Fructooligosaccharide (FOS)-related protein nucleic acid molecules and polypeptides and fragments and variants thereof are disclosed in the current invention. In addition, FOS-related fusion proteins, antigenic peptides, and anti-FOS-related antibodies are encompassed. The invention also provides recombinant expression vectors containing a nucleic acid molecule of the invention and host cells into which the expression vectors have been introduced. Methods for producing the polypeptides of the invention and methods for their use are further disclosed.

Inventors:  Barrangou; Rodolphe (Madison, WI), Klaenhammer; Todd R. (Raleigh, NC), Altermann; Eric (Apex, NC) 
Assignee: North Carolina State University (Raleigh, NC)
 
Appl. No.:  10/873,467
Filed:  June 22, 2004

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Related U.S. Patent Documents

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 Application Number Filing Date Patent Number Issue Date
 60480764 Jun., 2003  
 

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Current U.S. Class: 435/200 ; 435/252.3; 435/252.5; 435/320.1; 435/6; 435/69.1; 536/23.2
Current International Class:  C12N 9/24 (20060101); C07H 21/04 (20060101); C12N 15/74 (20060101); C12Q 1/68 (20060101)
Field of Search:  435/4,6,183,69.1,200,252.3,320.1 536/23.2,23.4,23.5,23.7 530/350 

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References Cited [Referenced By]

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U.S. Patent Documents
  
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6451584 September 2002 Tomita et al.
6476209 November 2002 Glenn et al.
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6635460 October 2003 Van Hijum et al.
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2003/0110535 June 2003 Barry et al.
2003/0138822 July 2003 Glenn et al.
2004/0009490 January 2004 Glenn et al.
2004/0208863 October 2004 Versalovic et al.
2005/0003510 January 2005 Change et al.
2005/0112612 May 2005 Klaenhammer et al.
 

Foreign Patent Documents
     
 0 888 118  Jan., 1999  EP
 WO 02/12506  Feb., 2002  WO
 WO 02/074798  Sep., 2002  WO
 WO 02/077021  Oct., 2002  WO
 WO 03/084989  Oct., 2003  WO
 WO 2004/020467  Mar., 2004  WO
 WO 2004/031389  Apr., 2004  WO
 WO 2004/069178  Aug., 2004  WO
 WO 2004/096992  Nov., 2004  WO
 WO 2005/001057  Jan., 2005  WO
 WO 2005/012491  Feb., 2005  WO
 

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Primary Examiner: Saidha; Tekchand
Assistant Examiner: Meah; Younus
Attorney, Agent or Firm: Alston & Bird LLP

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser. No. 60/480,764, filed Jun. 23, 2003, the contents of which is herein incorporated by reference in its entirety.
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Claims

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That which is claimed is:

1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein said polypeptide has fructosidase activity, or a full length complement thereof.

2. A vector comprising a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein said polypeptide has fructosidase activity.

3. The vector of claim 2, further comprising a nucleic acid molecule encoding a heterologous polypeptide.

4. An isolated host cell comprising the vector of claim 2.

5. The host cell of claim 4, wherein said cell is a bacterial host cell.

6. A method for producing a polypeptide comprising culturing a host cell comprising a heterologous nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein said polypeptide has fructosidase activity, wherein said host cell is cultured under conditions in which the nucleic acid molecule is expressed.

7. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 7 or a full length complement thereof.

8. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

9. The vector of claim 2, wherein said nucleic acid molecule comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7.

10. The vector of claim 2, wherein said nucleic acid molecule comprises nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

11. An isolated host cell comprising a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein said polypeptide has fructosidase activity and said nucleic acid molecule is heterologous to the host cell.

12. The host cell of claim 11, wherein said host cell is a bacterial host cell.

13. The host cell of claim 12, wherein said bacterial host cell comprises a Lactobacillus bacteria strain.

14. The host cell of claim 11, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 7.

15. The host cell of claim 11, wherein said nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

16. The host cell of claim 13, wherein said Lactobacillus bacterial strain is a mutated strain having an enhanced ability to colonize the gastrointestinal tract of a host compared to a wild-type Lactobacillus bacterial strain.

17. The host cell according to claim 16, wherein said Lactobacillus bacterial strain does not utilize frucooligosaccharide in the absence of said heterologous nucleic acid molecule.

18. A culture comprising the Lactobacillus bacterial strain of claim 16.

19. The host cell of claim 13, wherein said Lactobacillus bacterial strain has an enhanced ability to metabolize FOS or other complex carbohydrates compared to a wild-type Lactobacillus bacterial strain lacking said nucleic acid molecule.

20. A culture comprising the Lactobacillus bacterial strain of claim 19.
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Description

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FIELD OF THE INVENTION

This invention relates to polynucleotides isolated from lactic acid bacteria, namely Lactobacillus acidophilus, and polypeptides encoded by them, as well as methods for using the polypeptides and microorganisms expressing them.

BACKGROUND OF THE INVENTION

Lactobacillus acidophilus is a Gram-positive, rod-shaped, non-spore forming, homofermentative bacterium that is a normal inhabitant of the gastrointestinal and genitourinary tracts. Since its original isolation by Moro (1900) from infant feces, the "acid loving" organism has been found in the intestinal tract of humans, breast fed infants, and persons consuming high milk-, lactose-, or dextrin diets. Historically, L. acidophilus is the Lactobacillus species most often implicated as an intestinal probiotic capable of eliciting beneficial effects on the microflora of the gastrointestinal tract (Klaenhammer, T. R., and W. M. Russell. 2000. Species of the Lactobacillus acidophilus complex. Encyclopedia of Food Microbiology, Volume 2, pp 1151-1157. Robinson, R. K, Batt, C., and Patel, P. D (eds). Academic Press, San Diego). L. acidophilus can ferment hexoses, including lactose and more complex oligosaccharides (Kaplan and Hutkins (2000) Appl. Environ. Microbiol. 66, 2682-2684) to produce lactic acid and lower the pH of the environment where the organism is cultured. Acidified environments (e.g. food, vagina, and regions within the gastrointestinal tract) can interfere with the growth of undesirable bacteria, pathogens, and yeasts. The organism is well known for its acid tolerance, survival in cultured dairy products, and viability during passage through the stomach and gastrointestinal tract. Lactobacilli and other commensal bacteria, some of which are considered as probiotic bacteria that "favor life," have been studied extensively for their effects on human health, particularly in the prevention or treatment of enteric infections, diarrheal disease, prevention of cancer, and stimulation of the immune system.

SUMMARY OF THE INVENTION

Specifically, the present invention provides for isolated nucleic acid molecules encoding FOS-related polypeptides comprising the nucleotide sequences found in SEQ ID NOS:1-172 (it being understood that nucleic acids are given in odd-numbered sequence ID numbers only for SEQ ID NOS:1-172, while amino acid sequences are given in even numbers of SEQ ID NOS:1-172), and isolated nucleic acid molecules encoding the amino acid sequences found in SEQ ID NOS:1-172. Further provided are isolated nucleic acid molecules comprising the nucleotide sequences found in SEQ ID NOS:173, 174, 175, 353 and 354. Also provided are isolated or recombinant polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein. Variant nucleic acid molecules and polypeptides sufficiently identical to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and sufficiently identical fragments of the nucleotide and amino acid sequences are encompassed. Nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention are also encompassed.

