Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans
Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% ho...
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Veröffentlicht in: | The Journal of biological chemistry 1997-11, Vol.272 (45), p.28652-28659 |
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description | Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels inEscherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities. |
doi_str_mv | 10.1074/jbc.272.45.28652 |
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Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels inEscherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.272.45.28652</identifier><identifier>PMID: 9353332</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Base Sequence ; Caenorhabditis elegans - enzymology ; Caenorhabditis elegans - genetics ; Caenorhabditis elegans Proteins - biosynthesis ; Caenorhabditis elegans Proteins - chemistry ; Caenorhabditis elegans Proteins - genetics ; Chromosome Mapping ; Cloning, Molecular ; Exons ; Introns ; Isoelectric Focusing ; Molecular Sequence Data ; Superoxide Dismutase - biosynthesis ; Superoxide Dismutase - chemistry ; Superoxide Dismutase - genetics</subject><ispartof>The Journal of biological chemistry, 1997-11, Vol.272 (45), p.28652-28659</ispartof><rights>1997 © 1997 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c513t-b9c60dc5d0aa2f8ae7ec30c15ec189b0a7ed1a5aa52a87a5ba6d9273dd8cb43d3</citedby><cites>FETCH-LOGICAL-c513t-b9c60dc5d0aa2f8ae7ec30c15ec189b0a7ed1a5aa52a87a5ba6d9273dd8cb43d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,782,786,27931,27932</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9353332$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hunter, Thérèse</creatorcontrib><creatorcontrib>Bannister, William H.</creatorcontrib><creatorcontrib>Hunter, Gary J.</creatorcontrib><title>Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels inEscherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Caenorhabditis elegans - enzymology</subject><subject>Caenorhabditis elegans - genetics</subject><subject>Caenorhabditis elegans Proteins - biosynthesis</subject><subject>Caenorhabditis elegans Proteins - chemistry</subject><subject>Caenorhabditis elegans Proteins - genetics</subject><subject>Chromosome Mapping</subject><subject>Cloning, Molecular</subject><subject>Exons</subject><subject>Introns</subject><subject>Isoelectric Focusing</subject><subject>Molecular Sequence Data</subject><subject>Superoxide Dismutase - biosynthesis</subject><subject>Superoxide Dismutase - chemistry</subject><subject>Superoxide Dismutase - genetics</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUU1v1DAUtFBRuy3cuVTyAXFqFn_EG4dbFVpAKuJAkbhZL_bLxlUSL3ZCC78ew644IKG-y5PemxmNZgh5wdmas6p8fdfatajEulRroTdKPCErzrQspOJfj8iKMcGLWih9Qk5TumN5ypofk-NaKimlWJGhGcLkp-0FvXrYRUzJh-mCwuRo00MEO2P0P2HOVxo6ensf6EeYtjBhQvp52WEMD94hfevTuMyQMNEuhpE2gFOIPbTOzz5RHDBz0jPytIMh4fPDPiNfrq9um_fFzad3H5rLm8IqLueire2GOascAxCdBqzQSma5Qst13TKo0HFQAEqArkC1sHG1qKRz2raldPKMvNrr7mL4tmCazeiTxWHIvsOSTFVLrUpdPwrkGyFKXaoMZHugjSGliJ3ZRT9C_GE4M7-bMLkJk5swpTJ_msiU84P20o7o_hIO0ef_y_2_99v-3kc0rQ-2x_FfmTd7GObAvnuMJlmPk0WXKXY2Lvj_e_gFVw-mTQ</recordid><startdate>19971107</startdate><enddate>19971107</enddate><creator>Hunter, Thérèse</creator><creator>Bannister, William H.</creator><creator>Hunter, Gary J.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>19971107</creationdate><title>Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans</title><author>Hunter, Thérèse ; Bannister, William H. ; Hunter, Gary J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-b9c60dc5d0aa2f8ae7ec30c15ec189b0a7ed1a5aa52a87a5ba6d9273dd8cb43d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Caenorhabditis elegans - enzymology</topic><topic>Caenorhabditis elegans - genetics</topic><topic>Caenorhabditis elegans Proteins - biosynthesis</topic><topic>Caenorhabditis elegans Proteins - chemistry</topic><topic>Caenorhabditis elegans Proteins - genetics</topic><topic>Chromosome Mapping</topic><topic>Cloning, Molecular</topic><topic>Exons</topic><topic>Introns</topic><topic>Isoelectric Focusing</topic><topic>Molecular Sequence Data</topic><topic>Superoxide Dismutase - biosynthesis</topic><topic>Superoxide Dismutase - chemistry</topic><topic>Superoxide Dismutase - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hunter, Thérèse</creatorcontrib><creatorcontrib>Bannister, William H.</creatorcontrib><creatorcontrib>Hunter, Gary J.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hunter, Thérèse</au><au>Bannister, William H.</au><au>Hunter, Gary J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1997-11-07</date><risdate>1997</risdate><volume>272</volume><issue>45</issue><spage>28652</spage><epage>28659</epage><pages>28652-28659</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels inEscherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>9353332</pmid><doi>10.1074/jbc.272.45.28652</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Amino Acid Sequence Animals Base Sequence Caenorhabditis elegans - enzymology Caenorhabditis elegans - genetics Caenorhabditis elegans Proteins - biosynthesis Caenorhabditis elegans Proteins - chemistry Caenorhabditis elegans Proteins - genetics Chromosome Mapping Cloning, Molecular Exons Introns Isoelectric Focusing Molecular Sequence Data Superoxide Dismutase - biosynthesis Superoxide Dismutase - chemistry Superoxide Dismutase - genetics |
title | Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans |
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