP activity. Other amino acid residues, however, those that are

O

z not conserved or only semi-conserved in the domain having LMP activity) may not be C essential for activity and thus are likely to be amenable to alteration without altering LMP activity.

t Accordingly, another aspect of the invention pertains to nucleic acid molecules 0 encoding LMPs that contain changes in amino acid residues that are not essential for LMP activity. Such LMPs differ in amino acid sequence from a sequence yet retain at least one of the LMP activities described herein. In one embodiment, the isolated nucleic acid molecule

O

0 comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence encoded by a nucleic acid of the Figures and is capable of participation in the metabolism of compounds necessary for the production of seed storage compounds in Arabidopsis thaliana, or cellular membranes, or has one or more activities set forth in Table 4. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous as shown in SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, more preferably at least about 60-70% homologous as shown in SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, even more preferably at least about 70-80%, 80-90%, homologous as shown in SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, and most preferably at least about 96%, 97%, 98%, or 99% homologous as shown in SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, WO 02/099076 PCT/US02/17937 SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

O To determine the percent homology of two amino acid sequences the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID

O

z NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, CN SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, tI SEQ ID NO:46 or SEQ ID NO:48 and a mutant form thereof) or of two nucleic acids, the \sequences are aligned for optimal comparison purposes gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions Sor nucleotide positions are then compared. When a position in one sequence SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence a mutant or homolog of the sequence shown in SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48), then the molecules are homologous at that position as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences homology numbers of identical positions/total numbers of positions x 100).

An isolated nucleic acid molecule encoding a LMP homologous to a protein sequence encoded by a nucleic acid of the Figures can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the Figures such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the sequences of the Figures by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Preferably, conservative amino acid substitutions are made at one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is one in which the 21 WO 02/099076 PCT/US02/17937 amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art.

O

O These families include amino acids with basic side chains lysine, arginine, histidine), acidic side chains aspartic acid, glutamic acid), uncharged polar side chains

O

Sglycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains C1 alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains threonine, valine, isoleucine) and aromatic side chains t tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid \NO residue in a LMP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a LMP coding sequence, such as by saturation mutagenesis, and the 0 resultant mutants can be screened for a LMP activity described herein to identify mutants that retain LMP activity. Following mutagenesis of one of the sequences of the Figures, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Examples 9, 13,-14 of the Exemplification).

LMPs are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described herein) and the LMP is expressed in the host cell. The LMP can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a LMP or peptide thereof can be synthesized chemically using standard peptide synthesis techniques. Moreover, native LMP can be isolated from cells, for example using an anti-LMP antibody, which can be produced by standard techniques utilizing a LMP or fragment thereof of this invention.

The invention also provides LMP chimeric or fusion proteins. As used herein, a LMP "chimeric protein" or "fusion protein" comprises a LMP polypeptide operatively linked to a non-LMP polypeptide. An "LMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a LMP, whereas a "non-LMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the LMP, a protein which is different from the LMP and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the LMP polypeptide and the non-LMP polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-LMP polypeptide can be fused to the N-terminus or C-terminus of 22 WO 02/099076 PCT/US02/17937 the LMP polypeptide. For example, in one embodiment, the fusion protein is a GST-LMP r. (glutathione S-transferase) fusion protein in which the LMP sequences are fused to the C- 0 terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant LMPs. In another embodiment, the fusion protein is a LMP containing a z heterologous signal sequence at its N-terminus. In certain host cells mammalian host cells), expression and/or secretion of a LMP can be increased through use of a heterologous signal sequence.

tt Preferably, a LMP chimeric or fusion protein of the invention is produced by standard Srecombinant DNA techniques. For example, DNA fragments coding for the different c polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, Srestriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety a GST polypeptide). An LMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the LMP.

In addition to the nucleic acid molecules encoding LMPs described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto.

An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire LMP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a LMP. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues the entire coding region of Pk002b comprises nucleotides 1 to 2050). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding LMP. The term "noncoding region" WO 02/099076 PCT/US02/17937 refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids also referred to as 5' and 3' untranslated regions).

O

O Given the coding strand sequences encoding LMP disclosed herein the N sequences set forth in the Figures), antisense nucleic acids of the invention can be designed

O

z according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule N can be complementary to the entire coding region of LMP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of t LMP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of LMP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense or sense nucleic acid of the invention can be constructed using chemical synthesis 0 and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, (carboxyhydroxylmethyl) uracil, 5-carboxymethylamino-methyl-2-thiouridine, carboxymethylaminomethyluracil, dihydro-uracil, beta-D-galactosylqueosine, inosine, N-6isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine, 7methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-Dmannosylqueosine, 5'-methoxycarboxymethyl-uracil, 5-methoxyuracil, 2-methylthio-N-6isopentenyladenine, uracil-5-oxyacetic acid wybutoxosine, pseudouracil, queosine, 2thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, oxyacetic acid methylester, uracil-5-oxyacetic acid 5-methyl-2-thiouracil, 3-(3-anino-3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diamino-purine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

In another variation of the antisense technology, a double-strand interfering RNA construct can be used to cause a down-regulation of the LMP mRNA level and LMP activity 24 WO 02/099076 PCT/US02/17937 in transgenic plants. This requires transforming the plants with a chimeric construct containing a portion of the LMP sequence in the sense orientation fused to the antisense

O

O sequence of the same portion of the LMP sequence. A DNA linker region of variable length can be used to separate the sense and antisense fragments of LMP sequences in the construct.

O

z The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a LMP to thereby inhibit expression of the protein, by t inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an c antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it Sspecifically binds to a receptor or an antigen expressed on a selected cell surface, by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic including plant promoters are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the strands run parallel to each other (Gaultier et al. 1987, Nucleic Acids Res. 15:6625-6641).

The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.

1987, FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.

Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes hammerhead ribozymes (described in Haselhoff Gerlach 1988, Nature 334:585-591)) can be used to catalytically cleave LMP mRNA transcripts to thereby inhibit translation of LMP mRNA. A ribozyme having specificity for a LMP-encoding nucleic acid can be designed based upon the nucleotide sequence of a LMP cDNA disclosed herein Pk002b in the Figures) or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the WO 02/099076 PCT/US02/17937 nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a LMP-encoding mRNA (see, Cech et al., U.S. Patent No. 4,987,071 and O Cech et al., U.S. Patent No. 5,116,742). Alternatively, LMP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see,

O

z Bartel, D. Szostak J.W. 1993, Science 261:1411-1418).

NC Alternatively, LMP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a LMP nucleotide sequence a tI LMP promoter and/or enhancers) to form triple helical structures that prevent transcription of \O a LMP gene in target cells (See generally, Helene C. 1991, Anticancer Drug Des. 6:569-84; m Helene C. et al. 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. 1992, Bioassays 14:807-15).

O Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a LMP (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used inter-changeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors replication defective retroviruses, adenoviruses and adenoassociated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory 26 WO 02/099076 PCT/USO2/17937 sequence(s) in a manner which allows for expression of the nucleotide sequence and both sequences are fused to each other so that each fulfills its proposed function in an in vitro O transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other

O

z expression control elements polyadenylation signals). Such regulatory sequences are C described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) or see: Gruber and Crosby, in: Methods in Plant I Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Florida, eds.: Glick s Thompson, Chapter 7, 89-108 including the references therein. Regulatory sequences include ¢c those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells or O under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein LMPs, mutant forms of LMPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of LMPs in prokaryotic or eukaryotic cells. For example, LMP genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos M.A. et al. 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al. 1991, Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, Bennet Lasure, eds., p. 396-428:Academic Press: an Diego; and van den Hondel Punt 1991, Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al. 1999, Marine Biotechnology 1:239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a transformation method as described in WO 98/01572 and multicellular plant cells (see Schmidt Willmitzer 1988, High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.:583-586); Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, chapter 6/7, S.71-119 (1993); White, Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and 27 WO 02/099076 PCT/US02/17937 Wu, Academic Press 1993, 128-43; Potrykus 1991, Annu. Rev. Plant Physiol. Plant Mol.

Biol. 42:205-225 (and references cited therein) or mammalian cells. Suitable host cells are 0 discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA 1990). Alternatively, the recombinant expression vector can

O

z be transcribed and translated in vitro, for example using T7 promoter regulatory sequences C and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out with vectors containing tIn constitutive or inducible promoters directing the expression of either fusion or non-fusion IND proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to c the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve one or more of the O following purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith Johnson 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the LMP is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant LMP unfused to GST can be recovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. 1988, Gene 69:301-315) and pET lid (Studier et al. 1990, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, California 60-89).

Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 d vector relies on transcription from a T7 gnl0-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or WO 02/099076 PCT/US02/17937 HMS174(DE3) from a resident X prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

0 One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein

O

z (Gottesman S. 1990, Gene Expression Technology: Methods in Enzymology 185:119-128, CN Academic Press, San Diego, California). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual I codons for each amino acid are those preferentially utilized in the bacterium chosen for \expression (Wada et al. 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic n acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the LMP expression vector is a yeast expression vector.

O Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al.

1987, EMBO J. 6:229-234), pMFa (Kurjan Herskowitz 1982, Cell 30:933-943), pJRY88 (Schultz et al. 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel Punt 1991, "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge University Press: Cambridge.

Alternatively, the LMPs of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells Sf9 cells) include the pAc series (Smith et al. 1983, Mol. Cell Biol.

3:2156-2165) and the pVL series (Lucklow Summers 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed 1987, Nature 329:840) and pMT2PC (Kaufman et al. 1987, EMBO J.

6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, the LMPs of the invention may be expressed in uni-cellular plant cells (such as algae, see Falciatore et al. (1999, Marine Biotechnology 1:239-251 and references therein) and plant cells from higher plants the spermatophytes, such as crop 29 WO 02/099076 PCT/US02/17937 plants)). Examples of plant expression vectors include those detailed in: Becker, Kemper, Schell and Masterson (1992, "New plant binary vectors with selectable markers located O proximal to the left border", Plant Mol. Biol. 20:1195-1197) and Bevan (1984, "Binary Agrobacterium vectors for plant transformation, Nucleic Acids Res. 12:8711-8721; Vectors

O

Z for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and CN Utilization, eds.: Kung und R. Wu, Academic Press, 1993, S. 15-38).

A plant expression cassette preferably contains regulatory sequences capable to drive tI gene expression in plant cells and which are operably linked so that each sequence can fulfill I\D its function such as termination of transcription such as polyadenylation signals. Preferred Cc polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, O EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptional levels a plant expression cassette preferably contains other operably linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al. 1987, Nucleic Acids Res. 15:8693-8711).

Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferred are promoters driving constitutive expression (Benfey et al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al. 1980, Cell 21:285-294), the 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters like those from Rubisco small subunit described in US 4,962,028. Even more preferred are seed-specific promoters driving expression of LMP proteins during all or selected stages of seed development. Seed-specific plant promoters are known to those of ordinary skill in the art and are identified and characterized using seed-specific mRNA libraries and expression profiling techniques. Seedspecific promoters include the napin-gene promoter from rapeseed (US 5,608,152), the USPpromoter from Vicia faba (Baeumlein et al. 1991, Mol. Gen. Genetics 225:459-67), the oleosin-promoter from Arabidopsis (WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (US 5,504,200), the Bce4-promoter from Brassica (W09113980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233-239) as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice etc. Suitable promoters to note are the lpt2 or lptl-gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein-gene, the WO 02/099076 PCT/US02/17937 rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, the rye Ssecalin gene).

Plant gene expression can also be facilitated via an inducible promoter (for review see z Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is desired in a time specific manner.

Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a V' tetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404) and an ethanol i inducible promoter (WO 93/21334).

c Promoters responding to biotic or abiotic stress conditions are also suitable promoters such as the pathogen inducible PRPl-gene promoter (Ward et al., 1993, Plant. Mol. Biol.

0 22:361-366), the heat inducible hsp80-promoter from tomato (US 5,187,267), cold inducible alpha-amylase promoter from potato (WO 96/12814) or the wound-inducible pinll-promoter (EP 375091).

Other preferred sequences for use in plant gene expression cassettes are targetingsequences necessary to direct the gene-product in its appropriate cell compartment (for review see Kermode 1996, Crit. Rev. Plant Sci. 15:285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells. Also especially suited are promoters that confer plastidspecific gene expression, as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNApolymerase promoter are described in WO 95/16783 and WO 97/06250 and the clpPpromoter from Arabidopsis described in WO 99/46394.

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner, which allows for expression (by transcription of the DNA molecule) of an RNA molecule, which is antisense to LMP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which 31 WO 02/099076 PCT/US02/17937 can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (1986, Antisense 0 RNA as a molecular tool for genetic analysis, Reviews Trends in Genetics, Vol. 1) and Mol et al. (1990, FEBS Lett. 268:427-430).

O

z Another aspect of the invention pertains to host cells into which a recombinant NC expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such Sterms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can Sbe any prokaryotic or eukaryotic cell. For example, a LMP can be expressed in bacterial cells, insect cells, fungal cells, mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates or plant cells. Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", "conjugation" and' "transduction" are intended to refer to a variety of artrecognized techniques for introducing foreign nucleic acid DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation.

Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and other laboratory manuals such as Methods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, New Jersey.

For stable transfection of mammalian and plant cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, kanamycin and methotrexate or in plants that confer resistance towards an herbicide such as glyphosate or glufosinate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a LMP or can be introduced on a separate vector. Cells stably 32 WO 02/099076 PCT/US02/17937 transfected with the introduced nucleic acid can be identified by, for example, drug selection cells that have incorporated the selectable marker gene will survive, while the other Scells die).

To create a homologous recombinant microorganism, a vector is prepared which z contains at least a portion of a LMP gene into which a deletion, addition or substitution has ,1 been introduced to thereby alter, functionally disrupt, the LMP gene. Preferably, this LMP gene is an Arabidopsis thaliana LMP gene, but it can be a homologue from a related Splant or even from a mammalian, yeast, or insect source. In a preferred embodiment, the Svector is designed such that, upon homologous recombination, the endogenous LMP gene is functionally disrupted no longer encodes a functional protein; also referred to as a knock-out vector). Alternatively, the vector can be designed such that, upon homologous Srecombination, the endogenous LMP gene is mutated or otherwise altered but still encodes functional protein the upstream regulatory region can be altered to thereby alter the expression of the endogenous LMP). To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al. 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, American Scientist 87:240-247). Homologous recombination procedures in Arabidopsis thaliana are also well known in the art and are contemplated for use herein.

In a homologous recombination vector, the altered portion of the LMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the LMP gene to allow for homologous recombination to occur between the exogenous LMP gene carried by the vector and an endogenous LMP gene in a microorganism or plant. The additional flankinLMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene., Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas Capecchi 1987, Cell 51:503, for a description of homologous recombination vectors). The vector is introduced into a microorganism or plant cell via polyethyleneglycol mediated DNA). Cells in which the introduced LMP gene has homologously recombined with the endogenous LMP gene are selected using art-known techniques.

In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of a LMP gene on a vector placing it under control of the lac operon permits expression of the LMP gene only in the presence of IPTG. Such regulatory systems are well known in the art.

WO 02/099076 PCT/US02/17937 A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture can be used to produce express) a LMP. Accordingly, the invention further provides O methods for producing LMPs using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (into which a recombinant expression

O

z vector encoding a LMP has been introduced, or which contains a wild-type or altered LMP CN gene in it's genome) in a suitable medium until LMP is produced. In another embodiment, the method further comprises isolating LMPs from the medium or the host cell.

I Another aspect of the invention pertains to isolated LMPs, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language S"substantially free of cellular material" includes preparations of LMP in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of LMP having less than about 30% (by dry weight) of non-LMP (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-LMP, still more preferably less than about 10% of non-LMP, and most preferably less than about non-LMP. When the LMP or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of LMP in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of LMP having less than about 30% (by dry weight) of chemical precursors or non-LMP chemicals, more preferably less than about chemical precursors or non-LMP chemicals, still more preferably less than about chemical precursors or non-LMP chemicals, and most preferably less than about chemical precursors or non-LMP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the LMP is derived. Typically, such proteins are produced by recombinant expression of, for example, an Arabidopsis thaliana LMP in other plants than Arabidopsis thaliana or microorganisms, algae or fungi.

An isolated LMP or a portion thereof of the invention can participate in the metabolism of compounds necessary for the production of seed storage compounds in 34 WO 02/099076 PCT/US02/1 7937 Arabidopsis thaliana, or of cellular membranes, or has one or more of the activities set forth in Table 4. In preferred embodiments, the protein or portion thereof comprises an amino acid Ssequence which is sufficiently homologous to an amino acid sequence encoded by a nucleic acid of the Figures such that the protein or portion thereof maintains the ability to participate 0 z in the metabolism of compounds necessary for the construction of cellular membranes in Arabidopsis thaliana, or in the transport of molecules across these membranes. The portion of the protein is preferably a biologically active portion as described herein. In another tIt preferred embodiment, a LMP of the invention has an amino acid sequence encoded by a nucleic acid of the Figures. In yet another preferred embodiment, the LMP has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, hybridizes under r- stringent conditions, to a nucleotide sequence of the Figures. In still another preferred Sembodiment, the LMP has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%, 99% or more homologous to one of the amino acid sequences encoded by a nucleic acid of the Figures. The preferred LMPs of the present invention also preferably possess at least one of the LMP activities described herein. For example, a preferred LMP of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, to a nucleotide sequence of the Figures, and which can participate in the metabolism of compounds necessary for the construction of cellular membranes in Arabidopsis thaliana, or in the transport of molecules across these membranes, or which has one or more of the activities set forth in Table 4.

In other embodiments, the LMP is substantially homologous to an amino acid sequence encoded by a nucleic acid of the Figures and retains the functional activity of the protein of one of the sequences encoded by a nucleic acid of the Figures yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail above.

Accordingly, in another embodiment, the LMP is a protein which comprises an amino acid sequence which is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence and which has at least one of the LMP activities described herein. In another embodiment, the invention pertains to a full Arabidopsis thaliana protein which is substantially homologous to an entire amino acid sequence encoded by a nucleic acid of the Figures.

Homologues of the LMP can be generated by mutagenesis, discrete point mutation or truncation of the LMP. As used herein, the term "homologue" refers to a variant WO 02/099076 PCT/US02/17937 form of the LMP which acts as an agonist or antagonist of the activity of the LMP. An agonist of the LMP can retain substantially the same, or a subset, of the biological activities O of the LMP. An antagonist of the LMP can inhibit one or more of the activities of the naturally occurring form of the LMP, by, for example, competitively binding to a

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z downstream or upstream member of the cell membrane component metabolic cascade which M includes the LMP, or by binding to a LMP which mediates transport of compounds across such membranes, thereby preventing translocation from taking place.

I In an alternative embodiment, homologues of the LMP can be identified by screening O combinatorial libraries of mutants, truncation mutants, of the LMP for LMP agonist or n antagonist activity. In one embodiment, a variegated library of LMP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene 0library. A variegated library of LMP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential LMP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins for phage display) containing the set of LMP sequences therein. There are a variety of methods which can be used to produce libraries of potential LMP homologues from a degenerate oligonucleotide sequence.

Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential LMP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, Narang 1983, Tetrahedron 39:3; Itakura et al.

1984, Annu. Rev. Biochem. 53:323; Itakura et al. 1984, Science 198:1056; Ike et al. 1983, Nucleic Acids Res. 11:477).

In addition, libraries of fragments of the LMP coding sequences can be used to generate a variegated population of LMP fragments for screening and subsequent selection of homologues of a LMP. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a LMP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the

LMP.

WO 02/099076 PCT/USO2/17937 Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of LMP homologues. The most z widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the Scombinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify LMP Shomologues (Arkin Yourvan 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. 1993, Protein Engineering 6:327-331).

In another embodiment, cell based assays can be exploited to analyze a variegated LMP library, using methods well known in the art.

The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Arabidopsis thaliana and related organisms; mapping of genomes of organisms related to Arabidopsis thaliana; identification and localization of Arabidopsis thaliana sequences of interest; evolutionary studies; determination of LMP regions required for function; modulation of a LMP activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; and modulation of seed storage compound accumulation.

The plant Arabidopsis thaliana represents one member of higher (or seed) plants. It is related to other plants such as Brassica napus or soybean which require light to drive photosynthesis and growth. Plants like Arabidopsis thaliana and Brassica napus share a high degree of homology on the DNA sequence and polypeptide level, allowing the use of heterologous screening of DNA molecules with probes evolving from other plants or organisms, thus enabling the derivation of a consensus sequence suitable for heterologous screening or functional annotation and prediction of gene functions in third species. The ability to identify such functions can therefore have significant relevance, prediction of substrate specificity of enzymes. Further, these nucleic acid molecules may serve as reference points for the mapping of Arabidopsis genomes, or of genomes of related organisms.

WO 02/099076 PCT/US02/17937 The LMP nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Arabidopsis thaliana or a close relative thereof.

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O Also, they may be used to identify the presence of Arabidopsis thaliana or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences

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z of a number of Arabidopsis thaliana genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of an Arabidopsis thaliana gene which is unique to this organism, t one can ascertain whether this organism is present.

c\ Further, the nucleic acid and protein molecules of the invention may serve as markers Sfor specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of Arabidopsis thaliana proteins. For example, to identify the O region of the genome to which a particular Arabidopsis thaliana DNA-binding protein binds, the Arabidopsis thaliana genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of Arabidopsis thaliana, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related plants.

The LMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety ofprokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms ofmutagenesis without losing function.

Manipulation of the LMP nucleic acid molecules of the invention may result in the production of LMPs having functional differences from the wild-type LMPs. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.