The nucleotide sequences of the present invention provided in odd SEQ ID NOS:1-172 include non-coding region upstream of the start site. Therefore, nucleotide sequences comprising the coding region of odd SEQ ID NOS:1-172 are also provided. The coding region may be identified by reviewing the sequence listing, specifically odd SEQ ID NOS:1-172, where the amino acid translation provided beneath the nucleotide sequence is indicative of the coding portion.

Compositions further include vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as transgenic microbial populations comprising the vectors. Also included in the invention are methods for the recombinant production of the polypeptides of the invention, and methods for their use. Further are included methods and kits for detecting the presence of a nucleic acid or polypeptide sequence of the invention in a sample, and antibodies that bind to a polypeptide of the invention.

Nucleic acids of the present invention are useful for imparting better FOS-utilizing capacity to probiotic bacteria such as other lactic acid bacteria, including other Lactobacillus species, particularly those that do not otherwise utilize FOS (or other FOS-related compounds). Enhanced FOS-utilizing capacity in such probiotic bacteria is useful for enhancing the ability of such probiotic bacteria to compete with, colonize, or maintain their population position with respect to other bacteria in the gastrointestinal tract of subjects to whom prebiotics are fed, and to whom probiotic bacteria are administered. In addition, the nucleic acids of the present invention are useful as probes in screening other bacteria for the ability to utilize FOS. Other bacteria (particularly lactic acid bacteria and most particularly other species of genus Lactobacillus) found to carry FOS-related sequences like those of the present invention, as identified by probes of the present invention, are useful as probiotic bacteria for administration to human or animal subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Operon layout. The region upstream (SEQ ID NO: 355) of msmE is shown. The start and stop codons are in bold, the putative ribosome binding site is boxed, and the cre-like elements are underlined. Terminators are indicated by hairpin structures.

FIGS. 2A & 2B. Sugar induction and repression. FIG. 2A. Transcriptional induction of the msmE, and bfrA genes, monitored by RT-PCR (top) and RNA slot blots (bottom). Cells were grown on glucose (Glc), fructose (Fru), sucrose (Suc), FOS GF.sub.n, and FOS F.sub.n. Chromosomal DNA was used as a positive control for the probe. FIG. 2B. Transcriptional repression analysis of msmE and bfrA by variable levels of glucose (Glc) and fructose (Fru): 0.1% (5.5 mM), 0.5% (28 mM) and 1.0% (55 mM), in the presence of 1% Fn. Cells were grown in the presence of F.sub.n until OD.sub.600 nm approximated 0.5-0.6, glucose was added and cells were propagated for an additional 30 minutes.

FIG. 3. Growth curves. The two mutants, bfrA (top) and msmE (bottom) were grown on semi-synthetic medium supplemented with 0.5% w/v carbohydrate: fructose (.circle-solid.), GFn (.smallcircle.), Fn (), Fn for one passage (.box-solid.). The lacZ mutant grown on Fn was used as control (.gradient.).

FIG. 4. Operon architecture analysis. A. Alignment of the msm locus from selected bacteria. Regulators, white; .alpha.-galactosidases, blue; ABC transporters, gray; fructosidases, yellow; sucrose phosphorylase, red. B. Alignment of the sucrose locus from selected microbes. Regulators, white; fructosidases, yellow; PTS transporters, green; fructokinase, purple; putative proteins, black.

FIG. 5. Neighbor-joining phylogenetic trees. Lactobacillales, black; bacillales, green; clostridia, blue; thermotogae, yellow; proteobacteria, red. A, 16S; B, fructosidase; C, ABC; D, PTS; E, regulators; F, fructokinase. L. acidophilus proteins are boxed, and shaded when encoded by the msm locus. Bars indicate scales for computed pairwise distances.

FIG. 6. Co-expression of contiguous genes. Co-transcription of contiguous genes was monitored by RT-PCR using primers as shown on the lower panel. In each set of three bands, a negative control did not undergo reverse transcription (left), and a positive control was obtained from chromosomal DNA used as a template for PCR (right).

FIG. 7. Mutant growth on select carbohydrates. Strains were grown overnight (18 hours) on semi-synthetic medium supplemented with 0.5% w/v carbohydrates, either glucose (Glc), fructose (Fru), sucrose (Suc), FOS-GFn (GFn), FOS-Fn from Orafti (Fn), FOS-Fn from Rhone-Poulenc (FnRP), lactose (Lac), or galactose (Gal). Cell counts obtained after one passage of the bfrA mutant on FOS-Fn are shown in the lower graph.

FIGS. 8A & 8B. Motifs highly conserved amongst repressors and fructosidases. FIG. 8A, conserved helix-turn-helix motif of the regulators, * the consensus sequence was obtained from Nguyen et al., 1995 (26); FIG. 8B, conserved motifs of the .beta.-fructosidases.

FIG. 9. Biochemical pathways. Biochemical pathways describing the likely reactions carried out by the enzymes encoded in the raffinose, msm and sucrose gene clusters. Each enzymatic reaction depicted on the pathways is carried out by a protein encoded by the gene of the same color. For the raffinose operon, raffinose is transported across the membrane by an ABC transporter, the alpha-galactosidase hydrolyses the galactose moiety, and the sucrose phosphorylase hydrolyses sucrose into glucose-1-phosphate and fructose. For the msm operon, FOS is transported across the membrane by an ABC transporter, the fructosidase hydrolyses fructose moieties, and the sucrose phosphorylase hydrolyses sucrose into glucose-1-phosphate and fructose. For the sucrose operon, sucrose is transported across the membrane and phosphorylated by a PTS transporter, the sucrose phosphate hydrolase hydrolyses the phosphorylated sucrose molecule into fructose and glucose-6-phosphate, and fructose is phosphorylated by the fructokinase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to fructo-oligosaccharide (FOS)-related molecules from Lactobacillus acidophilus. Nucleotide and amino acid sequences of the molecules are provided. The sequences find use in modifying organisms to have enhanced benefits.

By "FOS-related molecules" is intended "FOS-utilization molecules" and "FOS-induced molecules." By "FOS-utilization molecules" is intended a protein that facilitates the utilization of a fructo-oligosaccharide (FOS) by a cell in any way, including but not limited to metabolic or catabolic pathway molecules that catalyze the splitting of fructo-oligosaccharides or components thereof into smaller saccharides for further utilization by the cell in energy pathways; a transport protein that facilitates the transport of a fructo-oligosaccharide into the cell for further metabolic utilization, etc. FOS-utilization molecules can be found, for example, in SEQ ID NOS:1, 3, 5, 7, 9, and 11. By "FOS-induced molecules" is intended molecules that are induced during FOS-utilization. The FOS-related molecules of the present invention include, in general, protein molecules from L. acidophilus, and variants and fragments thereof. The FOS-related molecules include the nucleic acid molecules listed in Table 1 and the polypeptides encoded by them.

