EP1255839A2 - Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport - Google Patents

Abstract

Isolated nucleic acid molecules, designated MCT nucleic acid molecules, which encode novel MCT proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing MCT nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated MCT proteins, mutated MCT proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of MCT genes in this organism.

Description

CORYNEBACTERIUM GLUTAMICUM GENES ENCODING PROTEINS INVOLVED IN MEMBRANE SYNTHESIS AND MEMBRANE TRANSPORT

Related Applications

This application claims priority to prior filed U.S. Provisional Patent Application Serial No. 60/141031, filed June 25, 1999. This application also claims priority to German Patent Application No. 19931454.3, filed July 8, 1999, German Patent Application No. 19931478.0, filed July 8, 1999, German Patent Application No. 19931563.9, filed July 8, 1999, German Patent Application No. 19932122.1, filed July 9, 1999, German Patent Application No. 19932124.8, filed 99709, German Patent Application No. 19932125.6, filed July 9, 1999, German Patent Application No. 19932128.0, filed July 9, 1999, German Patent Application No. 19932180.9, filed July 9, 1999, German Patent Application No. 19932182.5, filed July 9, 1999, German Patent Application No. 19932190.6, filed July 9, 1999, German Patent Application No.

19932191.4, filed July 9, 1999, German Patent Application No. 19932209.0, filed July 9, 1999, German Patent Application No. 19932212.0, filed July 9, 1999, German Patent Application No. 19932227.9, filed July 9, 1999, German Patent Application No. 19932228.7, filed July 9, 1999, German Patent Application No. 19932229.5, filed 99070, German Patent Application No. 19932230.9, filed July 9, 1999, German Patent Application No. 19932927.3, filed July 14, 1999, German Patent Application No. 19933005.0, filed July 14, 1999, German Patent Application No. 19933006.9, filed July 14, 1999, German Patent Application No. 19940764.9, filed August 27, 1999, German Patent Application No. 19940765.7, filed August 27, 1999, German Patent Application No. 19940766.5, filed August 27, 1999, German Patent Application No. 19940830.0, filed August 27, 1999, German Patent Application No. 19940831.9, filed August 27, 1999, German Patent Application No. 19940832.7, filed August 27, 1999, German Patent Application No. 19940833.5, filed August 27, 1999, German Patent Application No. 19941378.9 filed August 31, 1999, German Patent Application No. 19941379.7, filed August 31, 1999, German Patent Application No. 19941395.9, filed August 31 , 1999, German Patent Application No. 19942077.7, filed September 3, 1999, German Patent Application No. 19942078.5, filed September 3, 1999, German Patent Application No. 19942079.3, filed September 3, 1999, and German Patent Application No. 19942088.2, filed September 3, 1999. The entire contents of all of the above referenced applications are hereby expressly incorporated herein by this reference.

Background of the Invention

Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed 'fine chemicals', include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through the large-scale culture of bacteria developed to produce and secrete large quantities of one or more desired molecules. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.

Summary of the Invention

The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C. glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as membrane construction and membrane transport (MCT) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The MCT nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the MCT nucleic acids of the invention, or modification of the sequence of the MCT nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).

The MCT nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum 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 a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.

The MCT nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms. Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.

The MCT proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the metabolism (e.g., the biosynthesis or degradation) of compounds necessary for membrane biosynthesis, or of assisting in the transmembrane transport of one or more compounds either into or out of the cell. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al, U.S. Patent No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J Bacteriol. 162: 591-597 (1985); Katsumata et al, J. Bacteriol. 159: 306-311 (1984); and Santamaria et al, J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals. This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation.

There are a number of mechanisms by which the alteration of an MCT protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incorporating such an altered protein. Those MCT proteins involved in the export of fine chemical molecules from the cell may be increased in number or activity such that greater quantities of these compounds are secreted to the extracellular medium, from which they are more readily recovered. Similarly, those MCT proteins involved in the import of nutrients necessary for the biosynthesis of one or more fine chemicals (e.g., phosphate, sulfate, nitrogen compounds, etc.) may be increased in number or activity such that these precursors, cofactors, or intermediate compounds are increased in concentration within the cell. Further, fatty acids and lipids themselves are desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins of the invention which participate in the biosynthesis of these compounds, or by impairing the activity of one or more MCT proteins which are involved in the degradation of these compounds, it may be possible to increase the yield, production, and/or efficiency of production of fatty acid and lipid molecules from C. glutamicum.

The mutagenesis of one or more MCT genes of the invention may also result in MCT proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C .glutamicum. For example, MCT proteins of the invention involved in the export of waste products may be increased in number or activity such that the normal metabolic wastes of the cell (possibly increased in quantity due to the overproduction of the desired fine chemical) are efficiently exported before they are able to damage nucleotides and proteins within the cell (which would decrease the viability of the cell) or to interfere with fine chemical biosynthetic pathways (which would decrease the yield, production, or efficiency of production of the desired fine chemical). Further, the relatively large intracellular quantities of the desired fine chemical may in itself be toxic to the cell, so by increasing the activity or number of transporters able to export this compound from the cell, one may increase the viability of the cell in culture, in turn leading to a greater number of cells in the culture producing the desired fine chemical. The MCT proteins of the invention may also be manipulated such that the relative amounts of different lipid and fatty acid molecules are produced. This may have a profound effect on the lipid composition of the membrane of the cell. Since each type of lipid has different physical properties, an alteration in the lipid composition of a membrane may significantly alter membrane fluidity. Changes in membrane fluidity can impact the transport of molecules across the membrane, as well as the integrity of the cell, both of which have a profound effect on the production of fine chemicals from C. glutamicum in large-scale fermentative culture.

The invention provides novel nucleic acid molecules which encode proteins, referred to herein as MCT proteins, which are capable of, for example, participating in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. Nucleic acid molecules encoding an MCT protein are referred to herein as MCT nucleic acid molecules. In a preferred embodiment, the MCT protein participates in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. Examples of such proteins include those encoded by the genes set forth in Table 1.

Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an MCT protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of MCT- encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), forth as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%), 99% or more homologous to a nucleotide sequence set forth as an odd-numbered SEQ ID NO in the Sequence Listing (e.g. , SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth as an even-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8...). The preferred MCT proteins of the present invention also preferably possess at least one of the MCT activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an MCT activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%) and most preferably at least about 95%>, 96%, 97%, 98%), or 99% or more homologous to an amino acid sequence of the invention (e.g., an entire amino acid sequence selected from those having an even-numbered SEQ ID NO in the Sequence Listing). In another preferred embodiment, the protein is a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....). In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an MCT fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and is able to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or has one or more of the activities set forth in Table 1 , and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions. In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention (e.g., a sequence of an odd- numbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum MCT protein, or a biologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce an MCT protein by culturing the host cell in a suitable medium. The MCT protein can be then isolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically altered microorganism in which an MCT gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding wild-type or mutated MCT sequence as a transgene. In another embodiment, an endogenous MCT gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered MCT gene. In another embodiment, an endogenous or introduced MCT gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional MCT protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an MCT gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the MCT gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred. In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 676) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated MCT protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated MCT protein or portion thereof can participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. In another preferred embodiment, the isolated MCT protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes.

The invention also provides an isolated preparation of an MCT protein. In preferred embodiments, the MCT protein comprises an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing). In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) (encoded by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO: of the Sequence Listing A). In yet another embodiment, the protein is at least about 50%), preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%>, 96%, 97%, 98%, or 99%> or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated MCT protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and is able to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or has one or more of the activities set forth in Table 1.

Alternatively, the isolated MCT protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g. , hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%), more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%>, 97%), 98,%>, or 99% or more homologous, to a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred that the preferred forms of MCT proteins also have one or more of the MCT bioactivities described herein.

The MCT polypeptide, or a biologically active portion thereof, can be operatively linked to a non-MCT polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the MCT protein alone. In other preferred embodiments, this fusion protein participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates production of a desired compound from the cell. In another aspect, the invention provides methods for screening molecules which modulate the activity of an MCT protein, either by interacting with the protein itself or a substrate or binding partner of the MCT protein, or by modulating the transcription or translation of an MCT nucleic acid molecule of the invention.

Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an MCT nucleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of an MCT nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.

Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates MCT protein activity or MCT nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C. glutamicum metabolic pathways for cell membrane components or is modulated for the transport of compounds across such membranes, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates MCT protein activity can be an agent which stimulates MCT protein activity or MCT nucleic acid expression. Examples of agents which stimulate MCT protein activity or MCT nucleic acid expression include small molecules, active MCT proteins, and nucleic acids encoding MCT proteins that have been introduced into the cell. Examples of agents which inhibit MCT activity or expression include small molecules and antisense MCT nucleic acid molecules. Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant MCT gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can be random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is L-lysine.

Detailed Description of the Invention

The present invention provides MCT nucleic acid and protein molecules which are involved in the metabolism of cellular membrane components in C. glutamicum or in the transport of compounds across such membranes. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, such as C. glutamicum, either directly (e.g., where overexpression or optimization of a fatty acid biosynthesis protein has a direct impact on the yield, production, and/or efficiency of production of the fatty acid from modified C. glutamicum), or may have an indirect impact which nonetheless results in an increase of yield, production, and/or efficiency of production of the desired compound (e.g., where modulation of the metabolism of cell membrane components results in alterations in the yield, production, and/or efficiency of production or the composition of the cell membrane, which in turn may impact the production of one or more fine chemicals). Aspects of the invention are further explicated below.

L Fine Chemicals

The term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries. Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al, eds. VCH: Weinheim, and references contained therein), lipids, both saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol), carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research - Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term "amino acid" is art- recognized. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids may be in the D- or L- optical configuration, though L- amino acids are generally the only type found in naturally-occurring proteins. Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3^rd edition, pages 578-590 (1988)). The 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 1 1 'nonessential' amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine). Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for normal protein synthesis to occur. Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most commonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine. Glycine, L- methionine and tryptophan are all utilized in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ L- methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids - technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as N- acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH: Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann. Rev. Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of α- ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a three- step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase. Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate. Tryptophan is also produced from these two initial molecules, but its synthesis is an 11- step pathway. Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate. Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.

Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3^rd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesize them. Thus it is not suφrising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3^r ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p. 575-600 (1988)). Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell. B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses

Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman' s Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is art- recognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself. The group of vitamins may encompass cofactors and nutraceutical compounds. The language "cofactor" includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic. The term "nutraceutical" includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g., polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Ullman' s Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons; Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and

Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research - Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).

Thiamin (vitamin Bi) is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin B₂) is synthesized from guanosine-5'-triphosphate (GTP) and ribose-5 '-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed 'vitamin B₆' (e.g., pyridoxine, pyridoxamine, pyridoxa- 5 '-phosphate, and the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)-β-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of β-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to β- alanine and for the condensation to panthotenic acid are known. The metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in 5 enzymatic steps. Pantothenate, pyridoxal-5' -phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, (R)- panthenol (provitamin B₅), pantetheine (and its derivatives) and coenzyme A. Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6- methylpterin. The biosynthesis of folic acid and its derivatives, starting from the metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and p- amino-benzoic acid has been studied in detail in certain microorganisms. Corrinoids (such as the cobalamines and particularly vitamin B_J2) and poφhyrines belong to a group of chemicals characterized by a tetrapyrole ring system. The biosynthesis of vitamin B₁ is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed 'niacin'. Niacin is the precursor of the important coenzymes NAD

(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms. The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by large- scale culture of microorganisms, such as riboflavin, Vitamin B₆, pantothenate, and biotin. Only Vitamin Bι₂ is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The language "purine" or "pyrimidine" includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose), and phosphoric acid. The language "nucleoside" includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess. By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism (e.g. Christopherson, R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10: 505-548). Studies of enzymes involved in purine and pyrimidine metabolism have been focused on the development of new drugs which can be used, for example, as immunosuppressants or anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis." Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al, eds. VCH: Weinheim, p. 561- 612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.

The metabolism of these compounds in bacteria has been characterized (for reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine nucleotide biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound inosine-5'- phosphate (IMP), resulting in the production of guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5 '-triphosphate (CTP). The deoxy- forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules, bound in α, α-1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al, (1998) U.S. Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech. 16: 460- 467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.

II. Membrane Biosynthesis and Transmembrane Transport

Cellular membranes serve a variety of functions in a cell. First and foremost, a membrane differentiates the contents of a cell from the surrounding environment, thus giving integrity to the cell. Membranes may also serve as barriers to the influx of hazardous or unwanted compounds, and also to the efflux of desired compounds. Cellular membranes are by nature impervious to the unfacilitated diffusion of hydrophilic compounds such as proteins, water molecules and ions due to their structure: a bilayer of lipid molecules in which the polar head groups face outwards (towards the exterior and interior of the cell, respectively) and the nonpolar tails face inwards at the center of the bilayer, forming a hydrophobic core (for a general review of membrane structure and function, see Gennis, R.B. (1989) Biomembranes, Molecular Structure and Function, Springer: Heidelberg). This barrier enables cells to maintain a relatively higher concentration of desired compounds and a relatively lower concentration of undesired compounds than are contained within the surrounding medium, since the diffusion of these compounds is effectively blocked by the membrane. However, the membrane also presents an effective barrier to the import of desired compounds and the export of waste molecules. To overcome this difficulty, cellular membranes incoφorate many kinds of transporter proteins which are able to facilitate the transmembrane transport of different kinds of compounds. There are two general classes of these transport proteins: pores or channels and transporters. The former are integral membrane proteins, sometimes complexes of proteins, which form a regulated hole through the membrane. This regulation, or 'gating' is generally specific to the molecules to be transported by the pore or channel, rendering these transmembrane constructs selectively permeable to a specific class of substrates; for example, a potassium channel is constructed such that only ions having a like charge and size to that of potassium may pass through. Channel and pore proteins tend to have discrete hydrophobic and hydrophilic domains, such that the hydrophobic face of the protein may associate with the interior of the membrane while the hydrophilic face lines the interior of the channel, thus providing a sheltered hydrophilic environment through which the selected hydrophilic molecule may pass. Many such pores/channels are known in the art, including those for potassium, calcium, sodium, and chloride ions.

This pore and channel-mediated system of facilitated diffusion is limited to very small molecules, such as ions, because pores or channels large enough to permit the passage of whole proteins by facilitated diffusion would be unable to prevent the passage of smaller hydrophilic molecules as well. Transport of molecules by this process is sometimes termed 'facilitated diffusion' since the driving force of a concentration gradient is required for the transport to occur. Permeases also permit facilitated diffusion of larger molecules, such as glucose or other sugars, into the cell when the concentration of these molecules on one side of the membrane is greater than that on the other (also called 'uniport'). In contrast to pores or channels, these integral membrane proteins (often having between 6-14 membrane-spanning α-helices) do not form open channels through the membrane, but rather bind to the target molecule at the surface of the membrane and then undergo a conformational shift such that the target molecule is released on the opposite side of the membrane.

However, cells frequently require the import or export of molecules against the existing concentration gradient ('active transport'), a situation in which facilitated diffusion cannot occur. There are two general mechanisms used by cells for such membrane transport: symport or antiport, and energy-coupled transport such as that mediated by the ABC transporters. Symport and antiport systems couple the movement of two different molecules across the membrane (via permeases having two separate binding sites for the two different molecules); in symport, both molecules are transported in the same direction, while in antiport, one molecule is imported while the other is exported. This is possible energetically because one of the two molecules moves in accordance with a concentration gradient, and this energetically favorable event is permitted only upon concomitant movement of a desired compound against the prevailing concentration gradient. Single molecules may be transported across the membrane against the concentration gradient in an energy-driven process, such as that utilized by the ABC transporters. In this system, the transport protein located in the membrane has an ATP-binding cassette; upon binding of the target molecule, the ATP is converted to ADP + Pi, and the resulting release of energy is used to drive the movement of the target molecule to the opposite face of the membrane, facilitated by the transporter. For more detailed descriptions of all of these transport systems, see: Bamberg, E. et al. , (1993) "Charge transport of ion pumps on lipid bilayer membranes", Q. Rev. Biophys. 26: 1-25; Findlay, J.B.C. (1991) "Structure and function in membrane transport systems", Curr. Opin. Struct. Biol. 1 :804-810; Higgins, CF. (1992) "ABC transporters from microorganisms to man", Ann. Rev. Cell Biol. 8: 67-113; Gennis, R.B. (1989) "Pores, Channels and Transporters", in: Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 270-322; and Nikaido, H. and Saier, H. (1992)

"Transport proteins in bacteria: common themes in their design", Science 258: 936-942, and references contained within each of these references.

The synthesis of membranes is a well-characterized process involving a number of components, the most important of which are lipid molecules. Lipid synthesis may be divided into two parts: the synthesis of fatty acids and their attachment to sn- glycerol-3 -phosphate, and the addition or modification of a polar head group. Typical lipids utilized in bacterial membranes include phospholipids, glycolipids, sphingolipids, and phosphoglycerides. Fatty acid synthesis begins with the conversion of acetyl CoA either to malonyl CoA by acetyl CoA carboxylase, or to acetyl-ACP by acetyltransacylase. Following a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted by a series of condensation, reduction and dehydration reactions to yield a saturated fatty acid molecule having a desired chain length. The production of unsaturated fatty acids from such molecules is catalyzed by specific desaturases either aerobically, with the help of molecular oxygen, or anaerobically (for reference on fatty acid synthesis, see F.C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., p. 612-636 and references contained therein; Lengeler et al. (eds) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and references contained therein; and Magnuson, K. et al, (1993) Microbiological Reviews 57: 522-542, and references contained therein). The cyclopropane fatty acids (CFA) are synthesized by a specific CFA-synthase using SAM as a cosubstrate. Branched chain fatty acids are synthesized from branched chain amino acids that are deaminated to yield branched chain 2-oxo-acids (see Lengeler et al, eds. (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and references contained therein). Another essential step in lipid synthesis is the transfer of fatty acids onto the polar head groups by, for example, glycerol-phosphate-acyltransferases. The combination of various precursor molecules and biosynthetic enzymes results in the production of different fatty acid molecules, which has a profound effect on the composition of the membrane.

HI. Elements and Methods of the Invention

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as MCT nucleic acid and protein molecules, which control the production of cellular membranes in C. glutamicum and govern the movement of molecules across such membranes. In one embodiment, the MCT molecules participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. In a preferred embodiment, the activity of the MCT molecules of the present invention to regulate membrane component production and membrane transport has an impact on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the MCT molecules of the invention are modulated in activity, such that the C. glutamicum metabolic pathways which the MCT proteins of the invention regulate are modulated in yield, production, and/or efficiency of production and the transport of compounds through the membranes is altered in efficiency, which either directly or indirectly modulates the yield, production, and/or efficiency of production of a desired fine chemical by C. glutamicum.

The language, "MCT protein" or "MCT polypeptide" includes proteins which participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. Examples of MCT proteins include those encoded by the MCT genes set forth in Table 1 and by the odd-numbered SEQ ID NOs. The terms "MCT gene" or "MCT nucleic acid sequence" include nucleic acid sequences encoding an MCT protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions.

Examples of MCT genes include those set forth in Table 1. The terms "production" or "productivity" are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter). The term "efficiency of production" includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical). The term "yield" or "product/carbon yield" is art-recognized and includes the efficiency of the conversion of the carbon source into the product (i.e., fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms "biosynthesis" or a

"biosynthetic pathway" are art-recognized and include, the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process. The terms "degradation" or a "degradation pathway" are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The language "metabolism" is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, (e.g., the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.

In another embodiment, the MCT molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. There are a number of mechanisms by which the alteration of an MCT protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incoφorating such an altered protein. Those MCT proteins involved in the export of fine chemical molecules from the cell may be increased in number or activity such that greater quantities of these compounds are secreted to the extracellular medium, from which they are more readily recovered. Similarly, those MCT proteins involved in the import of nutrients necessary for the biosynthesis of one or more fine chemicals (e.g., phosphate, sulfate, nitrogen compounds, etc.) may be increased in number or activity such that these precursor , cofactor, or intermediate compounds are increased in concentration within the cell. Further, fatty acids and lipids themselves are desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins of the invention which participate in the biosynthesis of these compounds, or by impairing the activity of one or more MCT proteins which are involved in the degradation of these compounds, it may be possible to increase the yield, production, and/or efficiency of production of fatty acid and lipid molecules from C. glutamicum.

The mutagenesis of one or more MCT genes of the invention may also result in MCT proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C .glutamicum. For example, MCT proteins of the invention involved in the export of waste products may be increased in number or activity such that the normal metabolic wastes of the cell (possibly increased in quantity due to the oveφroduction of the desired fine chemical) are efficiently exported before they are able to damage nucleotides and proteins within the cell (which would decrease the viability of the cell) or to interfere with fine chemical biosynthetic pathways (which would decrease the yield, production, or efficiency of production of the desired fine chemical). Further, the relatively large intracellular quantities of the desired fine chemical may in itself be toxic to the cell, so by increasing the activity or number of transporters able to export this compound from the cell, one may increase the viability of the cell in culture, in turn leading to a greater number of cells in the culture producing the desired fine chemical. The MCT proteins of the invention may also be manipulated such that the relative amounts of different lipid and fatty acid molecules are produced. This may have a profound effect on the lipid composition of the membrane of the cell. Since each type of lipid has different physical properties, an alteration in the lipid composition of a membrane may significantly alter membrane fluidity. Changes in membrane fluidity can impact the transport of molecules across the membrane, as well as the integrity of the cell, both of which have a profound effect on the production of fine chemicals from C. glutamicum in large-scale fermentative culture.

The isolated nucleic acid sequences of the invention are contained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum MCT DNAs and the predicted amino acid sequences of the C. glutamicum MCT proteins are shown in the Sequence Listing as odd-numbered SEQ ID NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode proteins involved in the metabolism of cellular membrane components or proteins involved in the transport of compounds across such membranes.

The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention (e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing).. As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, e.g. , the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%), 80-90%, or 90-95%, and most preferably at least about 96%), 97%), 98%, 99%> or more homologous to the selected amino acid sequence.

The MCT protein or a biologically active portion or fragment thereof of the invention can participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or have one or more of the activities set forth in Table 1. Various aspects of the invention are described in further detail in the following subsections:

A . Isolated Nude ic A cid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules that encode MCT 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 MCT-encoding nucleic acid (e.g., MCT DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 100 nucleotides of sequence upstream from the 5' end of the coding region and at least about 20 nucleotides of sequence downstream from the 3 'end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., 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 MCT nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an "isolated" nucleic acid molecule, such as a DNA 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, e.g., a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C. glutamicum MCT DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T ^'. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence). For example, mRNA can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can be prepared using reverse transcriptase (e.g. , 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 Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an MCT nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the invention, as set forth in the Sequence Listing correspond to the Corynebacterium glutamicum MCT DNAs of the invention. This DNA comprises sequences encoding MCT proteins (i.e., the "coding region", indicated in each odd- numbered SEQ ID NO: in the Sequence Listing), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID NO: in the Sequence Listing. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the nucleic acid sequences of the Sequence Listing.

For the puφoses of this application, it will be understood that each of the nucleic acid and amino acid sequences set forth in the Sequence Listing has an identifying RXA, RXN, RXS, or RXC number having the designation "RXA", "RXN", "RXS" or "RXC" followed by 5 digits (i.e., RXA02099, RXN03097, RXS00148, or RXC01748). Each of the nucleic acid sequences comprises up to three parts: a 5' upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation "one of the odd-numbered sequences in of the Sequence Listing", then, refers to any of the nucleic acid sequences in the Sequence Listing, which may also be distinguished by their differing RXA, RXN, RXS, or RXC designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence. For example, the coding region for RXA03097 is set forth in SEQ ID NO: 1 , while the amino acid sequence which it encodes is set forth as SEQ ID NO:2. The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, RXS, or RXC designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequences designated RXA02099, RXN03097, RXS00148, and RXC01748 are translations of the coding region of the nucleotide sequences of nucleic acid molecules RXA02099, RXN03097, RXS00148, and RXC01748, respectively. The correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1. For example, as set forth in Table 1, the nucleotide sequence of RXA00104 is SEQ ID NO:5, and the amino acid sequence of RXA00104 is SEQ ID NO:6.