38 WO 02/099076 PCT/US02/17937 There are a number of mechanisms by which the alteration of a LMP of the invention may directly affect the accumulation of seed storage compounds. In the case of plants O expressing LMPs, increased transport can lead to altered accumulation of compounds and/or solute partitioning within the plant tissue and organs which ultimately could be used to affect

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z the accumulation of one or more seed storage compounds during seed development. An N example is provided by Mitsukawa et al. (1997, Proc. Natl. Acad. Sci. USA 94:7098-7102), where over expression of an Arabidopsis high-affinity phosphate transporter gene in tobacco I cultured cells enhanced cell growth under phosphate-limited conditions. Phosphate I\D availability also affects significantly the production of sugars and metabolic intermediates mc (Hurry et al. 2000, Plant J. 24:383-396) and the lipid composition in leaves and roots (Hartel et al. 2000, Proc. Natl. Acad. Sci. USA 97:10649-10654). Likewise, the activity of the plant O ACCase has been demonstrated to be regulated by phosphorylation (Savage Ohlrogge 1999, Plant J. 18:521-527) and alterations in the activity of the kinases and phosphatases (LMPs) that act on the ACCase could lead to increased or decreased levels of seed lipid accumulation. Moreover, the presence of lipid kinase activities in chloroplast envelope membranes suggests that signal transduction pathways and/or membrane protein regulation occur in envelopes (see, Miiller et al. 2000, J. Biol. Chem. 275:19475-19481 and literature cited therein). The ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C, which are regulators in abscisic acid signaling pathway, and thereby in early and late seed development Merlot et al. 2001, Plant J. 25:295-303). For more examples see also the section 'background of the invention'.

The present invention also provides antibodies which specifically binds to an LMPpolypeptide, or a portion thereof, as encoded by a nucleic acid disclosed herein or as described herein.

Antibodies can be made by many well-known methods (see, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an inunune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced (see, for example, Kelly et al. 1992, Bio/Technology 10:163-167; Bebbington et al. 1992, Bio/Technology 10:169-175).

The phrase "selectively binds" with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and 39 WO 02/099076 PCT/US02/1 7937 other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in Sthe sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats z may be used to select antibodies that selectively bind with a particular protein. For example, N solid-phase ELISA immunoassays are routinely used to select antibodies selectively N immunoreactive with a protein. See Harlow and Lane "Antibodies, A Laboratory Manual" t¢ Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.

SIn some instances, it is desirable to prepare monoclonal antibodies from various hosts.

rA description of techniques for preparing such monoclonal antibodies may be found in Stites Set al., editors, "Basic and Clinical Immunology," (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane ("Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York, 1988).

Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and Examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims included herein.

EXAMPLES

Example 1: General Processes a) General Cloning Processes: Cloning processes such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli and yeast cells, growth of bacteria and sequence analysis of recombinant DNA were carried out as described in Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969- WO 02/099076 PCT/US02/17937 309-6) or Kaiser, Michaelis and Mitchell (1994, "Methods in Yeast Genetics", Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3).

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b) Chemicals:

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z The chemicals used were obtained, if not mentioned otherwise in the text, in p.a.

CI quality from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared using purified, pyrogen-free t water, designated as H20 in the following text, from a Milli-Q water system water IND purification plant (Millipore, Eschbom). Restriction endonucleases, DNA-modifying Cc enzymes and molecular biology kits were obtained from the companies AGS (Heidelberg),

C-I

Amersham (Braunschweig), Biometra (Gdttingen), Boehringer (Mannheim), Genomed (Bad SOeynnhausen), New England Biolabs (Schwalbach/ Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, Netherlands). They were used, if not mentioned otherwise, according to the manufacturer's instructions.

c) Plant Material: For this study, in one series of experiments, root material of wild-type and pickle mutant plants ofArabidopsis thaliana were used. Thepkl mutation was isolated from an ethyl methanesulfonate-mutagenized population of the Columbia ecotype as described (Ogas et al.

1997, Science 277:91-94; Ogas et al. 1999, Proc. Natl. Acad. Sci. USA 96:13839-13844). In other series of experiments, siliques of individual ecotypes of Arabidopsis thaliana and of selected Arabidopsis phytohormone mutants were used. Seeds were obtained from the Arabidopsis stock center.

d) Plant Growth: Plants were either grown on Murashige-Skoog medium as described in Ogas et al.

(1997, Science 277:91-94; 1999, Proc. Natl. Acad. Sci. USA 96:13839-13844) or on soil under standard conditions as described in Focks Benning (1998, Plant Physiol. 118:91- 101).

WO 02/099076 PCT/US02/17937 Example 2: Total DNA Isolation from Plants t The details for the isolation of total DNA relate to the working up of one gram fresh Sweight of plant material.

CTAB buffer: 2% N-cethyl-N,N,N-trimethylanmmonium bromide (CTAB); 100

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z mM Tris HC1 pH 8.0; 1.4 M NaCI; 20 mM EDTA. N-Laurylsarcosine buffer:10% N- C' laurylsarcosine; 100 mM Tris HCI pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar to give a fine I powder and transferred to 2 ml Eppendorf vessels. The frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 1 of Nc laurylsarcosine buffer, 20 1 of -mercaptoethanol and 10 1 of proteinase K solution, mg/ml) and incubated at 60 0 C for one hour with continuous shaking. The homogenate Sobtained was distributed into two Eppendorf vessels (2 ml) and extracted twice by shaking with the same volume of chloroform/isoamyl alcohol For phase separation, centrifugation was carried out at 8000 g and RT for 15 min in each case. The DNA was then precipitated at -70 0 C for 30 min using ice-cold isopropanol. The precipitated DNA was sedimented at 4 0 C and 10,000 g for 30 min and resuspended in 180 1 of TE buffer (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA was treated with NaCI (1.2 M final concentration) and precipitated again at -70C for 30 min using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and subsequently taken up in 50 1 of

H

2 0 RNAse (50 mg/ml final concentration). The DNA was dissolved overnight at 4 0 C and the RNAse digestion was subsequently carried out at 37°C for 1 h. Storage of the DNA took place at 4 0

C.

Example 3: Isolation of Total RNA and poly-(A)+ RNA from Plants For the investigation of transcripts, both total RNA and poly-(A) RNA were isolated.

RNA is isolated from siliques of Arabidopsis plants according to the following procedure: RNA preparation from Arabidopsis seeds "hot" extraction: 1. Buffers, enzymes and solution 2M KCI Proteinase K Phenol (for RNA) Chloroform:Isoamylalcohol (Phenol:choloroform 1:1; pH adjusted for RNA) 4 M LiC1, DEPC-treated DEPC-treated water WO 02/099076 PCT/US02/I7937 3M NaOAc, pH 5, DEPC-treated Isopropanol 0 70% ethanol (made up with DEPC-treated water) 0 Resuspension buffer:0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC-treated water as this solution can not be DEPC-treated S- Extraction Buffer: 0.2M Na Borate mM EDTA C 30 mM EGTA 1% SDS (250ul of 10% SDS-solution for 2.5ml buffer) 1% Deoxycholate (25mg for 2,5ml buffer) 2% PVPP (insoluble 50mg for 2.5ml buffer) \O 2% PVP 40K (50mg for 2.5ml buffer) mM DTT N, 100 mM P-Mercaptoethanol (fresh, handle under fume hood use 35gl of 14.3M solution for 5ml buffer) 2. Extraction Heat extraction buffer up to 80 0 C. Grind tissue in liquid nitrogen-cooled mortar, transfer tissue powder to 1.5ml tube. Tissue should kept frozen until buffer is added so transfer the sample with pre-cooled spatula and keep the tube in liquid nitrogen all time. Add 350pl preheated extraction buffer (here for 100mg tissue, buffer volume can be as much as 500tl for bigger samples) to tube, vortex and heat tube to 80 0 C for -1 min. Keep then on ice. Vortex sample, grind additionally with electric mortar.

3. Digestion Add Proteinase K (0.15mg/100mg tissue), vortex and keep at 37°C for one hour.

4. First Purification Add 27tl 2M KC1. Chill on ice for 10 min. Centrifuge at 12.000 rpm for 10 minutes at room temperature. Transfer supernatant to fresh, RNAase-free tube and do one phenol extraction, followed by a choloroform:isoamylalcohol extraction. Add 1 vol.

isopropanol to supernatant and chill on ice for 10 min. Pellet RNA by centrifugation (7000 rpm for 10 min at RT). Resolve pellet in lml 4M LiCI by 10 to vortexing. Pellet RNA by 5min centrifugation.

Second Purification Resuspend pellet in 500pl Resuspension buffer. Add 500pl phenol and vortex. Add 2 50ptl chloroform:isoamylalcohol and vortex. Spin for 5 min. and transfer supernatant to fresh tube. Repeat choloform:isoamylalcohol extraction until interface is clear.

Transfer supernatant to fresh tube and add 1/10 vol 3M NaOAc, pH 5 and 600utl isopropanol. Keep at -20 for 20 min or longer. Pellet RNA by 10 min centrifugation.

Wash pellet once with 70% ethanol. Remove all remaining alcohol before resolving WO 02/099076 PCT/US02/17937 pellet with 15 to 201jl DEPC-water. Determine quantity and quality by measuring the absorbance of a 1:200 dilution at 260 and 280nm. 40utg RNA/ml 1 OD260 SRNA from roots of wild-type and the pickle mutant of Arabidopsis is isolated as described (Ogas et al. 1997, Science 277:91-94; Ogas et al. 1999, Proc. Natl. Acad. Sci. USA

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Z 96:13839-13844).

C The mRNA is prepared from total RNA, using the Amersham Pharmacia Biotech mRNA purification kit, which utilizes oligo(dT)-cellulose columns.

t Isolation of Poly-(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo, Norway) \0 following the instructions of the manufacturer's protocol. After determination of the mC concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70 0

C.

Example 4: cDNA Library Construction For cDNA library construction, first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12 0 C (2 16 0 C (1 h) and 22 0 C (1 The reaction was stopped by incubation at 65 0 C (10 min) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37 0 C (30 min).

Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns.

EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNAligase (Roche, 12 0 C, overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37°C, 30 min). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPH phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.

Example 5: Identification of LMP Genes of Interest The pickle Arabidopsis mutant was used to identify LMP-encoding genes. The pickle mutant accumulates seed storage compounds, such as seed storage lipids and seed storage proteins, in the root tips (Ogas et al. 1997, Science 277:91-94; Ogas et al. 1999, Proc. Natl.

Acad. Sci. USA 96:13839-13844). mRNA isolated from roots of wild-type and pickle plants was used to create a subtracted and normalized cDNA library (SSH library) containing 44 WO 02/099076 PCT/UJS02/17937 cDNAs that are only present in the pickle roots, but not in the wild-type roots. Clones from the SSH library were spotted onto nylon membranes and hybridized with radio-labeled pickle or wild-type root mRNA to ascertain that the SSH clones were more abundant in pickle roots compared to wild-type roots. These SSH clones were randomly sequenced and the sequences were annotated (see Example Based on the expression levels and on these initial functional annotations (see Table clones from the SSH library were identified as potential LMP-encoding genes.

Other LMP candidate genes were identified by selecting various Arabidopsis phytohormone mutants obtained from EMS treatment) from the Arabidopsis stock center. These mutants and control wild-type plants were grown under standard conditions in growth chambers and screened for the accumulation of seed storage compounds. Mutants showing altered levels of seed storage lipids were considered as having a mutation in a LMP candidate gene and were investigated further. Table X3 contains the phytohormone mutants that showed the largest decrease in seed storage lipids. Based on our finding that some mutants had significantly decreased seed oil content we selected the respective wild-type allele or other enzymes in the same pathway as LMP-encoding genes (see table 4, sequence codes pk095, pk096, pk097, pk098, pk099, pk199, pkl05 and pkl07)..

Table 3.

Total fatty acid content in seeds of selected phytohormone mutants Allele Phenotype FA FA (g/g) Analysis of the Analysis of the first generation next generation of seeds of seeds Ler-0 wild type 0.35±0.02 0.35±0.02 abil-l (in Ler background) ABA-insensitive 0.33±0.01 0.34±0.02 r abi2-1 (in Ler background) ABA-insensitive 0.34±0.03 0.37±0.02 abi3-1 (in Ler background) ABA-insensitive 0.29±0.02 0.29±0.02 Col wild type 0.35±0.02 0.35±0.01 axr2 (in Col background) Auxin-resistant 0.28±0.02 0.28±0.02 etrl-1 (in Col background) Ethylene-insensitive 0.2710.03 0.29±0.02 ein1J-1 (m olI oacKground) Ethylene-insensitive 0.29±0.03 0.30±0 Ein 7 (in Col background) Ethylene-insensitive 0.29±0.02 0.30±0 .03 .01 WO 02/099076 PCT/US02/17937 Table 4.

Putative LMP Functions Sequence Function SEQ ID code NO: Pk002b Hypothetical protein Arabidopsis thaliana 1 Pk020b Unknown protein conserved hypothetical protein SPCC330.09 3 Schizosaccharomyces pombe Pk033 Hypothetical protein- A. thaliana chromosome II BAC T08113 genomic Pk037 65 kDa regulatory subunit of protein phosphatase 2A, PP2A-A 7 Pk038 Beta-1,3-glucanase class I precursor 9 Pk042a ATP dependent copper transporter Arabidopsis 11 Pk053b unknown protein Arabidopsis thaliana 13 Pk060 Sequence of BAC T22H22 from Arabidopsis thaliana Pk063b putative malate dehydrogenase Arabidopsis thaliana 17 Pk064 late embryogenesis abundant protein Arabidopsis 19 Pk073 gene:"T 18B16.210"; product:"putative protein" 21 Pk082 Nucleic acid binding protein-like Arabidopsis thaliana 23 Pk084a A. thaliana, BAC F13K23.10, chromosome 1, Pk086 hypothetical protein A_IG002P16.9 Arabidopsis thaliana 27 Pk088b putative nonspecific lipid-transfer protein precursor 29 Pk095 A. thaliana AP2 domain family transcription factor homolog 31 (ABI4) Pk096 Arabidopsis thaliana putative ethylene receptor (ETR2) 33 Pk097a Arabidopsis ethylene-response gene ETR1 Pk097b Arabidopsis ethylene-response gene ETR1 like 37 Pk098 Arabidopsis thaliana IAA17 (F19P19.31), auxin response gene 39 Pk099 A. thaliana IAA7 (IAA7) gene, primary auxin-responsive genes 41 Pk100 ABI3 gene; abscisic acid insensitive gene 43 protein phosphatase 2C involved in ABA signal (abil) Pkl07 regulation of ethylene response pathway (ctrl) 47 WO 02/099076 WO 02/99076PCT/US02/1 7937 Table Grouping of LMPs based on Functional protein domains Functional SEQ SEQ Functional domain Domain category ID: Code: position DNA-binding 4 pkO2Ob IHMG-I and HMG-Y DNA-binding 206-226 proteins domain (A+T-hook) 8 pk03 LexA DNA binding domain 127-14 1 8 pk037 STE-like transcription factor 7-12 20 pk064 DM DNA-binding domain 20 1-240 20 pk6 Histone H2B 209-253 24 pk082 HMG-I and HMG-Y DNA-binding 174-192 domain (A+T-hook) 24 pk082 B-box zinc finger superfamily 2 12-225 26 pkO84a 11IMG-I and HiMG-Y DNA-binding 8-30 domain 26 pkOS4a High mobility group proteins HMG1 and 11-35 SHMG2 28 pk086 GATA-type zinc finger 38-70 32 pk095 Ethylene responsive element binding 55-66 protein 77-93 93-113 119-140 44 pklOO B3 DNA-binding domain 37-47 270-3 12 457-473 533-549 565-598 617-63 1 Kinases 36, pk097a, His-kinase A 35 1-360, 38 pkO97b 466-475, 511-5 19 48 pk1O7 Protein kinase C-terminal domain 666-700 48 pkO7 Tyrosine kinase catalytic domain 714-752 22 pk073 -Shikimate kinase 165-16 1 22 pk073 Diacyglycerol kinase accessory domain 138-16 1 16 pkO6O Glycerate kinase 156-18 1 Signal 2 pkOO2b Troponin Ca2± sensitive switch 336-37 1 Transduction 2 pkOO2b ITMG1 and HMG2 domain 352-376 14 pkOS3b Developmental signaling protein Wnt-1 8-69 family 34 pk096 Response regulator receiver domain 697-7 34 pk096 Inositol I ,4,5-triphosphate-binding 645-669 protein receptor_______ pk098 AUX/IAA family domain 7-22 82-95 112-143 181-210 WO 02/099076 PCT/US02/17937 42 pk099 AUX/IAA family domain 6-21 82-95 126-157 194-223 46 Pkl05 Protein phosphatase 2C family 135-420 Proteases 6 pk033 DnaJ chaperonin central domain 6-38 6 pk033 ATP-dependent protease La (LON) 535-569 domain 6 pk033 Protease-associated (PA) domain 375-405 12 pk042a Carboxypeptidase Taq metalloprotease 176-196 signature 12 pk042a Gamma-glutamyltranspeptidase 110-164 12 pk042a E1-E2 ATPase 127-156 12 pk042a X-His dipeptidase (M25) signature 3-20 Carbohydrate 10 pk038 Beta-1,3-glucanase, Glycoside hydrolase 24-334 metabolism family 17 18 pk063b Malic enzyme 45-555 pk088b Lipid transfer protein 42-56 70-95 pk088b Cellulose-binding domain 56-86 pk088b Mannose-6-phosphate isomerase type II 150-174 pk088b Glyceraldehyde dehydrogenase 3-phosphate 137-171 a i Classification of the proteins was done by Blasting against the BLOCKS database Henikoff J. G. Henikoff, "Protein family classification based on searching a database of blocks", Genomic, 19:97-107 (1994)).

Example 6 Cloning of full-length cDNAs and Binary Plasmidsfor Plant Transformation RACE-PCR to determine full-length sequences Full-length sequences of the Arabidopsis thaliana partial cDNAs (ESTs) that were identified in the SSH library and from MegaSort and MPSS EST sequencing in the Arabidopsis thaliana EST sequencing program using the annotation program EST-Max were isolated by RACE PCR using the SMART RACE cDNA amplification kit from Clontech allowing both and 3' rapid amplification of cDNA ends (RACE). The isolation of cDNAs and the RACE PCR protocol used were based on the manufacturer's conditions. The RACE product fragments were extracted from agarose gels with a QIAquick® Gel Extraction Kit (Qiagen) and ligated into the TOPO® pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into TOP10 cells (Invitrogen) using standard conditions (Sambrook et al. 1989). Transformed cells are grown overnight at 37°C WO 02/099076 PCT/US02/17937 on LB agar containing 50 gg/ml kanamycin and spread with 40 pl of a 40 mg/ml stock solution of X-gal in dimethylformamide for blue-white selection. Single white colonies are 0selected and used to inoculate 3 ml of liquid LB containing 50 upg/ml kanamycin and grown overnight at 37°C. Plasmid DNA was extracted using the QIAprep® Spin Miniprep Kit z (Qiagen) following manufacturer's instructions. Subsequent analyses of clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al.

1989). The sequences obtained from the RACE reactions were compiled to give the i nucleotide sequences for the LMP genes (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, S23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47).

m RT-PCR and cloning ofArabidopsis thaliana LMP genes C¢l Full-length LMP cDNAs were isolated by RT-PCR from Arabidopsis thaliana RNA.

0 The synthesis of the first strand cDNA was achieved using AMV Reverse Transcriptase (Roche, Mannheim, Germany). The resulting single-stranded cDNA was amplified via Polymerase Chain Reaction (PCR) utilizing two gene-specific primers. The conditions for the reaction were standard conditions with Expand High Fidelity PCR system (Roche). The parameters for the reaction were: five minutes at 94C followed by five cycles of 40 seconds at 94'C, 40 seconds at 50 0 C and 1.5 minutes at 72 0 C. This was followed by thirty cycles of seconds at 94°C, 40 seconds at 65 0 C and 1.5 minutes at 72 0 C. The fragments generated under these RT-PCR conditions were analyzed by agarose gel electrophoresis to make sure that PCR products of the expected length had been obtained.

