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(12) United States Patent (10) Patent No.: US 6,632,937 B1 Swanson Et Al

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(12) United States Patent (10) Patent No.: US 6,632,937 B1 Swanson Et Al USOO6632937B1 (12) United States Patent (10) Patent No.: US 6,632,937 B1 Swanson et al. (45) Date of Patent: Oct. 14, 2003 (54) NUCLEICACIDS AND PROTEINS FROM Preston et al., “A pSychrophilic crenarchaeon inhabits a CENARCHAEUM SYMBIOSUM marine Sponge: Cenarchaeum Symbiosum gen. nov., Sp. nov.” Proc. Natl. Acad. Sci. USA 93:6241-6246 (Jun. (75) Inventors: Ronald V. Swanson, La Jolla, CA 1996); & “Cenarchaeum symbiosum small subunit riboso (US); Robert A. Feldman, Poway, CA mal RNA gene Sequence,' Database Genbank Accession (US); Christa Schleper, Darmstadt No. U51469 (Aug. 13, 1996) XP002130621 (abstract). (DE) Schleper et al., “Genomic analysis reveals chromosomal (73) Assignee: Diversa Corporation, San Diego, CA variation in natural populations of the uncultured psychro (US) philic archaeon Cenarchaeum SymbioSum,” J. Bacteriol. 180(19):5003–5009, Database EMBL XP-002136935; & (*) Notice: Subject to any disclaimer, the term of this “Cenarchaeum Symbiosum strain B. Database EMBL patent is extended or adjusted under 35 Accession No. AF083072 (Sep. 23, 1998) XP002136935 U.S.C. 154(b) by 0 days. (abstract). (21) Appl. No.: 09/408,020 Schleper et al., “Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum (22) Filed: Sep. 29, 1999 Symbiosum,” Journal of Bacteriology 179(24):7803–7811 (Dec. 1997) XP00872756; & “Cenarchaeum symbiosum Related U.S. Application Data DNA polymerase gene.” Database Genbank Accession No. (60) Provisional application No. 60/102.294, filed on Sep. 29, 1998. AF028831 (Jan. 6, 1998) XP002130624 (abstract). Stein et al., “Characterization of Uncultivated Prokaryotes: (51) Int. Cl." ......................... C07H2/04; CO7K 14/195 Isolation and Analysis of a 40-Kilobase-Pair Genome Frag (52) U.S. Cl. ....................................... 536/23.7; 530/350 ment from a Planktonic Marine Archaeon,” Journal of (58) Field of Search .......................... 536/23.7; 530/350 Bacteriology 178(3):591–599 (Feb. 1996) XP002050143. Suzuki et al., “Pig gad65 mRNA forglutamic acid decar (56) References Cited boxylase,” Database EMBL Accession No. D31848 (Apr. PUBLICATIONS 28, 1995) XP002130623 (abstract). X57760, A. Fainsod Jul. 8, 1992.* AF016442, Wilson et al., Aug. 7, 1997.* * cited by examiner AF083071, Schleper et al., Oct. 6, 1998.* Baker et al. Protein Structure prediction and structural Primary Examiner Ardin H. Marschel genomics. Science. (Oct. 5, 2001) vol. 294, pp. 93-96.* Assistant Examiner Marjorie A. Moran Ainsworth et al., “T. aestivum AGP-S mRNA. Database EMBL Accession No. X66080 (May 13, 1992) (74) Attorney, Agent, or Firm-Fish & Richardson P.C. XP002136936 (abstract). (57) ABSTRACT DeLong et al., “Application of Gebomics for Understanding the Evolution of Hyperthermophilic and Nonthermophilic The present application relates to nucleic acids and polypep Crenarchaeota,” Biological Bulletin 196(3):363-365 (Jun. tides from Cenarchaeum Symbiosum. Methods of making 1999). the polypeptides and antibodies against the polypeptides are Krejci et al., “Rattus norvegicus acetylcholinesterase-asso also described. ciated collagen,” Database EMBL Accession No. AF007583 (Nov. 1, 1997) XP002130622 (abstract). 2 Claims, 7 Drawing Sheets U.S. Patent Oct. 14, 2003 Sheet 2 of 7 US 6,632,937 B1 92 /2 82 62 U.S. Patent Oct. 14, 2003 Sheet 3 of 7 US 6,632,937 B1 U.S. Patent Oct. 14, 2003 Sheet 4 of 7 US 6,632,937 B1 D 125A 1258 125C E" E" E. 100 COMPUTER SYSTEM PROCESSOR 115 NTERNA STORAGE 118 120 DATA RETREVING DISPLAY DEVICE FIGURE 3 U.S. Patent Oct. 14, 2003 Sheet 5 of 7 US 6,632,937 B1 20 NY START 2O2 STORE NEW SECRUENCE TO A MEMORY OPEN DATABASE OF SEOUENCES 206 READ FRS SEQUENCE NOAABASE 210 PERFORM COMPARISON OF NEW SEQUENCE AND STORED SECUENCE 212 YES 214 DSPLAY STORED SEQUENCE NAME TO USER NO 224 GO TO NEX SECUENCEN DAABASE MORE SECUENCES IN YES DATABASE NO 220 FIGURE 4 U.S. Patent Oct. 14, 2003 Sheet 6 of 7 US 6,632,937 B1 252 250 START 254 STORE A FIRST SEOUENCE TO A MEMORY 256 STORE A SECOND SECUENCE TO A MEMORY 260 READ FIRST CHARACTER OF FIRST SECUENCE 262 READ FRST CHARACTER OF SECOND SECUENCE 264 <sses READ NEXT CHARACTER OF FIRST AND SECOND YES SEQUENCES NO CHARACTERS TO NO 276 DSPLAY HOMOLOGY LEVEL BE WEEN THE FIRST AND SECOND SECUENCES FIGURE 5 U.S. Patent Oct. 14, 2003 Sheet 7 of 7 US 6,632,937 B1 302 300 NY 304 STORE AFRST SECUENCE TO MEMORY 306 OPEN DATABASE OF SECRUENCE FEATURES 308 READ FRS FEAURE FROM DAABASE 310 COMPARE FEATUREA TRIBUTES WITH THE FIRST SECUENCE 316 DSPLAY FOUND FEATURE TO THE USER 326 NO READ NEXT FSATURE IN DATABASE MORE FEATURES IN YES DAABASE NO 324 FIGURE 6 US 6,632,937 B1 1 2 NUCLEC ACIDS AND PROTEINS FROM 1995. Molecular phylogenetic analysis of a soil microbial CENARCHAEUMSYMBIOSUM community. Eur: J. Soil Sci. 46, 415-421; Hershberger, K. L. et al. 1996. Wide diversity of Crenarchaeota. Nature 384, RELATED APPLICATIONS 420; MacGregor, B.J. 1997. Crenarchaeota in Lake Michi gan sediment. Appl. Env, Microb. 