Several of the genes of the invention are "F-designated genes". An F-designated gene includes those genes set forth in Table 1 which have an 'F' in front of the RXA, RXN, RXS, or RXC designation. For example, SEQ ID NO: 11 , designated, as indicated on Table 1, as "F RXA02581", is an F-designated gene, as are SEQ ID NOs: 31, 33, and 43 (designated on Table 1 as "F RXA02487", "F RXA02490", and "F RXA02809", respectively).

In one embodiment, the nucleic acid molecules of the present invention are not intended to include those compiled in Table 2. In the case of the dapD gene, a sequence for this gene was published in Wehrmann, A., et al. (1998) J. Bacteriol. 180(12): 3159- 3165. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences shown in of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:)such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50%, 51%, 52%, 53%), 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%), 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. Ranges and identity values intermediate to the above-recited ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention, or a portion thereof.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an MCT protein. The nucleotide sequences determined from the cloning of the MCT genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning MCT homologues in other cell types and organisms, as well as MCT homologues from other Corynebacteria or related species. 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 nucleotide sequences of the invention (e.g., a sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone MCT homologues. Probes based on the MCT 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 diagnostic test kit for identifying cells which misexpress an MCT protein, such as by measuring a level of an MCT-encoding nucleic acid in a sample of cells, e.g., detecting MCT mRNA levels or determining whether a genomic MCT 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 sequence of the invention (e.g., a sequence of an even- numbered SEQ ID NO of the Sequence Listing) such that the protein or portion thereof maintains the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. 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 (e.g., an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the even-numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes. Protein members of such membrane component metabolic pathways or membrane transport systems, as described herein, may play a role in the production and secretion of one or more fine chemicals. Examples of such activities are also described herein. Thus, "the function of an MCT protein" contributes either directly or indirectly to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of MCT protein activities are set forth in Table 1. In another embodiment, the protein 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 of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).

Portions of proteins encoded by the MCT nucleic acid molecules of the invention are preferably biologically active portions of one of the MCT proteins. As used herein, the term "biologically active portion of an MCT protein" is intended to include a portion, e.g., a domain/motif, of an MCT protein that participates in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or has an activity as set forth in Table 1. To determine whether an MCT protein or a biologically active portion thereof can participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification. Additional nucleic acid fragments encoding biologically active portions of an

MCT protein can be prepared by isolating a portion of one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the MCT protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the MCT protein or peptide.

The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same MCT protein as that encoded by the nucleotide sequences of the invention. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In a still further embodiment, the nucleic acid molecule of the invention encodes a full length C. glutamicum protein which is substantially homologous to an amino acid sequence of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing). It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or 4). For example, the invention includes a nucleotide sequence which is greater than and/or at least 38%o identical to the nucleotide sequence designated RXA01420 (SEQ ID NO: 7), a nucleotide sequence which is greater than and/or at least 43% identical to the nucleotide sequence designated RXA00104 (SEQ ID NO:5), and a nucleotide sequence which is greater than and/or at least 45% identical to the nucleotide sequence designated RXA02173 (SEQ ID NO:25). One of ordinary skill in the art would be able to calculate the lower threshold of percent identity for any given sequence of the invention by examining the GAP-calculated percent identity scores set forth in Table 4 for each of the three top hits for the given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%), 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%), 99%> or more identical) are also encompassed by the invention. In addition to the C. glutamicum MCT nucleotide sequences set forth in the

Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by one of ordinary skill in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences of MCT proteins may exist within a population (e.g. , the C. glutamicum population). Such genetic polymoφhism in the MCT 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 an MCT protein, preferably a C. glutamicum MCT protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the MCT gene. Any and all such nucleotide variations and resulting amino acid polymoφhisms in MCT that are the result of natural variation and that do not alter the functional activity of MCT proteins are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural variants and non- glutamicum homologues of the C. glutamicum MCT DNA of the invention can be isolated based on their homology to the C. glutamicum MCT nucleic acid disclosed herein using the C. glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. 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 an odd-numbered SEQ ID NO: of the Sequence Listing. 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 60% 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 70%, 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 of ordinary skill 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°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention 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 (e.g. , encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C. glutamicum MCT protein.

In addition to naturally-occurring variants of the MCT sequence that may exist in the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the invention, thereby leading to changes in the amino acid sequence of the encoded MCT protein, without altering the functional ability of the MCT protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a nucleotide sequence of the invention. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the MCT proteins (e.g., an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said MCT protein, whereas an "essential" amino acid residue is required for MCT protein activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having MCT activity) may not be essential for activity and thus are likely to be amenable to alteration without altering MCT activity. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding MCT proteins that contain changes in amino acid residues that are not essential for MCT activity. Such MCT proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the MCT activities described herein. In one embodiment, the isolated nucleic acid molecule 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 of the invention and is capable of participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or has one or more activities set forth in Table 1. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing , more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98%, or 99%) homologous to one of the amino acid sequences of the invention..

To determine the percent homology of two amino acid sequences (e.g., one of the amino acid sequences of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (e.g., 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 or nucleotide positions are then compared. When a position in one sequence (e.g., one of the amino acid sequences of the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the amino acid sequence), then the molecules are homologous at that position (i.e., 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 (i.e., % homology = # of identical positions/total # of positions x 100).

An isolated nucleic acid molecule encoding an MCT protein homologous to a protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention 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 nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the 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. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an MCT protein 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 an MCT coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an MCT activity described herein to identify mutants that retain MCT activity. Following mutagenesis of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding MCT proteins 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, e.g., 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 MCT 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 an MCT protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g. , the entire coding region of SEQ ID NO: 5 (RXA00104) comprises nucleotides 1 to 756). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding MCT. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids ( . e. , also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding MCT disclosed herein (e.g., the sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of MCT mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of MCT mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of MCT 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 nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., 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, e.g., 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, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. 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 (i.e., 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).

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 an MCT protein to thereby inhibit expression of the protein, e.g. , by 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 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 specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., 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 promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-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 (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave MCT mRNA transcripts to thereby inhibit translation of MCT mRNA. A ribozyme having specificity for an MCT-encoding nucleic acid can be designed based upon the nucleotide sequence of an MCT DNA disclosed herein (i.e., SEQ ID NO. 5 (RXA00104). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an MCT-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742. Alternatively, MCT mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 :1411-1418.

Alternatively, MCT gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an MCT nucleotide sequence (e.g., an MCT promoter and/or enhancers) to form triple helical structures that prevent transcription of an MCT gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27- 36; and Maher, L.J. (1992) Bioassays 14(12):807-15. B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an MCT protein (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 (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., 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 interchangeably 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 (e.g. , replication defective retroviruses, adenoviruses and adeno- associated 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 sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro 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 expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include 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. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, tφ-, tet-, tφ-tet-, lpp-, lac-, lpp-lac-, lacl^q-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, λ-P_R- or λ P_L, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, φ28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by one of ordinary skill 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 (e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for expression of MCT proteins in prokaryotic or eukaryotic cells. For example, MCT genes can be expressed in bacterial cells such as C. glutamicum, 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, J.W. Bennet & L.L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al, eds., p. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to 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 three puφoses: 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, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (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 MCT protein 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 MCT protein 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) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III 113-B1, λgtl 1, pBdCl, and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89 ; and Pouwels et al, eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid tφ-lac fusion promoter. Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJlOl, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBLl, pSA77, or pAJ667 (Pouwels et al, eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

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 (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 1 19-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et α/. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the MCT protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), 2 μ, pAG-1, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), and pYES2 (Invitrogen Coφoration, 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, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al, eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al, eds. (1985) Cloning Vectors. Elsevier: New York (IBSN 0 444 904018).

Alternatively, the MCT proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In another embodiment, the MCT proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl Acid. Res. 12: 871 1-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al, eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

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, B. (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, J., Fritsh, E. F., and Maniatis, T. 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 recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue- specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

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 MCT 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 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, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but 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 be any prokaryotic or eukaryotic cell. For example, an MCT protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to one of ordinary skill in the art. Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.

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 art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other 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 can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. For stable transfection of mammalian 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 (e.g., 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 and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an MCT protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incoφorated the selectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an MCT gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the MCT gene. Preferably, this MCT gene is a Corynebacterium glutamicum MCT gene, but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous MCT gene is functionally disrupted ( . e. , no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous MCT gene is mutated or otherwise altered but still encodes functional protein (e.g. , the upstream regulatory region can be altered to thereby alter the expression of the endogenous MCT protein). In the homologous recombination vector, the altered portion of the MCT gene is flanked at its 5' and 3' ends by additional nucleic acid of the MCT gene to allow for homologous recombination to occur between the exogenous MCT gene carried by the vector and an endogenous MCT gene in a microorganism. The additional flanking MCT nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R., and Capecchi, M.R. (1987) Cell 51 : 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g. , by electroporation) and cells in which the introduced MCT gene has homologously recombined with the endogenous MCT 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 an MCT gene on a vector placing it under control of the lac operon permits expression of the MCT gene only in the presence of IPTG. Such regulatory systems are well known in the art.

In another embodiment, an endogenous MCT gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced MCT gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional MCT protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an MCT gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the MCT gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the described MCT gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an MCT protein. Accordingly, the invention further provides methods for producing MCT proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an MCT protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered MCT protein) in a suitable medium until MCT protein is produced. In another embodiment, the method further comprises isolating MCT proteins from the medium or the host cell.

C. Isolated MCT Proteins

Another aspect of the invention pertains to isolated MCT proteins, 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 "substantially free of cellular material" includes preparations of MCT protein 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 MCT protein having less than about 30% (by dry weight) of non-MCT protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-MCT protein, still more preferably less than about 10% of non-MCT protein, and most preferably less than about 5% non-MCT protein. When the MCT protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., 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 MCT protein 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 MCT protein having less than about 30% (by dry weight) of chemical precursors or non-MCT chemicals, more preferably less than about 20% chemical precursors or non-MCT chemicals, still more preferably less than about 10%) chemical precursors or non-MCT chemicals, and most preferably less than about 5% chemical precursors or non-MCT chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the MCT protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum MCT protein in a microorganism such as C. glutamicum. An isolated MCT protein or a portion thereof of the invention can participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or has one or more of the activities set forth in Table 1. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, 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 preferred embodiment, an MCT protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing.. In yet another preferred embodiment, the MCT protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing). In still another preferred embodiment, the MCT protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90%) identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred MCT proteins of the present invention also preferably possess at least one of the MCT activities described herein. For example, a preferred MCT protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can participate in the metabolism of compounds necessary for the construction of cellular membranes in C. glutamicum, or in the transport of molecules across these membranes, or which has one or more of the activities set forth in Table 1.

In other embodiments, the MCT protein is substantially homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the MCT protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%> or more homologous to an entire amino acid sequence of the invention and which has at least one of the MCT activities described herein. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In another embodiment, the invention pertains to a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention. Biologically active portions of an MCT protein include peptides comprising amino acid sequences derived from the amino acid sequence of an MCT protein, e.g., the an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an MCT protein, which include fewer amino acids than a full length MCT protein or the full length protein which is homologous to an MCT protein, and exhibit at least one activity of an MCT protein. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an MCT protein. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an MCT protein include one or more selected domains/motifs or portions thereof having biological activity. MCT proteins 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 above) and the MCT protein is expressed in the host cell. The MCT protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, an MCT protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native MCT protein can be isolated from cells (e.g., endothelial cells), for example using an anti-MCT antibody, which can be produced by standard techniques utilizing an MCT protein or fragment thereof of this invention.

The invention also provides MCT chimeric or fusion proteins. As used herein, an MCT "chimeric protein" or "fusion protein" comprises an MCT polypeptide operatively linked to a non-MCT polypeptide. An "MCT polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an MCT protein, whereas a "non-MCT polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the MCT protein, e.g., a protein which is different from the MCT protein 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 MCT polypeptide and the non-MCT polypeptide are fused in-frame to each other. The non-MCT polypeptide can be fused to the N-terminus or C- terminus of the MCT polypeptide. For example, in one embodiment the fusion protein is a GST-MCT fusion protein in which the MCT sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant MCT proteins. In another embodiment, the fusion protein is an MCT protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an MCT protein can be increased through use of a heterologous signal sequence.

Preferably, an MCT chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction 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 (e.g., a GST polypeptide). An MCT- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the MCT protein. Homologues of the MCT protein can be generated by mutagenesis, e.g. , discrete point mutation or truncation of the MCT protein. As used herein, the term "homologue" refers to a variant form of the MCT protein which acts as an agonist or antagonist of the activity of the MCT protein. An agonist of the MCT protein can retain substantially the same, or a subset, of the biological activities of the MCT protein. An antagonist of the MCT protein can inhibit one or more of the activities of the naturally occurring form of the MCT protein, by, for example, competitively binding to a downstream or upstream member of the cell membrane component metabolic cascade which includes the MCT protein, or by binding to an MCT protein which mediates transport of compounds across such membranes, thereby preventing translocation from taking place.

In an alternative embodiment, homologues of the MCT protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the MCT protein for MCT protein agonist or antagonist activity. In one embodiment, a variegated library of MCT variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of MCT variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential MCT sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of MCT sequences therein. There are a variety of methods which can be used to produce libraries of potential MCT 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 MCT sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et α/. (1984) Annu. Rev. Biochem. 53:323; Itakura et α/. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.

In addition, libraries of fragments of the MCT protein coding can be used to generate a variegated population of MCT fragments for screening and subsequent selection of homologues of an MCT protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an MCT 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 SI 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 MCT protein. 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 MCT homologues. The most 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 combinatorial 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 MCT homologues (Arkin and Yourvan (1992) PNAS £9:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331). In another embodiment, cell based assays can be exploited to analyze a variegated MCT library, using methods well known in the art.

D. Uses and Methods of the Invention

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 C. glutamicum and related organisms; mapping of genomes of organisms related to C. glutamicum; identification and localization of C. glutamicum sequences of interest; evolutionary studies; determination of MCT protein regions required for function; modulation of an MCT protein activity; modulation of the metabolism of one or more cell membrane components; modulation of the transmembrane transport of one or more compounds; and modulation of cellular production of a desired compound, such as a fine chemical.

The MCT nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum 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 a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells; the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease. Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of

Corynebacterium diphtheriae in the subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C. glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject. The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutamicum 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 C. glutamicum, 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 bacteria, such as Brevibacterium lactofermentum.

The MCT 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 of prokaryotic 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 of mutagenesis without losing function.

Manipulation of the MCT nucleic acid molecules of the invention may result in the production of MCT proteins having functional differences from the wild-type MCT proteins. 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.

The invention provides methods for screening molecules which modulate the activity of an MCT protein, either by interacting with the protein itself or a substrate or binding partner of the MCT protein, or by modulating the transcription or translation of an MCT nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more MCT proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the MCT protein is assessed.

There are a number of mechanisms by which the alteration of an MCT protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incoφorating such an altered protein. Recovery of fine chemical compounds from large-scale cultures of C. glutamicum is significantly improved if C. glutamicum secretes the desired compounds, since such compounds may be readily purified from the culture medium (as opposed to extracted from the mass of C. glutamicum cells). By either increasing the number or the activity of transporter molecules which export fine chemicals from the cell, it may be possible to increase the amount of the produced fine chemical which is present in the extracellular medium, thus permitting greater ease of harvesting and purification. Conversely, in order to efficiently oveφroduce one or more fine chemicals, increased amounts of the cofactors, precursor molecules, and intermediate compounds for the appropriate biosynthetic pathways are required. Therefore, by increasing the number and/or activity of transporter proteins involved in the import of nutrients, such as carbon sources ( . e. , sugars), nitrogen sources ( . e. , amino acids, ammonium salts), phosphate, and sulfur, it may be possible to improve the production of a fine chemical, due to the removal of any nutrient supply limitations on the biosynthetic process. Further, fatty acids and lipids are themselves desirable fine chemicals, so by optimizing the activity or increasing the number of one or more MCT proteins of the invention which participate in the biosynthesis of these compounds, or by impairing the activity of one or more MCT proteins which are involved in the degradation of these compounds, it may be possible to increase the yield, production, and/or efficiency of production of fatty acid and lipid molecules from C. glutamicum. The engineering of one or more MCT genes of the invention may also result in

MCT proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C .glutamicum. For example, the normal biochemical processes of metabolism result in the production of a variety of waste products (e.g., hydrogen peroxide and other reactive oxygen species) which may actively interfere with these same metabolic processes (for example, peroxynitrite is known to nitrate tyrosine side chains, thereby inactivating some enzymes having tyrosine in the active site (Groves, J.T. (1999) Curr. Opin. Chem. Biol. 3(2): 226-235). While these waste products are typically excreted, the C. glutamicum strains utilized for large-scale fermentative production are optimized for the oveφroduction of one or more fine chemicals, and thus may produce more waste products than is typical for a wild-type C. glutamicum. By optimizing the activity of one or more MCT proteins of the invention which are involved in the export of waste molecules, it may be possible to improve the viability of the cell and to maintain efficient metabolic activity. Also, the presence of high intracellular levels of the desired fine chemical may actually be toxic to the cell, so by increasing the ability of the cell to secrete these compounds, one may improve the viability of the cell. Further, the MCT proteins of the invention may be manipulated such that the relative amounts of various lipid and fatty acid molecules produced are altered. This may have a profound effect on the lipid composition of the membrane of the cell. Since each type of lipid has different physical properties, an alteration in the lipid composition of a membrane may significantly alter membrane fluidity. Changes in membrane fluidity can impact the transport of molecules across the membrane, which, as previously explicated, may modify the export of waste products or the produced fine chemical or the import of necessary nutrients. Such membrane fluidity changes may also profoundly affect the integrity of the cell; cells with relatively weaker membranes are more vulnerable in the large-scale fermentor environment to mechanical stresses which may damage or kill the cell. By manipulating MCT proteins involved in the production of fatty acids and lipids for membrane construction such that the resulting membrane has a membrane composition more amenable to the environmental conditions extant in the cultures utilized to produce fine chemicals, a greater proportion of the C. glutamicum cells should survive and multiply. Greater numbers of C. glutamicum cells in a culture should translate into greater yields, production, or efficiency of production of the fine chemical from the culture.

The aforementioned mutagenesis strategies for MCT proteins to result in increased yields of a fine chemical from C. glutamicum are not meant to be limiting; variations on these strategies will be readily apparent to one of ordinary skill in the art. Using such strategies, and incoφorating the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention may be utilized to generate C. glutamicum or related strains of bacteria expressing mutated MCT nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved. This desired compound may be any natural product of C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C. glutamicum strain of the invention.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patent applications, patents, published patent applications, Tables, and the sequence listing cited throughout this application are hereby incoφorated by reference.

TABLE 1 : GENES IN THE APPLICATION

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

1 2 RXN03097 W0062 3 557 AMMONIUM TRANSPORT SYSTEM

3 4 RXA02099 GR00630 6198 6470 AMMONIUM TRANSPORT SYSTEM

5 6 RXA00104 GR00014 15895 16650 CYSQ PROTEIN, ammonium transport protein

Polyketide Synthesis

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function SEQ ID NO SEQ ID NO

7 8 RXA01420 GR00416 775 17 4"-MYCAROSYL ISOVALERYL-COA TRANSFERASE (EC 2 ■

9 10 RXN02581 W0098 30482 28623 POLYKETIDE SYNTHASE

11 12 F RXA02581 GR00741 1 1527 POLYKETIDE SYNTHASE

13 14 RXA02582 GR00741 1890 6719 PROBABLE POLYKETIDE SYNTHASE CY338 20

15 16 RXA01138 GR00318 1656 2072 ACTINORHODIN POLYKETIDE DIMERASE (EC )

17 18 RXA01980 GR00573 1470 838 POLYKETIDE CYCLASE

19 20 RXN01007 W0021 2572 866 FRNA

21 22 RXN00784 W0103 27531 28265 FRNE o

Fatty acid and lipid synthesis

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

23 24 RXA02335 GR00672 550 2322 BIOTIN CARBOXYLASE (EC 6 3 4 14) 25 26 RXA02173 GR00641 7473 8924 ACETYL-COENZYME A CARBOXYLASE CARBOXYL TRANSFERASE SUBUNIT

BETA (EC 64 1 2)

27 28 RXA01764 GR00500 2178 3110 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC 1 1 1 100) 29 30 RXN02487 W0007 6367 4664 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3) 31 32 F RXA02487 GR00718 4937 4650 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3) 33 34 F RXA02490 GR00720 817 5 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3) 35 36 RXA01467 GR00422 920 1210 ACYL CARRIER PROTEIN 37 38 RXA00796 GR00212 202 5 Acyl carrier protein phosphodiesterase 39 40 RXA01897 GR00544 617 1159 Acyl carrier protein phosphodiesterase 41 42 RXN02809 W0342 380 6 Acyl carrier protein phosphodiesterase 43 44 F RXA02809 GR00790 277 5 Acyl carrier protein phosphodiesterase 45 46 RXN00113 W0129 103 5724 FATTY ACID SYNTHASE (EC 2 3 1 85) [INCLUDES EC 2 3 1 38, EC 2 3 1 39, EC

2 3 1 41 ,

47 48 F RXA00113 GR00017 2 3295 FATTY-ACID SYNTHASE (EC 2 3 1 85)

Table 1 (continued)

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

49 50 RXN03111 W0084 6040 5 FATTY ACID SYNTHASE (EC 2 3 1 85) [INCLUDES EC 2 3 1 38, EC 2 3 1 39, EC 2 3 1 41 , EC 1 1 1 100, EC 4 2 1 61. EC 1 3 1 10, EC 3 1 2 14]

51 52 F RXA00158 GR00024 2088 4 FATTY ACID SYNTHASE (EC 2 3 1 85)

53 54 F RXA00572 GR00155 2 3832 FATTY ACID SYNTHASE (EC 2 3 1 85)

55 56 RXA02582 GR00741 1890 6719 PROBABLE POLYKETIDE SYNTHASE CY338 20

57 58 RXA02691 GR00754 15347 14541 FATTY ACYL RESPONSIVE REGULATOR

59 60 RXA00880 GR00242 6213 8057 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3)

61 62 RXA01060 GR00296 9566 10489 OMEGA-3 FATTY ACID DESATURASE (EC 1 14 99 -)

63 64 RXN01722 W0036 2938 1214 MEDIUM-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 -)

65 66 F RXA01722 GR00488 5746 4022 MEDIUM-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 -)

67 68 RXA01644 GR00456 9854 8577 CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE (EC 2 1 1 79)

69 70 RXA02029 GR00618 356 1669 CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE (EC 2 1 1 79)

71 72 RXA01801 GR00509 3396 2380 ENOYL-COA HYDRATASE (EC 4 2 1 17)

73 74 RXN02512 W01 1 16147 15185 LIPID A BIOSYNTHESIS LAUROYL ACYLTRANSFERASE (EC 2 3 1 -)

75 76 F RXA02512 GR00721 3303 4259 LIPID A BIOSYNTHESIS LAUROYL ACYLTRANSFERASE (EC 2 3 1 -)

77 78 RXA00899 GR00245 1599 2864 CARDIOLIPIN SYNTHETASE (EC 2 7 8 -)

79 80 RXN00819 W0054 18127 19455 ACYL-COA DEHYDROGENASE (EC 1 3 99 -)

81 82 F RXA00819 GR00221 18 1007 ACYL-COA DEHYDROGENASE (EC 1 3 99 -)

83 84 F RXA01766 GR00500 4081 4371 ACYL-COA DEHYDROGENASE (EC 1 3 99 -)

85 86 RXN01762 W0054 15318 13783 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3)

87 88 F RXA01762 GR00500 1272 10 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6 2 1 3)

89 90 RXA00681 GR00179 3405 2662 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC 1 1 1 100)

91 92 RXA00802 GR00214 3803 4516 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC 1 1 1 100)

93 94 RXA02133 GR00639 3 308 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC 1 1 1 100)

95 96 RXN01114 W0182 9118 10341 3-KETOACYL-COA THIOLASE (EC 2 3 1 16)