Full-length LMP cDNA were isolated by using synthetic oligonucleotide primers (MWG-Biotech) designed based on the LMP gene specific DNA sequence that was determined by EST sequencing and by sequencing of RACE PCR products. All 5' PCR primers ("forward primer", F) contained an AscI restriction site 5' upstream of the ATG start codon. All 3' PCR primers ("reverse primers", R) contained a PacI restriction site 3' downstream of the stop codon. The restriction sites were added so that the RT-PCR amplification products could be cloned into the AscI and PacI restriction sites located in the multiple cloning site of the binary vector pBPS-GB1. The first 2 nucleotides are used as spacers so the restriction enzymes cut properly. The following "forward" and "reverse" primers were used to amplify the full-length Arabidopsis thaliana cDNAs by RT-PCR using RNA from Physcomitrella as original template: WO 02/099076 PCT/US02/ 17937 For amplification of SEQ ED) NO: I PkOO2F (5'-ATGGCGCGCCATGGTTCGTCCTGGATTCATTATGC-3') (SEQ ID NO:49) pkOO2R (5'-GCTTAATTAATCAGGCTTTGGATTTACCGGGAC-3') (SEQ ID Z Ni For amplification of SEQ ID) NO:3 pkO2OF (5'-ATGGCGCGCCGGGGAGGCCACCAAGAAAGTGCTG (SEQ ID NO:51) IND pkO2OR (5'-TCTTAATTAAGCTACATTTATCATTGCCGGCCTC (SEQ ID NO:52) For amplification of SEQ ID pkO33F (5'-ATGGCGCGCCGAGCCGTTATTAGTTTTCATTCAAGGG..3') (SEQ ID NO:53) pkO33R (5'-AGTTAATTAATCTTAACTTACCAAGGAGCCGACAAA-3') (SEQ ID NO:54) For amplification of SEQ ED NO:7 pkO37F -ATTGGCGCGCCTGGTGTTGCAGTGATTTGATTC-3') (SEQ ID pkO37R (3 (SEQ ID NO:56) For amplification of SEQ ID) NO:9 pkO3 8F -ATGGCGCGCCATGACCACGTTATTCCTCCTTATTGC-3') (SEQ ID NO:57) pkO3 8R (5 '-GCTTAATTAACATCACTCAACCGCCGTACCGTC-3') (SEQ ID NO:58) For amplification of SEQ ID) NO:1 1 pkO42F (5'-ATGGCGCGCCGGGCAAAGGCCATTGTCGAGT-3') (SEQ ID NO:59) pkO42R (5'-GCTTAATTAACCGGTACTCGGTTTAGTTCAATTT-3') (SEQ ID For amplification of SEQ ID NO: 13 pkO53F (5'-ATGGCGCGCCCATGGATGTCTTGTTTCTCGATCTG-3') (SEQ lID NO:61) pkO53R (5'-GCTTAATTAAGATTTCTGACTTTGAATGGAAATGG-3') (SEQ ID NO:62) WO 02/0990(76 PCT/US02/1 7937 For amplification of SEQ ID NO: pkO6OF -ATGGCGCGCCGAATCAGAAATTGGGCGAAGAAG-3') (SEQ ID NO:63) pkO6OR (5'-GCTTAATTAACACAACAATCACTAGCTCAGAAGAGC-3') (SEQ lID NO: 64) For amplification of SEQ ED NO: 17 pkO63F (5 '-ATGGCGCGCCATGGAGAAAGTGACCAACTCAGACT-3') (SEQ ID pkO63R (5 '-GCTTAATTAATTCAACGGTAGAGACGGTATGTGGG-3') (SEQ IID NO:66) For amplification of SEQ DD NO: 19 pkO64F (5'-ATGGCGCGCCAGAAAAAAGCAATGGCGTCAGAC-3') (SEQ ID NO:67) pkO64R (5 '-GCTTAATTAACTCACAGCTTTCCCTTATCTTTCC-3') (SEQ ID NO:68) For amplification of SEQ ID) NO:21 pkO73F ATGGCGCGCCGCCAGAGGACGAGACAAGGGG-3') (SEQ ID NO:69) pkO73R -GCTTAATTAACCATTGGGGATGGTCTAAGTTTCC (SEQ ID For amplification of SEQ ID NO:23 pkO82F (5'-ATGGCGCGCCATGGAAGGTGGAGCTGCTCTC (SEQ ID) NO:71) pkO82R (5 '-GCTTAATTAAGCCAAATAAAGACAGATCGAGGG-3') (SEQ ED NO:72) For amplification of SEQ ID pkO84F (5 '-ATGGCGCGCCATGGGGTCATCACAGTCTTCGC-3') (SEQ ID NO:73) pkOS4R -GCTTAATTAATCAATAGTGTAGAAACCTAACAAGTATTGG- 3')(SEQ ID) NO:74) For amplification of SEQ ID NO:27 pkO86F -ATGGCGCGCCCAAGGGGGGTCTCTGTCGGTTC (SEQ ED pkO86R (5 '-GCTTAATTAACATCATCATTGCTCTCCAAACCC 3')(SEQ ID NO:76) WO 02/099076 PCT/US02/I 7937 For amplification of SEQ ID) NO:29 pkO88F (5 '-ATGGCGCGCCGAGAAGAAGAATCAAAATCCCACACA (SEQ ID NO:77) Z pkO88R (5'-GCTTAATTAAGCGATAGATCGAATAAAAGGACCA c-I 3') (SEQ ID NO:78) For amplification of SEQ ID NO:31 IND pkO95F (5 '-ATGGCGCGCCCCTTCTTGATTCGTCCTCTCCACT-3') (SEQ ID NO:79) r- pkO95R (5 '-GCTTAATTAACCACCATCTCCTCCGATTCTCTTC- 3') (SEQ HI) For amplification of SEQ ID NO:33 pkO96F (5 '-ATGGCGCGCCATGGTTAAAGAAATAGCTTCTTGGTT-3') (SEQ ED NO: 81) pkO96R (5 '-GCTTAATTAATTAGAGAAGTTGGTCAGCTTGCAAC- 3') (SEQ ID NO: 82) For amiplification of SEQ ID pkO97F ATGGCGCGCCATGGAAGTCTGCAATTGTATTGAACC-3') (SEQ lED NO: 83) pkO97R GCTTAATTAATTACATGCCCTCGTACAGTACCCG- 3') (SEQ ID NO:84) For amplification of SEQ ID NO:37 pkO97F ATGGCGCGCCATGGAAGTCTGCAATTGTATTGAACC-3') (SEQ ID NO: pkO97R GCTTAATTAATTACATGCCCTCGTACAGTACCCG- 3') (SEQ ID NO:86) WO 02/099076 PCT/1JS02/I 7937 For amplification of SEQ ID) NO:39 pkO98F (5'-ATGGCGCGCCATGATGGGCAGTGTCGAGCTG (SEQ ID NO:87) pkO98R (5'-GCTTAATTAATCAAGCTCTGCTCTTGCACTTCT z 3') c-i (SEQ ID NO:88) For amplification of SEQ ID NO:41 PkO99F ATGGCGCGCCATGATCGGCCAACTTATGACCT-3') (SEQ ID NO: 89) pkO99R GCTTAATTAATCAAGATCTGTTCTTGCAGTACTTCTC- 3') (SEQ ID For amplification of SEQ II) NO:43 Pkl GOF (5 '-ATGGCGCGCCGGAGAAAATAGTTAGCTTTGGTCGG (SEQ ID NO:91) pkl GOR (5'-GCTTAATTAACCAAACGAGTGGTGCAATTACAC 3') (SEQ ID NO:92) For amiplification of SEQ ID ATGGCGCGCCATGGAGGAAGTATCTCCGGCGATC (SEQ ID NO:93) pklIO0R GCTTAATTAATCAGTTCAAGGGTTTGCTCTTGAG 3') (SEQ ID NO:94) For amplification of SEQ ID NO:47 PklO07F ATGGCGCGCCATGGAAATGCCCGGTAGAAGATC (SEQ ID pklO7R GCTTAATTAATTACAAATCCGAGCGGTTGGGCG 3') (SEQ IID NO:96) Example 7 Agrobacteriumi Mediated Plant Tranisformiation For plant transformation, binary vectors such as pBinAR can be used (H~fgen Willmitzer 1990, Plant Sci. 66: 221-230). Plant binary vectors encoding LMvlP genes were 53 WO 02/099076 PCT/US02/17937 constructed with the aim to achieve the overexpression of functionally active proteins in transgenic plants. All LMP gene candidates were cloned into the plant binary vector pBPS- SGB1 vector. The binary vector contains a selectable marker gene driven under the control of the AtAct2-I promoter (Ann Y-Q et al., 1996, Plant Journal 10:107-121) and a USP

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z (unknown seed protein, Biumlein et al., Mol Gen Genet 225: 459-467, 1991) seed-specific C, promoter driving the candidate LMP gene with the NOSpA terminator. Full-length LMP cDNA were cloned into AscI and PacI restriction sites in the multiple cloning site of pBPS- GB1 in sense orientation behind the USP seed-specific promoter. The recombinant binary \CO vectors (based on pBPS-GB1) containing the genes of interest were transformed into E. coli Cc Topl0 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing an antibiotic and grown overnight at 37°C. Plasmid DNA was extracted O using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions.

Analysis of subsequent clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual. 2 nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY). The nucleotide sequence of the inserted LMP genes was verified by sequencing (the insert DNA was sequence by determining the nucleotide sequence of one DNA stand with two independent sequence reactions and the complementary DNA strand with on sequencing reaction according to the Bermuda convention). The full length sequences are shown as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47.

Agrobacterium mediated plant transformation with binary vectors encoding the LMP nucleic acids described herein was performed using standard transformation and regeneration techniques (Gelvin, Stanton B. Schilperoort R.A, Plant Molecular Biology Manual, 2nd ed.

Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Zentrale Signatur: BT11-P; Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and Biotechnology, S. 360, CRC Press, Boca Raton 1993).

The Agrobacterium mediated transformation of Arabidopsis thaliana was performed using the GV3 (pMP90) (Koncz Schell, 1986, Mol. Gen. Genet. 204: 383-396) Agrobacterium tumefaciens strain. Arabidopsis thaliana ecotype Col-2 was grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316: 1194- 1199; Bent et al. 1994, Science 265: 1856-1860). Kanamycin was used as antibiotic selection marker for Agrobacterium transformation. The presence and correct orientation of the LMPencoding binary vectors in Agrobacterium cultures was verified by PCR using the LMP genespecific primers described in example 6. For the plant transformation flowering Arabidopsis 54 WO 02/099076 PCT/US02/17937 plants were dipped into the recombinant Agrobacterium cultures and allowed to go to seed.

Transgenic Arabidopsis T1 plants were identified by growing the seeds on Petri plates O containing the selection agent appropriate for the selection marker present on the T-DNA.

Surviving healthy seedlings were transferred to soil and grown in a growth chamber under

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z controlled conditions. T2 seeds were harvested from these T1 plants. The transgenic lines CN were propagated through successive generations and T2, T3 and T4 seeds were obtained. The segregation ratio of the presence or absence of the T-DNA was monitored in order to In determine whether the lines contained single-locus or multi-locus insertions and whether the Slines were homozygous or heterozygous for the T-DNA insertion. T2, T3 and T4 seeds were c analyzed for seed oil content (see also example 8).

Agrobacterium mediated plant transformation is also applicable to Brassica and other 0crops. In particular, seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% Clorox supplemented with 0.05 Tween for 20 minutes, at room temperature with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. The seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of their water content. The seeds are then stored at room temperature in a sealed Petri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics 100 mg/1 streptomycin, 50 mg/1 kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and re-suspended in MS (Murashige Skoog 1962, Physiol. Plant. 15: 473-497) medium supplemented with 100 pM acetosyringone. Bacteria cultures are incubated in this preinduction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 44% moisture content are imbibed for 2 hours at room temperature with the pre-induced Agrobacterium suspension culture. (The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes). The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/1 carbenicillin or 300 mg/1 cefotaxime to kill the WO 02/099076 PCT/US02/17937 agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25 0 C, under 440 pmol m 2 sec' 1 and 12 hours photoperiod. Once Sthe seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept Z under a plastic cover for 1 week to favor the acclimatization process. Then the plants are Stransferred to a growth room where they are incubated at 25 0 C, under 440 tmol m' 2 s light intensity and 12 h photoperiod for about 80 days.

ttn Samples of the primary transgenic plants (To) are analyzed by PCR to confirm the ^0 presence of T-DNA. These results are confirmed by Southern hybridization wherein DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to Sprepare a digoxigenin-labeled probe by PCR, and used as recommended by the manufacturer.

Transformation of soybean can be performed using for example a technique described in EP 424 047, U.S. Patent No. 5,322,783 (Pioneer Hi-Bred International) or in EP 0397 687, U.S. Patent No. 5,376,543 or U.S. Patent No. 5,169,770 (University Toledo). Soybean seeds are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% Clorox supplemented with 0.05% Tween for minutes with continuous shaking. Then the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours.

The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.

Example 8 Analysis of the hnpact of Recombinant LMPs on the Production of a Desired Seed Storage Compound: Fatty Acid production The total fatty acid content of Arabidopsis seeds was determined by saponification of seeds in 0.5 M KOH in methanol at 80 0 C for 2 h followed by LC-MS analysis of the free fatty acids. Total fatty acid content of seeds of control and transgenic plants was measured with bulked seeds (usually 5 mg seed weight) of a single plant. Three different types of controls have been used: Col-2 (Columbia-2, the Arabidopsis ecotypes LMP gene of interest have been transformed in), C-24 (an Arabidopsis ecotype found to accumulate high amounts of total fatty acids in seeds) and BPS empty (without LMP gene of interest) binary vector construct. The controls indicated in the tables below have been grown side by side with the 56 WO 02/099076 PCT/US02/17937 transgenic lines. Differences in the total values of the controls are explained either by differences in the growth conditions, which were found to be very sensitive to small Svariations in the plant cultivation, or by differences in the standards added to quantify the fatty acid content. Because of the seed bulking all values obtained with T2 seeds and in part

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z also with T3 seeds are the result of a mixture of homozygous (for the gene of interest) and heterozygous events, implying that these data underestimate the LMP gene effect.

l Table 6. Detennination of the T2 seed total fatty acid content of transgenic lines of pk002b (containing SEQ ID NO:1). Shown are the means standard deviation). (Average mean Cc values are shown standard deviation, number of individual measurements per plant line: 12-18; Col-2 is the Arabidopsis ecotype the LMP gene has been transformed in, C-24 is a O high-oil Arabidopsis ecotype used as another control) Genotype g total fatty acids/g seed weight C-24 wild-type seeds 0.513 0.042 Col-2 wild-type seeds 0.462 0.053 pk002b transgenic seeds 0.487 0.047 Table 7. Determination of the T2 seed total fatty acid content of transgenic lines of pk020b (containing SEQ ID NO:3). Shown are the means standard deviation) of 10 (Col-2) and 9 (pk020b) individual plants, respectively.

Genotype g total fatty acids/g seed weight Col-2 wild-type seeds 0.353 0.045 pk020b transgenic seeds 0.402 0.050 Table 8. Determination of the T2 seed total fatty acid content of transgenic lines of pk033 (containing SEQ ID NO:5). Shown are the means standard deviation) of 9 (Col-2) and 18 (pk033) individual plants, respectively.

Genotype g total fatty acids/g seed weight Col-2 wild-type seeds 0.481 0.034 Pk033 transgenic seeds 0.507 0.047 WO 02/099076 PCT/US02/17937 Table 9. Determination of the T2 seed total fatty acid content of transgenic lines of pk037 (containing SEQ ID NO:7). Shown are the means standard deviation) of 10 (Col-2) and 19 (pk037) individual plants, respectively.

Genotype Col-2 wild-type seeds pk037 transgenic seeds 0.301 0.028 0.328 0.026 Table 10. Determination of the T2 seed total fatty acid content of transgenic lines of pk038 (containing SEQ ID NO:9). Shown are the means standard deviation) of 12-20 individual plants per line.

Genotvne o i0 fatt17 Id A M aLtT r IU 1 C-24 wild-type seeds Col-2 wild-type seeds Pk038 transgenic seeds 0.366 0.025 0.319 0.026 0.382 0.028 Table 11. Determination of the T2 seed total fatty acid content of transgenic lines of pk053b (containing SEQ ID NO:13). Shown are the means standard deviation) of 13-19 individual plants per line.

Genotype g total fatty acids/E seed weight Col-2 wild-type seeds C-24 wild-type seeds pk053b transgenic seeds 0.483 0.040 0.483 0.040 0.472 0.042 Table 12. Determination of the T2 seed total fatty acid content of transgenic lines of pk060 (containing SEQ ID NO: 15). Shown are the means standard deviation) of 14-18 individual plants per line.

Genotvne Ge--n *T t tyULLyt aluLaLi nccU wvlIill Col-2 wild-type seeds pk060 transgenic seeds 0.406 0.047 0.435 0.034 WO 02/099076 PCT/US02/17937 Table 13. Determination of the T2 seed total fatty acid content of transgenic lines of pk063b (containing SEQ ID NO: 17). Shown are the means standard deviation) of 16-19 individual plants per line.

Genotype Col-2 wild-type seeds C-24 wild-type seeds pk063b transgenic seeds p total fatty naids/o sed wpioht 0.466 0.036 0.485 0.046 0.480 0.064 Table 14. Determination of the T2 seed total fatty acid content of transgenic lines of pk064 (containing SEQ ID NO:19). Shown are the means standard deviation) of 15-19 individual plants per line.

Genotvne P total fatty 2Pids2/a cppul wc~inIi* a tta ftt agrl/ pp, va

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Col-2 wild-type seeds C-24 wild-type seeds pk064 transgenic seeds 0.466 0.036 0.485 0.046 0.480 0.055 Table 15. Determination of the T2 seed total fatty acid content of transgenic lines of pk073 (containing SEQ ID NO:21). Shown are the means standard deviation) of 13-17 individual plants per line.

Genotvne G e -notvne___ C-24 wild-type seeds Col-2 wild-type seeds pk073 transgenic seeds g total fatty acids/g seed weight 0.513 0.042 0.462 0.053 0.498 0.035 Table 16a. Determination of the T2 seed total fatty acid content of transgenic lines of pk082 (containing SEQ ID NO:23). Shown are the means standard deviation) of 14-19 individual plants per line.

Genotvoe p total fatty ac~id.~/u ~pp~ ii~puht P~ toa at cdsase e i C-24 wild-type seeds Col-2 wild-type seeds pk082 transgenic seeds 0. 377 0.041 0.358 0.028 0.381 0.029 WO 02/099076 PCT/US02/17937 Table 16b. Determination of the T3 seed total fatty acid content of transgenic lines of pk082 (containing SEQ ID NO:23). Shown are the means standard deviation) of 14-19 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds pk082-4 transgenic seeds pk082-10 transgenic seeds pk082-11 transgenic seeds pk082-12 transgenic seeds g total fatty acids/g seed weight 0. 351 L 0.026 0.326 0.036 0.351 0.021 0.361 0.027 0.342 0.032 0.335 0.019 Table 17. Determination of the T2 seed total fatty acid content of transgenic lines of pk084 (containing SEQ ID NO:25). Shown are the means standard deviation) of 12-18 individual plants per line.

Genotype total fatty acids/s seed weight ii m i 1 iI 1 C-24 wild-type seeds Col-2 wild-type seeds pk084 transgenic seeds 0. 377 0.041 0.358 0.028 0.372 0.042 Table 18. Determination of the T2 seed total fatty acid content of transgenic lines of pk086 (containing SEQ ID NO:27). Shown are the means standard deviation) of 14-20 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk086 transgenic seeds g total fatty acids/g seed weight 0.496 0.049 0.443 0.036 0.472 0.049 WO 02/099076 PCT/US02/17937 Table 19. Determination of the T2 seed total fatty acid content of transgenic lines of pk088 (containing SEQ ID NO:29). Shown are the means standard deviation) of 14-20 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk088 transgenic seeds gtotal fattv arirla/a fpprl WPiOh~ 0.466 0.033 0.372 0.045 0.448 0.015 Table 20. Determination of the T2 seed total fatty acid content of transgenic lines of pk095 (containing SEQ ID NO:31). Shown are the means standard deviation) of 14-20 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk095 transgenic seeds g total fatty acids/g seed weight 0.439 0.036 0.419 0.034 0.442 0.037 Table 21. Determination of the T2 seed total fatty acid content of transgenic lines of pk096 (containing SEQ ID NO:33). Shown are the means standard deviation) of 11-19 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk096 transgenic seeds g total fatty acids/g seed weight 0.496 0.049 0.443 0.036 0.472 0.039 Table 22. Determination of the T2 seed total fatty acid content of transgenic lines of pk097a (containing SEQ ID NO:35). Shown are the means standard deviation) of 14-18 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk097a transgenic seeds Stotal fatty awr-igc/ pp 'wpirhl- 0.513 0.042 0.462 0.053 0.506 0.034 WO 02/099076 PCT/US02/17937 Table 23. Determination of the T2 seed total fatty acid content of transgenic lines of pk097b (containing SEQ ID NO:37). Shown are the means standard deviation) of 14-18 individual plants per line.

Genotvne a total fattv acid/o sPer wpioht C-24 wild-type seeds Col-2 wild-type seeds Pk097b transgenic seeds 0.513 0.042 0.462 0.053 0.506 0.034 Table 24. Determination of the T2 seed total fatty acid content of transgenic lines of pk098 (containing SEQ ID NO:39). Shown are the means (I standard deviation) of 12-19 individual plants per line.

Genotype C-24 wild-type seeds Col-2 wild-type seeds Pk098 transgenic seeds g total fatty acids/g seed weight 0.513 0.042 0.462 0.053 0.489 0.027 Table 25. Determination of the T2 seed total fatty acid content of transgenic lines of pk099 (containing SEQ ID NO:41). Shown are the means standard deviation) of 11-20 individual plants per line.

Genotvne 0 tntfll ftlv nid'a/o .ppi1 wpicrt o total fattv acids/a -PrI wpi h+ C-24 wild-type seeds Col-2 wild-type seeds Pk099 transgenic seeds 0.496 0.049 0.443 0.036 0.492 0.034 Table 26. Determination of the T2 seed total fatty acid content of transgenic lines of (containing SEQ ID NO:45). Shown are the means standard deviation) of 14-20 individual plants per line.

Genotype f total fatty acids/g seed weight I II I

I

C-24 wild-type seeds Col-2 wild-type seeds pp84 transgenic seeds 0.439 0.036 0.419 0.034 0.442 0.037 WO 02/099076 PCT/US02/17937 Table 27a. Determination of the T2 seed total fatty acid content of transgenic lines of pkl07

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0(containing SEQ ID NO:47). Shown are the means standard deviation) of 14-20 individual plants per line.

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C1 Genotype g total fatty acids/g seed weight C-24 wild-type seeds 0.496 0.049 Col-2 wild-type seeds 0.443 0.036 SPkl07 transgenic seeds 0.492 0.059 Table 27b. Determination of the T3 seed total fatty acid content of transgenic lines of pkl 07 0(containing SEQ ID NO:47). Shown are the means standard deviation) of 18-30 individual plants per line.