These novel FOS-related proteins include transport system proteins, including ATP-binding proteins, solute-binding proteins, and ABC transporters; sucrose phosphorylases; transcriptional repressors; phosphoribosylglycinamide synthetases (GARS); ribosomal proteins; elongation factor proteins; kinases; ATPases; transferases; isomerases; dehydrogenases; aldolases; ligases; peptidases; synthases; phosphatases; and DNA binding proteins.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF), particularly those encoding a FOS-related protein. Isolated nucleic acid molecules of the present invention comprise nucleic acid sequences encoding FOS-related proteins, nucleic acid sequences encoding the amino acid sequences set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, and 172 (hereinafter designated "even SEQ ID NOS:1-172"), the nucleic acid sequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, and 171 (hereinafter designated "odd SEQ ID NOS:1-172"), and variants and fragments thereof. Isolated nucleic acid molecules of the present invention also comprise nucleic acid sequences set forth in SEQ ID NOS:173, 174, 175, 353 and 354. The present invention also encompasses antisense nucleic acid molecules, as described below.

In addition, isolated polypeptides and proteins encoded by the nucleotide sequences set forth, and variants and fragments thereof, are encompassed, as well as methods for producing those polypeptides. For purposes of the present invention, the terms "protein" and "polypeptide" are used interchangeably. The polypeptides of the present invention have FOS-utilization activity. FOS-utilization activity refers to a biological or functional activity as determined in vivo or in vitro according to standard assay techniques (see, for example, Example 1). In one embodiment, the activity is catalyzing the splitting of fructooligosaccharides into smaller saccharides. In another embodiment, the activity is transport of fructooligosaccharides into cells carrying the FOS-related molecule.

In a third embodiment, the promoter sequence (SEQ ID NO:173) or fragments thereof (e.g., but not limited to SEQ ID NOS:353 and 354), or nucleic acid sequences comprising at least one of the catabolite response element (cre) sequences found in SEQ ID NOS:174 and 175 can be employed for controlled expression of heterologous genes and their encoded proteins.

The nucleic acid and protein compositions encompassed by the present invention are isolated or substantially purified. By "isolated" or "substantially purified" is intended that the nucleic acid or protein molecules, or biologically active fragments or variants, are substantially or essentially free from components normally found in association with the nucleic acid or protein in its natural state. Such components include other cellular material, culture media from recombinant production, and various chemicals used in chemically synthesizing the proteins or nucleic acids. Preferably, an "isolated" nucleic acid of the present invention is free of nucleic acid sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5' or 3' ends). However, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition. For example, in various embodiments, the isolated nucleic acid contains less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequence normally associated with the genomic DNA in the cells from which it was derived. Similarly, a substantially purified protein has less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein, or non-FOS-related protein. When the protein is recombinantly produced, preferably culture medium represents less than 30%, 20%, 10%, or 5% of the volume of the protein preparation, and when the protein is produced chemically, preferably the preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors, or non-FOS-related chemicals.

The compositions and methods of the present invention can be used to modulate the function of the FOS-related molecules of L. acidophilus. By "modulate", "alter", or "modify" is intended the up- or down-regulation of a target activity. Proteins of the invention are useful in modifying the abilities of lactic acid bacteria, and also in modifying the nutritional or health-promoting characteristics of foods fermented by such bacteria. Nucleotide molecules of the invention are useful in modulating protein expression by lactic acid bacteria. Up- or down-regulation of expression from a polynucleotide of the present invention is encompassed. Up-regulation may be accomplished by providing multiple gene copies, modulating expression by modifying regulatory elements, promoting transcriptional or translational mechanisms, or other means. Down-regulation may be accomplished by using known antisense and gene silencing techniques.

By "lactic acid bacteria" is intended bacteria from a genus selected from the following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera, Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001) Am. J. Clin. Nutr. 73:365S-373S; Bergey's Manual of Systematic Bacteriology, Vol 2. 1986. Williams and Wilkins, Baltimore. pp 1075-1079).

By "Lactobacillus" is meant any bacteria from the genus Lactobacillus, including but not limited to L. casei, L. rhamnosus, L. johnsonni, L. gasseri, L. acidophilus, L. plantarum, L. fermentum, L. salivarius, L. bulgaricus, and numerous other species outlined by Wood et al. (Holzapfel, W. H. N. The Genera of Lactic Acid Bacteria, Vol. 2. 1995. Brian J. B. Wood, Ed. Aspen Publishers, Inc.)

The polypeptides of the present invention or microbes expressing them are useful as nutritional additives or supplements, and as additives in dairy and fermentation processing. The polynucleotide sequences, encoded polypeptides and microorganisms expressing them are useful in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks and buttermilk. Microorganisms that express polypeptides of the invention may be probiotic organisms. By "probiotic" is intended a live microorganism that survives passage through the gastrointestinal tract and has a beneficial effect on the subject. By "subject" is intended a living organism that comes into contact with a microorganism expressing a protein of the present invention. Subject may refer to humans and other animals.

The polynucleotides and polypeptides of the present invention are useful in modifying milk-derived products. These uses include, but are not limited to, enhancing the ability of bacteria to colonize the gastrointestinal tract of a subject, stimulating the growth of beneficial commensal bacteria residing in the gastrointestinal tract, and altering the products produced during fermentation of FOS compounds.

The nucleic acid molecules of the invention encode FOS-related proteins having the amino acid sequences set forth in even SEQ ID NOS:1-172.

In addition to the FOS-related nucleotide sequences disclosed herein, and fragments and variants thereof, the isolated nucleic acid molecules of the current invention also encompass homologous DNA sequences identified and isolated from other organisms or cells by hybridization with entire or partial sequences obtained from the FOS-related nucleotide sequences disclosed herein, or variants and fragments thereof.

Fragments and Variants

The invention includes isolated nucleic acid molecules comprising nucleotide sequences regulating and encoding FOS-related proteins or variants and fragments thereof, as well as the FOS-related proteins encoded thereby. By "FOS-related protein" is intended proteins having the amino acid sequences set forth in even SEQ ID NOS:1-172, as well as fragments, biologically active portions, and variants thereof. By "fragment" of a nucleotide or protein is intended a portion of the nucleotide or amino acid sequence.