97 98 F RXA01114 GR00308 2 793 3-KETOACYL-COA THIOLASE (EC 2 3 1 16)

99 100 RXA01894 GR00542 1622 2476 PHOSPHATIDATE CYTIDYLYLTRANSFERASE (EC 2 7 7 41)

101 102 RXA02599 GR00742 3179 3655 PHOSPHATIDYLGLYCEROPHOSPHATASE B (EC 3 1 3 27)

103 104 RXN02638 W0098 54531 53656 1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (EC 2 3 1 51)

105 106 F RXA02638 GR00749 8 511 1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (EC 2 3 1 51)

107 108 RXA00856 GR00232 720 1256 CDP-DIACYLGLYCEROL-GLYCEROL-3-PHOSPHATE 3- PHOSPHATIDYLTRANSFERASE (EC 2 7 8 5)

109 110 RXA02511 GR00721 2621 3277 CDP-DIACYLGLYCEROL-GLYCEROL-3-PHOSPHATE 3- PHOSPHATIDYLTRANSFERASE (EC 2 7 8 5)

111 112 RXN02836 W0102 32818 33372 KETOACYL REDUCTASE HETN (EC 1 3 1 -)

113 114 F RXA02836 GR00827 106 411 KETOACYL REDUCTASE HETN (EC 1 3 1 -)

115 116 RXA02578 GR00740 2438 3541 PUTATIVE ACYLTRANSFERASE

117 118 RXA02150 GR00639 18858 19658 1 -ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (EC 2 3 1 51)

119 120 RXA00607 GR00160 1869 2249 POLY(3-HYDROXYALKANOATE) POLYMERASE (EC 2 3 1 -)

121 122 RXA02397 GR00698 1688 2683 POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC 2 3 1 -)

123 124 RXN03110 W0083 16568 17929 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

125 126 F RXA00660 GR00171 1027 5 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

127 128 RXA00801 GR00214 3138 3770 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

129 130 RXA00821 GR00221 1469 2311 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

131 132 RXN02966 W0143 12056 13462 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

133 134 F RXA01833 GR00517 1666 260 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

135 136 RXA01853 GR00525 5561 5010 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

Table 1 (continued)

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

137 138 RXN02424 W0116 10570 11169 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

139 140 F RXA02424 GR00706 808 428 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3 1 2 6)

141 142 RXN00419 W0112 1024 266 ACETOACETYL-COA REDUCTASE (EC 1 1 1 36)

143 144 F RXA00419 GR00095 3 464 ACETOACETYL-COA REDUCTASE (EC 1 1 1 36)

145 146 F RXA00421 GR00096 565 723 ACETOACETYL-COA REDUCTASE (EC 1 1 1 36)

147 148 RXN02923 W0088 3301 2564 ACETOACETYL-COA REDUCTASE (EC 1 1 1 36)

149 150 RXN02922 W0321 11407 10328 ACYL-COA DEHYDROGENASE, SHORT-CHAIN SPECIFIC (EC 1 3 992)

151 152 RXN03065 W0038 6237 6629 HOLO-[ACYL-CARRIER PROTEIN] SYNTHASE (EC 2 7 8 7)

153 154 RXN03132 W0127 39053 39472 POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC 2 3 1 -)

155 156 RXN03157 W0188 1607 1170 LIPOPOLYSACCHARIDE CORE BIOSYNTHESIS PROTEIN KDTB

157 158 RXN00934 W0171 15181 14099 (AE000805) LPS biosynthesis RfbU related protein [Methanobactenum thermoautotrophicum]

159 160 RXN00792 W0321 10328 9132 ACYL-COA DEHYDROGENASE, SHORT-CHAIN SPECIFIC (EC 1 3 99 2)

161 162 RXN00931 W0171 13011 12166 ACYL-COA THIOESTERASE II (EC 3 1 2 -)

163 164 F RXA00931 GR00253 4959 4114 thioesterase II

165 166 RXN01421 W0122 16024 15638 ACYLTRANSFERASE (EC 2 3 1 -)

167 168 RXN02342 W0078 3460 4266 BIOTIN-[ACETYL-COA-CARBOXYLASE] SYNTHETASE (EC 6 3 4 15)

169 170 RXN00563 W0038 1 2739 FATTY ACID SYNTHASE (EC 2 3 1 85) [INCLUDES EC 2 3 1 38, EC 2 3 1 39, EC

2 3 1 41 , EC 1 1 1 100, EC 4 2 1 61 , EC 1 3 1 10, EC 3 1 2 14]

171 172 RXN02168 W0100 2894 81 FATTY ACID SYNTHASE (EC 2 3 1 85) [INCLUDES EC 2 3 1 38, EC 2 3 1 39, EC

2 3 1 41 , EC 1 1 1 100, EC 4 2 1 61. EC 1 3 1 10, EC 3 1 2 14]

173 174 RXN01090 W0155 6483 5686 KETOACYL REDUCTASE HETN (EC 1 3 1 -)

175 176 RXN02062 W0222 3159 1990 Lipopolysacchande N-acetylglucosaminyltransferase

177 178 RXN02148 W0300 16561 17703 Lipopolysacchaπde N-acetylglucosaminyltransferase

179 180 RXN02595 W0098 11098 9935 Lipopolysacchande N-acetylglucosaminyltransferase

181 182 RXS00148 W0167 9849 12059 METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC 54 99 2)

183 184 RXS00149 W0167 7995 9842 METHYLMALONYL-COA MUTASE BETA-SUBUNIT (EC 5 4 99 2)

185 186 RXS02106 W0123 22649 21594 LIPOATE-PROTEIN LIGASE A (EC 6 - - -)

187 188 RXS01746 W0185 934 1686 LIPOATE-PROTEIN LIGASE B (EC 6 - - -)

189 190 RXS01747 W0185 1826 2869 LIPOIC ACID SYNTHETASE

191 192 RXC01748 W0185 3001 3780 protein involved in lipid metabolism

193 194 RXC00354 W0135 33604 32792 Cytosolic Protein involved in lipid metabolism

195 196 RXC01749 W0185 3953 5569 Membrane Spanning Protein involved in lipid metabolism

Fatty acid degradation

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function SEQ ID NO SEQ ID NO

197 198 RXA02268 GR00655 2182 3081 LIPASE (EC 3 1 1 3) 199 200 RXA02269 GR00655 3094 4065 LIPASE (EC 3 1 1 3) 201 202 RXA01614 GR00449 8219 7197 LYSOPHOSPHOLIPASE L2 (EC 3 1 1 5) 203 204 RXA01983 GR00573 3559 3053 LIPASE (EC 3 1 1 3) 205 206 RXN02947 W0078 1319 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3) 207 208 F RXA02320 GR00667 593 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 64 1 3) 209 210 F RXA02851 GR00851 524 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3) 211 212 RXN02321 W0078 3291 1663 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3)

Table 1 (continued)

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

213 214 F RXA02321 GR00667 1380 937 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3)

215 216 F RXA02343 GR00675 1403 1816 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3)

217 218 F RXA02850 GR00850 2 493 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3)

219 220 RXA02583 GR00741 6743 8290 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6 4 1 3)

221 222 RXA00870 GR00239 809 2320 METHYLMALONATE-SEMIALDEHYDE DEHYDROGENASE (ACYLATING) (EC 1 2 1 27) 2-Methyl-3-oxopropanoate NAD+ oxidoreductase (CoA-propanoylatmg)

223 224 RXA01260 GR00367 2381 1200 LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1 8 1 4)

225 226 RXA01261 GR00367 2607 2437 LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1 8 1 4)

227 228 RXA01136 GR00318 685 1116 ISOVALERYL-COA DEHYDROGENASE (EC 1 3 99 10)

229 230 RXN00559 W0103 7568 6552 PROTEIN VDLD

231 232 F RXA00559 GR00149 218 6 PROTEIN VDLD

233 234 RXA01580 GR00440 707 6 Glycerophosphoryl diester phosphodiesterase

235 236 RXA02677 GR00754 3119 3877 GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASE (EC 3 1 4 46)

237 238 RXS01166 W0117 18142 16838 EXTRACELLULAR LIPASE PRECURSOR (EC 3 1 1 3)

Terpenoid biosynthesis

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

239 240 RXA00875 GR00241 2423 1857 ISOPENTENYL-DIPHOSPHATE DELTA-ISOMERASE (EC 5332)

241 242 RXA01292 GR00373 1204 2388 PHYTOENE DEHYDROGENASE (EC 13 - -)

243 244 RXA01293 GR00373 2370 2696 PHYTOENE DEHYDROGENASE (EC 13 - -)

245 246 RXA02310 GR00665 1132 2394 GERANYLGERANYL HYDROGENASE

247 248 RXA02718 GR00758 18539 19585 GERANYLGERANYL PYROPHOSPHATE SYNTHASE (EC 2511)

249 250 RXA01067 GR00298 1453 2181 undecaprenyl-diphosphate synthase (EC 2 5 1 31)

251 252 RXA01269 GR00367 20334 19894 UNDECAPRENYL-PHOSPHATE GALACTOSEPHOSPHOTRANSFERASE (EC

2 7 8 6)

253 254 RXA01205 GR00346 3 533 PUTATIVE UNDECAPRENYL-PHOSPHATE ALPHA-N-

ACETYLGLUCOSAMINYLTRANSFERASE (EC 2 4 1 -)

255 256 RXA01576 GR00438 8053 8811 DOLICHYL-PHOSPHATE BETA-GLUCOSYLTRANSFERASE (EC 2 4 1 117)

257 258 RXN02309 W0025 28493 29542 OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2 5 1 -)

259 260 F RXA02309 GR00665 978 4 OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2 5 1 -)

261 262 RXN00477 W0086 38905 37262 PHYTOENE DEHYDROGENASE (EC 1 3 - -)

263 264 F RXA00477 GR00119 13187 11544 PHYTOENE DEHYDROGENASE (EC 1 3 - -)

265 266 RXA00478 GR00119 14020 13190 PHYTOENE SYNTHASE (EC 2 5 1 -)

267 268 RXA01291 GR00373 345 1277 PHYTOENE SYNTHASE (EC 2 5 1 -)

269 270 RXA00480 GR00119 17444 16329 FARNESYL DIPHOSPHATE SYNTHASE (EC 2 5 1 1) (EC 2 5 1 10)

271 272 RXS01879 W0105 1505 573 isopentenyl-phosphate kmase (EC 2 7 4 -)

273 274 RXS02023 W0160 3234 4001 P450 cytochrome.isopentenyltransf, ferπdox

275 276 RXS00948 W0107 4266 5384 12-oxophytodιenoate reductase (EC 1 3 1 42)

277 278 RXS02228 W0068 1876 2778 TRNA DELTA(2)-ISOPENTENYLPYROPHOSPHATE TRANSFERASE (EC 2 5 1 8)

279 280 RXC01971 W0105 4545 3715 Metal-Dependent Hydrolase involved in metabolism of terpenoids

281 282 RXC02697 W0017 31257 32783 membrane protein involved in metabolism of terpenoids

Table 1 (continued)

ABC-Transporter

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

283 284 RXN01946 W0228 2 1276 Hypothetical ABC Transporter ATP-Binding Protein

285 286 F RXA01946 GR00559 1849 575 (AL021184) ABC transporter ATP binding protein [Mycobactenum tuberculosis]

287 288 RXN00164 W0232 1782 94 Hypothetical ABC Transporter ATP-Binding Protein

289 290 F RXA00164 GR00025 1782 94 , P, G, R ATPase subunits of ABC transporters

291 292 RXN00243 W0057 28915 27899 , P, G, R ATPase subunits of ABC transporters

293 294 F RXA00243 GR00037 930 4 , P, G, R ATPase subunits of ABC transporters

295 296 RXA00259 GR00039 8469 6268 , P, G, R ATPase subunits of ABC transporters

297 298 RXN00410 W0086 51988 51323 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ

299 300 F RXA00410 GR00092 829 164 , P, G, R ATPase subunits of ABC transporters

301 302 RXN00456 W0076 6780 8156 , P, G, R ATPase subunits of ABC transporters

303 304 F RXA00456 GR00114 316 5 , P, G, R ATPase subunits of ABC transporters

305 306 F RXA00459 GR00115 1231 245 , P, G, R ATPase subunits of ABC transporters

307 308 RXN01604 W0137 8117 7470 , P, G, R ATPase subunits of ABC transporters

309 310 F RXA01604 GR00448 2 607 , P, G, R ATPase subunits of ABC transporters

311 312 RXN02547 W0057 27726 25588 , P, G, R ATPase subunits of ABC transporters

313 314 F RXA02547 GR00726 22055 19932 , P, G, R ATPase subunits of ABC transporters

315 316 RXN02571 W0101 12331 13359 MALTOSE/MALTODEXTRIN TRANSPORT ATP-BINDING PROTEIN MALK

317 318 F RXA02571 GR00736 1469 2497 , P, G, R ATPase subunits of ABC transporters

319 320 RXN02074 W0318 12775 11153 TRANSPORT ATP-BINDING PROTEIN CYDD

321 322 F RXA02074 GR00628 5798 4176 , P, G, R ATPase subunits of ABC transporters

323 324 RXA02095 GR00629 14071 15474 , P, G, R ATPase subunits of ABC transporters

325 326 RXA02225 GR00652 3156 2275 , P, G, R ATPase subunits of ABC transporters

327 328 RXA02253 GR00654 20480 21406 , P, G, R ATPase subunits of ABC transporters

329 330 RXN01881 W0105 529 95 Hypothetical ABC Transporter ATP-Binding Protein

331 332 F RXA01881 GR00537 3092 3532 ATPase components of ABC transporters with duplicated ATPase domains

333 334 RXA00526 GR00136 1353 664 Hypothetical ABC Transporter ATP-Binding Protein

335 336 RXN00733 W0132 1647 2531 Hypothetical ABC Transporter ATP-Binding Protein

337 338 F RXA00733 GR00197 411 4 Hypothetical ABC Transporter ATP-Binding Protein

339 340 RXA00735 GR00198 849 181 Hypothetical ABC Transporter ATP-Binding Protein

341 342 RXA00878 GR00242 3733 1871 Hypothetical ABC Transporter ATP-Bmdiπg Protein

343 344 RXN01191 W0169 10478 12067 Hypothetical ABC Transporter ATP-Binding Protein

345 346 F RXA01191 GR00341 1571 165 Hypothetical ABC Transporter ATP-Binding Protein

347 348 RXN01212 W0169 3284 4207 Hypothetical ABC Transporter ATP-Binding Protein

349 350 F RXA01212 GR00350 1 813 Hypothetical ABC Transporter ATP-Binding Protein

351 352 RXA02749 GR00764 4153 5028 Hypothetical ABC Transporter ATP-Binding Protein

353 354 RXA02224 GR00652 2271 475 Hypothetical ABC Transporter ATP-Binding Protein

355 356 RXN01602 W0229 1109 2638 Hypothetical ABC Transporter ATP-Binding Protein

357 358 RXN02515 W0087 962 1717 Hypothetical ABC Transporter ATP-Binding Protein

359 360 RXN00525 W0079 26304 27566 Hypothetical ABC Transporter Permease Protein

361 362 RXN02096 W0126 20444 22135 Hypothetical ABC Transporter Permease Protein

363 364 RXN00412 W0086 53923 52844 Hypothetical Am o Acid ABC Transporter ATP-Binding Protein

365 366 RXN00411 W0086 52844 52170 Hypothetical Ammo Acid ABC Transporter Permease Protein

367 368 RXN02614 W0313 5964 5236 TAURINE TRANSPORT ATP-BINDING PROTEIN TAUB

369 370 RXN02613 W0313 5223 4267 TAURINE-BINDING PERIPLASMIC PROTEIN PRECURSOR

Table 1 (continued)

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

371 372 RXN00368 W0226 2300 726 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA

373 374 F RXA00368 GR00076 1 579 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA

375 376 F RXA00370 GR00077 6 803 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA

377 378 RXN01285 W0215 1780 1055 FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING PROTEIN FEPC

379 380 RXN00523 W0194 1363 338 FERRIC ENTEROBACTIN TRANSPORT PROTEIN FEPG

381 382 RXN01142 W0077 5805 6302 NITRATE TRANSPORT ATP-BINDING PROTEIN NRTD

383 384 RXN01141 W0077 4644 5468 NITRATE TRANSPORT PROTEIN NRTA

385 386 RXN01002 W0106 8858 8055 PHOSPHONATES TRANSPORT ATP-BINDING PROTEIN PHNC

387 388 RXN01000 W0106 7252 6407 PHOSPHONATES TRANSPORT SYSTEM PERMEASE PROTEIN PHNE

389 390 RXN01732 W0106 9944 8895 PHOSPHONATES-BINDING PERIPLASMIC PROTEIN PRECURSOR

391 392 RXN03080 W0045 1670 2449 FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING PROTEIN FEPC

393 394 RXN03081 W0045 2476 2934 FERRIENTEROBACTIN-BINDING PERIPLASMIC PROTEIN PRECURSOR

395 396 RXN03082 W0045 3131 3451 FERRIENTEROBACTIN-BINDING PERIPLASMIC PROTEIN PRECURSOR

Other transporters

Nucleic Acid Am o Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

397 398 RXA02261 GR00654 30936 32291 AMMONIUM TRANSPORT SYSTEM 399 400 RXA02020 GR00613 1015 5 AROMATIC AMINO ACID TRANSPORT PROTEIN AROP 401 402 RXA00281 GR00043 4721 5404 BACITRACIN TRANSPORT ATP-BINDING PROTEIN BCRA 403 404 RXN00570 W0147 855 4 BENZOATE MEMBRANE TRANSPORT PROTEIN 405 406 F RXA00570 GR00153 1 498 BENZOATE MEMBRANE TRANSPORT PROTEIN 407 408 RXN00571 W0173 1298 42 BENZOATE MEMBRANE TRANSPORT PROTEIN 409 410 F RXA00571 GR00154 2 1 186 BENZOATE MEMBRANE TRANSPORT PROTEIN 411 412 RXA00962 GR00268 2 667 BENZOATE MEMBRANE TRANSPORT PROTEIN 413 414 RXA02811 GR00792 177 560 BENZOATE MEMBRANE TRANSPORT PROTEIN 415 416 RXA02115 GR00635 2 1 198 BENZOATE MEMBRANE TRANSPORT PROTEIN 417 418 RXN00590 W0178 5043 6230 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER PROTEIN 419 420 F RXA00590 GR00157 178 564 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER PROTEIN 421 422 F RXA01538 GR00427 5040 5429 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER PROTEIN 423 424 RXA01727 GR00489 1471 194 BRANCHED-CHAIN AMINO ACID TRANSPORT SYSTEM CARRIER PROTEIN 425 426 RXA00623 GR00163 6525 7862 C4-DICARBOXYLATE TRANSPORT PROTEIN 427 428 RXA01584 GR00441 55 597 CHROMATE TRANSPORT PROTEIN 429 430 RXA00852 GR00231 3137 2448 COBALT TRANSPORT ATP-BINDING PROTEIN CBIO 431 432 RXA00690 GR00181 1213 68 COBALT TRANSPORT PROTEIN CBIQ 433 434 RXA00827 GR00223 1319 567 COBALT TRANSPORT PROTEIN CBIQ 435 436 RXA00851 GR00231 2448 1840 COBALT TRANSPORT PROTEIN CBIQ 437 438 RXS03220 D-XYLOSE-PROTON SYMPORT 439 440 F RXA02762 GR00768 346 630 D-XYLOSE PROTON-SYMPORTER 441 442 RXN00092 W0129 27509 26844 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 443 444 F RXA00092 GR00014 1 204 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 445 446 RXN03060 W0030 6227 5376 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 447 448 F RXA02618 GR00745 1914 2351 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 449 450 F RXA02900 GR 10040 2979 2128 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ

Table 1 (continued)

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

451 452 RXS03212 GLYCINE BETAINE TRANSPORTER BETP

453 454 F RXA01591 GR00446 3 947 GLYCINE BETAINE TRANSPORTER BETP

455 456 RXN00201 W0096 197 6 HIGH AFFINITY RIBOSE TRANSPORT PROTEIN RBSD

457 458 F RXA00201 GR00032 191 6 HIGH AFFINITY RIBOSE TRANSPORT PROTEIN RBSD

459 460 RXA01221 GR00354 2108 2833 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT ATP-BINDING

PROTEIN BRAG

461 462 RXA01222 GR00354 2844 3542 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT ATP-BINDING

PROTEIN LIVF

463 464 RXA01219 GR00354 151 1032 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT PERMEASE

PROTEIN LIVH

465 466 RXA01220 GR00354 1032 2108 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT PERMEASE

PROTEIN LIVM

467 468 RXA00091 GR00013 7762 8514 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

469 470 RXA00228 GR00032 29232 28642 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

471 472 ^• RXA00346 GR00064 1054 1743 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

473 474 RXA00524 GR00135 779 1111 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

475 476 RXA01823 GR00516 591 1367 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

477 478 RXA02767 GR00770 1032 1814 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

479 480 RXA02792 GR00777 8581 7829 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE

481 482 RXN02929 W0090 36837 37874 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

483 484 F RXA01235 GR00358 1165 194 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

485 486 RXN02794 W0134 10625 9552 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

487 488 F XA01419 GR00415 888 1151 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

489 490 F RXA02794 GR00777 10172 9552 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD -fc

491 492 RXN03079 W0045 644 1660 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

493 494 F RXA02865 GR10007 3832 2816 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD

495 496 RXA00181 GR00028 3954 2383 PROLINE TRANSPORT SYSTEM

497 498 RXA00591 GR00158 229 1581 PROLINE/BETAINE TRANSPORTER

499 500 RXA01629 GR00453 3476 1965 PROLINE/BETAINE TRANSPORTER

501 502 RXA02030 GR00618 3072 1687 PROLINE/BETAINE TRANSPORTER

503 504 RXA00186 GR00028 12242 12988 SHORT-CHAIN FATTY ACIDS TRANSPORTER

505 506 RXA00187 GR00028 13097 13447 SHORT-CHAIN FATTY ACIDS TRANSPORTER

507 508 RXA01667 GR00464 703 1908 SODIUM/GLUTAMATE SYMPORT CARRIER PROTEIN

509 510 RXA02171 GR00641 6571 4919 SODIUM/PROUNE SYMPORTER

511 512 RXA00902 GR00245 4643 5875 SODIUM-DEPENDENT PHOSPHATE TRANSPORT PROTEIN

513 514 RXA00941 GR00257 1999 683 sodium-dependent phosphate transport protein

515 516 RXN00449 W0112 30992 32572 Sodium-Dicarboxylate Symport Protein

517 518 F RXA00449 GR00109 2040 1036 Sodium-Dicarboxylate Symport Protein

519 520 F RXA01755 GR00498 352 5 Sodium-Dicarboxylate Symport Protein

521 522 RXA00269 GR00041 1826 1038 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA

523 524 RXA00369 GR00076 583 1299 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA

525 526 RXA02073 GR00628 4176 2647 TRANSPORT ATP-BINDING PROTEIN CYDC

527 528 RXA01399 GR00409 1 1119 TRANSPORT ATP-BINDING PROTEIN CYDD

529 530 RXA01339 GR00389 8408 7164 TYROSINE-SPECIFIC TRANSPORT PROTEIN

531 532 RXA02527 GR00725 5519 6847 2-OXOGLUTARATE/MALATE TRANSLOCATOR PRECURSOR

533 534 RXN00298 W0176 40228 42072 HIGH-AFFINITY CHOLINE TRANSPORT PROTEIN

535 536 F RXA00298 GR00048 4459 6303 Ectoine/Proline/Glycme betame carrier ectP

Table 1 (continued)

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

537 538 RXA00596 GR00159 335 787 potassium efflux system protein phaE

539 540 RXA02364 GR00686 841 215 C4-DICARBOXYLATE-BINDING PERIPLASMIC PROTEIN PRECURSOR, transport protein

541 542 RXN01411 W0050 26015 26779 SHIKIMATE TRANSPORTER

543 544 RXN00960 W0075 1139 105 PROTON/SODIUM-GLUTAMATE SYMPORT PROTEIN

545 546 RXN02447 W0107 14297 13203 GALACTOSE-PROTON SYMPORT

547 548 RXN02395 W0176 16747 14858 GLYCINE BETAINE TRANSPORTER BETP

549 550 RXN02348 W0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE PROTEIN