Genotype g total fatty acids/g seed weight C-24 wild-type seeds 0.371 0.025 Col-2 wild-type seeds 0.321 0.028 Pkl07-10 transgenic seeds 0.375 0.023 Pk107-14 transgenic seeds 0.383 0.023 Pk107-17 transgenic seeds 0.399 0.024 Example 9: Analysis of the Impact of Recombinant Proteins on the Production of a Desired Seed Storage Compound The effect of the genetic modification in plants on a desired seed storage compound (such as a sugar, lipid or fatty acid) can be assessed by growing the modified plant under suitable conditions and analyzing the seeds or any other plant organ for increased production of the desired product a lipid or a fatty acid). Such analysis techniques are well known to one skilled in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman 1985, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and 443-613, VCH: Weinheim; Fallon, A. et al. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Product recovery and purification, Biotechnology, vol. 3, Chapter III, pp. 469-714, VCH: Weinheim; Belter, P.A. et al., 1988 Bioseparations: downstream processing for biotechnology, John WO 02/099076 PCT/US02/17937 Wiley Sons; Kennedy J.F. Cabral J.M.S. 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz J.A. Henry J.D. 1988, Biochemical separations Sin: Ulmann's Encyclopedia of Industrial Chemistry, Separation and purification techniques in biotechnology, vol. B3, Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow F.J. 1989).

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z Besides the above-mentioned methods, plant lipids are extracted from plant material as described by Cahoon et al. (1999, Proc. Natl. Acad. Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry 442:141-145). Qualitative and quantitative lipid or I fatty acid analysis is described in Christie, William Advances in Lipid Methodology.

Ayr/Scotland :Oily Press. (Oily Press Lipid Library; Christie, William Gas Chromatography and Lipids. A Practical Guide Ayr, Scotland :Oily Press, 1989 Repr. 1992.

IX,307 S. (Oily Press Lipid Library; and "Progress in Lipid Research, Oxford :Pergamon SPress, 1 (1952) 16 (1977) Progress in the Chemistry of Fats and Other Lipids CODEN.

Unequivocal proof of the presence of fatty acid products can be obtained by the analysis of transgenic plants following standard analytical procedures: GC, GC-MS or TLC as variously described by Christie and references therein (1997 in: Advances on Lipid Methodology 4th ed.: Christie, Oily Press, Dundee, pp. 119-169; 1998). Detailed methods are described for leaves by Lemieux et al. (1990, Theor. Appl. Genet. 80:234-240) and for seeds by Focks Benning (1998, Plant Physiol. 118:91-101) Positional analysis of the fatty acid composition at the C-l, C-2 or C-3 positions of the glycerol backbone is determined by lipase digestion (see, Siebertz Heinz 1977, Z.

Naturforsch. 32c:193-205, and Christie 1987, Lipid Analysis 2 nd Edition, Pergamon Press, Exeter, ISBN 0-08-023791-6).

A typical way to gather information regarding the influence of increased or decreased protein activities on lipid and sugar biosynthetic pathways is for example via analyzing the carbon fluxes by labeling studies with leaves or seeds using 14C-acetate or 1 4 C-pyruvate (see, e.g. Focks Benning 1998, Plant Physiol. 118:91-101; Eccleston Ohlrogge 1998, Plant Cell 10:613-621). The distribution of carbon-14 into lipids and aqueous soluble components can be determined by liquid scintillation counting after the respective separation (for example on TLC plates) including standards like 14C-sucrose and 14C-malate (Eccleston Ohirogge 1998, Plant Cell 10:613-621).

Material to be analyzed can be disintegrated via sonification, glass milling, liquid nitrogen and grinding or via other applicable methods. The material has to be centrifuged after disintegration. The sediment is re-suspended in distilled water, heated for 10 minutes at 100 0 C, cooled on ice and centrifuged again followed by extraction in 0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at 90 0 C leading to hydrolyzed oil and 64 WO 02/099076 PCT/US02/17937 lipid compounds resulting in transmethylated lipids. These fatty acid methyl esters are extracted in petrolether and finally subjected to GC analysis using a capillary column O (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperature gradient between 170 0 C and 240°C for 20 minutes and 5 min. at 240 0 C. The identity of resulting fatty

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z acid methylesters is defined by the use of standards available form commercial sources Ci Sigma).

In case of fatty acids where standards are not available, molecule identity is shown via Iderivatization and subsequent GC-MS analysis. For example, the localization of triple bond \O fatty acids is shown via GC-MS after derivatization via 4,4-Dimethoxy-oxazolin-Derivaten c (Christie, Oily Press, Dundee, 1998).

A common standard method for analyzing sugars, especially starch, is published by

O

SStitt Lilley R.Mc.C., Gerhardt R. and Heldt M.W. (1989, "Determination of metabolite levels in specific cells and subcellular compartments of plant leaves" Methods Enzymol.

174:518-552; for other methods see also Hartel et al. 1998, Plant Physiol. Biochem. 36:407- 417 and Focks Benning 1998, Plant Physiol. 118:91-101).

For the extraction of soluble sugars and starch, 50 seeds are homogenized in 500 pI of ethanol in a 1.5-ml polypropylene test tube and incubated at 70°C for 90 min.

Following centrifugation at 16,000 g for 5 min, the supernatant is transferred to a new test tube. The pellet is extracted twice with 500 p1 of 80% ethanol. The solvent of the combined supernatants is evaporated at room temperature under a vacuum. The residue is dissolved in ptl of water, representing the soluble carbohydrate fraction. The pellet left from the ethanol extraction, which contains the insoluble carbohydrates including starch, is homogenized in 200 pl of 0.2 N KOH, and the suspension is incubated at 95 0 C for 1 h to dissolve the starch.

Following the addition of 35 p of 1 N acetic acid and centrifugation for 5 min at 16,000 g, the supematant is used for starch quantification.

To quantify soluble sugars, 10 pl of the sugar extract is added to 990 ptl of reaction buffer containing 100 mM imidazole, pH 6.9, 5 mM MgCl 2 2 mM NADP, 1 mM ATP, and 2 units 2 ml' of Glucose-6-P-dehydrogenase. For enzymatic determination of glucose, fructose and sucrose, 4.5 units of hexokinase, 1 unit of phosphoglucoisomerase, and 2 pl of a saturated fructosidase solution are added in succession. The production of NADPH is photometrically monitored at a wavelength of 340 nm. Similarly, starch is assayed in 30 pl of the insoluble carbohydrate fraction with a kit from Boehringer Mannheim.

An example for analyzing the protein content in leaves and seeds can be found by Bradford M.M. (1976, "A rapid and sensitive method for the quantification of microgram WO 02/099076 PCT/US02/17937 quantities of protein using the principle of protein dye binding" Anal. Biochem. 72:248-254).

For quantification of total seed protein, 15-20 seeds are homogenized in 250 pl of acetone in Sa 1.5-ml polypropylene test tube. Following centrifugation at 16,000 g, the supematant is >discarded and the vacuum-dried pellet is resuspended in 250 ptl of extraction buffer

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Z containing 50 mM Tris-HC1, pH 8.0, 250 mM NaCI, 1 mM EDTA, and 1% SDS.

Following incubation for 2 h at 25 0 C, the homogenate is centrifuged at 16,000 g for 5 min and 200 ml of the supernatant will be used for protein measurements. In the assay, y-globulin n is used for calibration. For protein measurements, Lowry DC protein assay (Bio-Rad) or SBradford-assay (Bio-Rad) are used.

SEnzymatic assays of hexokinase and fructokinase are performed spectrophotometrically according to Renz et al. (1993, Planta 190:156-165), of phosphoglucoisomerase, ATP-dependent 6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase, Fructose-1,6-bisphosphate aldolase, triose phosphate isomerase, glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase are performed according to Burrell et al. (1994, Planta 194:95-101) and of UDP-Glucose-pyrophosphorylase according to Zrenner et al. (1995, Plant J. 7:97-107).

Intermediates of the carbohydrate metabolism, like Glucose-1-phosphate, Glucose-6phosphate, Fructose-6-phosphate, Phosphoenolpyruvate, Pyruvate, and ATP are measured as described in Hirtel et al. (1998, Plant PhysioL Biochem. 36:407-417) and metabolites are measured as described in Jelitto et al. (1992, Planta 188:238-244).

In addition to the measurement of the final seed storage compound lipid, starch or storage protein) it is also possible to analyze other components of the metabolic pathways utilized for the production of a desired seed storage compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound (Fiehn et al.

2000, Nature Biotech. 18:1447-1161).

For example, yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.

Similarly, plant expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soybean, rape, maize, wheat, Medicago truncatula, etc., using standard protocols. The resulting transgenic cells and/or plants derived therefrom can then be assayed for alterations in sugar, oil, lipid or fatty acid contents.

WO 02/099076 PCT/US02/17937 Additionally, the sequences disclosed herein, or fragments thereof, can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, O mammalian cells, yeast cells, and plant cells (Girke at al. 1998, Plant J. 15:39-48). The resultant knockout cells can then be evaluated for their composition and content in seed

O

z storage compounds, and the effect on the phenotype and/or genotype of the mutation. For CN other methods of gene inactivation include US 6004804 "Non-Chimeric Mutational Vectors" and Puttaraju et al. (1999, "Spliceosome-mediated RNA trans-splicing as a tool for gene t therapy" Nature Biotech. 17:246-252).

SExample 10: Northern-Hybridization For RNA hybridization, 20 g of total RNA or 1 g of poly-(A)+ RNA is separated Sby gel electrophoresis in 1.25% strength agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem. 152:304), transferred by capillary attraction using 10 x SSC to positively charged nylon membranes (Hybond Amersham, Braunschweig), immobilized by UV light and pre-hybridized for 3 hours at 68 0 C using hybridization buffer dextran sulfate w/v, 1 M NaC1, 1% SDS, 100 g/ml of herring sperm DNA). The labeling of the DNA probe with the Highprime DNA labeling kit (Roche, Mannheim, Germany) is carried out during the pre-hybridization using alpha- 3 2 P dCTP (Amersham, Braunschweig, Germany). Hybridization is carried out after addition of the labeled DNA probe in the same buffer at 68 0 C overnight. The washing steps are carried out twice for min using 2 x SSC and twice for 30 min using 1 x SSC, 1% SDS at 68 0 C. The exposure of the sealed filters is carried out at -70 0 C for a period of 1 day to 14 days.

Example 11: DNA Sequencing and Computational Functional Analysis of SSH library The SSH cDNA library as described in Examples 4 and 5 was used for DNA sequencing according to standard methods, in particular by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin- Elmer, Weiterstadt, Germany). Random sequencing was carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DHO1B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands). Plasmid DNA was prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing ampicillin (see Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNA preparation robot (Qiagen, Hilden) according to the manufacturer's protocols). Sequencing primers with the following nucleotide sequences were used: WO 02/099076 PCT/US02/17937 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO:97) '-CTAAAGGGAACAAAAGCTG-3' (SEQ ID NO:98) S5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO:99) Sequences were processed and annotated using the software package EST-MAX

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z commercially provided by Bio-Max (Munich, Germany). The program incorporates C practically all bioinformatics methods important for functional and structural characterization of protein sequences. For reference see http://pedant.mips.biochenm.mpg.de.

i The most important algorithms incorporated in EST-MAX are: FASTA: Very sensitive protein sequence database searches with estimates of statistical significance (Pearson W.R.

1990, Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98). BLAST: Very sensitive protein sequence database searches with 0 estimates of statistical significance (Altschul Gish Miller Myers E.W. and Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 215:403-410). PREDATOR: High-accuracy secondary structure prediction from single and multiple sequences. (Frishman Argos 1997, 75% accuracy in protein secondary structure prediction. Proteins 27:329-335).

CLUSTALW: Multiple sequence alignment (Thompson, Higgins, D.G. and Gibson, T.J.

1994, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22:4673-4680). TMAP: Transmembrane region prediction from multiply aligned sequences (Persson B. Argos P. 1994, Prediction of transmembrane segments in proteins utilizing multiple sequence alignments, J. Mol. Biol. 237:182-192).

ALOM2:Transmembrane region prediction from single sequences (Klein Kanehisa M., and DeLisi C. 1984, Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221-226. Version 2 by Dr. K. Nakai).

PROSEARCH: Detection of PROSITE protein sequence patterns. Kolakowski L.F. Jr., Leunissen J.A.M. and Smith J.E. 1992, ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13:919- 921). BLIMPS: Similarity searches against a database of ungapped blocks (Wallace Henikoff 1992, PATMAT:A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford).

Example 12: In vivo Mutagenesis In vivo mutagenesis of microorganisms can be performed by incorporation and passage of the plasmid (or other vector) DNA through E. coli or other microorganisms (e.g.

Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their 68 WO 02/099076 PCT/US02/17937 capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system mutHLS, mutD, mutT, etc.; for O reference, see Rupp W.D. 1996, DNA repair mechanisms, in: Escherichia coli and Sabnonella, p. 2277-2294, ASM: Washington.) Such strains are well known to those skilled

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z in the art. The use of such strains is illustrated, for example, in Greener and Callahan 1994, Strategies 7:32-34. Transfer of mutated DNA molecules into plants is preferably done after selection and testing in microorganisms. Transgenic plants are generated according to various t examples within the exemplification of this document.

SExample 13: Assessment of the mRNA Expression and Activity of a Recombinant Gene Product in the Transformed Organism SThe activity of a recombinant gene product in the transformed host organism can be measured on the transcriptional or/and on the translational level. A useful method to ascertain the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information at least partially demonstrates the degree of transcription of the transformed gene. Total cellular RNA can be prepared from plant cells, tissues or organs by several methods, all well-known in the art, such as that described in Bormann et al. (1992, Mol. Microbiol. 6:317-326).

To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (see, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.

The activity of LMPs that bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such LMP on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar H. et al. 1995, EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both 0 prokaryotic and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.

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z The determination of activity of lipid metabolism membrane-transport proteins can be CN performed according to techniques such as those described in Gennis R.B. (1989 Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, Springer: l Heidelberg, pp. 85-137, 199-234 and 270-322).

SExample 14: In vitro Analysis of the Function of Arabidopsis thaliana Genes in c Transgenic Plants The determination of activities and kinetic parameters of enzymes is well established Sin the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one skilled in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M. Webb, E.C. 1979, Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, ed. (1983) The Enzymes, 3 rd ed.

Academic Press: New York; Bisswanger, (1994) Enzymkinetik, 2 n d ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, Bergmeyer, GraB1, eds. (1983-1986) Methods of Enzymatic Analysis, 3 rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes. VCH: Weinheim, p. 352- 363.

Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge in Australia.

AO 752756.1 '7n

Claims (28)