Fragments of nucleic acid molecules can be used as hybridization probes to identify FOS-related-protein-encoding nucleic acids, or can be used as primers in PCR amplification or mutation of FOS-related nucleic acid molecules. Fragments of nucleic acids can also be bound to a physical substrate to comprise what may be considered a macro- or microarray (see, for example, U.S. Pat. No. 5,837,832; U.S. Pat. No. 5,861,242; WO 89/10977; WO 89/11548; WO 93/17126; U.S. Pat. No. 6,309,823). Such arrays of nucleic acids may be used to study gene expression or to identify nucleic acid molecules with sufficient identity to the target sequences. By "nucleic acid molecule" is intended 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. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleotide fragment of a FOS-related protein may encode a protein fragment that is biologically active, or it may be used as a hybridization probe or PCR primer as described below. A biologically active nucleotide fragment can be prepared by isolating a portion of one of the nucleotide sequences of the invention, expressing the encoded portion of the FOS-related protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the FOS-related protein. Fragments of FOS-related nucleic acid molecules comprise at least about 15, 20, 50, 75, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 nucleotides or up to the total number of nucleotides present in a full-length FOS-related nucleotide sequence as disclosed herein. (For example, 1314 for SEQ ID NO:1, 960 for SEQ ID NO:3, etc.).

Fragments of the nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the FOS-related protein and, hence, retain FOS-utilization protein activity. By "retains activity" is intended that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the activity of the FOS-related protein disclosed in even SEQ ID NOS:1-172. Methods for measuring FOS-utilization activity are well known in the art. See, for example, the Example section below as well as the section entitled "Methods of Use" for examples of functional assays.

Fragments of amino acid sequences include polypeptide fragments suitable for use as immunogens to raise anti-FOS-related antibodies. Fragments include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a FOS-related protein, or partial-length protein, of the invention and exhibiting at least one activity of a FOS-related protein, but which include fewer amino acids than the full-length FOS-related proteins disclosed herein. Typically, biologically active portions comprise a domain or motif with at least one activity of the FOS-related protein. A biologically active portion of a FOS-related protein can be a polypeptide which is, for example, 10, 25, 50, 100, 150, 200 contiguous amino acids in length, or up to the total number of amino acids present in a full-length FOS-related protein of the current invention. (For example, 415 for SEQ ID NO:2, 294 for SEQ ID NO:4, etc.). Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native FOS-related protein. As used here, a fragment comprises at least 5 contiguous amino acids of any of even SEQ ID NOS:1-172. The invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, or 9 amino acids.

Variants of the nucleotide and amino acid sequences are encompassed in the present invention. By "variant" is intended a sufficiently identical sequence. Accordingly, the invention encompasses isolated nucleic acid molecules that are sufficiently identical to the nucleotide sequences encoding FOS-related proteins in even SEQ ID NOS:1-172, or nucleic acid molecules that hybridize to a nucleic acid molecule of odd SEQ ID NOS:1-172, or a complement thereof, under stringent conditions. Variants also include polypeptides encoded by the nucleotide sequences of the present invention. In addition, polypeptides of the current invention have an amino acid sequence that is sufficiently identical to an amino acid sequence put forth in even SEQ ID NOS:1-172. By "sufficiently identical" is intended that one amino acid or nucleotide sequence contains a sufficient or minimal number of equivalent or identical amino acid residues as compared to a second amino acid or nucleotide sequence, thus providing a common structural domain and/or indicating a common functional activity. Conservative variants include those sequences that differ due to the degeneracy of the genetic code.

In general, amino acids or nucleotide sequences that have at least about 45%, 55%, or 65% identity, preferably about 70% or 75% identity, more preferably about 80%, 85% or 90%, most preferably about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid sequences of even SEQ ID NOS:1-172 or any of the nucleotide sequences of odd SEQ ID NOS:1-172, respectively, using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, FOS-utilization activity as described herein. By "retains activity" is intended that the variant will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the activity of the FOS-related protein disclosed in even SEQ ID NOS:1-172. Methods for measuring FOS-utilization activity are well known in the art. See, for example, the Example section below as well as the section entitled "Methods of Use" for examples of functional assays. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Naturally occurring variants may exist within a population (e.g., the L. acidophilus population). Such variants can be identified by using well-known molecular biology techniques, such as the polymerase chain reaction (PCR), and hybridization as described below. Synthetically derived nucleotide sequences, for example, sequences generated by site-directed mutagenesis or PCR-mediated mutagenesis which still encode a FOS-related protein, are also included as variants. One or more nucleotide or amino acid substitutions, additions, or deletions can be introduced into a nucleotide or amino acid sequence disclosed herein, such that the substitutions, additions, or deletions are introduced into the encoded protein. The additions (insertions) or deletions (truncations) may be made at the N-terminal or C-terminal end of the native protein, or at one or more sites in the native protein. Similarly, a substitution of one or more nucleotides or amino acids may be made at one or more sites in the native protein.

For example, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential" amino acid is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains are known 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). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity.

Alternatively, mutations can be made randomly along all or part of the length of the FOS-related coding sequence, such as by saturation mutagenesis. The mutants can be expressed recombinantly, and screened for those that retain biological activity by assaying for FOS-related activity using standard assay techniques. Methods for mutagenesis and nucleotide sequence alterations are known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. Molecular Biology (MacMillan Publishing Company, New York) and the references sited therein. Obviously the mutations made in the DNA encoding the variant must not disrupt the reading frame and preferably will not create complimentary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by comparing the activity of the modified sequence with the activity of the original sequence.

Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different FOS-related protein coding regions can be used to create a new FOS-related protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the FOS-related gene of the invention and other known FOS-related genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K.sub.m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Variants of the FOS-related proteins can function as either FOS-related agonists (mimetics) or as FOS-related antagonists. An agonist of the FOS-related protein can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the FOS-related protein. An antagonist of the FOS-related protein can inhibit one or more of the activities of the naturally occurring form of the FOS-related protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade that includes the FOS-related protein.

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

In addition, libraries of fragments of a FOS-related protein coding sequence can be used to generate a variegated population of FOS-related fragments for screening and subsequent selection of variants of a FOS-related protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of a FOS-related 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 S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, one can derive an expression library that encodes N-terminal and internal fragments of various sizes of the FOS-related 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 FOS-related proteins. 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 technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify FOS-related variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Regulatory Sequences

It will be appreciated that an embodiment of the present invention provides isolated DNAs that encode regulatory elements comprising the nucleotide sequences set forth in SEQ ID NO:173, 353 and 354, and isolated nucleic acid molecules comprising one or both of the cre elements provided in SEQ ID NOS:174 and 175. By "regulatory element" or "regulatory nucleotide sequence" as used herein is any DNA sequence that regulates nucleic acid expression at the transcriptional level (i.e., activates and/or suppresses), and is intended to include controllable transcriptional promoters, operators, enhancers, transcriptional terminators, and other expression control elements such as translational control sequences (e.g., Shine-Dalgarno consensus sequence, initiation and termination codons). By "promoter" is intended a regulatory region of DNA, generally comprising a TATA box that is capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a given coding sequence. A promoter may also comprise other recognition sequences, generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements. It is recognized that having identified the nucleotide sequences for the regulatory or promoter regions disclosed herein, it is within the ability of one skilled in the art to isolate and identify additional regulatory elements in the 5' untranslated region from the particular regulatory or promoter regions identified herein. By "catabolite responsive element," "cre sequence" or "cre-like sequence" is intended a cis-acting DNA sequence involved in catabolite repression. The regulatory elements disclosed herein that activate transcription of the nucleic acids, increase nucleic acid transcription by at least 50%, more preferably by at least 100%, 150%, 200%, or even 300%, regulatory elements disclosed herein that suppress transcription of the nucleic acids do so by at least 25%, more preferably by at least 35%, 50%, 60%, 75%, or even 85%, or more.