551 552 RXN00297 W0176 38630 39541 Hypothetical Malonate Transporter

553 554 RXN03103 W0070 845 1087 GLUTAMATE-BINDING PROTEIN PRECURSOR

555 556 RXN02993 W0071 736 65 GLUTAMINE-BINDING PROTEIN

557 558 RXN00349 W0135 35187 36653 Hypothetical Trehalose Transport Protein

559 560 RXN03095 W0057 4056 4424 CADMIUM EFFLUX SYSTEM ACCESSORY PROTEIN HOMOLOG

561 562 RXN03160 W0189 5150 5617 CHROMATE TRANSPORT PROTEIN

563 564 RXN02955 W0176 8666 9187 DICARBOXYLATE TRANSPORTER

565 566 RXN03109 W0082 659 6 HEMIN TRANSPORT SYSTEM PERMEASE PROTEIN HMUU

567 568 RXN02979 W0149 2150 2383 MERCURIC TRANSPORT PROTEIN PERIPLASMIC COMPONENT PRECURSOR

569 570 RXN02987 W0234 527 294 MERCURIC TRANSPORT PROTEIN PERIPLASMIC COMPONENT PRECURSOR

571 572 RXN03084 W0048 900 1817 IRON(III) DICITRATE-BINDING PERIPLASMIC PROTEIN PRECURSOR

573 574 RXN03183 W0372 1 417 TREHALOSE/MALTOSE BINDING PROTEIN

575 576 RXN01139 W0077 2776 1823 CATION EFFLUX SYSTEM PROTEIN CZCD

577 578 RXN00378 W0223 8027 5418 Cation transport ATPases

579 580 RXN01338 W0032 2 1903 CATION-TRANSPORTING ATPASE PACS (EC 361 -)

581 582 RXN00980 W0149 2635 4428 CATION-TRANSPORTING P-TYPE ATPASE B (EC 361 -) 0

583 584 RXN00099 W0129 18876 17704 CYANATE TRANSPORT PROTEIN CYNX

585 586 RXN02662 W0315 1461 1724 DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN DPPC

587 588 RXN02442 W0217 5970 6818 zinc transport system membrane protein

589 590 RXN02443 W0217 6818 7771 zinc-binding periplasmic protein precursor

591 592 RXN00842 W0138 8686 7487 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER PROTEIN

593 594 F RXA00842 GR00228 3208 2009 Permeases

595 596 RXN00832 W0180 3133 4182 CALCIUM/PROTON ANTIPORTER

597 598 RXN00466 W0086 63271 64266 Femchrome transport proteins

599 600 RXN01936 W0127 40116 41387 MACROLIDE-EFFLUX PROTEIN

601 602 RXN01995 W0182 2139 3476 PUTATIVE 3-(3-HYDROXYPHENYL) PROPIONATE TRANSPORT PROTEIN

603 604 RXN00661 W0142 9718 9029 PNUC PROTEIN

Permeases

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

605 606 RXN02566 W0154 11823 13031 NUCLEOSIDE PERMEASE NUPG

607 608 F RXA02561 GR00732 664 5 NUCLEOSIDE PERMEASE NUPG

609 610 F RXA02566 GR00733 782 345 NUCLEOSIDE PERMEASE NUPG

611 612 RXA00051 GR00008 5770 7173 PROLINE-SPECIFIC PERMEASE PROY

613 614 RXA01172 GR00334 2687 4141 SULFATE PERMEASE

615 616 RXA02128 GR00637 2906 4600 SULFATE PERMEASE

Table 1 (continued)

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

617 618 RXA02634 GR00748 6045 7655 SULFATE PERMEASE

619 620 RXN02233 W0068 6856 8142 URACIL PERMEASE

621 622 F RXA02233 GR00653 6856 8067 URACIL PERMEASE

623 624 RXN02372 W0213 9311 11197 XANTHINE PERMEASE

625 626 F RXA02372 GR00688 6 560 XANTHINE PERMEASE

627 628 F RXA02377 GR00689 3336 4526 XANTHINE PERMEASE

629 630 RXA02676 GR00754 2697 1309 GLUCONATE PERMEASE

631 632 RXN00432 W0112 14751 13267 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE

633 634 F RXA00432 GR00100 1 891 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE

635 636 F RXA00436 GR00101 45 569 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE

637 638 RXA00847 GR00230 1829 381 OLIGOPEPTIDE-BINDING PROTEIN APPA PRECURSOR (permease)

639 640 RXN01382 W0119 8670 9761 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR

641 642 F RXA01382 GR00405 1067 6 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR (permease)

643 644 RXA02659 GR00753 2 313 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR (permease)

645 646 RXN02933 W0176 30042 29233 DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN DPPC

647 648 RXN02991 W0072 618 4 GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP

649 650 RXN02992 W0072 842 621 GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP

651 652 RXN02996 W0069 1980 2648 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT PERMEASE

PROTEIN LIVH

653 654 RXN03126 W0112 9894 9001 TEICHOIC ACID TRANSLOCATION PERMEASE PROTEIN TAGG

655 656 RXN00443 W0112 21572 20769 MOLYBDATE-BINDING PERIPLASMIC PROTEIN PRECURSOR

657 658 RXN00444 W0112 20785 19949 MOLYBDENUM TRANSPORT SYSTEM PERMEASE PROTEIN MODB

659 660 RXN00193 W0371 1 594 POTENTIAL STARCH DEGRADATION PRODUCTS TRANSPORT SYSTEM

PERMEASE PROTEIN AMYD ON

661 662 RXN01298 W0116 2071 1142 POTENTIAL STARCH DEGRADATION PRODUCTS TRANSPORT SYSTEM

PERMEASE PROTEIN AMYD

Channel Proteins

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function

SEQ ID NO SEQ ID NO

663 664 RXA01737 GR00493 2913 3971 POTASSIUM CHANNEL PROTEIN

665 666 RXN02348 W0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE PROTEIN

667 668 RXA02426 GR00707 2165 633 PROBABLE NA(+)/H(+) ANTIPORTER

669 670 RXN03164 W0277 1586 2455 POTASSIUM CHANNEL BETA SUBUNIT

671 672 RXN00024 W0127 64219 63275 POTASSIUM CHANNEL BETA SUBUNIT

Lipoprotein and Lipopolysacchande synthesis

Nucleic Acid Ammo Acid Identification Code Contig NT Start NT Stop Function SEQ ID NO SEQ ID NO 673 674 RXN01164 W0117 15894 14260 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE (EC 24183) / APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 231 -)

675 676 RXN01168 W0117 14224 13415 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE (EC 24183) / APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 231 -)

4

o

o

TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention

ATCC: American Type Culture Collection, Rockville, MD, USA

FERM: Fermentation Research Institute, Chiba, Japan

NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA

CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain

NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK

CBS: Centraalbureau voor Schimmelcultures, Baarn, NL

NCTC: National Collection of Type Cultures, London, UK

DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany

For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (4^th edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.

Table 4: Alignment Results

ID# lenqth Genbank Hit Length Accession Name of Genbank Hit Source of Genbank Hit % homoloαv Date of

(NT) (GAP) Deposit rxa00051 1527 GBJHTG3 AC009685 210031 AC009685 Homo sapiens chromosome 15 clone 91_E_13 map 15, ^*** SEQUENCING IN Homo sapiens 34,247 29-Sep-

PROGRESS ^***, 27 unordered pieces

GBJHTG3 AC009685 210031 AC009685 Homo sapiens chromosome 15 clone 91_E_13 map 15, ^*** SEQUENCING IN Homo sapiens 34,247 29-Sep-

PROGRESS ^***, 27 unordered pieces

GB_HTG7 AC009511 271896 AC009511 Homo sapiens clone RP11-860B13, "' SEQUENCING IN PROGRESS ***, 59 Homo sapiens 35,033 09-DEC unordered pieces rxa00091 876 GB_BA1 D50453 146191 D50453 Bacillus subtilis DNA for 25-36 degree region containing the amyE-srfA region, Bacillus subtilis 54,452 10-Feb- complete eds

GB BA1 SCI51 40745 AL109848 Streptomyces coehcolor cosmid 151 Streptomyces coehcolor 36,806 16-Aug- A3(2)

GB_BA1 ECOUW93 338534 U14003 Escherichia coli K-12 chromosomal region from 92 8 to 00 1 minutes Escherichia coli 38,642 17-Apr- rxa00092 789 GB_BA1 SCH35 45396 AL078610 Streptomyces coehcolor cosmid H35 Streptomyces coehcolor 49,934 4-Jun-9

GB_HTG3 AC011498_ 312343 AC011498 Homo sapiens chromosome 19 clone CIT978SKB_50L17, ^*** SEQUENCING IN Homo sapiens 37,117 13-Dec-

0 PROGRESS ^***, 190 unordered pieces

GB_HTG3 AC011498_ 312343 AC011498 Homo sapiens chromosome 19 clone CIT978SKB_50L17, ^*** SEQUENCING IN Homo sapiens 37,117 13-Dec-

0 PROGRESS ^***, 190 unordered pieces rxa00104 879 GB_BA1 MTCY270 37586 Z95388 Mycobacterium tuberculosis H37Rv complete genome, segment 96/162 Mycobacterium 36,732 10-Feb- tuberculosis

GB_PL2 T24 8 68251 AF077409 Arabidopsis thahana BAC T24M8 Arabidopsis thaliana 37,150 3-Aug-9 GB_BA1 MTCY270 37586 Z95388 Mycobacterium tuberculosis H37Rv complete genome, segment 96/162 Mycobacterium 42,874 10-Feb- tuberculosis rxa00113 5745 GB_BA1 MAFASGEN 10520 X87822 B ammoniagenes FAS gene Corynebacterium 68,381 03-OC ammoniagenes

GB_BA1 BAFASAA 10549 X64795 B ammoniagenes FAS gene Corynebacterium 57,259 14-OC ammoniagenes

GB_BA1 MTCY159 33818 Z83863 Mycobacterium tuberculosis H37Rv complete genome, segment 111/162 Mycobacterium 39,870 17-Jun- tuberculosis rxa00164 1812 GB_HTG2 HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9, ^*** SEQUENCING IN Homo sapiens 35,714 03-DEC

PROGRESS ^***, in unordered pieces

GB_HTG2 HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9, *** SEQUENCING IN Homo sapiens 35,714 03-DEC

PROGRESS ^***, in unordered pieces

GBJHTG2 HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9, ^*** SEQUENCING IN Homo sapiens 35,334 03-DEC

PROGRESS ***, in unordered pieces rxa00181 1695 GB_BA1 CGPUTP 3791 Y09163 C glutamicum putP gene Corynebacterium 100,000 8-Sep-9 glutamicum

GB_BA2 U32814 10393 U32814 Haemophilus influenzae Rd section 129 of 163 of the complete genome Haemophilus influenzae 36,347 29-MA

Rd

GB_BA1 CGPUTP 3791 Y09163 C glutamicum putP gene Corynebacterium 37,454 8-Sep-9 glutamicum rxa00186 870 GB PR3 AC004843 136655 AC004843 Homo sapiens PAC clone DJ0612F12 from 7p12-p14, complete sequence Homo sapiens 37,315 5-N0V-9

Table 4 (continued)

GB_HTG2 HS745I14 133309 AL033532 Homo sapiens chromosome 1 clone RP4-745I14 map q23 1-24 3, ^*** Homo sapiens 38,129 03-DEC-

SEQUENCING IN PROGRESS ^***, in unordered pieces GB_HTG2 HS745I14 133309 AL033532 Homo sapiens chromosome 1 clone RP4-745I14 map q23 1-24 3, ^*** Homo sapiens 38,129 03-DEC-

SEQUENCING IN PROGRESS ^***, in unordered pieces rxa00187 474 GB_GSS10 AQ184082 506 AQ184082 HS_3216_A1_G08_T7 CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 37,297 1-N0V-98 sapiens genomic clone Plate=3216 Col=15 Row=M, genomic survey sequence GB_GSS1 CNS008ZZ 1101 AL052951 Drosophila melanogaster genome survey sequence T7 end of BAC # Drosophila melanogaster 34,120 3-Jun-99

BACR18L01 of RPCI-98 library from Drosophila melanogaster (fruit fly), genomic survey sequence

GB_GSS10 AQ 184082 506 AQ 184082 HS_3216_A1_G08_T7 CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 39,655 1 -Nov-9 sapiens genomic clone Plate=3216 Col=15 Row=M, genomic survey sequence rxa00201 292 GB_PR3 HSJ824F16 139330 AL050325 Human DNA sequence from clone 824F16 on chromosome 20, complete Homo sapiens 34,520 23-Nov- sequence

GB_BA1 RCSECA 2724 X89411 R capsulatus DNA for secA gene Rhodobacter capsulatus 38,163 6-Jan-96 GB_EST34 AV122904 242 AV122904 AV122904 Mus musculus C57BL/6J 10-day embryo Mus musculus cDNA clone Mus musculus 38,889 1-Jul-99

2610529H07, mRNA sequence rxa00228 714 GB_EST15 AA486042 515 AA486042 ab40c08 r1 Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone Homo sapiens 37,500 06-MAR-

IMAGE 843278 5', mRNA sequence

GB_EST15 AA486042 515 AA486042 ab40c08 r1 Stratagene HeLa cell s3 937216 Homo sapiens cDNA clone Homo sapiens 38,816 06-MAR-

IMAGE 843278 5', mRNA sequence rxa00243 1140 GB_PR2 CNS01 DS5 101584 AL121655 BAC sequence from the SPG4 candidate region at 2p21 -2p22, complete Homo sapiens 37,001 29-Sep- GB_HTG3 AC011408 79332 AC011408 Homo sapiens clone CIT978SKB_65D22, ^*** SEQUENCING IN PROGRESS ^***, Homo sapiens 38,040 06-OCT-

10 unordered pieces

GB_HTG3 AC011408 79332 AC011408 Homo sapiens clone CIT978SKB_65D22, "^* SEQUENCING IN PROGRESS ^***, Homo sapiens 38,040 06-OCT-

10 unordered pieces rxa00259 2325 GB_HTG1 CEY62E10 254217 AL031580 Caenorhabditis elegaπs chromosome IV clone Y62E10, ^*** SEQUENCING IN Caenorhabditis elegans 36,776 6-Sep-9

PROGRESS "*, in unordered pieces

GB_HTG1 CEY62E10 254217 AL031580 Caenorhabditis elegans chromosome IV clone Y62E10, ^*** SEQUENCING IN Caenorhabditis elegans 36,776 6-Sep-9

PROGRESS *^**, in unordered pieces

GB_PL2 YSCCHROMI 41988 L22015 Saccharomyces cerevisiae chromosome I centromere and right arm sequence Saccharomyces 39,260 05-MAR cerevisiae rxa00269 912 GB_HTG4 AC009974 219565 AC009974 Homo sapiens chromosome unknown clone NH0459I19, WORKING DRAFT Homo sapiens 37,358 29-OCT-

SEQUENCE, in unordered pieces

GB_HTG4 AC009974 219565 AC009974 Homo sapiens chromosome unknown clone NH0459I19, WORKING DRAFT Homo sapiens 37,358 29-OCT-

SEQUENCE, in unordered pieces

GB_BA1 AB017508 32050 AB017508 Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete eds Bacillus halodurans 44,622 14-Apr-9 rxa00281 766 GB_BA1 SCE8 24700 AL035654 Streptomyces coehcolor cosmid E8 Streptomyces coehcolor 36,328 11-MAR GB_BA1 SCU51332 3216 U51332 Streptomyces coehcolor histidine kinase homolog (absA1) and response Streptomyces coehcolor 39,089 14-Sep- regulator homolog (absA2) genes, complete eds

GB HTG4 AC011122 187123 AC011122 Homo sapiens chromosome 8 clone 23_D_19 map 8, ^*** SEQUENCING IN Homo sapiens 38,658 14-OCT-

PROGRESS ^***, 27 ordered pieces

Table 4 (continued) rxa00298 1968 GB_BA1 CGECTP 2719 AJ001436 Corynebacterium glutamicum ectP gene Corynebacterium 100,000 20-Nov- glutamicum

GB_BA1 CGECTP 2719 AJ001436 Corynebacterium glutamicum ectP gene Corynebacterium 100,000 20-Nov- glutamicum

GB_EST24 AI234006 432 AI234006 EST230694 Normalized rat lung, Bento Soares Rattus sp cDNA clone Rattus sp 46,552 31-Jan-9

RLUCU01 3' end, mRNA sequence rxa00346 813 GB_BA1 SC2E9 20850 AL021530 Streptomyces coehcolor cosmid 2E9 Streptomyces coehcolor 43,267 28-Jan-9

GB_BA1 SC9B1 24800 AL049727 Streptomyces coehcolor cosmid 9B1 Streptomyces coehcolor 44,613 27-Apr-9

GB_BA1 ECU70214 123171 U70214 Escherichia coli chromosome minutes 4-6 Escherichia coli 39,490 21-Sep- rxa00368 1698 GB BA2 AF065159 35209 AF065159 Bradyrhizobium japonicum putative arylsulfatase (arsA), putative soluble lytic Bradyrhizobium 40,409 27-OCT- transglycosylase precursor (sltA), dihydrodipicolinate synthase (dapA), MscL japonicum

(mscL), SmpB (smpB), BcpB (bcpB), RnpO (rnpO), RelA/SpoT homolog (relA),

PdxJ (pdxJ), and acyl earner protein synthase AcpS (acpS) genes, complete eds prokaryotic type I signal peptidase SipF (sipF) gene, sipF-sipS allele, complete eds, RNase III (rπc) gene, complete eds, GTP-bindmg protein Era (era) gene, partial eds, and unknown genes

GB_BA1 AEOCHIT1 6861 D63139 Aeromonas sp gene for chitinase, complete and partial eds Aeromonas sp 10S-24 38,577 13-Feb-

GB_EST4 D62996 314 D62996 HUM347G01 B Clontech human aorta polyA÷ mRNA (#6572) Homo sapiens Homo sapiens 41 ,613 29-Aug- cDNA clone GEN-347G01 5', mRNA sequence rxa00369 817 GB_BA1 YP102KB 119443 AL031866 Yersmia pestis 102 kbases unstable region from 1 to 119443 Yersmia pestis 35,396 4-Jan-9

GB_GSS8 AQ012142 501 AQ012142 8750H1A037010398 Cosmid library of chromosome II Rhodobacter sphaeroides Rhodobacter 54,800 4-Jun-98 genomic clone 8750H1A037010398, genomic survey sequence sphaeroides

G B_HTG2 AC005081 180096 AC005081 Homo sapiens clone RG270D13, ^*** SEQUENCING IN PROGRESS ^***, 18 Homo sapiens 45,786 12-Jun-9 unordered pieces rxa00410 789 GB_BA1 ATPLOCC 8870 Z30328 A tumefaciens Ti plasmid pTiAchδ genes for OccR, OccQ, OccM, OccP, OccT, Agrobacteπum 46,490 10-OCT-

OoxB, OoxA and ornithine cyclodeaminase tumefaciens

GB_BA2 U67591 9829 U67591 Methanococcus jannaschii section 133 of 150 of the complete genome Methanococcus 45,677 28-Jan- jannaschii

GB BA1 TIPOCCQMPJ 4350 M80607 Plasmid pTιA6 (from Agribacteπum tumefaciens) peπplasmic-type octopine Plasmid pTιA6 46,490 24-Apr-9 permease (occR, occQ, occM, occP, and occJ) and lysR-type regulatory protein

(occR) genes, complete eds rxa00419 882 GB_BA2 MSU46844 16951 U46844 Mycobacterium smegmatis catalase-peroxidase (katG), putative arabmosyl Mycobacterium 57,029 12-MAY transferase (embC, ermbA, embB), genes complete eds and putative propionyl- smegmatis coA carboxylase beta chain (pccB) genes, partial eds

GB_EST28 AI513245 471 AI513245 GH13311 3prιme GH Drosophila melanogaster head pOT2 Drosophila Drosophila melanogaster 37,696 16-MAR melanogaster cDNA clone GH13311 3prιme, mRNA sequence

GBJHTG4 AC010066 187240 AC010066 Drosophila melanogaster chromosome 3L/72A4 clone RPCI98-2501 , ^*" Drosophila melanogaster 39,607 16-OCT-

SEQUENCING IN PROGRESS ^***, 70 unordered pieces rxa00432 1608 GB_BA1 BSUB0015 218410 Z99118 Bacillus subtilis complete genome (section 15 of 21 ) from 2795131 to 3013540 Bacillus subtilis 49,810 26-NOV-

GB_PL1 CAC35A5 42565 AL033396 C albicans cosmid Ca35A5 Candida albicans 35,041 5-Nov-9

GB EST13 AA336266 378 AA336266 EST40981 Endometrial tumor Homo sapiens cDNA 5' end, mRNA sequence Homo sapiens 39,733 21-Apr-9

Table 4 (continued) rxa00449 1704 GB_HTG2 AC008199 124050 AC008199 Drosophila melanogaster chromosome 3 clone BACR01 K08 (D756) RPCI-98 Drosophila melanogaster 38,392 2-Aug-99

01 K 8 map 94D-94D strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ***,

83 unordered pieces

GB_HTG2 AC008199 124050 AC008199 Drosophila melanogaster chromosome 3 clone BACR01 K08 (D756) RPCI-98 Drosophila melanogaster 38,392 2-Aug-99

01 K 8 map 94D-94D strain y, en bw sp, ^*" SEQUENCING IN PROGRESS ***,

83 unordered pieces

GB_RO RATLNKP2 177 M22337 Rat link protein gene, exon 2 Rattus sp 40,678 27-Apr-9 rxa00456 1500 GB_GSS1 FR0030597 476 AL026966 Fugu rubπpes GSS sequence, clone 091C22aF9, genomic survey sequence Fugu rubπpes 47,407 25-Jun-9

GB_GSS5 AQ786587 556 AQ786587 HS_3086_B1_H05_MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 38,406 3-Aug-99 sapiens genomic clone Plate=3086 Col=9 Row=P, genomic survey sequence

GB_GSS14 AQ526586 434 AQ526586 HS_5198_B1_B03_SP6E RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 36,951 11-MAY- genomic clone Plate=774 Col=5 Row=D, genomic survey sequence rxa00477 1767 GB_EST17 AA610489 407 AA610489 np93e05 s1 NCI_CGAP_Thy1 Homo sapiens cDNA clone IMAGE 1133888 Homo sapiens 41 ,791 09-DEC- similar to gb M11353 HISTONE H3 3 (HUMAN),, mRNA sequence

GB_PR1 HSH33G4 1015 X05857 Human H3 3 gene exon 4 Homo sapiens 38,182 24-Jan-9

GB_EST30 AI637667 579 AI637667 tt10g11 x1 NCI_CGAP_GC6 Homo sapiens cDNA clone IMAGE 2240420 3', Homo sapiens 35,417 27-Apr-9 mRNA sequence rxa00478 954 GBJHTG3 AC008708 83932 AC008708 Homo sapiens chromosome 5 clone CIT978SKB_78F1 , *^** SEQUENCING IN Homo sapiens 38,769 3-Aug-99

PROGRESS ^***, 12 unordered pieces

GB_HTG3 AC008708 83932 AC008708 Homo sapiens chromosome 5 clone CIT978SKB_78F1 , ^*** SEQUENCING IN Homo sapiens 38,769 3-Aug-99

PROGRESS ^***, 12 unordered pieces

GB_HTG3 AC008708 83932 AC008708 Homo sapiens chromosome 5 clone CIT978SKB_78F1 , ^*** SEQUENCING IN Homo sapiens 36,797 3-Aug-99

PROGRESS *^**, 12 unordered pieces rxa00480 1239 GB_HTG1 HSJ575L21 94715 AL096841 Homo sapiens chromosome 1 clone RP4-575L21 , ^*** SEQUENCING IN Homo sapiens 38,138 23-Nov-9

PROGRESS ^***, in unordered pieces

GB_HTG1 HSJ575L21 94715 AL096841 Homo sapiens chromosome 1 clone RP4-575L21 , ^*** SEQUENCING IN Homo sapiens 38,138 23-Nov-9