1. 2. A method of producing a transgenic plant having a modified level of a seed storage compound comprising, transforming a plant cell with an expression vector comprising a LMP nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, and wherein the LMP nucleic acid comprises a polynucleotide sequence encoding a polypeptide selected from the group consisting of: SEQ ID NO:2, 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.
2. 3. A method of producing a transgenic plant having a modified level of a seed storage compound comprising, transforming a plant cell with an expression vector comprising a LMP nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, and wherein the LMP nucleic acid comprises a polynucleotide of least 60 consecutive nucleotides of the LMP nucleic acid of claim 1 or claim 2.
3. 4. A method of producing a transgenic plant having a modified level of a seed storage compound comprising, transforming a plant cell with an expression vector comprising a LMP nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, and wherein the LMP nucleic acid comprises a polynucleotide having at least 70% sequence identity with the LMP nucleic acid of claim 1 or claim 2. WO 02/099076 PCT/US02/17937 A method of producing a transgenic plant having a modified level of a seed storage 0 compound comprising, transforming a plant cell with an expression vector comprising a LMP C nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid >encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, z and wherein the LMP nucleic acid comprises a polynucleotide having at least 90% sequence N. identity with the LMP nucleic acid of claim 1 or claim 2.
4. 6. A method of producing a transgenic plant having a modified level of a seed storage compound comprising, transforming a plant cell with an expression vector comprising a LMP nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid c encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, and wherein the LMP nucleic acid comprises a first nucleic acid that hybridizes under Sstringent conditions to a second nucleic acid nucleic acid of claim 1 or claim 2.
5. 7. A method of producing a transgenic plant having a modified level of a seed storage compound comprising, transforming a plant cell with an expression vector comprising a LMP nucleic acid and generating from the plant cell the transgenic plant, wherein the nucleic acid encodes a polypeptide that functions as a modulator of a seed storage compound in the plant, and wherein the LMP nucleic acid comprises a polynucleotide complementary to the LMP nucleic acid of claim 1 or claim 2.
6. 8. A method of modulating a level of a seed storage compound in a plant comprising, modifying the expression of a LMP nucleic acid in the plant, wherein the LMP nucleic acid is selected from the group consisting of the LMP nucleic acids of claims 1, 2, 3, 4, 5, 6 or 7.
7. 9. The method of claim 8, wherein the plant is transgenic. The method of claim 8, wherein the plant is not transgenic.
8. 11. The method of claims 1, 2, 3, 4, 5, 6 or 8, wherein the nucleic acid encodes a polypeptide that contains a DNA-binding domain.
9. 12. The method of claim 11, wherein the nucleic acid encodes a polypeptide of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, or SEQ ID NO:44.
10. 13. The method of claims 1, 2, 3, 4, 5, 6 or 8, wherein the nucleic acid encodes a polypeptide that contains a protein kinase domain. WO 02/099076 PCT/US02/17937
11. 14. The method of claim 13, wherein the nucleic acid encodes a polypeptide of SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:48, SEQ ID NO:22 or SEQ ID NO:16. O 15. The method of claims 1, 2, 3, 4, 5, 6 or 8, wherein the nucleic acid encodes a Z polypeptide that contains a signal transduction domain.
12. 16. The method of claim 15, wherein the nucleic acid encodes a polypeptide of SEQ ID NO:2, SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:46. In
13. 17. The method of claims 1, 2, 3, 4, 5, 6 or 8, wherein the nucleic acid encodes a polypeptide that contains a protease domain.
14. 018. The method of claim 17, wherein the nucleic acid encodes a polypeptide of SEQ ID NO:6 or SEQ ID NO:12.
15. 19. The method of claims 1, 2, 3, 4, 5, 6 or 8, wherein the nucleic acid encodes a polypeptide that contains a carbohydrate metabolism domain. The method of claim 15, wherein the nucleic acid encodes a polypeptide of SEQ ID SEQ ID NO:18 or SEQ ID
16. 21. A transgenic plant made by the method of claims 1, 2, 3, 4, 5, 6 or 7 wherein expression of the LMP nucleic acid in the plant results in a modified level of a seed storage compound in the plant as compared to a wild type variety of the plant.
17. 22. The transgenic plant of claim 21, wherein the plant is a dicotyledonous plant.
18. 23. The transgenic plant of claim 21, wherein the plant is a monocotyledonous plant.
19. 24. The transgenic plant of claim 21, wherein the plant is an oil producing species. The transgenic plant of claim 21, wherein the plant is selected from the group consisting of rapeseed, canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, sugarbeet, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut.
20. 26. The transgenic plant of claim 21, wherein the level of the seed storage compound is increased.
21. 27. The transgenic plant of claim 21, wherein the seed storage compound is selected from the group consisting of a lipid, a fatty acid, a starch and a seed storage protein. 73 WO 02/099076 PCT/US02/17937
22. 28. A seed produced by the transgenic plant of claim 21, wherein the plant is true C breeding for a modified level of the seed storage compound as compared to a wild type variety of the plant. O
23. 29. A seed oil produced by the seed of claim 21. An isolated LMP nucleic acid comprising a polynucleotide sequence encoding a polypeptide that functions as a modulator of a seed storage compound in a plant, wherein the 0polynucleotide is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:9, SEQ me ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 or SEQ ID NO:47.
24. 31. An isolated LMP nucleic acid comprising a polynucleotide sequence encoding a polypeptide that functions as a modulator of a seed storage compound in a plant, wherein the polynucleotide is selected from the group encodes a polypeptide selected from the group consisting of a SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42 or SEQ ID NO:48.
25. 32. An isolated nucleic acid comprising a polynucleotide of least 60 consecutive nucleotides the LMP nucleic acid of claims 30 or 31.
26. 33. An isolated nucleic acid comprising a polynucleotide having at least 70% sequence identity with the LMP nucleic acid of claims 30 or 31.
27. 34. An isolated nucleic acid comprising a polynucleotide having at least 90% sequence identity with the LMP nucleic acid of claims 30 or 31. An isolated nucleic acid comprising a polynucleotide complementary to the LMP nucleic acid of claims 30 or 31.
28. 36. An isolated nucleic acid that hybridizes under stringent conditions to a second nucleic acid nucleic acid of claims 30 or 31. WO 02/099076 PCT/IJS02/17937 Figure IA SEQ ED NO: 1, Nucleotide sequence of the open reading framne of pkOO2b ATGGTTCGTCCTGGATTCATTATGCGGCCGCCTGGTACATCGGTGCTGTTCAAC z TAGCACCACGACCCCTTATTCCAGGAAMGCCTGGTCTCCGTCCTGTAATGCCTCCT ATGGTTAGACCGGCTTCTCTTCCTTTTGTAACACCTGCAGAAAAGCCCCAGACCA CAATTTACATTGGCAAGATAGCTACCGTGGAAAATGACTTTATGATGTCTATTCT TGAGTTTTGTGGCCATGTCAAAAGCTGTTTACGTGCGGAAGATCCTACCACpA AAACCTAAAGGTTTTGGATTCTATGAATTTGAATCAGCTGAAGGGATTCTCCGCG CAATACGCCTGCTGACCCAACGTACTATAGATGGACAAGAGCTTTTGGTGAATGT TAATCAAGCAACAAAGGAGTATTTGCTAATATGTTGAGAGATAGAGAC TGCAAAGAAAGCCAAGGAAAGTCAAGGAACCAAGAGAACCAAGCTGAAGGTC CTGAGAGTGAGCAAGACAAGCTTGAGAGTGCTGATAATGAGACAGGGAGGAT GGGACAGTAAAACTGAATCATCGTTCAC GATGAAGAAAGGGAAGCAGACAGAGAGGCTATGGAAGATTGAAACTGCTAT TGAAGAAAGGTTAAAGTCCAACCCTTTGCCTCCTCCACCACCACCACCTGCTGAT GGTTCAGGCATGGAATTTGCTTTCANATCTAAGGATGGTGACTCCAACACTGACG TAGCTAGGAGTGATGCCGCAGCAAATGATGTTGAGACTTCTGGAGACACAAJTA GGCCTGACACAAGCTCACCTGATTGGAGTAAGAGAAATGACCGAAGAGGCAGAG AAAGAGGTGAGAAGGAGCAAGAAATGGA&TAGATACGAGAGGGAGGCTGAAAGA GAACGGTCAAGGAAAGAGAGAGAGCAAAGGAGGA-AACTTGAGGATGCAGAGCG TGTACGCCTTCAATGACAGGAGGAAGAA GGAACGACAGTACGAGAAGGAGAAGAGAAGAGAAGAGCGCAGAGGAA AAGATCCAGGAGAAGAAGCAGTATAGA AAGATGGCATAGGGCTGCATTAGATGAGAGAAGAAGACGACAACTAGAGA AGGAGGATGACTTAGCTGATAGATTGAAAGAAGAGGAJAGAGGTTGCTGAGGCG AAGAGGAGTGCCGAGGAGCAAAATTTGCAGCAACAGCAATTAGATGCCTTAGA ATCCTATCGGGACAGGCAGCTATTGGAAGCGAAACGGTTCAGACATCACCTATT GAAAATGATCACAAGGCAACTCTCCACTGTCGGTGAATCTGCCATGAGCAC CATGCAGCAGATTTTGAAGAAAATGGTTCTGGTAATGAATCGATGGCTATCGATA ATAATAGTGGATCAGAGGCACATGCTCCCTCAAAGAAATTAGGATTTGGGCTTGT GGGATCCGGAAAGCGAACTTCTGTGCCTTCTGTTTTCTATGAGGAGGATGAGAT GAAGCACGTAAGGCTAANAAGATGAAACCTTTGGTTCCTATAGATTACTCAACC GAGGAACAAGAGGCTGTGGCCCATGGTGGCTCAGGGAATACACCACCTCATTTG GCTTTAGCCGCTGAATTTGCAACGAATTTCGAGTAcCAATCCCAGGAAGAG WO 02/099076 PCT/1JS02/I 7937 ACGATAGAAACCGAAAAACAAAGGAGCAGACGTTCTCATGATAAGGCAAGCCA CCGGGACAGGGAAAGGGAAAGGGAAAGGGACAGGGACAGGGATAGAGTCAGG GACCGAGGTGACGGGCATAGTGGTCCCACCAAGACGCCAAAGAGTCTGGAAAA GCAAAGATAATTGATACTAAGTTTCTGGATGCGAAACAATTGATAGATACAATCC z CAAAGACAAAGGAAGATTTATTTTCTTACGAGATAAACTGGGCTATGTATGACA AGCACCAAGTGCACGAAAGAATGAGACCATGGATCTCAAGJJAJJTATGGAGT TTCTCGGAGAAGAGGAAGCCACTCTGGTAGATTTCATCGTGTCAAACACTCAACA ACCTCGCTTAAGTGGTTGATATCAAGAA GCTGAGATGTTTGTGCTGAAGATGTGGAGAACGCTCATCTTTGAGATCAAGCGGG TTGAAGCTGGAGTCCCGGTAAAATCCAAAGCCTGA Figure lB SEQ ID NO:2, Amino acid sequence of the open reading frame of pkOO2b MVRPGFBVTRPGTIGAVQLAPRPLIPGMIPGLRPVMPPMYRPASLPFVTPAEKYQTTIYI GKIATVENDFMMSILEFCGIIVKSCLRAEDPTTKKPKGFGFYEFESAEGILRAIRLLTQ RTIIDGQELLVNVNQATKEYLLKYVEKIKIETAKKAICESQGTKENQAEGPESEQDKLES ADNETGKDGESKIKENIDIANSAVLTDEEREADREAM4EKIETAIEERLKSNPLPPPPPP PADGSGMEFAFKSKDGDSNTDVARSDMAANDVETSGEITNRPDTS SPDWSKRNDRR GRERGEKEQEMDRYEREAERRRSRKEREQRRJCIEDAERAYQTRILRQWERREREKE KERQYEKEKEKEKERKRKKEIRYEEEEEEDDDDSRRRWHRAADERRRRQLREKED DLADRLKEEEEVAEAKRSAEEQNLQQQQLDALR1LSGQAAIGSETVQTSPEENDHKA TLTGSNHADENSNSAINSSAASKGGVSKT VPSVFYEEDEDEAR KAKK'MKPLrPIDYSTEEQEAVA1{GGSGNTPPHLALAAEFAC2J S STNPKEETfITEKQRSRSKASDRRRRRDRVRGDGHSGPTCDAK ESGKAK]DTKFLDAQLDTIKTKEDLFSYEINWAAAYDKAQVHEPVIPWISKIME FLGEEEATLVDFIVSNTQQHVQASQMLELLQSLDEEAEMFVLKMVWRTLIFlEIKRVEA GVPVKSKA Figure 2A SEQ ID NO: 3, Nucleotide sequence of the open reading frame of pkO2Ob GTGCTGAAAAAGAAGAAACCCATACTTACTGACGAGCATTTCGTAGATGGTCGG TTTGGTTCGATGTTCCAAAATCCGGACTTCCAAATTGATAAGGACTCATATGAAT ATGGTGTCCTACACCCTGTTGCTTCTTCGAAGAAGCAACCTTCTCTGTTAGATGA WO 02/099076 PCTLJS02/I 7937 ACACTTTGAGCTGTATCAGATGATGACGAGAACAGTGATCTGATGCATCACAG CCTTCAGATGACGAGGCcGACGATGGAGACGCCTAGGCCAGCAGAAAGC GAGAACTCCGAAGTTGTATGAAGTGAAAGATGAGCGGCATGCCGCAGCTTATCA CAACCGCACTTCACTGGCTAAAGAAGATAGTCTTCCTATGGGCGAGCGTGTCA-AG z GCTATAGAGAACCGGCGTGGCAACTTTGGAGGCTCGAAGATATCJAAATTCGGT CCTGGAGGATCACGGGAGTTTTCTTTCAAGGCGAGAGGGTCATCJAAAGTACAAA GAAGATAGAGACGATGAGTATGAAGATGGGCAAGAAJACAGAGGAGAGGAGT TCAGTCTCTTGGACTGAAATCAACAAATATTAGAGGCGGTTTCAGAGGTAGAGG AGGTGGTGGTTTTAGAGGGAGAGGAGGCGGCGGTTCCCGGGGAAGGTGGCCG TGGTGGTGGGCGTGGAAGAGGCCGGCJ&ATGA Figure 2B SEQ ID NO:4, Amino acid sequence of the open reading frame of pkO2Ob VLKKKKETDEINFVDGRGSYQNPDFQISYEYGVLYVASSKQQPSLLDEHF EASDESSAQSDADDTRSKRPLEKEHAYNT LAESPGRKINRNGGKIFPGRFFAGSKYKLDRDDEY EDGQRNKRRGVQSLGLKSTNLRGGFRGRGGGGFRGRGGGGSRGKGGRGGGRGRGR Q Figure 3A SEQ lID NO:5, Nucleotide sequence of the open reading frame of pkO33 ATGGAGCAGATGATATTCATTAGAGACGACAAGTAGAGGTTTTTTCTGA GAAGAAGAGTTAAGGGTCTTATTACAGAGCGATTCTGGAGATACCAACG AAATCAGGACACAATAGCTTAGTTCGTTACTTGACGCAGCTCATGAACAC CGTTTGGCTCCTTTAACGGAATTCGTCGATCAGAGGTTCATTCGTCCTGTCCCGTC GGAGGATGTGAACGACGGCGTCGTTTTTGTAGAAGGCTTGATGGTCGACGCTTAT CTAAAGGGTGCGTTGTGAAAATGGAGGA TTTTTGGTTTACTTCGATTGCCCACCAGACATTATTCAGTTTGAGAAAGAAATT GAGGGTTCATCTTGATTGGACCGGCTTCAAATGGATCCGACCTGATATJAAGGA TTGGTCAAGTCTGTTTTTAGTTGCGGGACAATGGTGGAATTGAGATTTGATTGTG CTTGGATTCCGGTAATTGTCATTAAGGAGTTGGAGAAGGACAAGAGGTTTCTTGT WO 012/099076 PCT/US12/I 7937 CAAGTACTGGAATAAGTCCTATAGCTGCCGGGTCGAATTnATTGTTGAT r- ~TCCCTAAGACTAAGGCCTATGCAGCCTCTTTATCTGTTGGAGTATGAATTGC 0 TGGATCATGTAGAGGCGTTTAGTGGTTTTGAAkTGGCGTCAJAGGTGTGGTCAGGGG AATTGTCTTTGAGGGAAGGTACATGGTAAGTTTCGGGGCACKAAGGAGGCATC z GCAATTTAATCACTCTGATATTAGGCCTCCAATGGAGTGGGAAGATGGAGTTTGG CATAAAAGAACAAAGCCAAAACGCCAGAAAGAACTTCTTTAGACGGCACAG AAATGTGCAGACAAAGGAACCACCGGGAAATGAGATGGCTGATGATGTGAA AAGAATCTGGTTTACCTATAACCCTGGGGGTACTGCCGACACCC AAGGAAAGGTATCCCCTGTGCCAATGAAGAATGGCTTTGGAATGAGTCAACTC GAGAGAAGATGCCTGAGGAGCCTAAGATCAATATTATACTCGKAAGAGGAAAJA GAGGAGGTCTAAAGCTCAATTCATACATCAATAAGACTGTGTTATCCTCGGATCG GACCCCCAATGTGGTGAAGAATTCTGCTTCTAATGCTGAGGAAACCATGCAA ACACACAATAATGGTTTTGCCTTTTGCAAAGAAGTCACCGGTCTGGAAGACTTAT GAATCACTGGAGGTCTTCAAAAGTGTATCACACAGTCTTCATTTCAGCCCATTGT TTGAGACTAAGCAAGATTTCCGTGAAGGGTATGCAATAGGTATGATGGTGACTTA TTTTGGGTTACTGGAGAATTTAAGATCTTGAAGCCGACGTTCCTGTAGCCAA CTAAATAGCCTTAAAGATTCATTTTCGGAGCTCGAGAAACATGGCTTCAJATGTTA CAACTCCATTATCACGGATCGACAAGCTGTCAGCACTCAAAGATAGACAAJCTAT ATATAATGGAGGAACTAAAGGTTTTGACAAGGAGATGACAATGATTAGCA AGGCTAAACAAGAGTTTGATGACATGGAACAAAGATTCTTGAGGTGJAACACA AGTACACGAAGAGACGTTAAACAAGAC GAAAAAGAACAGAAAGATGCAGCCTGGAJAJAGATATGTCAGATGGAGTCATG CGCAAAAGATCTCAATGTAGAGCTTGAAGATGTGGAGTTTGAGTTTGAGACAATT TTGTCGGCTCCTTGGTAA Figure 3B SEQ ID NO:6, Amino acid sequence of the open reading frame of pk033 MEQMIIEKDDKVEVFSEEEELKGSYYRAILEDNPTKSGHNYKXTVRYLTQLEHRA PLTEFVDQRRPVPSEDVNGVVFVEGLMVDAYLKDGWWTGVVJKTMEDEKFLV YFCPHFKKRHDTFWRDNEVSFCTVL.DAEV VW.