Regulatory elements (SEQ ID NO:173, 353 and 354) of the present invention are located within the approximately 0.2 kb of DNA 5' to the msmE gene (SEQ ID NO:1) and is part of the 5' UTR of the msmE gene. It will be apparent that other sequence fragments from SEQ ID NO:173, longer or shorter than the foregoing sequence, e.g., including, but not limited to one or both of the cre sequences of SEQ ID NOS:174 and 175, SEQ ID NOS: 353 and 354, or with minor additions, deletions, or substitutions made thereto, as those that result from site-directed mutagenesis, as well as synthetically derived sequences, can be prepared which will also carry the FOS-related regulatory element, all of which are included within the present invention.

In one preferred embodiment of the invention, the isolated DNA encoding the regulatory element has the sequence given as SEQ ID NO:173, 353 or 354. In other preferred embodiments, the sequence of the isolated DNA encoding the regulatory element corresponds to a continuous segment of DNA within the DNA given as SEQ ID NO:173, 353 or 354, including but not limited to the continuous segment given as nucleotides 1 to 249 of SEQ ID NO:173, 1 to 204 of SEQ ID NO:353, and 1 to 198 of SEQ ID NO:354. Nucleic acid molecules that are fragments of a promoter or regulatory nucleotide sequence comprise at least 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 nucleotides, or up to the number of nucleotides present in the full-length regulatory nucleotide sequence disclosed herein (i.e., 249 for SEQ ID NO:173, 204 for SEQ ID NO:353, and 198 for SEQ ID NO:354). Fragments of a promoter sequence that retain their regulatory activity comprise at least 30, 35, 40 contiguous nucleotides, preferably at least 50 contiguous nucleotides, more preferably at least 75 contiguous nucleotides, still more preferably at least 100 contiguous nucleotides of the particular promoter or regulatory nucleotide sequence disclosed herein. Preferred fragment lengths depend upon the objective and will also vary depending upon the particular promoter or regulatory sequence.

The nucleotides of such fragments will usually comprise the TATA recognition sequence of the particular promoter sequence. Such fragments may be obtained by use of restriction enzymes to cleave the naturally occurring promoter nucleotide sequence disclosed herein; by synthesizing a nucleotide sequence from the naturally occurring sequence of the promoter DNA sequence; or may be obtained through the use of PCR technology. See, for example, Mullis et al. (1987) Methods Enzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Variants of these promoter fragments, such as those resulting from site-directed mutagenesis, are also encompassed by the compositions of the present invention.

Regulatory elements of the present invention include DNA molecules that regulate expression of nucleic acids encoding FOS-related molecules and have sequences that are substantially homologous to the DNA sequences comprising the regulatory elements disclosed herein, and particularly the regulatory elements disclosed herein as SEQ ID NOS:173, 353 and 354. Regulatory elements of the present invention also encompass DNA molecules that regulate expression of nucleic acids encoding FOS-related molecules and have sequences that are substantially homologous to DNA sequences located within SEQ ID NO:173, 353 and 354. This definition is intended to include natural variations in the DNA sequence comprising the regulatory element and sequences within SEQ ID NO:173, 353 and 354. As used herein, two regions of nucleotide sequences or polypeptides that are considered "substantially homologous" when they are at least about 50%, 60%, to 70%, generally at least about 75%, preferably at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

Regulatory elements include those which are at least about 75 percent homologous (and more preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% homologous) to the regulatory elements disclosed herein, in particular the regulatory element having the sequence given herein as SEQ ID NO:173, 353 and 354 and which are capable of regulating the transcription of nucleic acids encoding FOS-related molecules. Regulatory elements from other species also include those which are at least about 75 percent homologous (and more preferably 80%, 85%, 90% or even 95% homologous) to a continuous segment of the regulatory elements as defined herein as SEQ ID NO:173, 353 and 354, and which are capable of regulating the transcription of nucleic acids encoding FOS-related molecules, including but not limited to the continuous segment given herein as nucleotides 1 to 249 of SEQ ID NO:173, nucleotides 1 to 204 of SEQ ID NO:353, and nucleotides 1 to 198 of SEQ ID NO:354.

The present invention also provides recombinant DNAs comprising a regulatory element operably associated with heterologous DNA. The regulatory element is operably associated with the heterologous DNA such that the regulatory element is functionally linked to the heterologous DNA, and can thereby alter transcription of the heterologous DNA. Typically, the regulatory element will be located 5' to the heterologous DNA, but it may also be located 3' to the heterologous DNA as long as it is operably associated therewith. There are no particular upper or lower limits as to the distance between the regulatory element and the heterologous DNA, as long as the two DNA segments are operably associated with each other.

The heterologous DNA segment may encode any protein or peptide which is desirably expressed by the host cell. Typically, the heterologous DNA includes regulatory segments necessary for the expression of the protein or peptide in the host cell (i.e, promoter elements). Suitable heterologous DNA may be of prokaryotic or eukaryotic origin. Illustrative proteins and peptides encoded by the heterologous DNAs of the present invention include enzymes, hormones, growth factors, and cytokines. Preferably, the heterologous DNA encodes a FOS-related protein.

Alternatively, the heterologous DNA can be used to express antisense RNAs. In general, "antisense" refers to the use of small, synthetic oligonucleotides to inhibit gene expression by inhibiting the function of the target mRNA containing the complementary sequence. Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). Gene expression is inhibited through hybridization to coding (sense) sequences in a specific mRNA target by hydrogen bonding according to Watson-Crick base pairing rules. The mechanism of antisense inhibition is that the exogenously applied oligonucleotides decrease the mRNA and protein levels of the target gene. Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). See also Helene, C. and Toulme, J., Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S., Ed., OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press:Boca Raton, Fla. (1987).

As described above for the FOS-related sequences, the regulatory nucleotide sequences of the invention can be used to isolate other homologous sequences in other species. In these techniques all or part of the known promoter is used as a probe, which selectively hybridizes to other promoters present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.

Sequence Identity

The FOS-related sequences are members of multiple families of molecules, with conserved functional features. By "family" is intended two or more proteins or nucleic acid molecules having sufficient nucleotide or amino acid sequence identity. A family that contains deeply divergent groups may be divided into subfamilies. A clan is a group of families that are thought to have common ancestry. Members of a clan often have a similar tertiary structure.