PROGRESS ^***, in unordered pieces

GB_RO AC005960 158414 AC005960 Mus musculus chromosome 17 BAC cιtb20h22 from the MHC region, complete Mus musculus 38,712 01-DEC- sequence rxa00524 433 GB_BA1 SCI51 40745 AL109848 Streptomyces coehcolor cosmid 151 Streptomyces coehcolor 40,284 16-Aug-

A3(2)

GB_BA2 AF082879 3434 AF082879 Yersmia enterocolitica ABC transporter enterochelin/enterobactin gene cluster, Yersmia enterocolitica 55,634 20-OCT- complete sequence

GB_BA1 BSP132617 5192 AJ132617 Burkholdena sp P-transporter operon and flanking genes Burkholdena sp 40,793 13-Jul-9 rxa00526 813 GB_BA1 BSUB0008 208230 Z99111 Bacillus subtilis complete genome (section 8 of 21 ) from 1394791 to 1603020 Bacillus subtilis 54,534 26-NOV-

GB_BA2 AF012285 46864 AF012285 Bacillus subtilis mobA-nprE gene region Bacillus subtilis 54,534 l-Jul-98

GB_BA1 D90725 13796 D90725 Escherichia coli genomic DNA (19 7 - 20 0 mm) Escherichia coli 51 ,481 7-Feb-99 rxa00559 1140 GB BA2 CAU77910 3385 U77910 Corynebacterium ammoniagenes sequence upstream of the 5-phosphorιbosyl-1- Corynebacterium 39,007 1-Jan-98 pyrophosphate amidotransferase (purF) gene ammoniagenes

GB EST4H34952 382 H34952 EST108261 Rat PC-12 cells, untreated Rattus sp cDNA clone RPCCK07 similar Rattus sp 39,267 2-Apr-98 to NADH-ubiquinone oxidoreductase complex I 23 kDa precursor (iron-sulfur protein), mRNA sequence

GB BA2AE000963 22014 AE000963 Archaeoglobus fulgidus section 144 of 172 of the complete genome Archaeoglobus fulgidus 38,338 15-DEC-

Table 4 (continued) rxa00570 852 GB_GSS12 AQ422451 AQ422451 RPCI-11-185C3 TV RPCI-11 Homo sapiens genomic clone RPCI-11-185C3, Homo sapiens 38,767 23-MAR- genomic survey sequence GB_EST28 AI504741 AI504741 vl16c01 x1 Stratagene mouse Tcell 937311 Mus musculus cDNA clone Mus musculus 37,900 11-MAR-

IMAGE 972384 3' similar to gb Z14044 M musculus mRNA for valosin-containmg protein (MOUSE),, mRNA sequence

GB_EST18 AA712043 AA712043 vu29f10 r1 Barstead mouse myotubes MPLRB5 Mus musculus cDNA clone Mus musculus 42,647 24-DEC-

IMAGE 1182091 5' similar to gb L05093 60S RIBOSOMAL PROTEIN L18A

(HUMAN),, mRNA sequence rxa00571 1280 GB BA1 MTCY78 33818 Z77165 Mycobacterium tuberculosis H37Rv complete genome, segment 145/162 Mycobacterium 38,468 17-Jun-9 tuberculosis

GB_PR3 AC005788 36224 AC005788 Homo sapiens chromosome 19, cosmid R26652, complete sequence Homo sapiens 36,911 06-OCT-

GB_PR3 AC005338 34541 AC005338 Homo sapiens chromosome 19, cosmid R31646, complete sequence Homo sapiens 36,911 30-Jul-9 rxa00590 1288 GB_HTG6 AC010932 203273 AC010932 Homo sapiens chromosome 15 clone RP11 -296E22 map 15, ^*** SEQUENCING Homo sapiens 37,242 30-Nov-

IN PROGRESS ^***, 36 unordered pieces

GB_HTG6 AC010932 203273 AC010932 Homo sapiens chromosome 15 clone RP11-296E22 map 15, ^*** SEQUENCING Homo sapiens 36,485 30-Nov-

IN PROGRESS ^**^*, 36 unordered pieces

GB_BA1 MSGB26CS 37040 L78816 Mycobacterium leprae cosmid B26 DNA sequence Mycobacterium leprae 39,272 15-Jun-9 rxa00591 1476 GBJN1 CEK09E9 30098 Z79602 Caenorhabditis elegans cosmid K09E9, complete sequence Caenorhabditis elegans 34,092 2-Sep-9

GB_PR4 AF135802 4965 AF135802 Homo sapiens thyroid hormone receptor-associated protein complex component Homo sapiens 36,310 9-Apr-99

TRAP170 mRNA, complete eds

GB_PR4 AF104256 4365 AF 104256 Homo sapiens transcriptional co-activator CRSP150 (CRSP150) mRNA, Homo sapiens 36,617 4-Feb-9 complete eds rxa00596 576 GB_PR3 AC004659 129577 AC004659 Homo sapiens chromosome 19, CIT-HSP-87m17 BAC clone, complete Homo sapiens 34,321 02-MAY-

GB_PR3 AC004659 129577 AC004659 Homo sapiens chromosome 19, CIT-HSP-87m17 BAC clone, complete Homo sapiens 35,739 02-MAY-

GB_PR1 HUMCBP2 2047 D83174 Human mRNA for collagen binding protein 2, complete eds Homo sapiens 40,404 6-Feb-9 rxa00607 504 GB_BA1 MTV010 3400 AL021186 Mycobacterium tuberculosis H37Rv complete genome, segment 119/162 Mycobacterium 40,862 23-Jun-9 tuberculosis

GB_BA1 MTV010 3400 AL021186 Mycobacterium tuberculosis H37Rv complete genome, segment 119/162 Mycobacterium 38,833 23-Jun-9 tuberculosis rxa00623 1461 GB BA1 MTCY428 26914 Z81451 Mycobacterium tuberculosis H37Rv complete genome, segment 107/162 Mycobacterium 60,552 17-Jun- tuberculosis

GB_BA1 RSPNGR234 34010 Z68203 Rhizobium sp plasmid NGR234a DNA Rhizobium sp 51 ,992 8-Aug-9 GB BA2 AE000101 10057 AE000101 Rhizobium sp NGR234 plasmid pNGR234a, section 38 of 46 of the complete Rhizobium sp NGR234 51 ,992 12-DEC- plasmid sequence rxa00681

rxa00690 1269 GB HTG5 AC008338 136685 AC008338 Drosophila melanogaster chromosome X clone BACR30J04 (D908) RPCI-98 Drosophila melanogaster 35,341 15-NOV- 30 J 4 map 19C-19E strain y, en bw sp, *** SEQUENCING IN PROGRESS _* ' _** 93 unordered pieces

GB HTG4 AC009766 170502 AC009766 Homo sapiens chromosome 11 clone 404_A_03 map 11 , "^* SEQUENCING IN Homo sapiens 37,984 19-OCT- PROGRESS ^***, 27 unordered pieces

Table 4 (continued)

GB HTG4 AC009766 170502 AC009766 Homo sapiens chromosome 11 clone 404_A_03 map 11 , ^*** SEQUENCING IN Homo sapiens 37,984 19-OCT-

PROGRESS ^***, 27 unordered pieces rxa00733 1008 GB EST30 AU054038 245 AU054038 AU054038 Dictyostehum discoideum SL (H Urushihara) Dictyostehum Dictyostehum discoideum 43,265 28-Apr-9 discoideum cDNA clone SLK472, mRNA sequence

GB EST30 AU054038 245 AU054038 AU054038 Dictyostehum discoideum SL (H Urushihara) Dictyostehum Dictyostehum discoideum 43,265 28-Apr-9 discoideum cDNA clone SLK472, mRNA sequence rxa00735 692 GB BA1 MTCY50 36030 Z77137 Mycobacterium tuberculosis H37Rv complete genome, segment 55/162 Mycobacterium 36,819 17-Jun-9 tuberculosis

GB_BA1 D90904 150894 D90904 Synechocystis sp PCC6803 complete genome, 6/27, 630555-781448 Synechocystis sp 52,585 7-Feb-99

GB_BA1 D90904 150894 D90904 Synechocystis sp PCC6803 complete genome, 6/27, 630555-781448 Synechocystis sp 39,699 7-Feb-99 rxa00796 298 GB_GSS14 AQ579838 651 AQ579838 T135342b shotgun sub-library of BAC clone 31 P06 Medicago truncatula Medicago truncatula 37,153 27-Sep-9 genomic clone 31-P-06-C-054, genomic survey sequence

GB_PR4 AC007625 174701 AC007625 Genomic sequence of Homo sapiens clone 2314F2 from chromosome 18, Homo sapiens 38,014 30-Jun-9 complete sequence

GB EST14 AA427576 580 AA427576 zw54b04 s1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA clone Homo sapiens 42,731 16-OCT-

IMAGE 773839 3' similar to gb M86852 PEROXISOME ASSEMBLY FACTOR-1

(HUMAN),, mRNA sequence rxa00801 756 GB_BA1 MTV022 13025 AL021925 Mycobacterium tuberculosis H37Rv complete genome, segment 100/162 Mycobacterium 59,350 17-Jun-9 tuberculosis

GB_RO AC002109 160048 AC002109 Genomic sequence from Mouse 9, complete sequence Mus musculus 39,398 9-Sep-97 GB BA1 MTV022 13025 AL021925 Mycobacterium tuberculosis H37Rv complete genome, segment 100/162 Mycobacterium 36,842 17-Jun-9 tuberculosis rxa00802 837 GB_GSS14 AQ563349 642 AQ563349 HS_5335_B2_A09_T7A RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 37,649 29-MAY- genomic clone Plate=911 Col=18 Row=B, genomic survey sequence

GB_BA1 DIHCLPBA 2441 M32229 B nodosus clpB gene encoding a regulatory subunit of ATP-dependent protease Dichelobaeter nodosus 41 ,140 26-Apr-9

GB_GSS3 B61538 698 B61538 T17M17TR TAMU Arabidopsis tha ana genomic clone T17M17, genomic survey Arabidopsis thahana 36,946 21-N0V- sequence rxa00819 1452 GB_HTG3 AC008691_ 110000 AC008691 Homo sapiens chromosome 5 clone CIT978SKB_63A22, ^*** SEQUENCING IN Homo sapiens 38,270 3-Aug-99

1 PROGRESS ^***, 253 unordered pieces

GB_HTG3 AC008691 _ 110000 AC008691 Homo sapiens chromosome 5 clone CIT978SKB_63A22, *** SEQUENCING IN Homo sapiens 38,270 3-Aug-9

1 PROGRESS ^***, 253 unordered pieces

GB_HTG3 AC009127 186591 AC009127 Homo sapiens chromosome 16 clone RPCI-11_498D10, ^*** SEQUENCING IN Homo sapiens 38,947 3-Aug-9

PROGRESS ***, 49 unordered pieces rxa00821 966 GBJHTG1 HS32B1 271488 AL023693 Homo sapiens chromosome 6 clone RP1-32B1 , ^*** SEQUENCING IN Homo sapiens 36,565 23-NOV-9

PROGRESS ^***, in unordered pieces

GB_HTG1 HS32B1 271488 AL023693 Homo sapiens chromosome 6 clone RP1-32B1 , ^*** SEQUENCING IN Homo sapiens 36,565 23-Nov-9

PROGRESS ^***, in unordered pieces

GB_PR3 AC004919 75547 AC004919 Homo sapiens PAC clone DJ0895B23 from UL, complete sequence Homo sapiens 34,346 19-Sep-9 rxa00827 876 GB EST6 W06539 300 W06539 T2367 MVAT4 bloodstream form of serodeme WRATatl 1 Trypanosoma brucei Trypanosoma brucei 40,000 12-Aug-9 rhodesiense cDNA 5', mRNA sequence rhodesiense

GB PR4 AC008179 181745 AC008179 Homo sapiens clone NH0576F01 , complete sequence Homo sapiens 35,903 28-Sep-9

Table 4 (continued)

GB_EST18 AA710415 533 AA710415 vt53f08 r1 Barstead mouse irradiated colon MPLRB7 Mus musculus cDNA clone Mus musculus 41 ,562 24-DEC-

IMAGE 1 166823 5', mRNA sequence rxa00842 1323 GB_PR2 AC002379 118595 AC002379 Human BAC clone GS165I04 from 7q21 , complete sequence Homo sapiens 36,321 23-Jul-97

GB_PR2 AC002379 118595 AC002379 Human BAC clone GS165I04 from 7q21 , complete sequence Homo sapiens 37,284 23-Jul-97

GBJN1 CEF02D8 31624 Z78411 Caenorhabditis elegans cosmid F02D8, complete sequence Caenorhabditis elegans 38,163 23-NOV-9 rxa00847 1572 GB_OV XELRDS38A 1209 L79915 Xenopus laevis rds/peπpherin (rds38) mRNA, complete eds Xenopus laevis 36,044 30-Jul-97

GB_HTG4 AC007920 234529 AC007920 Homo sapiens chromosome 3q27 clone RPCI11-208N14, **^* SEQUENCING IN Homo sapiens 33,742 21-OCT-

PROGRESS ***, 51 unordered pieces

GB_HTG4 AC007920 234529 AC007920 Homo sapiens chromosome 3q27 clone RPCI1 1-208N14, *** SEQUENCING IN Homo sapiens 33,742 21-OCT-

PROGRESS ***, 51 unordered pieces rxa00851 732 GB_HTG2 AC004064 185000 AC004064 Homo sapiens chromosome 4, *** SEQUENCING IN PROGRESS "^*, 10 Homo sapiens 39,833 9-Jul-98 unordered pieces

GB_HTG2 AC004064 185000 AC004064 Homo sapiens chromosome 4, ^*** SEQUENCING IN PROGRESS ^***, 10 Homo sapiens 39,833 9-Jul-98 unordered pieces

GB_PR3 HSJ824F16 139330 AL050325 Human DNA sequence from clone 824F16 on chromosome 20, complete Homo sapiens 39,833 23-Nov-9 sequence rxa00852 813 GB_HTG3 AC010120 121582 AC010120 Drosophila melanogaster chromosome 3 clone BACR22N13 (D1061 ) RPCI-98 Drosophila melanogaster 36,855 24-Sep-9

22 N 13 map 96F-96F strain y, en bw sp, *** SEQUENCING IN PROGRESS ***,

83 unordered pieces

GB_HTG3 AC010120 121582 AC010120 Drosophila melanogaster chromosome 3 clone BACR22N13 (D1061 ) RPCI-98 Drosophila melanogaster 36,855 24-Sep-

22 N 13 map 96F-96F strain y, en bw sp, *" SEQUENCING IN PROGRESS **^*,

83 unordered pieces

GB HTG2 AC006898 299308 AC006898 Caenorhabditis elegans clone Y73B6x, ^*** SEQUENCING IN PROGRESS ^***, 9 Caenorhabditis elegans 36,768 24-Feb-9 unordered pieces rxa00856

rxa00870 1635 GB BA1 STMMSDA 3986 L48550 Streptomyces coehcolor methylmalonic acid semialdehyde dehydrogenase Streptomyces coehcolor 63,743 09-MAY-

(msdA) gene, complete eds

GB_PAT 192043 713 I92043 Sequence 10 from patent US 5726299 Unknown 38,850 01-DEC- GB_PAT I78754 713 I78754 Sequence 10 from patent US 5693781 Unknown 38,850 3-Apr-98 rxa00875 690 GB_BA2 AF1 19715 549 AF1 19715 Escherichia coli isopentyl diphosphate isomerase (idi) gene, complete eds Escherichia coli 54,827 22-Apr-9 GB_BA2 AE000372 12144 AE000372 Escherichia coli K-12 MG1655 section 262 of 400 of the complete genome Escherichia coli 51 ,416 12-N0V- GB_BA1 ECU28375 55175 U28375 Escherichia coli K-12 genome, approximately 64 to 65 minutes Escherichia coli 51 ,416 08-DEC- rxa00878 1986 GB HTG2 AC007472 1 14003 AC007472 Drosophila melanogaster chromosome 2 clone BACR30D19 (D587) RPCI-98 Drosophila melanogaster 36,592 2-Aug-9

30 D 19 map 49E-49F strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ***,

79 unordered pieces

GB HTG2 AC007472 114003 AC007472 Drosophila melanogaster chromosome 2 clone BACR30D19 (D587) RPCI-98 Drosophila melanogaster 36,592 2-Aug-9

30 D 19 map 49E-49F strain y, en bw sp, *** SEQUENCING IN PROGRESS ***,

79 unordered pieces

GB HTG2 AC006798 207370 AC006798 Caenorhabditis elegans clone Y51 F8, *** SEQUENCING IN PROGRESS ***, 30 Caenorhabditis elegans 36,699 25-Feb-9 unordered pieces

Table 4 (continued) rxa00880 1968 GB_EST4 H22888 468 H22888 ym54e12 r1 Soares infant brain 1 NIB Homo sapiens cDNA clone IMAGE 52158 Homo sapiens 37,179 6-Jul-95 5', mRNA sequence

GB_GSS13 AQ426858 516 AQ426858 CITBI-E1-2578F1 TF CITBI-E1 Homo sapiens genomic clone 2578F1 , genomic Homo sapiens 38,447 24-MAR- survey sequence

GB_PR1 AB002335 6289 AB002335 Human mRNA for KIAA0337 gene, complete eds Homo sapiens 35,799 13-Feb-9 rxa00899 1389 GB_BA1 NGU58849 2401 U58849 Neisseria gonorrhoeae pιlS6 silent pilus locus Neisseria gonorrhoeae 40,623 20-Jun-9

GB_BA1 PLPDHOS 3119 L06822 Plasmid pSa (from Escherichia coli) dihydropteroate synthase gene, 3' end Plasmid pSa 38,966 20-MAR-

GB BA1 PDGINTORF 6747 L06418 Integron In7 (from Plasmid pDGO100 from Escherichia coli) integrase (int), Plasmid pDGOI 00 38,966 20-MAR- aminoglycoside adenylyltransferase (aad), quaternary ammonium compound- resistance protein, dihydrofolate reductase (dhfrX), and dihydropteroate synthase (sull) genes rxa00902 1333 GB_GSS15 AQ606873 581 AQ606873 HS_5404_B2_H05_T7A RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 37,900 10-Jun-9 genomic clone Plate=980 Col=10 Row=P, genomic survey sequence

GB_GSS9 AQ 163442 658 AQ 163442 nbxb0007A07f CUGI Rice BAC Library Oryza sativa genomic clone Oryza sativa 41 ,885 12-Sep- nbxb0007A07f, genomic survey sequence

GB_PL1 PSST70 4974 X69213 P sativum Psst.70 gene for heat-shock protein Pisum sativum 36,866 3-Jul-96 rxa00931 969 GB_GSS1 FR0025208 612 AL018047 F rubπpes GSS sequence, clone 145D10aA8, genomic survey sequence Fugu rubnpes 37,815 10-DEC-

GB_GSS1 FR0021844 252 AL014715 F rubnpes GSS sequence, clone 069K22aG5, genomic survey sequence Fugu rubnpes 37,698 10-DEC-

GB_GSS12 AQ403344 593 AQ403344 HS_2257_B1_B03_T7C CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 31 ,552 13-MAR- sapiens genomic clone Plate=2257 Col=5 Row=D, genomic survey sequence rxa00941 1440 GB_BA1 MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv complete genome, segment 85/162 Mycobacterium 37,902 17-Jun-9 tuberculosis

GB_BA1 MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv complete genome, segment 85/162 Mycobacterium 39,140 17-Jun-9 tuberculosis

GB_BA2 MSGKATG 1745 L 14268 Mycobacterium tuberculosis ethyl methane sulphonate resistance protein (katG) Mycobacterium 42,517 26-Aug-9 gene, 3'end tuberculosis rxa00962 689 GB_HTG6 AC010998 144338 AC010998 Homo sapiens clone RP11-95116, ^*** SEQUENCING IN PROGRESS "^*, 17 Homo sapiens 39,497 08-DEC- unordered pieces

GB_GSS1 GGA340111 990 AJ232089 Gallus gallus anonymous sequence from Cosmid mapping to chromosome 2 Gallus gallus 37,970 25-Aug-

(Cosmid 34 - Contig 15), genomic survey sequence

GB_HTG6 AC010998 144338 AC010998 Homo sapiens clone RP11-95I16, ^*** SEQUENCING IN PROGRESS ^***, 17 Homo sapiens 38,226 08-DEC- unordered pieces rxa01060 1047 GB_BA1 ECTTN7 2280 AJ001816 Escherichia coli left end of transposon Tn7 including type 2 integron Escherichia coli 38,822 4-NOV-9

GBJN2 AF1 6377 8220 AF176377 Caenorhabditis briggsae CES-1 (ces-1 ) gene, complete eds, and CPN-1 (cpn-1 ) Caenorhabditis briggsae 39,921 09-DEC- gene, partial eds

GB_GSS10 AQ 196728 429 AQ 196728 CIT-HSP-2381 F4 TR CIT-HSP Homo sapiens genomic clone 2381 F4, genomic Homo sapiens 39,019 16-Sep- survey sequence rxa01067 852 GB_BA1 U00016 42931 U00016 Mycobacterium leprae cosmid B1937 Mycobacterium leprae 58,303 01 -MAR-

GB_BA1 SYCGROESL 3256 D12677 Synechocystis sp groES and groEL genes Synechocystis sp 34,593 3-Feb-99

GB_BA1 D90905 139467 D90905 Synechocystis sp PCC6803 complete genome, 7/27, 781449-920915 Synechocystis sp 34,593 7-Feb-99 rxa01114 1347 GB BA1 PSEFAOAB 3480 D10390 P fragi faoA and faoB genes, complete eds Pseudomonas fragi 51 ,919 2-Feb-99

Table 4 (continued)

GB_BA1 AB014757 6057 AB014757 Pseudomonas sp 61-3 genes for PhbR, acetoacetyl-CoA reductase, beta- Pseudomonas sp 61-3 50,573 26-DEC-1 ketothiolase and PHB synthase, complete eds

GB_BA1 SC8D9 38681 AL035569 Streptomyces coehcolor cosmid 8D9 Streptomyces coehcolor 42,200 26-Feb-9 rxa01136 555 GB_EST11 AA244557 379 AA244557 mx07a01 r1 Soares mouse NML Mus musculus cDNA clone IMAGE 679464 5', Mus musculus 39,050 10-MAR- mRNA sequence

GB_EST14 AA407673 306 AA407673 EST01834 Mouse 7 5 dpc embryo ectoplacental cone cDNA library Mus Mus musculus 38,562 26-Aug-9 musculus cDNA clone C0014F02 3', mRNA sequence

GB_EST26 AI390328 604 AI390328 mx07a01 y1 Soares mouse NML Mus musculus cDNA clone IMAGE 679464 5', Mus musculus 33,136 2-Feb-99 mRNA sequence rxa01138 540 GB_OV XLXINT1 1278 X13138 Xenopus laevis ιnt-1 mRNA for ιnt-1 protein Xenopus laevis 40,038 31-MAR-

GB_PR4 AC006054 143738 AC006054 Homo sapiens Xq28 BAC RPCI11 -382P7 (Roswell Park Cancer Institute Human Homo sapiens 37,996 1-Apr-99

BAC Library) complete sequence

GB_PR4 AC006054 143738 AC006054 Homo sapiens Xq28 BAC RPCI11-382P7 (Roswell Park Cancer Institute Human Homo sapiens 36,053 1-Apr-99

BAC Library) complete sequence rxa01172 1578 GB_BA1 SCE39 23550 AL049573 Streptomyces coehcolor cosmid E39 Streptomyces coehcolor 62,357 31 -MAR-

GB_BA1 MSU50335 5193 U50335 Mycobacterium smegmatis phage resistance (mpr) gene, complete eds Mycobacterium 37,853 1-Feb-97 smegmatis

GB_BA1 BACTHRTRN 15467 D84213 Bacillus subtilis genome, trnl-feuABC region Bacillus subtilis 53,807 6-Feb-99 A rxa01191 1713 GB PR2 HS1191 B2 60828 AL022237 Human DNA sequence from clone 1191 B2 on chromosome 22q13 2-13 3 Homo sapiens 38,366 23-NOV-9 Contains part of the BIK (NBK, BP4, BIP1 ) gene for BCL2-ιnteractιng killer (apoptosis-mducing), a 40S Ribososmal Protein S25 pseudogene and part of an alternatively spliced novel Acyl Transferase gene similar to C elegans C50D2 7 Contains ESTs, STSs, GSSs, two putative CpG islands and genomic marker D22S1151 , complete sequence