-ELEKDKRPLVKYWvNKSYS CRESKNL1VDSLRLR-PMQPPLSVGKYELLDHYBAFS GFEWRQGVVRGJVFEGRYMVSFGATKEASQFNIISDRPPMEWEDGVWTXJRTKPIJR QKTLGRVTEPNMDVKEGPTGTTNTGVPPK 4/29 WO 02/099076 PCT/1JS02/I 7937 GFGNESTREKMPEEPKKYTRRKRGGLKNSYINKTVLSSDRTPNVKNSASNAE ENHAKTVLPFAKKSPVWKTYESLEVFKSVSHSLBSPLFETKQDFGYAGMM1~ 0 IIEELKGFDKEMTNFSKQEFDDMEQKHKJLQEKLQEAEQEQ z IKDAAWKKICQMESCAKDLNVELEDVEFEFETILSAPW Figure 4A SEQ ID NO:7, Nucleotide sequence of the open reading framne of pk037 ATGGCTATGGTAGATGAACCGTTGTATCCCATTGCTGTGCTTATAGATGAGCTTA AGAATGATGATATTCAGCTTCGTTTGAACTCGATCCGTCGCTTATCTACTATAGCT CGTGCTCTTGGAGAGGAGCGTACAAGGAAGGAGTTAATCCCTTTTTTGAGTGAGA ATAGTGACGATGACGATGAGGTGCTTCTTGCAATGGCTGAGGAGTTAGGAGTTTT TATTCCGTTTGTTGGAGGAATTGAGTTTGCGCATGTTCTTCTTCCTCCTTTGGAAT CTTTTCGTAGGCTTTAAGAACGGATGTT TAAGATTGGATCTCAGATGAAAGAGAATGATCTTGTTGAATCTTTTGTTCCTCTTG TGAAGAGGTTAGCGGGTGGTGAATGGTTTGCAGCAAGAGTTTCTGCATGTGGTAT ATTTCATGTTGCATACCAAGGTTGCACTGATGTTTTGAAGACTGAGTTACGGGCT ACTTATAGCCAGTTGTGCAJ\AGATGATATGCCAATGGTGCGAAGAGCTGCTGCAT CTAACCTGGGGAAATTTGCTACAACTGTCGAGTCTACCTTTTTGATTGCTGAGAT CATGACTATGTTCGATGATCTTACTAAAGATGACCAAGATTCTGTGAGACTATTG GCGTAGGGGACCTGAGTTGACTAGTGGT CACGCATTTTACCTGTTATTGTTAATTTCTCTCAGGATAJAATCTTGGAGGGTGCGC TACATGGTTGCkATCAGCTATATGAACTTTGTAGGCAGTGGGTCCTGATTGCA CGAGGACGGATTTGGTTCCAGCATATGTAAGATTGCTAAGGGACI&AJGAGGCTG AAGTGCGAATAGCAGCAGCGGGAAGTGACCAAGTTCTGTCGGCTTTTGAATC CAGAGCTTGCGATTCAGCACATCCTTCCTTGTGTGAAGGAATTATCATCGGATTC TTCTCAACATGTCCGCTCTGCTCTAGCTTCAGTAATAATGGGGATGGCTCCTATCC TTGGGAAGGACTCAACCATTGAGCATCTGTTACCAJATTTTTCTTTCCCTTTTGA GAGATCTAGGGCTAACTACATAACATAC AGGTTATTGGAATTGATCTACTATCTCAATCCTTGTTACCGGCCATTGTAGAAJCTT GCTGAGGATCGGCACTGGAGAGTCCGACTTGCAATAATAGAGTATGTTCCACTGT TGGCCAGCCAGTTAGGTATAGGATTTTTCGATTACAAGCTCGGAGCCCTTTGCAT 5/29 WO 0)2/099076 PCT/US02/I 7937 GCAATGGCTGCAGACAGGTCTACTCTATCCGCGAGCTGCAGCTGCAGCAAA CACCTAAGCGCCTCGCAGAGGAGTTTGGTCCTGAATGGGCATGCAGCACTT AGTTCCCCAGGTATTGGACATGGTCAACAATCCGCACTACCTACACAGGATGATG GTTCTACGCGCAATATCTCTCATGGCGCCTGTAATGGGATCAGAATCACATGCT zCTAAGTTTCTTCCTGTGGTTGTTGGCATCAGACAGAGCCACATCAA GTTCAACGTTGCCAAACTTCTGCAATCCCTCATCCCCATAGTCGACCATCAGTG GTGGACAAAACAATCCGTCAGTGTTTGGTGGACCTGAGCGAAGACCCTGATGTT GATGTTCGTTATTTTGCAAATCAAGCACTTAATTCCATCGATGGTTCCACAGCAG N CACAATCCTGA Figure 4B CI SEQ ID NO: 8, Amino acid sequence of the open reading frame of pkO37 MAVELPALDLNDQRNHRSLRLERREIFSND DDEVLLAMAEELGVFIPFVGGIEPAVLLPPLESLCTVEETQSJRG}JVESLCKIGSQM KENDLVESFVPLVKRAGGEWFAARVSACGIFJIVAYQGCTDVLKTELRATYSQLCK DDTiRAA~LKATETLAEMMDLKDDVLAECA GKLPDVRLVVFQKWVYMAQ-'LKVPCRDVA VRLLRDNEAEVRkAGKVTKFCRILNPELAQHLPCKLSSDSSQHVJRSALASVI MGAEGDTELPFSLDFDVLUKDVQIELSSLAV LAEDRI{WRVRAImYVPLLASQLGIGFFDYLGACMQ WLQDKVYSIIIA A A A NLRAEGEAQLPVDVNHLkMLASMPMSIC KFLPVVVEASKDRXKIICYNVAKLLQSLPIVDQSVVDKTRQCLVDLSEDPDVDVR1Y FANQALNSJIDGSTAAQS Figure SEQ ID NO:9, Nucleotide sequence of the open reading frame of pkO38 ATACCTATCCTATCCTTCTAACACTACCA AGTGGAGAATCAGTAGGTGTATGCTATGGATGATGGGGAACACCTTCCTTCT CAATCAGACACAATCGCTCTCTTTAGACMLACAACATCCGACGTGTTAGACTCT ACGATCCAAACCAAGCCGCTTTAACGCTCTTAGAAACACGGGTATCGAAGTCA TCATCGGCGTTCCAAACACCGATCTTCGTTCACTCACTAACCCTTCTTCCGCTAGA TCATGGCTCCAAAACAACGTCCTCAACTATTACCCCGCCGTTAGCTTCAAGTACA TCGCCGTAGGTAACGAAGTATCTCCGTCGAACGGCGGTGATGTTGTGCTCCCTGC 6/29 WO (02/099076 PCT/IJS02/1 7937 CATGCGTAACGTTTACGATGCTCTGAGGTGCATCTTCAGATCGTATTAA GTTTCTACCGCCATTGATATGACTTTGATTGGAAACTCTTTCCCTCCTTCCTCCGG AGAGTTTCGTGGTGACGTTAGATGGTATATCGATCCCGTCATCGGGTTTCTTACG AGTACGAACTCAGCGTTACTAGCCAACATCTATCCTTACTTCAGCTACGTTGACA z ATCCACGTGACATATCTCTCTCTTACGCTCTCTTCACTTCTCCTTCCGTCGTCGTAT GGGACGGCTCTCGTGGCTACCAAAACCTCTTTGACGCTTTACTTGACGTTGTTTAC TCTGCCGTTGAACGCTCAGGCGGTGGATCTCTCCCAGTGGTTGTTTCCGAGAGCG GATGGCCTTCTAACGGTGGAAACGCCGCGAGTTTCGATAACGCGCGAGCTTTTTA CAGACTCTGGGGGGGACGGACCGAAACG AAGAGGAGTGGAACGTATTTGTTCGCTATGTTTGATGAGATCAAGAGTCCT r- GAGATCGAGAAGAATTTTGGTTTGTTTTTTCCTATAAACAACCAAATTTCCGA 0 TCACATTCTCTGCCGCGAGAGACGGTACGGCGGTTGAGTGA Figure SEQ EID NO: 10, Amino acid sequence of the open reading frame of pkO38 MTLLILITLPSEVVYMGNPQDILRNIRRYP QALARTIVIVNDRLNSASLNVMYASKIVN VSPSNGGDVVLPAMRNVYDARGANQDRIKVSTI1MTLIGNSFPPSSGEFRGD\JR WYIDPVIGFLTSTNSALLANYPIFSiVDNPRDISLSYALFTSPSVVVWDJGSRGYQNL FDALLDVVYSAVERSGGGSLPVVVSESGWPSNGGNASFDNAAFYTNLASRVEN RGPRGGEYFMDNKPIKFLFNQKPTSADTV Figure 6A SEQ ID NO: 11, Nucleotide sequence of the open reading frame of pkO42a GTGTCCATCACTGGAAAGGGAGTGAAGCCACCGTTJAAAGGTAGAGAGATTATG GTGGGGAACAAGAATCTGATGAATGATCATAAAGTTATTATTCCAGATGATGCTG AAGAGTTGCTAGCTGACTCTGAAGATATGGCCCAGACCGGATTCTTGTCTCCAT AAACAGTGAACTGATTGGAGTTTTGTCTGTTTCGGATCCTCTAACCGAGTGCT CGAGAAGCCATCTCCATTCTAATCCATGAATATCAAGCATCATGGTAACTG GTGGCAACTGGGGAACAGCAAACTCAATTGCTAGAGAAGTCGGTATCGACTCTG TTATCGCAGAAGCTAACCTGAGCAAGCAGAGAAGTCAAGGATTACAGG CTGCGGGACATGTTGTGGCAATGGTAGGTGACGGAATCAAJTGACTCACCGGCTCT WO 02/0990176 PCTIIJS02II7937 CGTGGCAGCGGATGTAGGTATGGCGATAGGTGCAGGAACAGACATTGCTATAGA r-ACGGAAATCGTGAGACTGAAGGTAACA TGATCTTTCAAGGAAJACGTTCTCAAGAATCCGTCTCACTACGTATGGGCTCTC GGGTATAACCTCATGGGGATACCGATCGCTGCGGGAGTGCTTTTCCCAGGGACAC z GTTTCAGGTTGCCTCCATGGATTGCAGGTGCTGCAATGGCTGCTTCTTCTGTTAGT N GTTGTGTGTTGCTCTCTCTTGCTTAAGAACTACAGCGACCTAGAAGCTTGATC ATCTGGAGATTCGGGAGATTCAGGTGGAGCGAGTTTAAk r- Figure 6B SEQ ID NO: 12, Amino acid sequence of the open reading frame of pkO042a VSTKVAVGEWGKLNHVIDAELDEMQGLSN ELGLVDLPAEIIKMISvVGNGASAEGDVAAP QKAEKVKELQAAGHVVAMVGDGNDSPALVAAJDVGMAIGAGTDAEADWLMES NLEDVITAIDLSRKTFSRINYVWALGYNLMGIPJAGVLFPGTRYPIPPWAGAAM AASS VS VVCCS LLLKNYKLRPKIKLDHLEIREIQ VER V Figure 7A SEQ ID NO: 13, Nucleotide sequence of the open reading frame of pkO53b GTGGCCATGGAAGGAGAATTTCAGGATGAGCTTGTGGTTGTTGGAGATGGAGTG GATTCAGCTTCTTTGATTATGGCCTTAAGGAAGAOCATGTCATGTCACTCTTG AGACTCTTGAAGAAGTGAAGAAGCCACAGGTCGAAGAGAAGTCTATTACACCGC ATTGCTGCATAGCTCAATGTCCTGTGGTTAGCAATGAGCAGCCAAGGCCTGAGGT TTATAGAATAGTGCATGATTCTTATGGTCCAACCACTGGGTGCTTAGTTATGTA Figure 7B SEQ ID NO: 14, Amino acid sequence of the open reading frame of pkO5 3b 8/29 WO 02/099076 PCT/11S02/1 7937 VAMEGEFQDELVVVGDGVDSASLBIALRKKACHVTLETLEEVKKPQVEEKSJTPHC CJAQCPVVSNEQPRPEVYRJVHDSYGPTTGCLVM CI Figure 8A SEQ ID NO: 15, Nucleotide sequence of the open reading frame of pkO6O ATGGTCTTGATTACTAAGATGTCTCTCTCTTTCTACATCATTCATCTTCTCATTTTC TCCTTGATTTCCACTTGTGTTGTCTCCAACCAGGCCGAGGATAATCTTCTTCAAGG CCTAAACAGCTACCGAACTGCTCAAAGAGTTCCTCCATTTGCCAAGAATGAGAA C~I GGCTGATTGTGTGGCTGATGAGATCGCCGACAAGCTCGAAGATCAGCCATGCAC AAACCACACCACAGCGAGCACGGTTACTCCTGGCTCGGTACCTCCACGGCTGAC GAACTACCAGGACATTCTCTCTGAATGCAAAATCGACCCAAACACTACCCGTGAC GGATTGATCTTACCTGTCTGTATCCCTAACCGGATCCCGACTTTGGCTTTAACTAA TTACACCCAAACTGGTTATGCTCGGTATCTTAATGATTCGAGGTATGTCGGGGCT GGTGTTGGGTCGGAGAAAGAGTGGATGGTGGTTGTATTGACGACAAGTACTCCA GGTGGAAGCTTTACAGCTGGTGTTGCTGCTGGCAAGGCGACGTCTGTGAGAGTA ATGGCTGGTTTAGGGCTAATGGGGTTGTTGTTTAGTTGCCTTGTGCTCTTCTGA Figure 8B. SEQ ID NO: 16, Amino acid sequence of the open reading frame of pkO6O MVLITKMSLSFYIIHLLIIFSLISTCVVSNQAEDNLLQGLNSYRTAQRVPPFAKNEKADC VADEIADKLEDQPCTNIITTASTVTPGSVPPRLTNYQDILSECKIDPNTTRDGLJLPVCI PNRIPTLALTNYTQTGYARYLNDSRYiVGAGVGSEKEWMVVVLTTSTPGGSFTAGVA AGKATSVRVMAGLGLMGLLFSCLVLF Figure 9A SEQ ID NO: 17, Nucleotide sequence of the open reading frame of pkO63b ATGGAGAAAGTGACCAACTCAGACTTGAAATCCTCTGTTGATGGTGGCGTTGTTG ATGTGTATGGAGAAGATTCAGCCACCATTGAGCACAACATAACTCCTTGGTCTCT CTCTGTTTCTAGTGGATATTCATTGCTGAGAGATCCTCGCTACAACAAAGGACTT 9/29 WO 02/099076 PCT/US02I 17937 GCTTTCACTGAGAAAGAGAGAGACACTCATTACTTGCGTGGTCTTCTCCCTCCAG TTGTTCTTGATCAAGCTTCAGGAGAGAGGCTGTTGACATATCCGACAATA TCAATTCCCATTACAAGTACATGGCTCTGACAGACTTCAGGAGAAACGA GAGACTGTTTTACGAGCTATTGATAGATATGTTGAGGAGCTACTTCCTATTGTTT zATACTCCA CTGTTGGTGAAGCTTGTCAGAAATTTGGAGTATTTTCAGGCGACC N TCAGGGTTTATTCATCAGTTTAAGACAAGGGAAGATTCTAGATGTGTTAAA AACTGGCCTGAAAGGAACATACAGGTTATTGTTGTTACTGACGGTGAAAGGATTT tn ~TAGGATTAGGAGATCTTGGATGTCAGGGGATGGGTATACCGTTGGTAGTTGG 1-10 CGTTATATTCAGCACTTGGAGGTGTTCGTCCTTCAGCGTGTTTACCTGTCACCATT GATGTGGGAACAAACAATGAGAAACTGTTGAATGATGAGTTCTACATAGGACTC r- ~AGGCAAAAGAGAGCAACGGGACAGGAATATAGTGACTCTTGATGAATTCATG C) AGTGCTGTGAAACAGAACTATGGTGAIAAAGTTCTTATTCAGTTTGALGATTTTG CTAATCATAATGCCTTTGAGTTGCTTGCAJA4ATACAGCGATACTCATCTCGTCTTC AACGATGATATACAGGGGACAGCATCAGTTGTTTTAGCAGGATTAGTTTCCGCAC AGAAGTTAACGAATAGCCCACTTGCAGAGCATACCTTCCTCTTTCTTGGTGCTGG TGAAGCTGGAACTGGAATAGCAGAACTCATAGCTCTCTATATGTCAAAACAGAT GAATGCTTCGGTAGAGGAGCCGCAAGAATCTGGCTTGTTGATTCCAAGGG ATTGATTGTTAACTCCCGCAAGATTCACTTCAGACTTTAAGAACCATGGGCT CAGAAGACGCAGCTTTGTCACAGATAAC ACTGTTCTGATTGGATCTTCTGGCGTTGGACGGTCTTTTACAAGAGTGATAG AAGCCATGTCCTCCATTAATGAGAGACCACTGATATGGCTCTCTCTAACCCCAC AAAATTATTCGCAGAGTAATGATAGCGG CATTTTTGCTAGTGGAAGCCCTTTTGATCCAGTTGAGTATGAGGAAAGGTGTTT GTATCTAGACAGGCGAACAATGCGTAGATATTCCCGGGCTTTGGAGTTGGTTTGG TTATCTCTGGAGCAATACGGGTACATGACGATATGCTTCTAGCTGCTGCTGAGGC ATACGAAGAGAAAACTTAAAGAGTTTCT ATTCTCTTCCATCCGGAAATATCAGCTCAGATTGCAGCCATGTAGCACTAG GCGTATGAACTAGGATTGGCAGGGCGGCTTCCACGGCCGAAGATATTGTCAA TGTGCAGAGAGTAGCATGTACAGCCCCACATACCGTCTCTACCGTTGA Figure 9B SEQ ID NO: 18, Amino acid sequence of the open reading frame of pkO63b 10/29 WO 02/099076 PCT/US02/I 7937 MEVTNSDLKS SVD GGV VD VYGED SATTEHNTPWSLS VS SGYSLLRDPRYNKGLAF TEEDHLGLPVDKQKLNIQQPQYATLENRF V1VVTDGERLGLGDLGCQGMGIPVGKLALYSAGGVYACLPVTDVGTNEKLL Z NDEFYIGLRQKRATGQEYSELLNEFMSAVKQNYGEKVLIQFEDFANHNAFELLAKYS DT HLVFNDDIQGTASVVLAGLVSAQKITNSPL.HTFLFLGAGEAGTGIAELILYM SKQMNASVEESRKJWLVDSKGLIVNSRDSLQDFKKWA1EHPVKLLGAYKAIK PTVLIGSSGVGRSFTKEVAMSSERLISNPTTQSECTAEAYTWSKGAFA SGSPFDPVEYEGKFVSTQANAYPGFGLGLVISGIVDDMLLAAALAGQV SKNEGIPFSZKSQANATAEGARPPDVCESYP r- YRLYR Figure SEQ ID NO: 19, Nucleotide sequence of the open reading frame of pk064 ATGGCGTCAGACAAACAAAAGGCGGAGAGAGCCGAGGTTGCGGCGAGGCTAGC GGTAGCTCTAATAAACGTGGTAGCCAGA AAGGTGACGGAGAGAACAACTGAACATCCACCGGAGCAAGATAGGCCCGGTGT GATAGGTTCAGTGTTCAGGGCTGTCCAAGGAACGTATGAGCATGCGAGAGACGC TGTAGTTGGAAAAACCCACGAAGCGGCTGAGTCTACCAGAGGAGCTCAGAT AGCTTCAGAGAAAGCGGTTGGAGCAAGGACGCAACCGTCGAGAAGCTAGG AAACCGCTGATTATACTGCGGAGAAGGTGGGTGAGTATAAAGACTATACGGTTG ATAGTAGGCAGAAACTCGGAGG-GAATC AATTATACTGCGGATAAGGCGGTGGAAGCAAAGGATAAGACGGCGGAGAAGAT TGGTGAGTACAAAGACTATGCGGTGGATAAGGCAGTAGAGCTAAAGATAGAC AGCGGAGAAGGCGAAGGAGACTTCGAATTATACGGCGGATAGGCTAAAGAGG CTAAGGACAAGACGGCTGAGAAGGTTGGTGAGTATAAGGATTACACGGTGGACA AGGCCGTGGAAGCTAGGGATTACACAGCGGAGAAGGCTATTGAGCAAGGAT AAGACAGCTGAGAAGACTGGAGAGTATAAGGACTATACGGTGGAGAAGGCGAC GGAGGGGAAAGATGTTACGGTGAGTAAGCTAGGAGAGCTGAAGGATAGTGCCGT TGAGACAGCGAAGAGAGCTATGGGTTTCTTGTCGGGGAAGACAGAGGAGGCCAA AGGAAAAGCTGTGGAGACCAAAGATACTGCCAAGGAACATGGAGAAAGCTG GAGAAGTAACAAGACAAAAGATGGAGGAAATGAGATTGGAAGGTAAGAGCTC AAAGAAGAAGCTGGAGCAAA-AGCCCAAGAGGCATCTCAAAAGACTAGGGAGAG 11/29 WO 02I4099076 PCT/US02/1 7937 TACTGAGTCGGGAGCTCAAAAAGCCGAAGAGACCAAAGATTCTCCTGCCGTGAG GGGAAATGAAGCGAAAGGGACTATTTTTGGTGCATTAGGG&JGTACGGAGC AATAAAGAGCAAACTGACAATGCCATCAGACATTGTGGAGGAALCACGCGCGGC ACGTGAGCATGGAGGGACGGGTAGGACTGTGGTTGAAGTCAAGGTCGAGGATTC z AAAGCCGGGTAAGGTGGCGACTTCACTGAAGGCGTCGGATCAAGACCGGTCA AACATTCAACGACGTTGGACGGATGGATGATGATGCTCGGAAAGATAAGGGAAA GCTGTGA Figure SEQ lID NO:20, Amino acid sequence of the open reading frame of pk064 MASDKQKAERAEVAARLAAEDLHDINKSGGADVTMYiKVTERTTEHPPEQDRPGVIG SVFRAVQGTYEHARDAVVGKTHEAkESTKEGAQIASEKAVGACDATVEKAKETAD YTAEKVGEYKDYTVDKAKEAKDTTAEKAKETANYTADKAVEAKrDKTAEKIGEYK DYVKVADTEAESYAKKADTEVE7DTDAE RDYTAEKAILEA1DKTAKTGEYKDYTVEKATEGKDVTVSLGELKDSAVETAKRA MGLGTEKK'TTDAEMEAETQMEREKLEAA AQEASQKTRESTESGAQKEETKDSPAVRGAGTIFGALGNVTEAIKSKTMPSDI VEETRAAREHGGTGRTVVEVKVEDSKPGKVATSLKASDQMTGQTFSDiVGRMTDDD ARKDKGKL Figure 11A SEQ ID NO:21, Nucleotide sequence of the open reading frame of pk073 ATGCTCTCTGACGCCGGGGGTGGTTCCGATTGCCGGCGTCGGGACTTATCAAJCTC CCATCAACCTTCATGTGTTCTATATAAGCTTTATCTTCATTGAATCTTCCTCCGTT ATCTCAAATCTCTCAAAATATCTAAATCTCCTTTTTTATGTGAGCTTCTTCACTGA AAGTTTTCTTTGTGACGGAAGAGTATATCGATGCTCTATTGGCTCCGATCTGACTC AGTTGAGACCGCTGACAACAAATCCAAT CAACAGCTCCTCTTCTCAACATCAGAGCAAGACTTCATCAACTTATCAAGC TCTAGATCTGGACTAGCACCAACACCACCTTTGGTTTCTTCTCACCGGTTTTCGTT