By "sequence identity" is intended the nucleotide or amino acid residues that are the same when aligning two sequences for maximum correspondence over a specified comparison window. By "comparison window" is intended a contiguous segment of the two nucleotide or amino acid sequences for optimal alignment, wherein the second sequence may contain additions or deletions (i.e., gaps) as compared to the first sequence. Generally, for nucleic acid alignments, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. For amino acid sequence alignments, the comparison window is at least 6 contiguous amino acids in length, and optionally can be 10, 15, 20, 30, or longer. Those of skill in the art understand that to avoid a high similarity due to inclusion of gaps, a gap penalty is typically introduced and is subtracted from the number of matches.

Family members may be from the same or different species, and can include homologues as well as distinct proteins. Often, members of a family display common functional characteristics. Homologues can be isolated based on their identity to the L. acidophilus FOS-related nucleic acid sequences disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below.

To determine the percent identity of two amino acid or nucleotide sequences, an alignment is performed. Percent identity of the two sequences is a function of the number of identical residues shared by the two sequences in the comparison window (i.e., percent identity=number of identical residues/total number of residues.times.100). In one embodiment, the sequences are the same length. Methods similar to those mentioned below can be used to determine the percent identity between two sequences. The methods can be used with or without allowing gaps. Alignment may also be performed manually be inspection.

When amino acid sequences differ in conservative substitutions, the percent identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are known in the art. Typically the conservative substitution is scored as a partial, rather than a full mismatch, thereby increasing the percentage sequence identity.

Mathematical algorithms can be used to determine the percent identity of two sequences. Non-limiting examples of mathematical algorithms are the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; and the search-for-local-alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.

Various computer implementations based on these mathematical algorithms have been designed to enable the determination of sequence identity. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 2 15:403 are based on the algorithm of Karlin and Altschul (1990) supra. Searches to obtain nucleotide sequences that are homologous to nucleotide sequences of the present invention can be performed with the BLASTN program, score=100, wordlength=12. To obtain amino acid sequences homologous to sequences encoding a protein or polypeptide of the current invention, the BLASTX program may be used, score=50, wordlength=3. Gapped alignments may be obtained by using Gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25:33 89. To detect distant relationships between molecules, PSI-BLAST can be used. See Altschul et al. (1997) supra. For all of the BLAST programs, the default parameters of the respective programs can be used. See the website at ncbi.nlm.nih.gov.

Another program that can be used to determine percent sequence identity is the ALIGN program (version 2.0), which uses the mathematical algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with this program when comparing amino acid sequences.

In addition to the ALIGN and BLAST programs, the BESTFIT, GAP, FASTA and TFASTA programs are part of the Wisconsin Genetics Software Package (available from Accelrys Inc., 9685 Scranton Rd., San Diego, Calif., USA), and can be used for performing sequence alignments. The preferred program is GAP version 10, which used the algorithm of Needleman and Wunsch (1970) supra. Unless otherwise stated the sequence identity values provided herein refer to those values obtained by using the GAP program with the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

Identification and Isolation of Homologous Sequences

FOS-related nucleotide sequences identified based on their sequence identity to the FOS-related nucleotide sequences set forth herein, or to fragments and variants thereof, are encompassed by the present invention. Methods such as PCR or hybridization can be used to identify sequences from a cDNA or genomic library, for example, that are substantially identical to the sequence of the invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY). Methods for construction of such cDNA and genomic libraries are generally known in the art and are also disclosed in the above reference.

In hybridization techniques, the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may consist of all or part of a known nucleotide sequence disclosed herein. In addition, they may be labeled with a detectable group such as .sup.32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known FOS-related nucleotide sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known FOS-related nucleotide sequence or encoded amino acid sequence can additionally be used. The hybridization probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 10, preferably about 20, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of a FOS-related nucleotide sequence of the invention or a fragment or variant thereof. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among FOS-related protein sequences. Preparation of probes for hybridization is generally known in the art and is disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by reference.

In one embodiment the entire nucleotide sequence encoding a FOS-related protein is used as a probe to identify novel FOS-related sequences and messenger RNAs. In another embodiment, the probe is a fragment of a nucleotide sequence disclosed herein. In some embodiments, the nucleotide sequence that hybridizes under stringent conditions to the probe can be at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or more nucleotides in length.

Substantially identical sequences will hybridize to each other under stringent conditions. By "stringent conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Generally, stringent conditions encompasses those conditions for hybridization and washing under which nucleotides having at least about 60%, 65%, 70%, preferably 75% sequence identity typically remain hybridized to each other. Stringent conditions are known in the art and can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6. Hybridization typically occurs for less than about 24 hours, usually about 4 to about 12 hours.

Stringent conditions are sequence-dependent and will differ in different circumstances. Full-length or partial nucleic acid sequences may be used to obtain homologues and orthologs encompassed by the present invention. By "orthologs" is intended genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.

When using probes, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides).

The post-hybridization washes are instrumental in controlling specificity. The two critical factors are ionic strength and temperature of the final wash solution. For the detection of sequences that hybridize to a full-length or approximately full-length target sequence, the temperature under stringent conditions is selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions would encompass temperatures in the range of 1.degree. C. to 20.degree. C. lower than the T.sub.m, depending on the desired degree of stringency as otherwise qualified herein. For DNA-DNA hybrids, the T.sub.m can be determined using the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6 (logM)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.

The ability to detect sequences with varying degrees of homology can be obtained by varying the stringency of the hybridization and/or washing conditions. To target sequences that are 100% identical (homologous probing), stringency conditions must be obtained that do not allow mismatching. By allowing mismatching of nucleotide residues to occur, sequences with a lower degree of similarity can be detected (heterologous probing). For every 1% of mismatching, the T.sub.m is reduced about 1.degree. C.; therefore, hybridization and/or wash conditions can be manipulated to allow hybridization of sequences of a target percentage identity. For example, if sequences with .gtoreq.90% sequence identity are preferred, the T.sub.m can be decreased by 10.degree. C. Two nucleotide sequences could be substantially identical, but fail to hybridize to each other under stringent conditions, if the polypeptides they encode are substantially identical. This situation could arise, for example, if the maximum codon degeneracy of the genetic code is used to create a copy of a nucleic acid.

Exemplary low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. PCR primers are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

Assays

Diagnostic assays to detect expression of the disclosed polypeptides and/or nucleic acid molecules as well as their disclosed activity in a sample are disclosed. An exemplary method for detecting the presence or absence of a disclosed nucleic acid or protein comprising the disclosed polypeptide in a sample involves obtaining a sample from a food/dairy/feed product, starter culture (mother, seed, bulk/set, concentrated, dried, lyophilized, frozen), cultured food/dairy/feed product, dietary supplement, bioprocessing fermentate, or a subject that has ingested a probiotic material, and contacting the sample with a compound or an agent capable of detecting the disclosed polypeptides or nucleic acids (e.g., an mRNA or genomic DNA comprising the disclosed nucleic acid or fragment thereof) such that the presence of the disclosed sequence is detected in the sample. Results obtained with a sample from the food, supplement, culture, product or subject may be compared to results obtained with a sample from a control culture, product or subject.