GB PR2 HS1191B2 60828 AL022237 Human DNA sequence from clone 1191B2 on chromosome 22q13 2-13 3 Homo sapiens 39,595 23-NOV-9 Contains part of the BIK (NBK, BP4, BIP1 ) gene for BCL2-ιnteractιng killer (apoptosis-mducing), a 40S Ribososmal Protein S25 pseudogene and part of an alternatively spliced novel Acyl Transferase gene similar to C elegans C50D2 7 Contains ESTs, STSs, GSSs, two putative CpG islands and genomic marker D22S1151 , complete sequence rxa01205 554 GB_BA1 MTCY373 35516 Z73419 Mycobacterium tuberculosis H37Rv complete genome, segment 57/162 Mycobacterium 57,762 17-Jun-9 tuberculosis

GB_PL1 ATY12776 38483 Y12776 Arabidopsis thahana DNA, 40 kb surrounding ACS1 locus Arabidopsis thahana 32,971 7-Sep-98

GB_PL2 ATT6K21 99643 AL021889 Arabidopsis thahana DNA chromosome 4, BAC clone T6K21 (ESSA project) Arabidopsis thahana 35,273 16-Aug-9 rxa01212 1047 GB_BA2 SCD25 41622 AL118514 Streptomyces coehcolor cosmid D25 Streptomyces coehcolor 39,654 21-Sep-9

A3(2)

GB_BA1 SLGLYUB 2576 X65556 S lividans tRNA-GlyU beta gene Streptomyces lividans 54,493 20-DEC-

GB_BA1 SCH10 39524 AL049754 Streptomyces coehcolor cosmid H10 Streptomyces coehcolor 44,638 04-MAY- rxa01219 1005 GB_PAT A68024 520 A68024 Sequence 19 from Patent WO9743409 unidentified 42,553 05-MAY-

GB_PAT A68025 193 A68025 Sequence 20 from Patent WO9743409 unidentified 43,229 05-MAY-

GB PAT A68027 193 A68027 Sequence 22 from Patent WO9743409 unidentified 38,342 05-MAY-

Table 4 (continued) rxa01220 1200 GB_PR3 HS512B11 64356 AL031058 Human DNA sequence from clone 512B11 on chromosome 6p24-25 Contains Homo sapiens 35,478 23-Nov- the Desmoplakin I (DPI) gene, ESTs, STSs and GSSs, complete sequence GB_EST6 N99239 424 N99239 zb76h11 s1 Soares_senescent_fιbroblasts_NbHSF Homo sapiens cDNA clone Homo sapiens 39,623 20-Aug-

IMAGE 309573 3', mRNA sequence GB EST16 AA554268 400 AA554268 nk36c09 s1 NCI_CGAP_GC2 Homo sapiens cDNA clone IMAGE 1015600 3' Homo sapiens 36,111 8-Sep-9 similar to gb X01677 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE,

LIVER (HUMAN),, mRNA sequence rxa01221 849 GB_PR4 AF179633 96371 AF179633 Homo sapiens chromosome 16 map 16q23 3-q24 1 sequence Homo sapiens 40,199 5-Sep-9 GB_VI EHVU20824 184427 U20824 Equine herpesvirus 2, complete genome Equine herpesvirus 2 37,001 2-Feb-96 GB_BA2 AE000407 10601 AE000407 Escherichia coli K-12 MG1655 section 297 of 400 of the complete genome Escherichia coli 39,471 12-Nov- rxa01222 822 GB_PAT AR068625 28804 AR068625 Sequence 1 from patent US 5854034 Unknown 40,574 29-Sep- GB BA2 SSU51197 28804 U51197 Sphingomonas S88 sphmgan polysaccharide synthesis (spsG), (spsS), (spsR), Sphingomonas sp S88 40,574 16-MAY- glycosyl transferase (spsQ), (spsl), glycosyl transferase (spsK), glycosyl transferase (spsL), (spsJ), (spsF), (spsD), (spsC), (spsE), Urf 32, Urf 26,

ATP-binding cassette transporter (atrD), ATP-bindmg cassette transporter

(atrB), glucosyl-isoprenylphosphate transferase (spsB), glucose-1-phosphate thymidylyltransferase (rhsA), dTDP-6-deoxy-D-glucose -3,5-epιmerase (rhsC) dTDP-D-glucose-4,6-dehydratase (rhsB), dTDP-6-deoxy-L-mannose- dehydrogenase (rhsD), Urf 31 , and Urf 34 genes, complete eds

GBJN1 BBU44918 2791 U44918 Babesia bovis ATP-binding protein (babe) mRNA, complete eds Babesia bovis 39,228 9-Aug-9 rxa01260 1305 GB_BA1 CGLPD 1800 Y16642 Corynebacterium glutamicum Ipd gene, complete CDS Corynebacterium 99,923 1-Feb-9 glutamicum

GB_BA1 MTV038 16094 AL021933 Mycobacterium tuberculosis H37Rv complete genome, segment 24/162 Mycobacterium 59,056 17-Jun-9 tuberculosis

GB_PR3 AC005618 176714 AC005618 Homo sapiens chromosome 5, BAC clone 249h5 (LBNL H149), complete Homo sapiens 36,270 5-Sep-9 sequence rxa01261 294 GB_BA1 CGLPD 1800 Y16642 Corynebacterium glutamicum Ipd gene, complete CDS Corynebacterium 100,000 1-Feb-9 glutamicum

GB_HTG4 AC010045 164829 AC010045 Drosophila melanogaster chromosome 3L/75A1 clone RPCI98-17C17, *** Drosophila melanogaster 50,512 16-OCT-

SEQUENCING IN PROGRESS ^***, 50 unordered pieces

GBJHTG4 AC010045 164829 AC010045 Drosophila melanogaster chromosome 3L/75A1 clone RPCI98-17C17, *** Drosophila melanogaster 50,512 16-OCT-

SEQUENCING IN PROGRESS ^***, 50 unordered pieces rxa01269 564 GB_BA2 AF125164 26443 AF125164 Bacteroides fragihs 638R polysaccharide B (PS B2) biosynthesis locus, complete Bacteroides fragi s 56,071 01-DEC- sequence, and unknown genes

GB_BA1 AB002668 24907 AB002668 Actinobacillus actinomycetemcomitans DNA for glycosyltransferase, lytic Actinobacillus 46,679 21-Feb- transglycosylase, dTDP-4-rhamnose reductase, complete eds actinomycetemcomitans

GB_BA1 AB010415 23112 AB010415 Actinobacillus actinomycetemcomitans gene cluster for 6-deoxy-L-talan Actinobacillus 46,679 13-Feb- synthesis, complete eds actinomycetemcomitans rxa01291 1056 GB_STS AU027820 238 AU027820 Rattus norvegicus, OTSUKA clone, OT78 02/918b07, microsatelhte sequence, Rattus norvegicus 34,874 02-MAR sequence tagged site

GB STS AU027820 238 AU027820 Rattus norvegicus, OTSUKA clone, OT78 02/918b07, microsatelhte sequence, Rattus norvegicus 34,874 02-MAR- sequence tagged site

Table 4 (continued)

GBJHTG3 AC006445 174547 AC006445 Homo sapiens chromosome 4, ^*** SEQUENCING IN PROGRESS ***, 7 Homo sapiens 34,812 15-Sep-9 unordered pieces rxa01292 1308 GB_BA1 BSUB0017 217420 Z99120 Bacillus subtilis complete genome (section 17 of 21 ) from 3197001 to 3414420 Bacillus subtilis 37,802 26-Nov-9

GB_HTG3 AC010580 121119 AC010580 Drosophila melanogaster chromosome 3 clone BACR48J06 (D1102) RPCI-98 Drosophila melanogaster 35,637 01-OCT-1

48 J 6 map 96F-96F strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ***,

71 unordered pieces GB_HTG3 AC010580 121119 AC010580 Drosophila melanogaster chromosome 3 clone BACR48J06 (D1102) RPCI-98 Drosophila melanogaster 35,637 01-OCT-1

48 J 6 map 96F-96F strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ***,

71 unordered pieces rxa01293 450 GB_GSS8 AQ001809 705 AQ001809 CIT-HSP-2290D17 TF CIT-HSP Homo sapiens genomic clone 2290D17, Homo sapiens 42,021 26-Jun-9 genomic survey sequence GB_GSS8 AQ001809 705 AQ001809 CIT-HSP-2290D17 TF CIT-HSP Homo sapiens genomic clone 2290D17, Homo sapiens 40,323 26-Jun-9 genomic survey sequence rxa01339 1111 GB_PL1 MGU60290 4614 U60290 Magnaporthe gnsea nitrogen regulatory protein (NUT1) gene, complete eds Magnaporthe gnsea 38,707 3-Jul-96 GBJHTG3 AC011371 189187 AC011371 Homo sapiens chromosome 5 clone CIT978SKB 07C20, ^*** SEQUENCING IN Homo sapiens 39,741 06-OCT-

PROGRESS ***, 31 unordered pieces

GBJ-TTG3 AC011371 189187 AC011371 Homo sapiens chromosome 5 clone CIT978SKB_107C20, SEQUENCING IN Homo sapiens 39,741 06-OCT-

PROGRESS ^***, 31 unordered pieces rxa01382 1192 GBJHTG4 AC009892 138122 AC009892 Homo sapiens chromosome 19 clone CIT978SKB_83J4, * SEQUENCING IN Homo sapiens 40,154 31-OCT-

PROGRESS ***, 6 ordered pieces

GBJHTG4 AC009892 138122 AC009892 Homo sapiens chromosome 19 clone CIT978SKB_83J4, ^* SEQUENCING IN Homo sapiens 40,154 31-OCT-

PROGRESS ^***, 6 ordered pieces

GB_PR3 AC002416 128915 AC002416 Human Chromosome X, complete sequence Homo sapiens 37,521 29-Jan-9 rxa01399 1142 GB_EST9 AA096601 524 AA096601 mo03b09 r1 Stratagene mouse lung 937302 Mus musculus cDNA clone Mus musculus 40,525 15-Feb-9

IMAGE 552473 5' similar to gb L0650560S RIBOSOMAL PROTEIN L12

(HUMAN), gb L04280 Mus musculus ribosomal protein (MOUSE),, mRNA

GB_EST37 AI982114 626 AI982114 pat pk0074 e9 f chicken activated T cell cDNA Gallus gallus cDNA clone Gallus gallus 37,785 15-Sep-9 pat pk0074 e9 f 5' similar to H-ATPase B subunit, mRNA sequence

GB_OV GGU20766 1645 U20766 Gallus gallus vacuolar H+-ATPase B subunit gene, complete eds Gallus gallus 38,244 07-DEC- rxa01420 1065 GBJHTG2 AC005690 193424 AC005690 Homo sapiens chromosome 4, *^** SEQUENCING IN PROGRESS ^***, 7 Homo sapiens 37,464 11-Apr-9 unordered pieces

GB_HTG2 AC005690 193424 AC005690 Homo sapiens chromosome 4, ^*** SEQUENCING IN PROGRESS ^***, 7 Homo sapiens 37,464 11-Apr-9 unordered pieces

GB_HTG2 AC006637 22092 AC006637 Caenorhabditis elegans clone F41 B4, ^*** SEQUENCING IN PROGRESS ^**^*, Caenorhabditis elegans 37,488 23-Feb-9 unordered pieces rxa01467 414 GB_HTG1 CEY102G3_ 110000 AL020985 Caenorhabditis elegans chromosome V clone Y102G3, ^*** SEQUENCING IN Caenorhabditis elegans 35,437 3-Dec-98 GBJHTG1 CEY102G3_ 110000 AL020985 Caenorhabditis elegans chromosome V clone Y102G3, ^*** SEQUENCING IN Caenorhabditis elegans 35,437 3-Dec-98 GB_HTG1 CEY113G7_ 110000 AL031113 Caenorhabditis elegans chromosome V clone Y113G7, ^*** SEQUENCING IN Caenorhabditis elegans 35,437 12-Jan-9 rxa01576 882 GB_BA2 AF030975 2511 AF030975 Aeromonas salmonicida chaperonm GroES and chaperonin GroEL genes, Aeromonas salmonicida 41 ,516 2-Apr-98 complete eds

GB BA2 AF030975 2511 AF030975 Aeromonas salmonicida chaperonin GroES and chaperonin GroEL genes, Aeromonas salmonicida 38,171 2-Apr-98 complete eds

Table 4 (continued)

GB EST22 AI068560 965 AI068560 mgae0003aC11 f Magnaporthe gnsea Appressonum Stage cDNA Library Pyπcularia gnsea 40,073 09-DEC-1

Pyriculaπa gnsea cDNA clone mgae0003aC11f 5', mRNA sequence rxa01580 840 GB GSS14 AQ554460 681 AQ554460 RPCI-11-419F2 TV RPCI-1 1 Homo sapiens genomic clone RPCI-1 1-419F2, Homo sapiens 36,522 28-MAY- genomic survey sequence

GB IN2 AC005449 85518 AC005449 Drosophila melanogaster, chromosome 2R, region 44C4-44C5, P1 clone Drosophila melanogaster 36,609 23-DEC-1

DS06765, complete sequence

GB IN2 AC005449 85518 AC005449 Drosophila melanogaster, chromosome 2R, region 44C4-44C5, P1 clone Drosophila melanogaster 33,612 23-DEC-1

DS06765, complete sequence rxa01584

rxa01604 771 GB. HTG3 AC011352 160167 AC011352 Homo sapiens chromosome 5 clone CIT-HSPC_327F10 SEQUENCING IN Homo sapiens 33,688 06-OCT-1

PROGRESS ^***, 15 unordered pieces

GB. HTG3 AC011352 160167 AC011352 Homo sapiens chromosome 5 clone CIT-HSPC_327F10 SEQUENCING IN Homo sapiens 33,688 06-OCT-

PROGRESS ^***, 15 unordered pieces

GB. HTG3 AC011402 168868 AC011402 Homo sapiens chromosome 5 clone CIT978SKB_38B5, ^*** SEQUENCING IN Homo sapiens 33,688 06-OCT-

PROGRESS ^***, 7 unordered pieces rxa01614 1146 GB. BA1 CGA224946 2408 AJ224946 Corynebacterium glutamicum DNA for L-Malate qumone oxidoreductase Corynebacterium 42,284 11 -Aug-9 glutamicum

GB. EST17 AA608825 439 AA608825 af03g07 s1 Soares_testιs_NHT Homo sapiens cDNA clone IMAGE 1030620 3' Homo sapiens 40,092 02-MAR- similar to TR G976083 G976083 HISTONE H2A RELATED „ mRNA sequence

GB. PR4 AC005377 102311 AC005377 Homo sapiens PAC clone DJ1136G02 from 7q32-q34, complete sequence Homo sapiens 37,811 28-Apr-9 rxa01629 1635 GB BA1 CGPROPGEN 2936 Y12537 C glutamicum proP gene Corynebacterium 100,000 17-Nov-9 glutamicum

GB_BA1 CGPROPGEN 2936 Y12537 C glutamicum proP gene Corynebacterium 100,000 17-Nov-9 glutamicum

GB_PR4 AF191071 88481 AF191071 Homo sapiens chromosome 8 clone BAC 388D06, complete sequence Homo sapiens 35,612 11-OCT- rxa01644 1401 GB_BA1 MSGB577CO 37770 L01263 M leprae genomic dna sequence, cosmid b577 Mycobacterium leprae 55,604 14-Jun-9

S

GB_BA1 MLCB2407 35615 AL023596 Mycobacterium leprae cosmid B2407 Mycobacterium leprae 36,416 27-Aug-9

GB_BA1 MTV025 121125 AL022121 Mycobacterium tuberculosis H37Rv complete genome, segment 155/162 Mycobacterium 55,844 24-Jun-9 tuberculosis rxa01667 1329 GB_BA1 CGU43536 3464 U43536 Corynebacterium glutamicum heat shock, ATP-bindmg protein (clpB) gene, Corynebacterium 100,000 13-MAR- complete eds glutamicum

GB_HTG4 AC009841 164434 AC009841 Drosophila melanogaster chromosome 3L/77E1 clone RPCI98-13F11 , *** Drosophila melanogaster 33,205 16-OCT-

SEQUENCING IN PROGRESS ^***, 70 unordered pieces

GB_HTG4 AC009841 164434 AC009841 Drosophila melanogaster chromosome 3L/77E1 clone RPCI98-13F11 , ^*** Drosophila melanogaster 33,205 16-OCT-

SEQUENCING IN PROGRESS ^***, 70 unordered pieces rxa01722 1848 GB_GSS1 FR0022586 522 AL015452 F rubnpes GSS sequence, clone 077P23aB10, genomic survey sequence Fugu rubnpes 40,192 10-DEC-

GB_GSS1 FR0022584 485 AL015450 F rubnpes GSS sequence, clone 077P23aB11 , genomic survey sequence Fugu rubnpes 35,876 10-DEC-

GB IN1 CET26H2 37569 Z82055 Caenorhabditis elegans cosmid T26H2, complete sequence Caenorhabditis elegans 34,759 19-NOV-9

Table 4 (continued) rxa01727 1401 GB BA2 CORCSLYS 2821 M89931 Corynebacterium glutamicum beta C-S lyase (aecD) and branched-chain ammo Corynebacterium 99,929 4-Jun-98 acid uptake carrier (brnQ) genes, complete eds, and hypothetical protein Yhbw glutamicum

(yhbw) gene, partial eds

GB_HTG6 AC011037 167849 AC011037 Homo sapiens clone RP11 -7F18, WORKING DRAFT SEQUENCE, 19 Homo sapiens 36,903 30-Nov-9 unordered pieces

GB_HTG6 AC011037 167849 AC011037 Homo sapiens clone RP11 -7F18, WORKING DRAFT SEQUENCE, 19 Homo sapiens 35,642 30-Nov-9 unordered pieces rxa01737 1182 GB_BA1 SCGD3 33779 AL096822 Streptomyces coehcolor cosmid GD3 Streptomyces coehcolor 38,054 8-Jul-99

GB_HTG1 CNS01 DSB 222193 AL121768 Homo sapiens chromosome 14 clone R-976B16, ^*** SEQUENCING IN Homo sapiens 35,147 05-OCT-

PROGRESS ^***, in ordered pieces

GBJ-TTG1 CNS01DSB 222193 AL121768 Homo sapiens chromosome 14 clone R-976B16, ^*** SEQUENCING IN Homo sapiens 35,147 05-OCT-

PROGRESS ^***, in ordered pieces rxa01762 1659 GB_BA1 MTCI28 36300 Z97050 Mycobacterium tuberculosis H37Rv complete genome, segment 10/162 Mycobacterium 49,574 23-Jun-9 tuberculosis

GB_BA1 SC6G10 36734 AL049497 Streptomyces coehcolor cosmid 6G10 Streptomyces coehcolor 44,049 24-MAR-

GB_BA1 SCE29 26477 AL035707 Streptomyces coehcolor cosmid E29 Streptomyces coehcolor 40,246 12-MAR- rxa01764 1056 GB_PL2 SPAC343 42947 AL109739 S pombe chromosome I cosmid c343 Schizosaccharomyces 37,084 6-Sep-99 pombe

GB_PL2 SPAC343 42947 AL109739 S pombe chromosome I cosmid c343 Schizosaccharomyces 34,890 6-Sep-99 pombe rxa01801 1140 GB_EST38 AW066306 334 AW066306 687009D03 y1 687 - Early embryo from Delaware Zea mays cDNA, mRNA Zea mays 46,108 12-OCT- sequence

GB_GSS13 AQ484750 375 AQ484750 RPCI-11 -248N4 TV RPCI-11 Homo sapiens genomic clone RPCI-11 -248N4, Homo sapiens 32,000 24-Apr-9 genomic survey sequence

GB_GSS13 AQ489971 252 AQ489971 RPCI-11 -247N23 TV RPCI-11 Homo sapiens genomic clone RPCI-11 -247N23, Homo sapiens 36,111 24-Apr-9 genomic survey sequence rxa01823 900 GB_BA1 SCI51 40745 AL109848 Streptomyces coehcolor cosmid 151 Streptomyces coehcolor 35,779 16-Aug-9

A3(2)

GB_BA1 ECU82598 136742 U82598 Escherichia coli genomic sequence of minutes 9 to 12 Escherichia coli 39,211 15-Jan-9

GB_BA1 BSUB0018 209510 Z99121 Bacillus subtilis complete genome (section 18 of 21) from 3399551 to 3609060 Bacillus subtilis 36,999 26-Nov-9 rxa01853 675 GB_BA1 MTCY227 35946 Z77724 Mycobacterium tuberculosis H37Rv complete genome, segment 114/162 Mycobacterium 37,612 17-Jun-9 tuberculosis

GBJHTG3 AC010189 265962 AC010189 Homo sapiens clone RPCI11-296K13, "^* SEQUENCING IN PROGRESS ^***, 80 Homo sapiens 39,006 16-Sep-9 unordered pieces

GB_HTG3 AC010189 265962 AC010189 Homo sapiens clone RPCI1 1-296K13, ^*** SEQUENCING IN PROGRESS ^***, 80 Homo sapiens 39,006 16-Sep-9 unordered pieces rxa01881 558 GB_HTG4 AC011117 148447 AC011117 Homo sapiens chromosome 4 clone 173_C_09 map 4, ^*** SEQUENCING IN Homo sapiens 39,130 14-OCT- PROGRESS ^***, 10 ordered pieces

GB_HTG4 AC011117 148447 AC011117 Homo sapiens chromosome 4 clone 173_C_09 map 4, ^*** SEQUENCING IN Homo sapiens 39,130 14-OCT- PROGRESS ^***, 10 ordered pieces

GB BA1 MTCY2B12 20431 Z81011 Mycobacterium tuberculosis H37Rv complete genome, segment 61/162 Mycobacterium 37,893 18-Jun-9 tuberculosis

Table 4 (continued) rxa01894 978 GB_BA1 MTCY274 39991 Z74024 Mycobacterium tuberculosis H37Rv complete genome, segment 126/162 Mycobacterium 37,229 19-Jun-98 tuberculosis GBJN1 CELF46H5 38886 U41543 Caenorhabditis elegans cosmid F46H5 Caenorhabditis elegans 38,525 29-Nov-9

GB_HTG3 AC009204 115633 AC009204 Drosophila melanogaster chromosome 2 clone BACR03E19 (D1033) RPCI-98 Drosophila melanogaster 31 ,579 18-Aug-9

03 E 19 map 36E-37C strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ^***,

94 unordered pieces rxa01897 666 GBJHTG1 CEY48B6 293827 AL021151 Caenorhabditis elegans chromosome II clone Y48B6, ^*** SEQUENCING IN Caenorhabditis elegans 34,703 1-Apr-99

PROGRESS ^***, in unordered pieces GBJHTG1 CEY48B6 293827 AL021151 Caenorhabditis elegans chromosome II clone Y48B6, ^*** SEQUENCING IN Caenorhabditis elegans 34,703 1-Apr-99

PROGRESS ^***, in unordered pieces GBJHTG1 CEY53F4_2 110000 Z92860 Caenorhabditis elegans chromosome II clone Y53F4, ^*** SEQUENCING IN Caenorhabditis elegans 33,333 15-Oct-99

PROGRESS ^***, in unordered pieces rxa01946 1298 GB_BA1 MTV007 32806 AL021184 Mycobacterium tuberculosis H37Rv complete genome, segment 64/162 Mycobacterium 65,560 17-Jun-98 tuberculosis GB_BA1 SC5F2A 40105 AL049587 Streptomyces coehcolor cosmid 5F2A Streptomyces coehcolor 50,648 24-MAY-1