GATGGCAGGAGTATCTCTTGGACCATCAGATGTGCTTCTTCCGCTGGGAACGTCG 12/29 WO 012A)99076 PCT/[US02/I 7937 r- CGCCCATCATTCCATTCTTCAGAATGACGGAGGCGCTTTCCTTATCCCGACAAC ATCAACCCTAG CI Figure lI B SEQ lID NO:22, Amino acid sequence of the open reading frame of pk073 MLSDAGGGSDCRRRDLSTPINLHVFYISFIFIES SSVISNLSKYLNLLFYV7SFFTESFLCD GRVYRCSIGSDLTQILDASLSSNPKQENSQQSNSSSSQTSEQDFINLSKSSRSGLAPTPP LVSSBFSLMAGVSLGPSDVLLPLGTSTAIDELKRWQWSPYMISPSFQFFVTEA LSLSRQHQP Figure 12A SEQ ID NO:23, Nucleotide sequence of the open reading frame of pk082 ATGGAAGGTGGAGCTGCTCTCTACAATCCTCGAACTGTCGAAGAAGTTTTCAAGG ATTTCAAAGGTCGTCGTACTGCCATTGTCAAAGCTCTCACCACCGATGTTCAAGA GTTTTACCAACAATGTGACCCTGAGAAGGAGAATCTTTGCTTGTATGGGTTACCG AATGAAGAATGGGAAGTGAATTTACCAGCTGAAGAAGTGCCTCCTGAGTTACCA GAGCCAGCTCTTGGTATTAACTTTGCTAGGGATGGGCTCTCTGAAAGGAATGGC TTTCGCTTGTTGCTATTCACAGTGACGCTTGGTTACTGTCTGTCTCGTTTTACTTTG GCCAGTTTTCCAGAAAGACTTTCAAGTA TGATGTTCCTACTATATTTGAAGTAGTGACTGGATGGCTAGCAAGGACAA GTCATCTGCTGCAAATCAACGGACAAJCCAGTCTACTCTAAGTTAG AATCGGGAAGTAAACACGCAAAGGAGA AAATAGATGAAGATGATGAGGATGACCACGGGGAAJACCCTTTGTGGAGCCTGTG GAGACAGTGATGGTGCTGATGAATTCTGGATCTGCTGTGACCTTTGTGAGAGTG GTTCCATGGCAAGTGTGTGAAGATCACTCCAGCTAGAGCTGAGCATATCAACA ATACAAGTGCCCTTCATGCAGCAACAAAAGAGCTCGAGCTTA Figure 12B SEQ ED NO:24, Amino acid sequence of the open reading frame of pk082 13/29 WO 02/099076 PCT/JS0217937 ffGGALYPRTVEEVFKDFGRRTATKALTTDVQEFYQQCDPEKENLCLYGLPN 0 SSKTKQPKEEDEEIEDDEDDHGETLCGACGDSDGADEFWICCDLCEKWFHGKCVK Z ITPARAEHIKQYKCPSCSNKJR4JA Figure 13A SEQ TD NO:25, Nucleotide sequence of the open reading framne of pkO84a GCGAATTGAATAACAGAAGATCGAGCTGGATATCTCTTGGTCAGAAAGTTC TCGAGCAAGAGCCTGAGATGCTTCCTTGTCACGCCTCTGCTTCTCCACTCTCTCCT CAGCTCTCTTCTCTTGGAACTCCTCGAATCGGACCTTCCATTAGTCTGGGATCC TTACAACGTCCTCGCGCCACCTCCTCCTTCTTCTCCGCCTCTTTTCTCTCGTATCTC CTCCGCCGCGGAACACGATCGCTCCGCCGTGACGGAAGTCTATTTCATCAGCCAC GGCGAGTGTGATCTCAATCTCAGGCCTGATCTGATTGGAGGTAGATGCCACGTGG CTACTCTCACACCCAACGGGAACGTCAGGCGAGAGCTCTAGCCGTATTTTTAA CTCTGAAGGTGTTCGATTCACCTCCGTCTTCTCTTCGCCTCTGGATCGAGCTAGAT CCATGGCTGTTTCTGTTTGCCAGGAAAkTGAATTTTCCTGAGGAGCATTTGCAGT CTCGGATGCTGTGGTTGAGATGAGTTTAGGGGACTGGGAAAGCTGTCATCGGTCA GAGATTTACACACCTGAACTCTAAGTTTATAGAGATGCCACCTGATTTCT CACCACGTATATAAAGAATTGAGTCGTC GAATGGGACAGTCTCAGGACTTTCGGAGAAGCTCAGGTCAGAACTTCTTCCATCT ACACAGCACACAAATTCCAGAGGGTTCTCGTTAGCTACTTCTATTCATCGCCCA TTTAAGAGATTGAGGCGTTAGGTATCAT GTACCAGTGGAAAATTGATAATAGAACC TAGGTGATATAAACATCAAGAGTTCTTCTTCTCACTCTCAACCTGCATTGGAGT TTTCACTCACTCTTTACCTATAAAGTGTCTTCTTACCGGTATCCTTGGATGCAGCC CGGTAATGACACATAAGATCTGTGTGGAGGATTCCTCTGTGACCGTATTACAGCA TTCGTGGAAAACCGGGTGGCAGGTAAAGCGGT1TAAATGACACCGCTCATCTTAG ACTGTTGTAG Figure 13B 14/29 WO 02/4)994176 PCT/US02/I 7937 SEQ ID N0:26, Amino acid sequence of the open reading frame of pkOS4a 0 GSSQLEEEDASGEEEEEEENRILNLKVEE SAAEHDRS z AVTEVYFISHGECDLNLRPDLIGGRCHVATLTPNGKQAAAFLNSEGVRPJTSVFS SPLDRARSMAVSVCQEMNFEEHLQVSDAVVEMSLGDWESCHRSEWYTPETLSLIER CQPDFSAPSGESLRQVEFRVQFLNGTVSGLSEyTLRSELLPSTQHTNSRGFSLATS]JR PELTRKKSGKSRFQVMNATGDHEGSEE1SHDEQHLGDINISSSQLSTCIGJFTH SLPII(CLLTGILGCSPVMTHKICVEDS SVTVLQHSWKTGWQVKRLNDTAHLRLL Figure 14A SEQ ID NO:27, Nucleotide sequence of the open reading frame of pk086 ATGCTAGATCACAGTGAAGGTCTTATTGGTTGATTCAGACCATGAAACA AGGTAGTTACACGAACCATTACAAGAA GACTTGTGCTGATTGTGGAACCAGTAAAACTCCTCTTTGGCGTGGTGGTCCTGTT GGTCCAAAGTCGTTGTGTAACGCGTGTGGGATCAGCAGAGAAGAGAAGA GGAGGAACAGAAGATAATAAGAiTTAAAGAATCGAGTTCTGGCGGCGGA CCGTAAATTTGGTGAATCGTTAACAGAGTTTGATGGATTTGGGGATAGGA GAACAGTGGACAGCGACTGGAAGAAGC CTGTGTTACTCATGGCTCTTTCTTATGGCTCTGTTTACGCTTAG Figure 14B SEQ ID NO:28, Amino acid sequence of the open reading frame of pk086 MLDHSEKVLLVDSETMTRDMIEQNNTSVDKKTCJCGTSKTPLW[IGGPVG PKSLCNACGRINRKKRRGGTEDNKKLKKSSS GGGNRKFGESLKQSLMDLGIIPJRSTV EKQRQKLGEEEQAAVLLMIALSYGSVYA Figure SEQ ID NO:29, Nucleotide sequence of the open reading frame of pkO88b 15/29 WO 0P2/099076 PCT/US02/I 7937 ATCCCTATCCTCCGTCTCAGTTCTCGGCGCATCTCACCACCACGCAJCGGCGCCG GCTCCGTCTGTAGACTGTTCGACTCTCATACTCAACATGGCTGACTGTTTATCCTT CGTTTCGAGCGGAGGCACGGTGGCGAAACCGGAAGGTACATGTTGCTCTGGTCTT Z GACGGTGCTTAAGCTGACTCTCAGTGTCTATGTGAGCGTTTAGAGCAGTG N CTTCTCTTGGAGTTACTTTGAJ\TATCACTAAGGCTTCTACTCTTCCCGCCGCATGC AAGCTTCACGCTCCTTCTATCGCTACTTGTGGATGTCTTGCTCCAGGAGTAGCTGC tn ~TGCTGGACCTGAGACAGCCGGATTTCTAGCTCCAATCCTTCTTCAGGGAACGAT GGATCTTCTTTGATTCCGACCTCGTTCACAACTGTACTCAGTGCCGTACTGTTCGT TTTGTTCTTCTCTAGTGCGTAA Figure SEQ lID NO:30, Amino acid sequence of the open reading frame of pkO88b MATHS SFTATTPLFLJVLLSLSSVSVLGASHM1TATAASV7DCSTLILNMDCLSFVSS GGTVAKPEGTCCSGLKTVLKADSQCLCEAYKSSASLGVTLNITAS TLPAACKLHAP SIATCGLSVAPSTAPGLAPGVAAGPETAGFLANPSSGNGSSLIPTSFTTVLSAVLF VLFFSSA Figure 16A SEQ ED NO: 31, Nucleotide, sequence of the open reading frame of pk095 ATGCCTACTCACTACCACTTGAAATACA CCCTAACCCATAATAATCCTCAATCCGATTCCACCACCGACTCATCAJACTTCCTCC GCTCAACGCAAACGCAAGGCAGGTGGTCCGGACAACTCCAGTTCCGTTAC CGTGGCGTTCGACAAAGAAGCTGGGGCAAATGGGTCGCCGAGATCCGAGAGCCA CGAGGATGAGGCTGACTCCACCGAAGCC CGTGCCTACGACCGGGCTGCCGTTTACCTATACGGGTCACGTGCTCAGCTCAACT TAACCCCTTCGTCTCCTTCCTCCGTCTCTTCCTCTTCCTCCTCCGTCTCCGCCGCTT CTTCTCCTTCCACCTCCTCTTCCTCCACTCAACCCTAGACCTCTCCTCCCTCGC CCCGCCGCCGCCACCGTAGGAGGAGGAGCCAACTTTGGTCCGTACGGTATCCCTT TTAACAACAACATCTTCCTTAATGGTGGGACCTCTATGTTATGCCCTAGTTATGGT TTTCCCAACAACAAATAAGTCGTGAATC 16/29 WO 0P2/099076 PCTIUS02I 17937 AAACAATTAATTCTCATCACAACAATCG r-CTGGTATAGTCGACTATGCTGTCTAGGT GCTAGCGACAGGAGGGTTAATAATCAGA 0 GATTTGAACTCTCTAGCTGGTTCGGTGGGTTCGAGTCTATCATAACTCATCCAC z CGCCGTTGGTTGATCCGGTATGTTCTATGGGTCTGGATCCGGGTTATATGGTTGG AGATGGATCTTCGACCATTTGGCCTTTTGGAGGAGAGAGATATAGTCATA-T TGGGGGAGTATTTGGGATTTTATTGATCCCATCTTGGGGGATTCTATTAA Figure 16B (Ni SEQ ID NO:32, Amino acid sequence of the open reading frame of pk095 0 ~MDPLASQHQHNHLEDNNQTLTHNPQSDSTTDSSTSSAQJJKQKGGPDNSyKpRYR GVQSGWAIERRRWGFTEARYRAYYSALL PSPSSSSVASPTSSQLPLRAAVGAFPGPNNF NGGTSMLCPSYGFFPQQQQQQNQMQMGQFQHQQYQHSNNKSDILTDV PVTNSTSFJIHEVAIGQEQGGSGCNNNSSMEDLNSLAGS\JGSSLSITBPPPLVDPVCS MGLDPGYMVGDGSSTIWPFGGEEEYSITNWGSIW1JFDPELGEFY Figure 1 7A SEQ ID NO:33, Nucleotide sequence of the open reading frame of pk096 ATGGTTAAAGAAATAGCTTCTTGGTTATTGATACTATCAA~TGGTGGTGTTTGTTTC TCGTTACAAAGCGGTTTCCAGACGGAAG- GGAAACAGTTTCTGGAGTACAGAGAACATTCTAGCTAAGAGTAAGCGAT TTCTTAATCGCAGTAGCTTATTTCTCAATCCCTATTGAGTTACTTTACTTCGTGAG TTGTTCCAATGTTCCATTCAAATGGGTTCTCTTTGAGPITATCGCCTTCATTGTTCT TTGTGGTATGACTCATCTTCTTCATGGTTGGACTTACTCTGCTCATCCATTTAGAT TAATGATGGCGTTTACTGTTTTCAAGATGTTGACTGCTTTAGTCTCTTGTGCTACT GCGATTACGCTTATTACTTTGATTCCTCTGCTTTTGAAAGTTAAAGTTAGAGAGTT TATGCTTAAGAAGAAAGCTCATGAGCTTGGTCGTGAAGTTGGTTTGATTTTGATT AAGAAAGAGACTGGCTTTCATGTTCGTATGCTTACTCAAGAGATTCGTAAGTCTT TGGATCGTCATACGATTCTTTATACTACTTTGGTTGAGCTTTCGAAJGACTTTAGGG TTGCAGAATTTTGCGGTTTGGATGCCGAATGACGGTGGAACGGAGATGGATTTGA CTCATGAGTTGAGAGGGAGAGGTGGTTATGGTGGTTGTTCTGTTTCTATGGAGGA 17/29 WO 0~2/099076 PCTIJS4)2/I7937 TCGTCCATTGCTCGAGCTAGTGGTGGTGGTGGGGATGTTAGTGAGATTGGTGCCG TGGCTGCTATTAGAATGCCGATGCTTCGTGTTTCGGATTTTATGGAGAGCTAG 0 TTATGCGATACTTGTTTGTGTTTTACCGGGCGGGACGCCTCGGGATTGGACTTATC Z AGGAGATTGAGATTGTTAGTTGTGGCGGATCAGTAJCCGTTGCGTTAGATCA N ~TGCAGCGGTTCTTGAAGAGTCTCAGCTATGAGGGAGAAGCTGGCGGAACAGAA CAGGGCGTTGCAGATGGCGAAkGAGAGACGCGTTGAGAGCGAGCCAAGCGAGGA ATGCGTTTCAGAAAACGATGAGCGAAGGGATGAGGCGTCCTATGCATTCGATAC TATGGATGTGCCTGACGGTAGATTTGGTACGGAGATGKAACCATTTAGTCTGCAT 0 ~CGTACGATCCATGAAGCAGCTTGTATGGCGAGATGTTTGTGTCTATGCATGGA TTAGGTTCTTGGTTGACGCGGAGAAGTCTCTACCTGATAATGTAGTAGGTGATGA AAGAAGGGTCTTTCAAGTGATACTTCATATAGTTGGTAGTTTAGTMAAGCCTAGA TTGGATAGGAGTGATCATAGATGGGCTGCTTGGAGATCACCGGCTTCTTCAGCAG ATGGAGATGTGTATATAAGATTTGATGAATGTAGAGATGATGATTCAAGTTC TCAATCATTTGCTTCTGTTTCCTCCAGAGATCAJAGAAGTTGGTGATGTTAGATTCT CCGGCGGCTATGGGTTAGGACAAGATCTAAGCTTTGGTGTTTGTJ&AGAAAGTGGT GCAGTTGATTCATGGGAATATCTCGGTGGTCCCTGGCTCGGATGGTTCACCGGAG ACCATGTCGTTGCTCCCTCGGTTTCGACGTAGACCCTCCATATCTGTCCATGGATC CAGCGAGTCGCCAGCTCCTGACCACCACGCTCACCCACATTCGATTCTCTGTTA CGTGGCTTACAAGTTTTATTGGTAGACACCAACGATTCGAACCGGGCAGTTACAC GTAAACTCTTAGAGAAACTCGGGTGCGATGTAACCGCGGTTTCCTCTGGATTCGA TTGCCTTACCGCCATTGCTCCCGGCTCGTCCTCGCCTTCTACTTCGTTTCAGTGG TGGTGCTTGATCTTCAAATGGCAGAGATGGACGGTTATGAAGTGGCCATGAGGA TCAGGAGTCGATCTTGGCCGTTGATTGTGGCGACGACAGTGGGCTTGGATGAJG AAATGTGGGACAAGTGTGCACAGATTGGAATCAATGGAGTTGTGAGAAAGCCAG TGTTAGGTTGGGGGTCGAATTGTCACGC AACTTCTCTAA Figure 17B SEQ ID NO:34, Amino acid sequence of the open reading frame of pk096 18/29 WO 012/099076 PCT/US02/1 7937 MYKIEIASWLLILSMVVFVSPVLAINGGGYPRCNCEDEGNSFWSTEMILETQRVSDFLI VRMLTQERKSLDRTITThYTLSTLELSKTLGLQTNGGTEMDLTHLRGRG VSDFNGELSYALVCVLPGGTPRWTYQEVVVAQVTVALDHAVLEESQLM REKLAEQNRALQMAKRDALRASQARNAFQKTMSEGMRRPM{SILGLLSMIQDEKJLS DEKIDMKGVSLGSDPGFTMPSHTHACAC CLNIFVAKLD-VDRVQVLIGLKRRESLFVK RGSLDRSDJRWAAWRSPASSADGDVYEVENDSSSQSFASVSSRQEVGDV RFGYLQLFVKVQ]iNSVPSGPTSLRFRPIVG SEPPHAPSSLRLVLDNSRVTKLKGDTVSGFDCL TAIAPGSSSPSTSFQVVVLDLQMMGYEVARISRSWxPLwATTVGLDEEMWD KCAQIGINGVVRKPVVLRAMESELRRVLLQAD-QLL Figure 18A SEQ ID NO:35, Nucleotide sequence of the open reading frame of pkO97a ATGGAAGTCTGCAATTGTATTGACCGCAATGGCCAGCGGATGATTGTTAATGA AATACCAATACATCTCCGATTTCTTCATTGCGATTGCGTATTTTTCGATTCCTCTT GATGTTCTGGAAACGCTTTCTTGTGTCT TTCAGTTTGGTGCTTTTATCGTTCTTTGTGGAGCACTCATCTTATTAACTTATGG ACTTTCACTACGCATTCGAGAJACCGTGGCGCTTGTGATGACTACCGCGAGGTGT TAACCGCTGTTGTCTCGTGTGCTACTGCGTTGATGCTTGTTCATATTATTCCTGAT CTTTTGAGTGTTAAGACTCGGGAGCTTTTCTTGA A.TAAAGCTGCTGAGCTCG ATAGAGAATGGGATTGATTCGACTCAGGAGAACCGGAGGCATGTGAGA ATGTTGACTCATGAGATTAGAAGCACTTTAGATAGACATACTATTTTAQACTA CACTTGTTGAGCTTGGTAGGACATTAGCTTTGGAGGAGTGTGCATTGTGGATGCC TACTAGAACTGGGTTAGAGCTACAGCTTTCTTATACACTTCGTCATCAACATCCC GTGGAGTATACGGTTCCTATCATTACCGGTGATTA.CCAGTGTTTGGTACTA GTAGGGCTGTKAAATATCTCCTAATTCTCCTGTGGCTAGGTTGAGACCTGTTTCT GGGAAATATATGCTAGGGGAGGTGGTCGCTGTGAGGGTTCCGCTTCTCCACCTTT CTAATTTTCAGATTAATGACTGGCCTGAGCTTTCACAAGAGATATGCTTTGAT GGTTTTGATGCTTCCTTCAGATAGTGCAAGGCAATGGCATGTCCATGAGTTGGA 19/29 WO 0P2/099076 PCT/US02/I 7937 CTCGTTGAAGTCGTCGCTGATCAGGTGGCTGTAGCTCTCTCACATGCTGCGATCC TAGAAGAGTCGATGCGAGCTAGGGACCTTCTCATGGAGCAGAATGTTGCTCTTGA TCTAGCTAGACGAGAAGCAGAAACAGCAATCCGTGCCCGCAATGATTTCCTAGC GGTTATGAACCATGAAATGCGAACACCGATGCATGCGATTATTGCACTCTCTTCC z TTACTCCAAGAAACGGAACTAACCCCTGAACAAAGACTGATGGTGGAACAATA CTTAAAAGTAGTAACCTTTTGGCAACTTTGATGAATGATGTCTTAGATCTTTCAA GGTTAGAAGATGGAAGTCTTCAACTTGAACTTGGGACATTCAATCTTCATACATT ATTTAGAGAGGTCCTCAATCTGATAAGCCTATAGCGGTTGTTAAGAAATTACCC ATCACACTAATCTTGCACC-AGATTTGCCAGATTTGTTGTTGGGGATGAGIAAAC GGCTAATGCAGATAATATTAATATAGTTGGTAATGCTGTGAATTCTCCAACA AGGTAGTATCTCCGTAACCGCTCTTGTCACCAAGTCAGACACACGAGCTGCTGAC TTTTTTGTCGTGCCAACTGGGAGTCATTTCTACTTGAGAGTGAGGTAAAGACT CTGGAGCAGGAATAAATCCTCAAGACATTCCA4AGATTTTCACTAAATTTGCTCA AACACAATCTTTAGCGACGAGAAGCTCGGGTGGTAGTGGGCTTGGCCTCGCCATC TCCAAGAGGTTTGTGAATCTGATGGAGGGTAACATTTGGATTGAGAGCGATGGTC TTGGAAAAGGATGCACGGCTATCTTTGATGTTAAACTTGGGATCTCAGAACGTTC AAACGAATCTAAACAGTCGGGCATACCGAAAGTTCCAGCCATTCCCCGACATTC AAATTTCACTGGACTTAAGGTTCTTGTCATGGATGAGAACGGGGTAAGTAGAATG GTGACGAAGGGACTTCTTGTACACCTTGGGTGCGAAGTGACCACGGTGAGTTCA AACGAGGAGTGTCTCCGAGTTGTGTCCCATGAGCACAAAGTGGTCTTCATGGACG TGTGCATGCCCGGGGTCGAAACTACCAAATCGCTCTCCGTATTCACGAGKAATT CACAAAACAACGCCACCAACGGCCACTACTTGTGGCACTCAGTGGTAACACTGA CAACAAAGGATCTACTTGCAAGTTTGTA ACCCGTATCACTAGACAACATAAGAGATGTTCTGTCTGATCTTCTCGAGCCCCGG GTACTGTACGAGGGCATGTAA Figure 18B SEQ ID NO:36, Amino acid sequence of the open reading frame of pkO97a MEVCNCIEPQWPADELLMKYQYISDFFIAIAYFSIPLELIYFVKKSAVFPYRWVLVQF GAFJVLCGATHLJNLWTFTTHSRTVALVMTTAKVLTAVVS CATALMLVHITPDLLSV KTRELFLKZNKAAELDREMGLIRTQEETGRHVRMLTHEIRSTLDRJILKTTLVELGRT LALEECALWMNPTRTGLELQLSYTLRHQHPVEYTVPIQLPVINQVFGTSRAVKISPNSP VARLRPVSGKYMLGEVVA\TRVPLLHLSNFQIINDWPELSTKRYALMVLMLPSDSARQ 20/29 WO 0J2/099076 PCT/US(02/I 7937 WHVTELELVEVADQVAVALSHAAILEESMADLLMQNVADLARREA-ETAIR ARINDFLAVMNHEMRTPIVIIAIIALSSLLQETELTPEQRLMVETILKSSNLLATLMNDV LDLSRLEDGSLQLELGTFNLHTLFREVLNLIIKPIAVVKKLPITLNLAPDLPEFVVGDEK RLMQIILNJVGNA\TKFSKQGSISVTAIVTKSDTRAADFFVVPTGSHFYLRVKVIRDSGA Z GIiNPQDIPKWTKFAQTQSLATRSSGGSGLGLMISKRjFVNLMEGNJWIESDGLGKGCTA WFDVKLGISERSNESKQSGLPKVPAIPRHSNFTGLKVLVMDENGVSRMVTKGLLVHLG CEVTTVSSNIEECLRVVSHEHKVVFMDVCMYGVENYQIALRIIIEKFTKQPIIQRPLLV in ALSGNTDKSTKEKCMSFGLDGVLLKPVSLDN11znVLSDLLEPRVLYEGM Figure 18C SEQ IDl NO:37, Nucleotide sequence of the open reading framne of pkO97b ATGGAGTCTGCAATTGTATTGA\CCGCAATGGCCAGCGGATGAAJTTGTTAJATGA AATACCAATACATCTCCGATTTCTTCATTGCGATTGCGTATTTTTCGATTCCTCTT GAGTTGATTTACTTTGTGAAGAAATCAGCCGTGTTTCCGTATAGATGGGTACTTG TTCAGTTTGGTGCTTTTATCGTTCTTTGTGGAGCAACTCATCTTATTAACTTATGG ACTTTCACTACGCATTCGAGAACCGTGGCGCTTGTGATGACTACCGCGAAGGTGT TAACCGCTGTTGTCTCGTGTGCTACTGCGTTGATGCTTGTTCATATTATTCCTGAT CTTTTGAGTGTTAAGACTCGGGAGCTTTTCTTGKAAAATAAAGCTGCTGAGCTCG ATAGAGAAATGGcJATTGATTCGAACTCAGGAAGAAACCGGAAGGCATGTGAGA ATGTTGACTCATGAGATTAGAAGCACTTTAGATAGACATACTATTTTAA4AGACTA CACTTGTTGAGCTTGGTAGGACATTAGCTTTGGAGGAGTGTGCATTGTGGATGCC