One agent for detecting the mRNA or genomic DNA comprising a disclosed nucleotide sequence is a labeled nucleic acid probe capable of hybridizing to the disclosed nucleotide sequence of the mRNA or genomic DNA. The nucleic acid probe can be, for example, a disclosed nucleic acid molecule, such as the nucleic acid of odd SEQ ID NOS:1-172, or a portion thereof, such as a nucleic acid molecule of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or genomic DNA comprising the disclosed nucleic acid sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein.

One agent for detecting a protein comprising a disclosed polypeptide sequence is an antibody capable of binding to the disclosed polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be used. The term "labeled," with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

The term "sample" is intended to include tissues, cells, and biological fluids present in or isolated from a subject, as well as cells from starter cultures or food products carrying such cultures, or derived from the use of such cultures. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA comprising a disclosed sequence in a sample both in vitro and in vivo. In vitro techniques for detection of mRNA comprising a disclosed sequence include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a protein comprising a disclosed polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of genomic DNA comprising the disclosed nucleotide sequences include Southern hybridizations. Furthermore, in vivo techniques for detection of a protein comprising a disclosed polypeptide include introducing into a subject a labeled antibody against the disclosed polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the sample contains protein molecules from a test subject that has consumed a probiotic material. Alternatively, the sample can contain mRNA or genomic DNA from a starter culture.

The invention also encompasses kits for detecting the presence of disclosed nucleic acids or proteins comprising disclosed polypeptides in a sample. Such kits can be used to determine if a microbe expressing a specific polypeptide of the invention is present in a food product or starter culture, or in a subject that has consumed a probiotic material. For example, the kit can comprise a labeled compound or agent capable of detecting a disclosed polypeptide or mRNA in a sample and means for determining the amount of a the disclosed polypeptide in the sample (e.g., an antibody that recognizes the disclosed polypeptide or an oligonucleotide probe that binds to DNA encoding a disclosed polypeptide, e.g., even SEQ ID NOS:1-172). Kits can also include instructions detailing the use of such compounds.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a disclosed polypeptide; and, optionally, (2) a second, different antibody that binds to the disclosed polypeptide or the first antibody and is conjugated to a detectable agent. For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, that hybridizes to a disclosed nucleic acid sequence or (2) a pair of primers useful for amplifying a disclosed nucleic acid molecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container, and all of the various containers are within a single package along with instructions for use.

In one embodiment, the kit comprises multiple probes in an array format, such as those described, for example, in U.S. Pat. Nos. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, herein incorporated by reference. Probes for use in the array may be synthesized either directly onto the surface of the array, as disclosed in PCT Publication No. WO 95/00530, or prior to immobilization onto the array surface (Gait, ed., Oligonucleotide synthesis a practical approach, IRL Press: Oxford, England, 1984). The probes may be immobilized onto the surface using techniques well known to one of skill in the art, such as those described in U.S. Pat. No. 5,412,087. Probes may be a nucleic acid or peptide sequence, preferably purified, or an antibody.

The arrays may be used to screen organisms, samples, or products for differences in their genomic, cDNA, polypeptide or antibody content, including the presence or absence of specific sequences or proteins, as well as the concentration of those materials. Binding to a capture probe is detected, for example, by signal generated from a label attached to the nucleic acid molecule comprising the disclosed nucleic acid sequence, a polypeptide comprising the disclosed amino acid sequence, or an antibody. The method can include contacting the molecule comprising the disclosed nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having a different plurality of capture probes. The results of each hybridization can be compared to analyze differences in expression between a first and second sample. The first plurality of capture probes can be from a control sample, e.g., a wild type lactic acid bacteria, or control subject, e.g., a food, dietary supplement, starter culture sample or a biological fluid. The second plurality of capture probes can be from an experimental sample, e.g., a mutant type lactic acid bacteria, or subject that has consumed a probiotic material, e.g., a starter culture sample or a biological fluid.

These assays may be especially useful in microbial selection and quality control procedures where the detection of unwanted materials is essential. The detection of particular nucleotide sequences or polypeptides may also be useful in determining the genetic composition of food, fermentation products, or industrial microbes, or microbes present in the digestive system of animals or humans that have consumed probiotics.

Antisense Nucleotide Sequences

The present invention also encompasses antisense nucleic acid molecules, i.e., molecules that are 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 FOS-related coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding a FOS-related protein. The noncoding regions are the 5' and 3' sequences that flank the coding region and are not translated into amino acids. Antisense nucleotide sequences are useful in disrupting the expression of the target gene. Antisense constructions having 70%, preferably 80%, more preferably 85% sequence identity to the corresponding sequence may be used.

Given the coding-strand sequence encoding a FOS-related protein disclosed herein (e.g., even SEQ ID NOS:1-172), 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 a FOS-related mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a FOS-related mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a FOS-related mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length, or it can be 100, 200 nucleotides, or greater in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation 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, including, but not limited to, for example e.g., phosphorothioate derivatives and acridine substituted nucleotides. 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).

An antisense nucleic acid molecule of the invention can be an .alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .beta.-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).

The invention also encompasses ribozymes, which are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave FOS-related mRNA transcripts to thereby inhibit translation of FOS-related mRNA. A ribozyme having specificity for a FOS-related-encoding nucleic acid can be designed based upon the nucleotide sequence of a FOS-related cDNA disclosed herein (e.g., odd SEQ ID NOS:1-172). See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742. Alternatively, FOS-related mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triple helical structures. For example, FOS-related gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the FOS-related protein (e.g., the FOS-related promoter and/or enhancers) to form triple helical structures that prevent transcription of the FOS-related gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807.

In some embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid-phase peptide synthesis protocols as described, for example, in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670.

PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA-directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996), supra); or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of a FOS-related molecule can be modified, e.g., to enhance their stability, specificity, or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra; Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

Fusion Proteins

The invention also includes FOS-related chimeric or fusion proteins. A FOS-related "chimeric protein" or "fusion protein" comprises a FOS-related polypeptide operably linked to a non-FOS-related polypeptide. A "FOS-related polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a FOS-related protein, whereas a "non-FOS-related polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially identical to the FOS-related protein, and which is derived from the same or a different organism. Within a FOS-related fusion protein, the FOS-related polypeptide can correspond to all or a portion of a FOS-related protein, preferably including at least one biologically active portion of a FOS-related protein. Within the fusion protein, the term "operably linked" is intended to indicate that the FOS-related polypeptide and the non-FOS-related polypeptide are fused in-frame to each other. The non-FOS-related polypeptide can be fused to the N-terminus or C-terminus of the FOS-related polypeptide.

Expression of the linked coding sequences results in two linked heterologous amino acid sequences which form the fusion protein. The carrier sequence (the non-FOS-related polypeptide) encodes a carrier polypeptide that potentiates or increases expression of the fusion protein in the bacterial host. The portion of the fusion protein encoded by the carrier sequence, i.e., the carrier polypeptide, may be a protein fragment, an entire functional moiety, or an entire protein sequence. The carrier region or polypeptide may additionally be designed to be used in purifying the fusion protein, either with antibodies or with affinity purification specific for that carrier polypeptide. Likewise, physical properties of the carrier polypeptide can be exploited to allow selective purification of the fusion protein.