GB_BA1 SCARD1GN 2321 X84374 S capreolus ardl gene Streptomyces capreolus 44,973 23-Aug-9 rxa01980 756 GB_PL2 AC008262 99698 AC008262 Genomic sequence for Arabidopsis thahana BAC F4N2 from chromosome I, Arabidopsis thahana 35,310 21-Aug-9 complete sequence GB_PL1 AB013388 73428 AB013388 Arabidopsis thahana genomic DNA, chromosome 5, TAC clone K19E1 , Arabidopsis thahana 35,505 20-Nov-9 complete sequence

GB_PL1 AB013388 73428 AB013388 Arabidopsis thahana genomic DNA, chromosome 5, TAC clone K19E1 , Arabidopsis thahana 39,973 20-Nov-9 complete sequence rxa01983 630 GBJHTG4 AC006467 175695 AC006467 Drosophila melanogaster chromosome 2 clone BACR03L08 (D532) RPCI-98 Drosophila melanogaster 36,672 27-OCT-1

03 L 8 map 40A-40C strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ^***, 9 unordered pieces GBJHTG4 AC006467 175695 AC006467 Drosophila melanogaster chromosome 2 clone BACR03L08 (D532) RPCI-98 Drosophila melanogaster 36,672 27-OCT-1

03 L 8 map 40A-40C strain y, en bw sp, ^*** SEQUENCING IN PROGRESS ^***, 9 unordered pieces GB_HTG4 AC006467 175695 AC006467 Drosophila melanogaster chromosome 2 clone BACR03L08 (D532) RPCI-98 Drosophila melanogaster 32,367 27-OCT-1

03 L 8 map 40A-40C strain y, en bw sp, ^*** SEQUENCING IN PROGRESSo ^***, 9 unordered pieces rxa02020 1111 GB_BA1 CGDNAAROP 2612 X85965 C glutamicum ORF3 and aroP gene Corynebacterium 100,000 30-Nov-9 glutamicum GB_PAT A58887 1612 A58887 Sequence 1 from Patent WO9701637 unidentified 100,000 06-MAR-1

GB_BA1 STYCARABA 4378 M95047 Salmonella typhimurium transport protein, complete eds, and transfer RNA-Arg Salmonella typhimurium 50,547 13-MAR-1 rxa02029 1437 GBJHTG2 AC003023 104768 AC003023 Homo sapiens chromosome 11 clone pDJ363p2, ^*** SEQUENCING IN Homo sapiens 35,820 21-OCT-1

PROGRESS ^***, 22 unordered pieces

GBJHTG2 AC003023 104768 AC003023 Homo sapiens chromosome 11 clone pDJ363p2, ^*** SEQUENCING IN Homo sapiens 35,820 21-OCT-1

PROGRESS ^***, 22 unordered pieces

GBJHTG2 HS118B18 104729 AL034344 Homo sapiens chromosome 6 clone RP1-118B18 map p24 1-25 3, ^*** Homo sapiens 34,355 03-DEC-1

SEQUENCING IN PROGRESS ^***, in unordered pieces

Table 4 (continued) rxa02030 1509 GB PR4 AC007695 63247 AC007695 Homo sapiens 12q24 BAC RPCI11-124N23 (Roswell Park Cancer Institute Homo sapiens 38,681 1-Sep-9

Human BAC Library) complete sequence

GB_PR4 AC006464 99908 AC006464 Homo sapiens BAC clone NH0436C12 from 2, complete sequence Homo sapiens 35,445 22-OCT- GB_PR4 AC006464 99908 AC006464 Homo sapiens BAC clone NH0436C12 from 2, complete sequence Homo sapiens 35,968 22-OCT- rxa02073 1653 GB_BA1 CGGDHA 2037 X72855 C glutamicum GDHA gene Corynebacterium 39,655 24-MAY- glutamicum

GB_BA1 CGGDH 2037 X59404 Corynebacterium glutamicum, gdh gen for glutamate dehydrogenase Corynebacterium 44,444 30-Jul-9 glutamicum

GB BA2 SC2H4 25970 AL031514 Streptomyces coehcolor cosmid 2H4 Streptomyces coehcolor 38,452 19-OCT-

A3(2) rxa02074

rxa02095 1527 GB_EST18 AA703380 471 AA703380 zj12b06 s1 Soares_fetal_hver_spleen_1 NFLS_S1 Homo sapiens cDNA clone Homo sapiens 36,518 24-DEC-

IMAGE 450035 3' similar to contains LTR5 t3 LTR5 repetitive element ,, mRNA sequence

GB_HTG6 AC009769 122911 AC009769 Homo sapiens chromosome 8 clone RP11-202112 map 8, LOW-PASS Homo sapiens 35,473 07-DEC-

SEQUENCE SAMPLING

GB_EST7 W70175 436 W70175 zd52c02 r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone Homo sapiens 34,174 16-OCT-

IMAGE 344258 5' similar to contains LTR5 b2 LTR5 repetitive element,, mRNA sequence rxa02099 373 GB_BA1 CAJ10319 5368 AJ010319 Corynebacterium glutamicum amtP, glnB, glnD genes and partial ftsY and srp Corynebacterium 100,000 14-MAY- genes glutamicum

GBJHTG3 AC011509 111353 AC011509 Homo sapiens chromosome 19 clone CITB-H1_2189E23, ^*** SEQUENCING IN Homo sapiens 33,423 07-OCT-

PROGRESS ^***, 35 unordered pieces

GBJHTG3 AC011509 111353 AC011509 Homo sapiens chromosome 19 clone CITB-H1_2189E23, ^*** SEQUENCING IN Homo sapiens 33,423 07-OCT-

PROGRESS ^***, 35 unordered pieces rxa02115 1197 GBJHTG5 AC010126 175986 AC010126 Homo sapiens clone GS502B02, ^*** SEQUENCING IN PROGRESS ^***, 3 Homo sapiens 36,717 13-NOV- unordered pieces

GBJHTG5 AC010126 175986 AC010126 Homo sapiens clone GS502B02, *^** SEQUENCING IN PROGRESS ^***, 3 Homo sapiens 36,092 13-NOV- unordered pieces

GB_PR1 HUMHM145 2214 D 10925 Human mRNA for HM145 Homo sapiens 39,171 3-Feb-9 rxa02128 1818 GB_BA1 MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv complete genome, segment 98/162 Mycobacterium 38,682 17-Jun- tuberculosis

GB_BA1 MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv complete genome, segment 98/162 Mycobacterium 35,746 17-Jun-9 tuberculosis

GB_GSS10 AQ161109 738 AQ161109 nbxb0006D03r CUGI Rice BAC Library Oryza sativa genomic clone Oryza sativa 38,482 12-Sep- nbxb0006D03r, genomic survey sequence rxa02133 329 GB_BA2 MPAE000058 28530 AE000058 Mycoplasma pneumoniae section 58 of 63 of the complete genome Mycoplasma 32,317 18-Nov- pneumoniae

GBJHTG4 AC008308 151373 AC008308 Drosophila melanogaster chromosome 3 clone BACR10M16 (D743) RPCI-98 Drosophila melanogaster ^• 34,579 20-OCT- 10 M 16 map 93C-93D strain y, en bw sp, ^*** SEQUENCING IN PROGRESS *^** 186 unordered pieces

Table 4 (continued)

GB HTG4 AC008308 151373 AC008308 Drosophila melanogaster chromosome 3 clone BACR10M16 (D743) RPCI-98 Drosophila melanogaster 34,579 20-OCT-

10 M 16 map 93C-93D strain y, en bw sp, ^*" SEQUENCING IN PROGRESS ^*

186 unordered pieces rxa02150 924 GB EST37 AW012260 358 AW012260 um06e09 y1 Sugano mouse kidney mkia Mus musculus cDNA clone Mus musculus 39,385 10-Sep-9

IMAGE 2182312 5' similar to SW AMPL_BOVIN P00727 CYTOSOL

AMINOPEPTIDASE „ mRNA sequence

GB GSS3 B87734 389 B87734 RPCI11-30D24 TP RPCI-11 Homo sapiens genomic clone RPCI-11-30D24, Homo sapiens 37,629 9-Apr-99 genomic survey sequence

GB_PR4 AC005042 192218 AC005042 Homo sapiens clone NH0552E01 , complete sequence Homo sapiens 36,901 14-Jan-9 rxa02171 1776 GB_BA2 AF010496 189370 AF010496 Rhodobacter capsulatus strain SB1003, partial genome Rhodobacter capsulatus 53,714 12-MAY-

GB_EST24 AH 70522 367 AH 70522 EST216450 Normalized rat lung, Bento Soares Rattus sp cDNA clone Rattus sp 44,186 20-Jan-9

RLUC075 3' end, mRNA sequence

GB_PL1 PHVDLECA 1441 K03288 P vulgans phytohemagglutinin gene encoding erythroagglutinating Phaseolus vulgans 39,103 27-Apr-9 phytohemagglutinin (PHA-E), complete eds rxa02173 1575 GB_BA1 CGGLTG 3013 X66112 C glutamicum git gene for citrate synthase and ORF Corynebacterium 44,118 17-Feb- glutamicum

GB_BA1 CGGLTG 3013 X66112 C glutamicum git gene for citrate synthase and ORF Corynebacterium 36,189 17-Feb- glutamicum

GB_BA2 AE000104 10146 AE000104 Rhizobium sp NGR234 plasmid pNGR234a, section 41 of 46 of the complete Rhizobium sp NGR234 38,487 12-DEC- plasmid sequence rxa02224 1920 GB_BA2 CXU21300 8990 U21300 Corynebacterium stnatum hypothetical protein YbhB gene, partial eds, ABC Corynebacterium 37,264 9-Apr-99 transporter TetB (tetB), ABC transporter TetA (tetA), transposase, 23S rRNA stnatum methyltransferase, and transposase genes, complete eds, and unknown genes

GB_HTG3 AC009185 87184 AC009185 Homo sapiens chromosome 5 clone CIT-HSPC_248019, ^*** SEQUENCING IN Homo sapiens 36,459 07-OCT-

PROGRESS ^**^*, 2 ordered pieces

GB_HTG3 AC009185 87184 AC009185 Homo sapiens chromosome 5 clone CIT-HSPC_248019, ^**^* SEQUENCING IN Homo sapiens 36,459 07-OCT-

PROGRESS ^***, 2 ordered pieces rxa02225 905 GB_BA2 MPAE000058 28530 AE000058 Mycoplasma pneumoniae section 58 of 63 of the complete genome Mycoplasma 35,498 18-NOV- pneumoniae

GB_EST26 AI337275 618 AI337275 tb96h11 x1 NCI_CGAP_Co16 Homo sapiens cDNA clone IMAGE 2062245 3' Homo sapiens 35,589 18-MAR similar to TR Q15392 Q15392 ORF, COMPLETE CDS „ mRNA sequence

GB_EST26 AI337275 618 AI337275 tb96h11 x1 NCI_CGAP_Co16 Homo sapiens cDNA clone IMAGE 2062245 3' Homo sapiens 42,786 18-MAR sιmιlar to TR Q15392 Q15392 ORF, COMPLETE CDS „ mRNA sequence rxa02233 1410 GB_BA1 ERWPNLB 1291 M65057 Erwmia carotovora pectin lyase (pnl) gene, complete eds Erwmia carotovora 37,780 26-Apr-9

GB_EST30 AV021947 313 AV021947 AV021947 Mus musculus 18-day embryo C57BL/6J Mus musculus cDNA clone Mus musculus 39,423 28-Aug-

1190024M23, mRNA sequence

GB_EST33 AV087117 251 AV087117 AV087117 Mus musculus tongue C57BL/6J adult Mus musculus cDNA clone Mus musculus 47,410 25-Jun-9

2310028C15, mRNA sequence rxa02253 1050 GB_EST11 AA250210 532 AA250210 mx79g10 r1 Soares mouse NML Mus musculus cDNA clone IMAGE 692610 5' Mus musculus 36,136 12-MAR- sιmιlar to TR E236517 E236517 F44G4 1 „ mRNA sequence

GB EST11 AA250210 532 AA250210 mx79g10 r1 Soares mouse NML Mus musculus cDNA clone IMAGE 692610 5' Mus musculus 36,202 12-MAR- similar to TR E236517 E236517 F44G4 1 „ mRNA sequence

Table 4 (continued) rxa02261 1479 GB_BA1 CGL007732 4460 AJ007732 Corynebactenum glutamicum 3' ppc gene, secG gene, amt gene, ocd gene and Corynebacterium 100,000 7-Jan-99

5' soxA gene glutamicum

GB_BA1 CGAMTGENE 2028 X93513 C glutamicum amt gene Corynebacterium 100,000 29-MAY- glutamicum

GB_BA1 CORPEPC 4885 M25819 C glutamicum phosphoenolpyruvate carboxylase gene, complete eds Corynebacterium 100,000 15-DEC- π yl liu itiaαriY ii Jiiiu"iu imi ( i rxa02268 1023 GB_PL2 AF087130 3478 AF087130 Neurospora crassa siderophore regulation protein (sre) gene, complete eds Neurospora crassa 39,268 22-OCT- GB EST30 AI663709 408 AI663709 ud47a06 y1 Soares mouse mammary gland NbMMG Mus musculus cDNA clone Mus musculus 41 ,523 10-MAY- IMAGE 1449010 5' similar to TR 075585 075585 MITOGEN- AND STRESS- ACTIVATED PROTEIN KINASE-2 „ mRNA sequence

GB_RO AF074714 3120 AF074714 Mus musculus mitogen- and stress-activated protein kιnase-2 (mMSK2) mRNA, Mus musculus 38,347 24-OCT- complete eds rxa02269 1095 GB_GSS4 AQ742825 847 AQ742825 HS_5482_B2_A04_T7A RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 37,703 16-Jul-9 genomic clone Plate=1058 Col=8 Row=B, genomic survey sequence

GB_HTG3 AC009293 162944 AC009293 Homo sapiens chromosome 18 clone 53_l_06 map 18, ^*** SEQUENCING IN Homo sapiens 37,006 13-Aug- PROGRESS ^***, 15 unordered pieces

GB_HTG3 AC009293 162944 AC009293 Homo sapiens chromosome 18 clone 53_l_06 map 18, ^*** SEQUENCING IN Homo sapiens 37,006 13-Aug- PROGRESS ^***, 15 unordered pieces rxa02309 1173 GB_BA1 MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv complete genome, segment 28/162 Mycobacterium 52,344 17-Jun-9 tuberculosis

GB_BA1 MSGY224 40051 AD000004 Mycobacterium tuberculosis sequence from clone y224 Mycobacterium 52,344 03-DEC- tuberculosis

GBJHTG2 AC007163 186618 AC007163 Homo sapiens clone NH0091M05, ^*** SEQUENCING IN PROGRESS ^***, 1 Homo sapiens 37,263 23-Apr-9 unordered pieces rxa02310 1386 GB_BA1 MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv complete genome, segment 28/162 Mycobacterium 36,861 17-Jun-9 tuberculosis

GB BA1 MSGY224 40051 AD000004 Mycobacterium tuberculosis sequence from clone y224 Mycobacterium 36,861 03-DEC- tuberculosis

GB_PR3 HS279N11 169998 Z98255 Human DNA sequence from PAC 279N 11 on chromosome Xq11 2-13 3 Homo sapiens 34,516 23-NOV- rxa02321 1752 GB_BA1 AB018531 4961 AB018531 Corynebacterium glutamicum dtsR1 and dtsR2 genes, complete eds Corynebacterium 99,030 19-OCT- glutamicum

GB_PAT E17019 4961 E17019 Brevibacterium lactofermentum dtsR and dtsR2 genes Corynebacterium 98,973 28-Jul-9 glutamicum

GB BA1 AB018530 2855 AB018530 Corynebacterium glutamicum dtsR gene, complete eds Corynebacterium 99,030 19-OCT- glutamicum rxa02335 1896 GB BA1 CGU35023 3195 U35023 Corynebacterium glutamicum thiosulfate sulfurtransferase (thtR) gene, partial Corynebacterium 99,947 16-Jan-9 eds, acyl CoA carboxylase (accBC) gene, complete eds glutamicum

GB_BA1 U00012 33312 U00012 Mycobacterium leprae cosmid B1308 Mycobacterium leprae 40,247 30-Jan-9

GB_BA1 MTCY71 42729 Z92771 Mycobacterium tuberculosis H37Rv complete genome, segment 141/162 Mycobacterium 67,568 10-Feb- tuberculosis rxa02364 750 GB_BA1 AP000006 319000 AP000006 Pyrococcus honkoshii OT3 genomic DNA, 1166001-1485000 nt position (6/7) Pyrococcus horikoshii 36,130 8-Feb-99

GB BA1 AP000006 319000 AP000006 Pyrococcus honkoshii OT3 genomic DNA, 1166001 -1485000 nt position (6/7) Pyrococcus honkoshii 34,543 8-Feb-99

Table 4 (continued) rxa02372 2010 GB_HTG3 AC011461 100974 AC011461 Homo sapiens chromosome 19 clone CIT-HSPC_429L19 SEQUENCING IN Homo sapiens 36,138 07-OCT-1999

PROGRESS ^***, 4 ordered pieces

GB_HTG3 AC011461 100974 AC011461 Homo sapiens chromosome 19 clone CIT-HSPC_429L19, ^*** SEQUENCING IN Homo sapiens 36,138 07-OCT-1999

PROGRESS ^***, 4 ordered pieces

GB_EST21 AA992021 279 AA992021 Ot36c01 s1 Soares_testιs_NHT Homo sapiens cDNA clone IMAGE 1618848 3', Homo sapiens 41 ,219 3-Jun-98 mRNA sequence rxa02397 1119 GB_HTG4 AC009273 76175 AC009273 Arabidopsis thahana chromosome 1 clone T1 N6, ^*** SEQUENCING IN Arabidopsis thahana 38,566 12-OCT-1999

PROGRESS ^***, 2 ordered pieces

GB_HTG4 AC009273 76175 AC009273 Arabidopsis thahana chromosome 1 clone T1 N6, ^*** SEQUENCING IN Arabidopsis thahana 38,566 12-OCT-1999

PROGRESS ***, 2 ordered pieces

GB_BA1 D90826 19493 D90826 E coli genomic DNA, Kohara clone #335(40 9-41 3 mm ) Escherichia coli 39,600 21-MAR-1997 rxa02424 723 GB_EST13 AA334108 275 AA334108 EST38262 Embryo, 9 week Homo sapiens cDNA 5' end, mRNA sequence Homo sapiens 38,603 21-Apr-97

GB_PR3 AC005224 166687 AC005224 Homo sapiens chromosome 17, clone hRPK 214_0_1 , complete sequence Homo sapiens 36,111 14-Aug-98

GB_PR3 AC005224 166687 AC005224 Homo sapiens chromosome 17, clone hRPK 214_0_1 , complete sequence Homo sapiens 33,427 14-Aug-98 rxa02426 1656 GB_PAT A06664 1350 A06664 B stearothermophilus let gene Bacillus 39,936 29-Jul-93 stearothermophilus

GB_PAT A04115 1361 A04115 B stearothermophilus recombinant let gene synthetic construct 40,042 17-Feb-97

GB_BA1 BACLDHL 1361 M14788 B stearothermophilus let gene encoding L-lactate dehydrogenase, complete eds Bacillus 40,338 26-Apr-93 stearothermophilus rxa02487 1827 GB_BA2 AF007101 32870 AF007101 Streptomyces hygroscopicus putative ptendine-dependent dioxygenase, PKS Streptomyces 43,298 13-Jan-98 modules 1 ,2,3 and 4, and putative regulatory protein genes, complete eds and hygroscopicus putative hydroxylase gene, partial eds

GB_BA1 MTCI364 29540 Z93777 Mycobacterium tuberculosis H37Rv complete genome, segment 52/162 Mycobacterium 44,352 17-Jun-98 tuberculosis

GB_BA2 AF 119621 15986 AF119621 Pseudomonas abietaniphila BKME-9 Ditl (ditl), dioxygenase DitA oxygenase Pseudomonas 43,611 28-Apr-99 component small subunit (dιtA2), dioxygenase DitA oxygenase component large abietaniphila subunit (dιtA1 ), DitH (ditH), DitG (ditG), DitF (ditF), DitR (ditR), DitE (ditE), DitD

(ditD), aromatic diterpenoid extradiol ring-cleavage dioygenase (ditC), DitB

(ditB), and dioxygenase DitA ferredoxin component (dιtA3) genes, complete eds, and unknown genes rxa02511 780 GB_PR4 AC002470 235395 AC002470 Homo sapiens Chromosome 22q11 2 BAC Clone b135h6 In BCRL2-GGT Homo sapiens 37,971 30-NOV-99

Region, complete sequence

GB_PR4 AC002472 147100 AC002472 Homo sapiens Chromosome 22q11 2 PAC Clone p_n5 In BCRL2-GGT Region, Homo sapiens 38,239 13-Sep-99 complete sequence

GB EST34 AI806938 118 AI806938 wf24b07 x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone Homo sapiens 38,983 7-Jul-99

IMAGE 2356501 3' similar to SW PLZF_HUMAN Q05516 ZINC FINGER

PROTEIN PLZF ,, mRNA sequence rxa02512 1086 GB BA1 MTCY1A10 25949 Z95387 Mycobacterium tuberculosis H37Rv complete genome, segment 117/162 Mycobacterium 37,407 17-Jun-98 tuberculosis

GB_BA1 MLCL581 36225 Z96801 Mycobacterium leprae cosmid L581 Mycobacterium leprae 43,193 24-Jun-97

GBJDV GGU43396 2738 U43396 Gallus gallus tropomyosin receptor kinase A (ctrkA) mRNA, complete eds Gallus gallus 38,789 18-Jan-96 rxa02527 1452 GB BA2 AF008220 220060 AF008220 Bacillus subtilis rrnB-dnaB genomic region Bacillus subtilis 37,395 4-Feb-98

Table 4 (continued)

GB_BA2 AF008220 220060 AF008220 Bacillus subtilis rrnB-dnaB genomic region Bacillus subtilis 36,218 4-Feb-98

GBJHTG2 AC005861 112369 AC005861 Arabidopsis thahana clone F23B24, ^*** SEQUENCING IN PROGRESS ^***, i Arabidopsis thahana 38,407 29-Apr-99 unordered pieces rxa02547 2262 GB_PL1 AB006530 7344 AB006530 Citrullus lanatus Sat gene for serine acetyltransferase, complete eds and 5'- Citrullus lanatus 35,449 20-Aug-97 flankmg region

GB_PL1 CNASA 5729 D85624 Citrullus vulgans serine acetyltransferase (Sat) DNA, complete eds Citrullus lanatus 35,449 6-Feb-99

GB_PL1 AB006530 7344 AB006530 Citrullus lanatus Sat gene for serine acetyltransferase, complete eds and 5'- Citrullus lanatus 34,646 20-Aug-97 flankmg region rxa02566 1332 GB_EST32 AI727189 619 AI727189 BNLGHι7498 Six-day Cotton fiber Gossypium hirsutum cDNA 5' similar to Gossypium hirsutum 35,099 11-Jun-99

(AB020715) KIAA0908 protein [Homo sapiens], mRNA sequence

GB_BA1 CGPUTP 3791 Y09163 C glutamicum putP gene Corynebacterium 38,562 8-Sep-97 glutamicum

GB_PL2 SPAC13G6 33481 Z54308 S pombe chromosome I cosmid c13G6 Sch izosaccha romyces 35,774 18-OCT-1999 pombe rxa02571 1152 GB_BA1 CGU43535 2531 U43535 Corynebacterium glutamicum multidrug resistance protein (cmr) gene, complete Corynebacterium 41 ,872 9-Apr-97 eds glutamicum

GB_EST35 AI857385 488 AI857385 wl55e03 x1 NCI_CGAP_Brn25 Homo sapiens cDNA clone IMAGE 2428828 3', Homo sapiens 39,139 26-Aug-99 mRNA sequence

GB_BA1 CGU43535 2531 U43535 Corynebacterium glutamicum multidrug resistance protein (cmr) gene, complete Corynebacterium 38,552 9-Apr-97 eds glutamicum rxa02578 1227 GB_PL1 AB016871 79109 AB016871 Arabidopsis thahana genomic DNA, chromosome 5, TAC clone K16L22, Arabidopsis thahana 34,213 20-Nov-99 complete sequence

GB_PL1 AB025602 55790 AB025602 Arabidopsis thahana genomic DNA, chromosome 5, BAC clone F14A1 , complete Arabidopsis thahana 36,461 20-Nov-99 sequence