TACTAGAACTGGGTTAGAGCTACAGCTTTCTTATACACTTCGTCATCAACATCCC GTGGAGTATACGGTTCCTATTCAATTACCGGTGATTAACCAAGTGTTTGGTACTA GTAGGGCTGTAAAAATATCTCCTAATTCTCCTGTGGCTAGGTTGAGACCTGTTTCT GGGAAATATATGCTAGGGGAGGTGGTCGCTGTGAGGGTTCCGCTTCTCCACCTTT CTAATTTTCAGATTAATGACTGGCCTGAGCTTTCAACAAAGAGATATGCTTTGAT GGTTTTGATGCTTCCTTCAGATAGTGCAAGGCAATGGCATGTCGATGAGTTGGAJA CTCGTTGAAGTCGTCGCTGATCAGGTGGCTGTAGCTCTCTCACATGCTGCGATCC TAGAAGAGTCGATGCGAGCTAGGGACCTTCTCATGGAGCAGAATGTTGCTCTTGA TCTAGCTAGACGAGAAGCAGAAACAGCAATCCGTGCCCGCAATGATTTCCTAGC GGTTATGA-ACCATGAAATGCGAACACCGATGCATGCGATTATTGCACTCTCTTCC TTACTCCAAGAAACGGAACTAACCCCTGAACAAAGACTGATGGTGGAAJACTA CTTAAAAGTAGTAACCTTTTGGCAACTTTGATGAATGATGTCTTAGATCTTTCAA GGTTAGAAGATGGAAGTCTTCAACTTGAACTTGGGACATTCAATCTTCATACATT 2 1/29 WO 02/099076 PCT/US02/I 7937 ATTTAGAGAGGTCCTCAATCTGATAAAGCCTATAGCGGTTGTTAAGAAATTACCC ATCACACTAAATCTTGCACCAGATTTGCCAGAATTTGTTGTTGGGGATGAGAAAC GGCTAATGCAGATAATATTAAATATAGTTGGTAATGCTGTGAAATTCTCCAAACA AGGTAGTATCTCCGTAACCGCTCTTGTCACCAAGTCAGACACACGAGCTGCTGAC Z TTTTTTGTCGTGCCAACTGGGAGTCATTTCTACTTGAGAGTGAAGGTAAAAGACT N CTGGAGCAGGAATAAATCCTCAAGACATTCCAAAGATTTTCACTAAA\TTTGCTCA AACACAATCTTTAGCGACGAGAAGCTCGGGTGGTAGTGGGCTTGGCCTCGCCATC TCCAAGAGGTTTGTGAATCTGATGGAGGGTAACATTTGGATTGAGAGCGATGGTC 1-10 TTGGAAAAGGATGCACGGCTATCTTTGATGTTAAACTTGGGATCTCAGAACGTTC AAACGAATCTAAACAGTCGGGCATACCGAAAGTTCCAGCCATTCCCCGACATTC AAATTTCACTGGACTTAAGGTTCTTGTCATGGATGAGAACGGGGTAAGTAGAATG GTGACGAAGGGACTTCTTGTACACCTTGGGTGCGAAGTGACCACGGTGAGTTCA AACGAGGAGTGTCTCCGAGTTGTGTCCCATGAGCACAAAGTGGTCTTCATGGACG TGTGCATGCCCGGGGTCGAAAACTACCAAATCGCTCTCCGTATTCACGAGAAATT CACAAAACAACGCCACCAACGGCCACTACTTGTGGCACTCAGTGGTAACACTGA CAAATCCACAAAAGAGAAJ\TGCATGAGCTTTGGTCTAGACGTGTGTTGCTCAAA CCCGTATCACTAG Figure 18D SEQ ID NO:38, Amino acid sequence of the open reading frame of pkO97b MEVCNCIEPQWPADELLMKYQYISDFFIAYFSTPLELIYFTKKSAVFPYRWVLVQF GAFIVLCGATHLINLWTFTTHSRTVALVMTTAKVLTAVVSCATALMLVHIIPDLLSV KTRELFLKNKAAELDREMGLIRTQEETGRIJVRMLTHEIRSTLDRHTILKTTLVELGRT LALEECALWMIPTRTGLELQLSYTLRHQJIYVEYTVPIQLPVINQVFGTSRAVKISPNSP VARLRPVSGKYMLGEVNAVRVPLLLILSNFQINDWPELSTKRYALMVLMLPSDSARQ WHVDELELVEVVADQVAVALSHAAILEESMRtkRDLLMEQNVALDLARREAETAIR ARNDFLAVNqHEMRTPMHAIJALSSLLQETELTPEQRLMYETILKSSNLLATLMNDv LDLSRLEDGSLQLELGTFNLHTLFREVLNLIKPJAVYKKLPITLNLAPDLPEFVVGDEK RLMQIILNJVGNAV7KFSKQGSISVTALVTKSDTRAADFFVVPTGSHFYLR\FKVKDSGA GINPQDIPKEWTKFAQTQSLATRSSGGSGLGLAISKRFVNLMEGNTWIESDGLGKGCTA IFD\TKLGISERSNESKQSGIPKVPAJPRIISNFTGLKVLVMDENGVSRMVTKGLLVHLG CEVTTVSSNEECLRVVSIJEHKVVFMDVCMPGVENYQIALRIHEKFTKQRHQRPLLV ALSGNTDKSTKIEKCMSFGLDVCCSNPYI{ 22/29 WO 02/099076 WO 02/99076PCT/US02II 7937 Figure 19A SEQ 1D NO:39, Nucleotide sequence of the open reading frame of pk098 ATGATGGGCAGTGTCGAGCTGAATCTGAGGGAGACTGAGCTGTGTCTTGGTCTTC CCGGTGGAGATACAGTGGCTCCGGTAACCGGAAACAAGAGAGGGTTCTCAGAGA CGGTTGATCTGAAGCTAATCTGAATAATGAGCCTGCACAAGGAGGATCTA CGACTCATGACGTCGTGACTTTTGATTCCAAGGAGAAGAGTGCTTGTCCTAAGA TCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATC ATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGC GGCGGCGTTCGTGAAGGTATCAATGGACGGAGCACCGTACTTGAGGAATCGA TTGGAGAAAGTCAGGTTTAGTTTCAAGT AGCTCTTTTACCATGGGCAAACATGGAGGAGAAGAGGAJTGATAGACTTCATG AATGAGAGGAAATTGATGGATTTGGTGAATAGCTGGGACTATGTTCCCTCTTATG AAGACAAAGACGGTGATTGGATGCTCGTCGGCGACGTTCCTTGGCCAATGTTCGT CGATACATGCAAGCGTTTACGTCTCATGAAAGGATCGGATGCCATTGGTCTCGCT CCGAGGGCGATGGAGAAGTGCAAGAGCAGAGCTTGA Figure 19B SEQ H) NO:40, Amino acid sequence of the open reading frame of pkO98 MMSENRTLLLGDVPTGKGSTDKNNEAKGT HDVVTFDSKEKSACPKDPAKYPACAQVVGWPPXTRSYPYJNV1MJSCQKSSGGPEAAA FVVMGPLKDRYSDLNLNFSTGHGEMDME KLDVSDVSEKGWLGVPPFDCRRMGDILP AMEKCKSRA Figure SEQ ID NO:41, Nucleotide sequence of the open reading frame of pk099 ATGATCGGCCAACTTATGAACCTCAAGGCCACGGAGCTCTGTCTCGGCCTCCCCG GCGGCGCTGAAGCAGTTGAGAGTCCTGCCAATCGGCGGTGGGAGCAAGAGAG GCTTCTCCGAAACCGTTGATCTCATGCTCAATCTTCAATCTAACAGAAGGCTC CGTTGATCTCAAAAACGTTTCTGCTGTTCCCAAGGAGAAGACTACCCTTAGAT 23/29 WO 02/0990176 PCT/US02/1 7937 CCTTCTAAGCCTCCTGCTMIAGCACAAGTGGTGGGATGGCCACCTGTGAGGAACT r- ACAGGAAGAACATGATGACTCAGCAGAAGACCAGTAGTGGTGCGGAGGAGGCC 0 AGCAGTGAGAAGGCCGGGAACTTTGGTGGAGGAGCAGCCGGAGCCGGCTTGGTG AAGGTCTCCATGGACGGTGCTCCATATCTGAGGAKAAGTTGACCTCAAGATGTACA z AAAGCTACCAGGATCTTTCTGATGCATTGGCCAAAATGTTCAGCTCCTTTACTAT GGGAAACTATGGAGCACAAGGAATGATAGATTTCATGAACGAGAGCAAGCTAAT GAATCTGCTGAATAGCTCTGAGTATGTGCCAAGCTACGAGGACAAAGATGGTGA CTGGATGCTCGTTGGCGATGTCCCATGGGAAATGTTTGTCGAGTCTTGCAAACGT TTGCGCATTATGAAGGGATCTGAAGCAGTTGGACTTGCTCCGAGAGCPApJGGAG AAGTACTGCAAGAACAGATCTTGA c-i Figure SEQ H) NO:42, Amino acid sequence of the open reading frame of pk099 MIGQLM1\WKATELCLGLPGGAEAVESPAKSAVGSKRGFSEVDLMLNLQSKGSV DLKNVSAVPKEKTTLKDPSKPPAKAQVVGWPPVRNYRKNMMVTQQKTSSGMEEASS EKAGNFGGGAAGAGLVKVSMDGAPYLPJVDLKYKSYQDLSDJAKMFSSFTMG NYGAQGMIDFMNESKLNLLNSSEYVPSYEDKDGDWyMLVGDVPWEMFVESCKRLJ RIM4KGSEAVGLAPRAMIEKYCKNRS Figure 21A SEQ ID NO:43, Nucleotide sequence of the open reading frame of pklOO ATGAAAAGCTTGCATGTGGCGGCCAACGCCGGAGATCTGGCTGAGGATTGTGGA ATACTCGGTGGAGACGCTGATGATACTGTTTTGATGGATGGAATTGATGAAGTTG GTAGAGAGATCTGGTTAcJATGACCATGGAGGAGATAATAATCATGTTCATGGTC ATCAAGATGATGATTTGATTGTTCATCATGACCCTTCAATCTTCTATGGAGATCTC CCAACGCTTCCTGATTTCCCATGCATGTCGTCTTCATCATCGTCTTCAACATCTCC AGCTCCTGTCAACGCAATCGTCTCCTCAGCCTCTTCTTCTTCGGCAGCTTCP'CCT CCACTTCCTCAGCTGCTTCTTGGGCTATATTGAGATCAGATGGAGAAGATCCGAC TCCAAACCAAAACCAATACGCATCAGGAAACTGTGACGACTCTTCTGGTGCATTG CAATCCACAGCTTCCATGGAGATTCCATTAGACAGCAGTCAAGGTTTTGGTTGCG GCGAAGGCGGTGGTGATTGCATTGATATGATGGAGACTTTCGGGTACATGGATCT ACTTGATAGCAACGAGTTCTTTGACACCTCAGCTATATTTAGCCAAGACGACGAC 24/29 WO 02/0990)76 PCT/US02/I 7937 ACGCAAAACCCTAACTTGATGGACCACCCTTGAGAGACAAGAGAccAGGTC GTTGTTCCGATGTTGGAGAATAACAGTGGTGGAGACATGCATGATGAATTCTT CCTTGGAACAGGACGATGATCTCGCTGCTGTGTTTTTGGAGTGGCTGAC N CAAGGAGACTGTGTCGGCTGAGGATTTGAGGGTAAAGATAAGAAAGCTAC zGATTGATCAGCGGCAGAAGACTAGGCGGTGGTAGAGCGATGAGCAGC TTTAGTATTGAGGCAATACCTCAGAAGA CACCACCACACCCTTACACTCACAACAT CAACCACCATAACACATCACTCACACTT TCTCACCTTCAACATGGGTTCCTCCACCACCACACACAGCTTTTGTCTCGGA CCCGGGTTTTGGATACATGCCTGCTCCAAACTATCCGCCACAGCCAGAGTTCCTT CCTTTACTTGAATCTCCACCGTCATGGCCACCACCACCACAGTCTGGTCCCATGC CAACAATCCAGCCACTGATTACATGAAC N ~AACAGGTTTCAATGGATACAACATGAATCCGTACCAATATCCTTATGTTCCTGCA GGACAAATGAGAGATCAGAGATTACTCCGTTTGTGTTCCTCAGCACTAAAGAG GCAAGAAAGAAACGGATGGCGAGACAGAGGAGGTTCTTGTCTCATCACCACAGA CAACAACAACAACAATACGACACCATG AGAAACCTGTGCCGCGGTGGCTCCACAACTTACCCCGTGGCCACAACCGCCAC GGGAGGGACCTGGATGTATTGGCCTAATGTCCCGGCAGTGCCGCCTC 2 &JJTACCG CCAGTGATGGAGACTCAGTTACCCACCATGGACCGAGCTGGCTCAGCTTCTGCTA TGCCACGTCAGCAGGTGGTACCAGATCGCCGGCAGGGATGGAACCAGAAGA ATTTGCGGTTTCTCTTGCAGAAGTCTTGAGCAAGCGACGTGGGTIAACCTCGG AAGGATCGTTTTGCCAAJAAAGAAGCTGAGACACACTTGCCGGAGCTAGAGGC AGAGACGGCATCTCTCTGGCCATGGAAGACATCGGAACCTCTCGTGTTTGGAAC ATGCGCTACAGGTTTTGGCCTAACAACAAAAGCAGGATGTATCTCCTCGAGAC ACCGGCGATTTTGTGAAAACCAATGGGCTCCAAGAAGGTGATTTCATAGTCATAT ACCGCTAATAAGGGTTAGAGCACACGCA AGCGGCCACTGCGACTCAGGCAAAGCCA GGAACATAAACAATAACTCTCCGTCGGCGAATGTGGTGGTCGCTTCACCACTTC TCAAACTGTTAAATGA Figure 21B SEQ lID NO:44, Amino acid sequence of the open reading frame of pkl100 2.5/29 WO 02/099076 PCT/US02/17937 r-HQDDDL1VLIiTDPSIFYGDLPTLPDFPCMSSSSSSSTSPA&y\TNh4VSSASSSSSSTS AASWAILRSDGEDPTPNQNQYASGNCDDSSGALQSTASMEIPLDSSQGFGCGEGGGD CIDMETFGYMDLLDSNEFFDTSAFSQDDDTQNPNLMDQTLERQEDQVVVPMLEN z GMMWSEQDLAFELKNEVADRKKKTEAR N ~LGGGKEAMKQLLKILEWVQTN{LQRTTTTTTSYQQSFQQDPFQPNN LIPPSDQTCFSPSTWVPPPPQQQAVSDPGFGYWAPYPPQPEFLPLLESPPSWVJPPP TTATGGTWMYWPNVPAVPPQLPPVMETQLPTMDRAGSASAPRQQV\/PDPJRQGW r-KENRLQVKSVNGILPKATLEERGSAEITR WINMRYRFWPNNKSRMYLLENTGDFVrKTNGLQEGDFLVIYSDVKIRIGVKVRQPSGQ KPEAPPSSAATKRQNKSQRNTNNSPSANVVVASPTSQTVK Figure 22A SEQ lID NO:45, Nucleotide sequence of the open-reading frame of ATGGAGACCGCACCGTCTCGCATTCAAC AGATGGATTTCACCGGGATCAGATTGGGTAAGGTTACTGCATACCAATACTC AAATCAAGATTCCGAGAACGGAGATCTAATGGTTTCGTTACCGGAGACTTCATCA TGCTCTGTTTCTGGGTCACATGGTTCTGATCTAGGAAGTTTTGATTTCTCGGAT CAATTCTCCTAATTTAAACATGAAGGAATCAGCAGCTGCTGATATAGTCGTCGTT GATATCTCCGCCGGAGATGAGATCACGGCTCAGATGTTACTAGCGAGAGAG ATGATCAGCAGAACAGAGAGTAGGAGTTTGTTTGAATTCAAGAGTGTGCCTTTGT ATGGTTTCACTTCGATTTGTGGAAGAAGACCAGAGATGGAAGATGCTGTTTCGAC TATACCAAGATTCCTTCAATCTTCTTCTGGTTCCATGTTAGATGGTCGGTTTGATC CTCAATCCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGGTTCTCAGGTA GCGAACTATTGTAGAGAGAGGATGCATTTGGCTTTGGCGGAGGAGATAGCTAG GAGAAACCGATGCTCTGCGATGGTGATACGTGGCTGGAGAGTGGAAGAAAGCT CTTTTCAACTCGTTCCTGAGAGTTGACTCGGAGATTGAGTCAGTTGCGCCGGAGA CGGTTGGGTCAACGTCGGTGGTTGCCGTTGTTTTCCCGTCTCACATCTTCGTCGCT AACTGCGGTGACTCTAGAGCCGTTCTTTGCCGCGGCAAAACTGCACTTCCATTAT 26/29 WO 0~2/0990l76 PCT/US02/I 7937 CCGTTGACCATAAACCGGATAGAGAAGATGAAGCTGCGAGGATTGAAGCCGCAG GAGGGAAAGTGATTCAGTGGAATGGAGCTCGTGTTTTCGGTGTTCTCGCCATGTC GAGATCCATTGGCGATAGATACTTGAAACCATCCATCATTCCTGATCCGGAAGTG ACGGCTGTGA-AGAGAGTAAAAGAAGATGATTGTCTGATTTTGGCGAGTGACGGG Z GTTTGGGATGTAATGACGGATGAAGAAGCGTGTGAGATGGCAAGGAAGCGGATT CTCTTGTGGCACAAGAAAAACGCGGTGGCTGGGGATGCATCGTTGCTCGCGGAT GAGCGGAGAAAGGAAGGGAAAGATCCTGCGGCGATGTCCGCGGCTGAGTATTTG TCAGTGGTCGGGAGAAGCAAAGGGTGGT GATTTGAAGCCTCGGAGGPLAACTCAAGAGCAAACCCTTGAACTGA Figure 22B SEQ lID NO:46, Amino acid sequence of the open reading frame of MEEVSPAIAGPFRPFSETQMLDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCS VSGSHGSESRKVLISRISPNLNMKESAAADIVVVDISAGDENGSDVTSEKCJ\ISRTE SRSLFEFKSVPLYGFTSICGRRPEMEDAVSTWPRFLQSSSGSMLDGRFDPQSAAMFFGV YDGHGGSQVANYCRERIARLALAEEIAKEKYMLCDGDTWLEKWKKAIFNSFLRVDS EIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAA RIEAAGGKVIQWNGAR\TFGVLAMSRSIGDRYLKISJIPDPEVTAVKRVKEDDCLILAS DGWVTEAEAKILHKAVGALAEREKPASA YLSKLAIQRGSKDNJSVVVVDLKPRRKIKSKPLN Figure 23A SEQ ID NO:47, Nucleotide sequence of the open reading frame of pk1O7 ATGGAAATGCCCGGTAGAAGATCTAATTACACTTTGCTTAGTCAATTTTCTGACG ATCAGGTGTCAGTTTCCGTCACCGGAGCTCCTCCGCCTCACTATGATTCCTTGTCG AGCGAAAACAGGAGCAACCATAACAGCGGGAACACCGGGAAAGCTAAGGCGGA GAGAGGCGGATTTGATTGGGATCCTAGCGGTGGTGGTGGTGGTGATCATAGGTT GAATAATCAACCGAATCGGGTTGGGAATKATATGTATGCTTCGTCTCTAGGGTTG CAAAGGCAATCCAGTGGGAGTAGTTTCGGTGAGAGCTCTTTGTCTGGGGATTATT ACATGCCTACGCTTTCTGCGGCGGCTAACGAGATCGAATCTGTTGGATTTCCTCA AGATGATGGG'TTTAGGCTTGGATTTGGTGGTGGTGGAGGAGATTTGAGGATACA GATGGCGGCGGACTCCGCTGGAGGGTCTTCATCTGGGAAGAGCTGGGCGCAGCA 27/29 WO 02/099076PTU0/73 GCTACTTGTGCCGACGATCCGAACTTTCTGGATCCTGTACCGGACGAGTCTGCTT TACGGACTTCGCCAAGTTCAGCCGAACCGTTTCACATCGTTTCTGGGTTAAJTGG 0 CTGCTTATCGTACTATGATAAGTTCCTGATGGGTTTTATATGATGAATGGTCTGG z ATCCCTATATTTGGACCTTATGCATCGACCTGCATGAGTGGTCGCATCCCTTC AATTGAATCATTAAGAGCTGTTGATTCTGGTGTTGATTCTTCGCTTGAGCGATC ATAGTTGATAGGCGTAGTGATCCAGCCTTCAGGACTTCACATAGAGTCCACG ACATATCTTGTAGCTGCATACCACAAAGAGGTTGTTGATCAGCTGGCAAGCT TATCTGCAATCGTATGGGGGGTCCAGTTATCATGGGGGJ&AGATGAGTTGGTTCCC ATGTGGAAGGAGTGCATTGATGGTCTAGAATCTTTAAGTGGTGGTTCCCA r- TAGGTAGCCTCTCTGTTGGACTCTGCAGACATCGAGCTTTACTCTTCAAAGTACT 0 GGCTGACATAATTGATTTACCCTGTCGTTGCCAGGATGTAATATTGTAAT AGAGACGATGCCGCTTCGTGCCTTGTCAGGTTTGGGCTTGATAGGGAGTACCTGG TTGATTTAGTAGGAAAGCCAGGTCAC TTATGGGAGCCTGATTCCTTGCTAATGG TCCTTCATCTATCTCAATTTCTTCTCCTCTGCGGTTTCCACGACCAAAGCCAGTTG AACCCGCAGTCGATTTTAGGTTACTAGCCACATATTTCTCCGATAGCCAGTC TCTTAATCTTGTTTTCGATCCTGCATCAGATGATATGGGATTCTCAATGTTTCATA GGCAATATGATAATCCGGGTGGAGAGATGACGCATTGGCAGAATGGTGGTG GGTCTTTGCCACCCAGTGCTAATATGCCTCCACAGACATGATGCGTCGTCAAA TCAAATTGAAGCAGCACCTATGAATGCCCCACCAJATCAGTCAGCCAGTTCCAAC AGGGCAAATAGGGAACTTGGAkCTTGATGGTGATGATATGGACATCCCGTGGTGT GATCTTAATATAAAAGAAAAGATTGGAGCAGGTTCCTTTGGCACTGTCCACCGTG CTGAGTGGCATGGCTCGGATGTTGCTGTGAAATTCTCATGGAGCAAGACTTCCA TGCTGAGCGTGTTAATGAGTTCTTAGAGAGGTTGCGATATGAACGCCTTCGC CACCCTAACATTGTTCTCTTCATGGGTGCGGTCACTCACCTCCAATTTGTCAAT AGTGACAGAATATTTGTCAAGAGGTAGTTTATACAGACTTTTGCATAAAGTGGA GCAAGGGAGCAATTAGATGAGAGACGTCGCCTGAGTATGGCTTATGATGTGGCT AAGGGAATGATTATCTTCACAATCGCATCCTCCAATTGTGCATAGAGATCTA AATCTCCAAACTTATTGGTTGACAAATATACAGTCAGGTTTGTGATTTTGG TCTCTCGCGATTGAAGGCCAGCACGTTTCTTTCCTCGAGTCAGCAGCTGGACC CCCGAGTGGATGGCACCAGAAGTCCTGCGAGATGAGCCGTCTATGAAAGTCA GATGTGTACAGCTTCGGGGTCATCTTGTGGGAGCTTGCTACATTGCACACCAT GGGGTAACTTAATCCGGCTCAGGTTGTAGCTGCGGTTGGTTTCAGTGTAAACG GCTGGAGATCCCGCGTAATCTGAATCCTCAGGTTGCAGCCATAATCGAGGGTTGT 28/29 WO 02/099076 PCT/USEI2/I 7937 Figure 23B Z SEQ ID NO:48, Amino acid sequence of the open reading frame of pk1O7 MEMiPGRRSNYTLLSQFSDDQVSVSVTGMPPPHYDSLS SENRSNHNSGNTGKAKAER GGFDWDPSGGGGGDLN±NQPNVGNMYASSLGLQRQSSGSSFGESSLSGDYYM PTLSAAANEffSVGFPQDDGFIGFGGGGGDLIQAASAGGSSSGKSWAQQTEES IN QQAARSETADNLPPDSLTPSEVB-WNCSY VPDGFYM ANGLDPYI WTLCTDLHES GRIPS IESLRAVS GVDSSLEAI1VDRRSDPAy KELH R VID IS CS CITTKEV VDQLAKLICNRMGGP V]MGEDEL VPMWKCIDGLKI FKVPGLVLRRLFVA]DLCLKCYNDASLRGD EYLVDLVGKPGHLWEPDSLLNGPSSISISSPLRPPPVEPAJFJLAKQYFSDS QS LNVDADMFMHQDPGNAANGSPSNPQMRS QIEAAPMNAPPISQPVPNRANRLGLDGDDMPWCDLKKGAGSFGTVRAEW HGDAKLEDHEVELEAHRRPILMATPNSVE LSGLRLKGRQDRRSAYVKMYHRPIHDKPL VDKTKCFLRKSFSKAGPWAELDPNKDYFV LWLTQPGLPQVAGKKLIRLPVAIECTEWR SFATEMDLLRPL1TKSAVPPPNRSDL 29/29