63, 1178–1181 et al.; The present application claims benefit of U.S. Provisional Schleper, C.et al. 1997. Recovery of crenarchaeotal riboso Patent Application Serial No. 60/102,294, filed Sep. 29, mal DNA sequences from freshwater-lake Sediments. Appl. 1998, the disclosure of which is incorporated herein by Env: Microb. 63, 321-323) The ecological distribution of reference in its entirety. these organisms was initially Surprising, Since their closest BACKGROUND OF THE INVENTION cultivated relatives are all thermophilic or hyperthermo philic. No representative of this new archaeal group has yet The identification and characterization of organisms been obtained in pure culture, So the phenotypic and meta which inhabit a diverse range of ecosystems leads to a bolic properties of these organisms, as well as their impact greater understanding of the operation of Such ecosystems. on the environment and global nutrient cycling, remain In addition, because the physiology of Such organisms is 15 unknown. Since growth temperature and habitat character adapted to function in the particular habitat which the istics vary So widely between non-thermophilic and the organism inhabits, the enzymes which carry out the organ hyperthermophilic Creanarchaeota, these groups are likely ism's physiological processes may possess characteristics to differ greatly with respect to their specific physiology and which provide advantages when they are utilized in thera metabolism. peutic procedures, industrial applications, or research appli To gain a better perspective on the genetic and physi cations. Furthermore, by determining the Sequences of these ological characteristics of non-thermophilic crenarchaeotes, organisms genes, insight into their biochemical pathways a genomic Study of Cenarchaeum Symbiosum was begun. and processes may be gained without the necessity of This archaeon lives in Specific association with the marine culturing the organisms in the laboratory, thereby enabling Sponge Axinella mexicana off the coast of California, allow the physiological characterization of organisms which are 25 ing access to relatively large amounts of biomass from this recalcitrant to growth in the laboratory. species. (Preston, C. M. et al. 1996. A psychrophilic crenar Molecular phylogenetic Surveys have recently revealed an chaeon inhabits a marine Sponge: Cenarchaeum Symbiosum ecologically widespread Crenarchaeal group that inhabits gen. nov., sp. nov. Proc. Natl. Acad. Sci. USA 93, cold and temperate terrestrial and marine environments. To 6241-6246) The approach taken herein differs in several date these organisms have resisted isolation in pure culture, respects from now Standard genomic characterization of So their phenotypic and genotypic characteristics remain cultivated organisms, and also from comparable Studies of largely unknown. In order to characterize the physiology of uncultivated obligate parasites or Symbionts. C. Symbiosum these archaea, to develop methodological approaches for has not been completely physically separated from the characterizing uncultivated microorganisms and identifying tissueS of its metazoan host. Therefore, its genetic material their presence in a Sample, and to identify enzymes produced 35 needs to be identified within the context of complex by these archae which may be useful in therapeutic, genomic libraries that contain Significant amounts of eucary industrial, or laboratory applications, genomic analyses of otic DNA, as well as DNA derived from members of the non-thermophilic crenarchaeote Cenarchaeum Symbio Bacteria. Sum was undertaken. Molecular phylogenetic Surveys of mixed microbial Non-thermophilic Crenarchaeota are one of the more 40 populations have revealed the existence of many new lin abundant, widespread and frequently recovered prokaryotic eages undetected by classical microbiological approaches. groups revealed by molecular phylogenetic approaches. (DeLong, E. F. 1997. Marine microbial diversity: the tip of These microorganisms were originally detected in high the iceberg. Tibtech 15, 2-9.; Pace, N. R. 1997. A molecular abundance in temperate ocean waters and polar Seas. view of microbial diversity and the biosphere. Science 276, (DeLong, E. F. 1992. Archaea in coastal marine environ 45 734–740) Furthermore, quantitative rRNA hybridization ments. Proc. Natl. Acad. Sci. 89,5685–5689; DeLong, E. F experiments demonstrate that Some of these novel prokary et al. 1994. High abundance of Archaea in Antarctic marine otic groups represent major components of natural microbial picoplankton. Nature 371, 695-697;
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