GBJN1 CELF36H9 35985 AF016668 Caenorhabditis elegans cosmid F36H9 Caenorhabditis elegans 35,977 8-Aug-97 rxa02581 1983 GB_BA1 MTV005 37840 AL010186 Mycobacterium tuberculosis H37Rv complete genome, segment 51/162 Mycobacterium 38,517 17-Jun-98 tuberculosis

GB_BA1 MTV005 37840 AL010186 Mycobacterium tuberculosis H37Rv complete genome, segment 51/162 Mycobacterium 39,173 17-Jun-98 tuberculosis rxa02582 4953 GB_BA1 MTV026 23740 AL022076 Mycobacterium tuberculosis H37Rv complete genome, segment 157/162 Mycobacterium 38,548 24-Jun-99 tuberculosis

GB_BA1 MTCY338 29372 Z74697 Mycobacterium tuberculosis H37Rv complete genome, segment 127/162 Mycobacterium 46,263 17-Jun-98 tuberculosis

GB_BA1 SEERYABS 20444 X62569 S erythraea eryA gene for 6-deoxyerythronolyde B synthase II & III Saccharopolyspora 45,053 28-Feb-92 erythraea rxa02583 1671 GB_BA2 AF113605 1593 AF 113605 Streptomyces coehcolor propionyl-CoA carboxylase complex B subunit (pccB) Streptomyces coehcolor 58,397 08-DEC-1999 gene, complete eds

GB_BA1 SC1 C2 42210 AL031124 Streptomyces coehcolor cosmid 1 C2 Streptomyces coehcolor 52,916 15-Jan-99

GB_BA1 AB018531 4961 AB018531 Corynebacterium glutamicum dtsR1 and dtsR2 genes, complete eds Corynebacterium 58,809 19-OCT-1998 glutamicum rxa02599 600 GB BA1 AEMML 2585 X99639 Ralstonia eutropha mmlH, mmll & mmlj genes Ralstonia eutropha 35,264 22-Jan-98

Table 4 (continued)

GB_EST15 AA508926 422 AA508926 MBAFCW1C08T3 Brugia malayi adult female cDNA (SAW96MLW-BmAF) Brugia malayi 43,377 8-Jul-97

Brugia malayi cDNA clone AFCW1C08 5', mRNA sequence

GB_BA1 AEMML 2585 X99639 Ralstonia eutropha mmlH, mmll & mmlj genes Ralstonia eutropha 41 ,148 22-Jan-98 rxa02634 1734 GB_BA1 SYNPOO 1964 X17439 Synechocystis ndhC, psbG genes for NDH-C, PSII-G and ORF157 Synechocystis PCC6803 38,145 10-Feb-99

GB_GSS9 AQ101527 184 AQ 101527 HS_2265_A1_E11_MF CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 38,798 27-Aug-98 sapiens genomic clone Plate=2265 Col=21 Row=l, genomic survey sequence

GBJN1 MNE133341 399 AJ133341 Melarhaphe nentoides partial caM gene, exons 1-2 Melarhaphe nentoides 39,098 2-Jun-99 rxa02638 999 GB_BA2 AE001756 10938 AE001756 Thermotoga maπtima section 68 of 136 of the complete genome Thermotoga maritima 40,104 2-Jun-99

GB_GSS12 AQ423878 689 AQ423878 CITBI-E1-2575E20 TF CITBI-E1 Homo sapiens genomic clone 2575E20, Homo sapiens 36,451 23-MAR-199 genomic survey sequence

GB_HTG2 AC006765 274498 AC006765 Caenorhabditis elegans clone Y43H11 , ^*** SEQUENCING IN PROGRESS^***, 7 Caenorhabditis elegans 39,072 23-Feb-99 unordered pieces rxa02659 335 GB_EST36 AI900317 436 AI900317 sc04a02 y1 Gm-c1012 Glycine max cDNA clone GENOME SYSTEMS CLONE Glycine max 41 ,566 06-DEC-1999

ID Gm-c1012-1155 5' similar to SW PRS6_SOLTU P54778 26S PROTEASE

REGULATORY SUBUNIT 6B HOMOLOG „ mRNA sequence

GB_GSS12 AQ342831 683 AQ342831 RPCI11-122K17 TJ RPCI-11 Homo sapiens genomic clone RPCI-11-122K17, Homo sapiens 34,762 07-MAY-199 genomic survey sequence

GB_EST36 AI900856 779 AI900856 sb95c11 y1 Gm-c1012 Glycine max cDNA clone GENOME SYSTEMS CLONE Glycine max 39,063 06-DEC-199

ID Gm-c1012-429 5' similar to SW PRS6_SOLTU P54778 26S PROTEASE

REGULATORY SUBUNIT 6B HOMOLOG „ mRNA sequence rxa02676 1512 GBJN2 CELB0213 39134 AF039050 Caenorhabditis elegans cosmid B0213 Caenorhabditis elegans 35,814 2-Jun-99

GB_GSS1 CNS00PZB 364 AL085157 Arabidopsis thahana genome survey sequence SP6 end of BAC F10D11 of IGF Arabidopsis thahana 38,462 28-Jun-99 library from strain Columbia of Arabidopsis thahana, genomic survey sequence

GB_RO RNITPR2R 10708 X61677 Rat ITPR2 gene for type 2 inositol triphosphate receptor Rattus norvegicus 37,543 21-OCT-1991 rxa02677 882 GB_RO D89728 5002 D89728 Mus musculus mRNA for LOK, complete eds Mus musculus 38,829 7-Feb-99

GB_GSS8 AQ062004 362 AQ062004 CIT-HSP-2346014 TR CIT-HSP Homo sapiens genomic clone 2346014, Homo sapiens 36,565 31 -Jul-98 genomic survey sequence

GB_GSS14 AQ555818 462 AQ555818 HS_5230_B1_G06_SP6E RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 36,534 29-MAY-199 genomic clone Plate=806 Col=11 Row=N, genomic survey sequence rxa02691 930 GBJN1 DME9736 7411 AJ009736 Drosophila melanogaster Idefix retroelement gag, pol and env genes, partial Drosophila melanogaster 36,522 19-Jan-99

GB_PR4 AC004801 193561 AC004801 Homo sapiens 12q13 1 PAC RPCI1-228P16 (Roswell Park Cancer Institute Homo sapiens 39,341 2-Feb-99

Human PAC Library) complete sequence

GB_PR4 AC004801 193561 AC004801 Homo sapiens 12q13 1 PAC RPCI1-228P16 (Roswell Park Cancer Institute Homo sapiens 37,037 2-Feb-99

Human PAC Library) complete sequence rxa02718 1170 GB_EST34 AV132028 258 AV132028 AV132028 Mus musculus C57BL/6J 11 -day embryo Mus musculus cDNA clone Mus musculus 43,529 1-Jul-99

2700087F01 , mRNA sequence

GB_GSS10 AQ240654 452 AQ240654 CIT-HSP-2385D24 TFB 1 CIT-HSP Homo sapiens genomic clone 2385D24, Homo sapiens 40,044 30-Sep-98 genomic survey sequence

GB GSS11 AQ309500 576 AQ309500 CIT-HSP-2384D24 TFD CIT-HSP Homo sapiens genomic clone 2384D24, Homo sapiens 38,869 22-DEC-199 genomic survey sequence

Table 4 (continued) rxa02749 999 GB BA2 AF086791 37867 AF086791 Zymomonas mobilis strain ZM4 clone 67E10 carbamoylphosphate synthetase Zymomonas mobilis 39,024 4-Nov-98 small subunit (carA), carbamoylphosphate synthetase large subunit (carB), transcription elongation factor (greA), enolase (eno), pyruvate dehydrogenase alpha subunit (pdhA), pyruvate dehydrogenase beta subunit

(pdhB), ribonuclease H (rnh), homoserine kinase homolog, alcohol dehydrogenase II (adhB), and excmuclease ABC subunit A (uvrA) genes, complete eds, and unknown genes

GB_BA1 SYCSLRB 146271 D64000 Synechocystis sp PCC6803 complete genome, 19/27, 2392729-2538999 Synechocystis sp 34,573 13-Feb-99

GB_BA2 AE001306 13316 AE001306 Chlamydia trachomatis section 33 of 87 of the complete genome Chlamydia trachomatis 38,940 2-Sep-98 rxa02767 906 GB_BA2 AF 126953 1638 AF126953 Corynebacterium glutamicum cystathionine gamma-synthase (metB) gene, Corynebacterium 100,000 10-Sep-99 complete eds glutamicum

GB_BA1 SCI5 6661 AL079332 Streptomyces coehcolor cosmid 15 Streptomyces coehcolor 37,486 16-Jun-99

GB_PR3 HS90L6 190837 Z97353 Human DNA sequence from clone 90L6 on chromosome 22q11 21 -11 23 Homo sapiens 34,149 23-Nov-99

Contains an RPL15 (60S Ribosomal Protein L15) pseudogene, ESTs, STSs and

GSSs, complete sequence rxa02792 876 GB_BA2 AF099015 5000 AF099015 Streptomyces coehcolor strain A3(2) integrase (int), Fe-contaming superoxide Streptomyces coehcolor 36,721 1-Jun-99 dismutase II (sodF2), Fe uptake system permease (ftrE), and Fe uptake system integral membrane protein (ftrD) genes, complete eds

GB_BA1 ECOUW93 338534 U14003 Escherichia coli K-12 chromosomal region from 92 8 to 00 1 minutes Escherichia coli 38,787 17-Apr-96

GB_HTG3 AC011361 186148 AC011361 Homo sapiens chromosome 5 clone CIT-HSPC_482N19, ^*** SEQUENCING IN Homo sapiens 43,577 06-OCT-1999

PROGRESS ^***, 69 unordered pieces rxa02794 1197 GB_PR4 AC005998 96556 AC005998 Homo sapiens clone DJ0622E21 , complete sequence Homo sapiens 37,298 29-Juf-99

GB_PR4 AC006008 57554 AC006008 Homo sapiens clone DJ0820A21 , complete sequence Homo sapiens 36,638 17-Jun-99

GB_PR3 HSDJ73H14 95556 AL080272 Human DNA sequence from clone 73H14 on chromosome Xq26 3-28, complete Homo sapiens 39,726 23-Nov-99 sequence rxa02809 375 GB_RO MUSSPCTLT 3172 M22527 Mouse cytotoxic T lymphocyte-specific serine protease CCPII gene, complete Mus musculus 47,518 19-Jan-96

GB_RO MUSGRC 894 M 18459 Mouse granzyme C serine esterase mRNA, complete eds Mus musculus 44,939 12-Jun-93

GB_RO RNU57062 880 U57062 Rattus norvegicus natural killer cell protease 4 (RNKP-4) mRNA, complete eds Rattus norvegicus 41 ,554 31-JUI-96 rxa02811 484 GB_GSS6 AQ832862 476 AQ832862 HS_5261_A2_E10_SP6E RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 35,610 27-Aug-99 genomic clone Plate=837 Col=20 Row=l, genomic survey sequence

GB_GSS5 AQ784593 515 AQ784593 HS_3248_A2_F02_T7C CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 38,956 3-Aug-99 sapiens genomic clone Plate=3248 Col=4 Row=K, genomic survey sequence

GB_GSS13 AQ473140 397 AQ473140 CITBI-E1-2589G6 TF CITBI-E1 Homo sapiens genomic clone 2589G6, genomic Homo sapiens 34,761 23-Apr-99 survey sequence rxa02836 678 GB_EST18 AA696785 316 AA696785 GM08392 5prιme GM Drosophila melanogaster ovary BlueScnpt Drosophila Drosophila melanogaster 40,604 28-Nov-98 melanogaster cDNA clone GM08392 5prιme, mRNA sequence

GB EST18 AA696785 316 AA696785 GM08392 5prιme GM Drosophila melanogaster ovary BlueScnpt Drosophila Drosophila melanogaster 38,281 28-NOV-98 melanogaster cDNA clone GM08392 5prιme, mRNA sequence rxs03212 1452 GB_BA1 CGBETPGEN 2339 X93514 C glutamicum betP gene Corynebacterium 99,931 8-Sep-97 glutamicum

GB_BA1 SC5F2A 40105 AL049587 Streptomyces coehcolor cosmid 5F2A Streptomyces coehcolor 57,557 24-MAY-1999

A3(2)

Table 4 (continued)

GB_BA2 AF008220 220060 AF008220 Bacillus subtilis rrnB-dnaB genomic region Bacillus subtilis 40,000 4-Feb-98 rxs03220 725 GB_PL1 CKHUP2 2353 X66855 C kesslen HUP2 mRNA Chlorella kesslen 45,328 17-Feb-9

GB_EST38 AW048153 383 AW048153 UI-M-BH1-alq-h-05-0-UI s1 NIH_BMAP_M_S2 Mus musculus cDNA clone Ul-M- Mus musculus 41 ,758 18-Sep-9

BH1-alq-h-05-0-UI 3', mRNA sequence

GB PL1 CKHUP2 2353 X66855 C kesslen HUP2 mRNA Chlorella kesslen 38,106 17-Feb-9

-

I s en

I

Exemplification

Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30°C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture — all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/1 sucrose, 2.46 g/1 MgSO₄ x 7H₂O, 10 ml/1 KH₂PO₄ solution ( 100 g/1, adjusted to pH 6.7 with KOH), 50 ml/1 Ml 2 concentrate (10 g/1 (NH₄)₂SO₄, 1 g/1 NaCl, 2 g/1 MgSO₄ x 7H₂O, 0.2 g/1 CaCl₂, 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/1 FeSO₄ x H₂O, 10 mg/1 ZnSO₄ x 7 H₂O, 3 mg/1 MnCl₂ x 4 H₂O, 30 mg/1 H₃BO₃ 20 mg/1 CoCl₂ x 6 H₂O, 1 mg/1 NiCl₂ x 6 H₂O, 3 mg/1 Na₂MoO₄ x 2 H₂O, 500 mg/1 complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/1 biotin, 0.2 mg/1 folic acid, 20 mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/1 ca-panthothenate, 140 mg/1 nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37°C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) are added. After adding of proteinase K to a final concentration of 200 μg/ml, the suspension is incubated for ca.18 h at 37°C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform- isoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20°C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing 20 μg/ml RNase A and dialysed at 4°C against 1000 ml TE-buffer for at least 3 hours. During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. After a 30 min incubation at -20°C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared by this procedure could be used for all purposes, including southern blotting or construction of genomic libraries.

Example 2: Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.

Using DNA prepared as described in Example , cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al. (1989) "Molecular Cloning : A Laboratory Manual", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons.)

Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol 134:1141-1 156), plasmids of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene, LaJolla, USA) or Loristό (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnoi. 4: 256-263).

Example 3: DNA Sequencing and Computational Functional Analysis

Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R.D. et al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496- 512). Sequencing primers with the following nucleotide sequences were used: 5'- GGAAACAGTATGACCATG-3' or 5'-GTAAAACGACGGCCAGT-3'.

Example 4: In vivo Mutagenesis In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of 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 capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D. (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well known to those of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 1: 32-34.

Example 5: DNA Transfer Between Escherichia coli and Corynebacterium glutamicum

Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as e.g., pHM1519 or pBLl) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al (1994) "Current Protocols in Molecular Biology", John Wiley & Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones —

Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E. coli and C. glutamicum, and which can be used for several purposes, including gene over- expression (for reference, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162:591-597, Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al (1991) Gene, 102:93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schafer, A et al (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol Biol. 166:1-19).

Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCGl (U.S. Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, CM. (1980) Proc. Natl Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J Microbiol. Biotechnoi. 4: 256-263).

Aside from the use of replicative plasmids, gene overexpression can also be achieved by integration into the genome. Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or through the use of transposons. It is also possible to modulate the activity of a gene of interest by modifying the regulatory regions (e.g., a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI). Nucleic acid sequences which function as transcriptional terminators may also be inserted 3' to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology. VCH: Weinheim.

Example 6: Assessment of the Expression of the Mutant Protein

Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein. A useful method to ascertain the level of transcription of the mutant gene (an indicator of the amount of mRNA available for translation to the gene - I l l - 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 is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by several methods, all well-known in the art, such as that described in Bormann, E.R. 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.

Example 7: Growth of Genetically Modified Corynebacterium glutamicum — Media and Culture Conditions

Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al (19S9) Appl. Microbiol. Biotechnoi., 32:205-210; von der Osten et al (1998) Biotechnology Letters, 11 :11-16; Patent DE 4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al, eds. Springer- Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH₄C1 or (NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.

All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121°C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.

Culture conditions are defined separately for each experiment. The temperature should be in a range between 15°C and 45°C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH₄OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.

If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD_6υo of O.5 - 1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2,5 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 agar, pH 6.8 with 2M NaOH) that had been incubated at 30°C. Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.

Example 8 - In vitro Analysis of the Function of Mutant Proteins

The determination of activities and kinetic parameters of enzymes is well established in 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 of ordinary skill 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., and 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, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3^rd ed. Academic Press: New York; Bisswanger, H., (1994) Enzymkinetik, 2^nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H.U., Bergmeyer, J., GraM, M., 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.

The activity of proteins which 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 proteins 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 pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.

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

Example 9: Analysis of Impact of Mutant Protein on the Production of the Desired Product

The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product (i.e., an amino acid). Such analysis techniques are well known to one of ordinary skill 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, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al, (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.)

In addition to the measurement of the final product of fermentation, it is also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound. Analysis methods include measurements of nutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references cited therein.

Example 10: Purification of the Desired Product from C. glutamicum Culture

Recovery of the desired product from the C. glutamicum cells or supernatant of the above-described culture can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C. glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supernate fraction is retained for further purification.

The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 : 27- 32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540- 547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

Example 11: Analysis of the Gene Sequences of the Invention

The comparison of sequences and determination of percent homology between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al (1990) J. Mol Biol 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to MCT nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to MCT protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al , (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence being analyzed.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci. 4: 11- 17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM, described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.

A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis (e.g., a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search (e.g., a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP (global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information. It will further be understood by one of ordinary skill in the art that the GAP alignment homology percentages set forth in Table 4 under the heading "% homology (GAP)" are listed in the European numerical format, wherein a ',' represents a decimal point. For example, a value of "40,345" in this column represents "40.345%".

Example 12: Construction and Operation of DNA Microarrays

The sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA arrays are well known in the art, and are described, for example, in Schena, M. et al. (1995)

Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L. et al (1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).

The sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470).

Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al (1997) Nature Biotechnology 15: 1359- 1367. By photolithographic methods, precisely defined regions of the matrix are exposed to light. Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.

The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. These nucleic acid molecules can be labeled according to standard methods. In brief, nucleic acid molecules (e.g., mRNA molecules or DNA molecules) are labeled by the incorporation of isotopically or fluorescently labeled nucleotides, e.g., during reverse transcription or DNA synthesis. Hybridization of labeled nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incoφorated label. Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

The application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C. glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.

Example 13: Analysis of the Dynamics of Cellular Protein Populations (Proteomics)

The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed 'proteomics'. Protein populations of interest include, but are not limited to, the total protein population of C. glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.

Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety of electrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221 ; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art. Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., S-methionine, S-cysteine, C-labelled amino acids, N-amino acids, NO₃ or ¹⁵NH₄ ⁺ or C-labelled amino acids) in the medium of C. glutamicum permits the labeling of proteins from these cells prior to their separation. Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.

Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such techniques are well-known in the art.

To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g. , Langen et al. (1997) Electrophoresis 18: 1184-1192)). The protein sequences provided herein can be used for the identification of C. glutamicum proteins by these techniques. The information obtained by these methods can be used to compare patterns of protein presence, activity, or modification between different samples from various biological conditions (e.g., different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals. Equivalents

Those of ordinary skill 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.

Claims

What is claimed:

1. An isolated nucleic acid molecule from Corynebacterium glutamicum encoding an MCT protein, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

2. The isolated nucleic acid molecule of claim 1 , wherein said nucleic acid molecule encodes an MCT protein involved in the production of a fine chemical.

3. An isolated Corynebacterium glutamicum nucleic acid molecule selected from the group consisting of those sequences set forth as odd-numbered SEQ ID NOs of the Sequence Listing, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

4. An isolated nucleic acid molecule which encodes a polypeptide sequence selected from the group consisting of those sequences set forth as even-numbered SEQ ID NOs of the Sequence Listing, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

5. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide selected from the group of amino acid sequences consisting of those sequences set forth as even-numbered SEQ ID NOs of the Sequence Listing, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

6. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleotide sequence selected from the group consisting of those sequences set forth as odd-numbered SEQ ID NOs of the Sequence Listing, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

7. An isolated nucleic acid molecule comprising a fragment of at least 15 nucleotides of a nucleic acid comprising a nucleotide sequence selected from the group consisting of those sequences set forth as odd-numbered SEQ ID NOs of the Sequence Listing, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.

8. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule of any one of claims 1-7 under stringent conditions.

9. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1 -8 or a portion thereof and a nucleotide sequence encoding a heterologous polypeptide.

10. A vector comprising the nucleic acid molecule of any one of claims 1-9.

11. The vector of claim 10, which is an expression vector.

12. A host cell transfected with the expression vector of claim 11.

13. The host cell of claim 12, wherein said cell is a microorganism.

14. The host cell of claim 13, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

15. The host cell of claim 12, wherein the expression of said nucleic acid molecule results in the modulation in production of a fine chemical from said cell.

16. The host cell of claim 15, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes.

17. A method of producing a polypeptide comprising culturing the host cell of claim 12 in an appropriate culture medium to, thereby, produce the polypeptide.

18. An isolated MCT polypeptide from Corynebacterium glutamicum, or a portion thereof.

19. The polypeptide of claim 18, wherein said polypeptide is involved in the production of a fine chemical production.

20. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of those sequences set forth as even-numbered SEQ ID NOs of the Sequence Listing, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.

21. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of those sequences set forth as even-numbered SEQ ID NOs of the Sequence

Listing, or a portion thereof, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.

22. The isolated polypeptide of any of claims 18-21, further comprising heterologous amino acid sequences.

23. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleic acid selected from the group consisting of those sequences set forth as odd-numbered SEQ ID NOs of the Sequence Listing, provided that the nucleic acid molecule does not consist of any of the F-designated nucleic acid molecules set forth in Table 1.

24. An isolated polypeptide comprising an amino acid sequence which is at least 50% homologous to an amino acid sequence selected from the group consisting of those sequences set forth as even-numbered SEQ ID NOs of the Sequence Listing, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.

25. A method for producing a fine chemical, comprising culturing a cell containing a vector of claim 12 such that the fine chemical is produced.

26. The method of claim 25, wherein said method further comprises the step of recovering the fine chemical from said culture.

27. The method of claim 25, wherein said method further comprises the step of transfecting said cell with the vector of claim 11 to result in a cell containing said vector.

28. The method of claim 25, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

29. The method of claim 25, wherein said cell is selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium, lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum,

Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum, and those strains set forth in Table 3.

30. The method of claim 25, wherein expression of the nucleic acid molecule from said vector results in modulation of production of said fine chemical.

31. The method of claim 25, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes.

32. The method of claim 25, wherein said fine chemical is an amino acid.

33. The method of claim 32, wherein said amino acid is drawn from the group consisting of: lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan.

34. A method for producing a fine chemical, comprising culturing a cell whose genomic DNA has been altered by the inclusion of a nucleic acid molecule of any one of claims 1-9.

35. A method for diagnosing the presence or activity of Corynebacterium diphtheriae in a subject, comprising detecting the presence of one or more of SEQ ID NOs 1 through 676 of the Sequence Listing in the subject, provided that the sequences are not or are not encoded by any of the F-designated sequences set forth in Table 1, thereby diagnosing the presence or activity of Corynebacterium diphtheriae in the subject.

36. A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth as odd-numbered SEQ ID NOs of the Sequence

Listing, wherein the nucleic acid molecule is disrupted.

37. A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth as odd-numbered SEQ ID NOs in the Sequence Listing, wherein the nucleic acid molecule comprises one or more nucleic acid modifications from the sequence set forth as odd-numbered SEQ ID NOs of the Sequence Listing s.

38. A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth as odd-numbered SEQ ID NOs of the Sequence Listing, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild-type regulatory region of the molecule.