Particular carrier polypeptides of interest include superoxide dismutase (SOD), maltose-binding protein (MBP), glutathione-S-transferase (GST), an N-terminal histidine (His) tag, and the like. This list is not intended to be limiting, as any carrier polypeptide that potentiates expression of the FOS-related protein as a fusion protein can be used in the methods of the invention.

In one embodiment, the fusion protein is a GST-FOS-related fusion protein in which the FOS-related sequences are fused to the C-terminus of the GST sequences. In another embodiment, the fusion protein is a FOS-related-immunoglobulin fusion protein in which all or part of a FOS-related protein is fused to sequences derived from a member of the immunoglobulin protein family. The FOS-related-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-FOS-related antibodies in a subject, to purify FOS-related ligands, and in screening assays to identify molecules that inhibit the interaction of a FOS-related protein with a FOS-related ligand.

In one embodiment of the invention, the fusion protein has the ability to modify the functional properties of a bacterial cell. By "functional properties" is intended the ability of a bacterium ability to perform certain non-native functions, such as those related to adhesion, immune stimulation, or lysis. The non-FOS-related protein may include, but is not limited to, an antibody, an enzyme, a vaccine antigen, a protein with bactericidal activity, or a protein with receptor-binding activity. By "bactericidal activity" is intended the ability to kill one or more bacteria. By "receptor-binding activity" is intended the ability to bind to a receptor on a cell membrane, cell surface, or in solution. Methods to assess the ability of a fusion protein expressed on the surface of gram-positive bacteria to be used as a vaccine are known in the art (see, for example, Fischetti et al. (1996) Curr. Opin. Biotechnol. 7:659-666; Pouwels et al. (1998) Int. J. Food Microbiol. 41:155-167).

One of skill in the art will recognize that the particular carrier polypeptide is chosen with the purification scheme in mind. For example, His tags, GST, and maltose-binding protein represent carrier polypeptides that have readily available affinity columns to which they can be bound and eluted. Thus, where the carrier polypeptide is an N-terminal His tag such as hexahistidine (His.sub.6 tag), the FOS-related fusion protein can be purified using a matrix comprising a metal-chelating resin, for example, nickel nitrilotriacetic acid (Ni-NTA), nickel iminodiacetic acid (Ni-IDA), and cobalt-containing resin (Co-resin). See, for example, Steinert et al. (1997) QIAGEN News 4:11-15, herein incorporated by reference in its entirety. Where the carrier polypeptide is GST, the FOS-related fusion protein can be purified using a matrix comprising glutathione-agarose beads (Sigma or Pharmacia Biotech); where the carrier polypeptide is a maltose-binding protein (MBP), the FOS-related fusion protein can be purified using a matrix comprising an agarose resin derivatized with amylose.

Preferably, a chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences may be ligated together in-frame, or the fusion gene can be synthesized, such as with automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley-Interscience, NY). Moreover, a FOS-related-protein-encoding nucleic acid can be cloned into a commercially available expression vector such that it is linked in-frame to an existing fusion moiety.

The fusion protein expression vector is typically designed for ease of removing the carrier polypeptide to allow the FOS-related protein to retain the native biological activity associated with it. Methods for cleavage of fusion proteins are known in the art. See, for example, Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.). Chemical cleavage of the fusion protein can be accomplished with reagents such as cyanogen bromide, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine, hydroxylamine, or low pH. Chemical cleavage is often accomplished under denaturing conditions to cleave otherwise insoluble fusion proteins.

Where separation of the FOS-related polypeptide from the carrier polypeptide is desired and a cleavage site at the junction between these fused polypeptides is not naturally occurring, the fusion construct can be designed to contain a specific protease cleavage site to facilitate enzymatic cleavage and removal of the carrier polypeptide. In this manner, a linker sequence comprising a coding sequence for a peptide that has a cleavage site specific for an enzyme of interest can be fused in-frame between the coding sequence for the carrier polypeptide (for example, MBP, GST, SOD, or an N-terminal His tag) and the coding sequence for the FOS-related polypeptide. Suitable enzymes having specificity for cleavage sites include, but are not limited to, factor Xa, thrombin, enterokinase, remin, collagenase, and tobacco etch virus (TEV) protease. Cleavage sites for these enzymes are well known in the art. Thus, for example, where factor Xa is to be used to cleave the carrier polypeptide from the FOS-related polypeptide, the fusion construct can be designed to comprise a linker sequence encoding a factor Xa-sensitive cleavage site, for example, the sequence IEGR (see, for example, Nagai and Thogersen (1984) Nature 309:810-812, Nagai and Thogersen (1987) Meth. Enzymol. 153:461-481, and Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309-319, herein incorporated by reference). Where thrombin is to be used to cleave the carrier polypeptide from the FOS-related polypeptide, the fusion construct can be designed to comprise a linker sequence encoding a thrombin-sensitive cleavage site, for example the sequence LVPRGS or VIAGR (see, for example, Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309-319, and Hong et al. (1997) Chin. Med. Sci. J. 12(3):143-147, respectively, herein incorporated by reference). Cleavage sites for TEV protease are known in the art. See, for example, the cleavage sites described in U.S. Pat. No. 5,532,142, herein incorporated by reference in its entirety. See also the discussion in Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Chapter 16.

Antibodies

An isolated polypeptide of the present invention can be used as an immunogen to generate antibodies that specifically bind FOS-related proteins, or stimulate production of antibodies in vivo. The full-length FOS-related protein can be used as an immunogen or, alternatively, antigenic peptide fragments of FOS-related proteins as described herein can be used. The antigenic peptide of an FOS-related protein comprises at least 8, preferably 10, 15, 20, or 30 amino acid residues of the amino acid sequence shown in even SEQ ID NOS:1-172 and encompasses an epitope of an FOS-related protein such that an antibody raised against the peptide forms a specific immune complex with the FOS-related protein. Preferred epitopes encompassed by the antigenic peptide are regions of a FOS-related protein that are located on the surface of the protein, e.g., hydrophilic regions.

Recombinant Expression Vectors

The nucleic acid molecules of the present invention may be included in vectors, preferably expression vectors. "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Expression vectors include one or more regulatory sequences and direct the expression of genes to which they are operably linked. By "operably linked" is intended that the nucleotide sequence of interest is linked to the regulatory sequence(s) such that expression of the nucleotide sequence is allowed (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" or "regulatory element" is intended to include controllable transcriptional promoters, operators, enhancers, transcriptional terminators, and other expression control elements such as translational control sequences (e.g., Shine-Dalgamo consensus sequence, initiation and termination codons). These regulatory sequences will differ, for example, depending on the host cell being used.

The vectors can be autonomously replicated in a host cell (episomal vectors), or may be integrated into the genome of a host cell, and replicated along with the host genome (non-episomal mammalian vectors). Integrating ve