Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis
Identifieur interne : 000186 ( Istex/Corpus ); précédent : 000185; suivant : 000187Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis
Auteurs : K. J. Laing ; J. Holland ; S. Bonilla ; C. Cunningham ; C. J. SecombesSource :
- Developmental and Comparative Immunology [ 0145-305X ] ; 2000.
English descriptors
- KwdEn :
Abstract
The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.
Url:
DOI: 10.1016/S0145-305X(00)00061-6
Links to Exploration step
ISTEX:B557C68F569F0AD361DAD61798D70DC248EEE113Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title><author><name sortKey="Laing, K J" sort="Laing, K J" uniqKey="Laing K" first="K. J." last="Laing">K. J. Laing</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>1 Equal contribution to the work.</mods:affiliation></affiliation></author><author><name sortKey="Holland, J" sort="Holland, J" uniqKey="Holland J" first="J." last="Holland">J. Holland</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>1 Equal contribution to the work.</mods:affiliation></affiliation></author><author><name sortKey="Bonilla, S" sort="Bonilla, S" uniqKey="Bonilla S" first="S." last="Bonilla">S. Bonilla</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>2 Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</mods:affiliation></affiliation></author><author><name sortKey="Cunningham, C" sort="Cunningham, C" uniqKey="Cunningham C" first="C." last="Cunningham">C. Cunningham</name><affiliation><mods:affiliation>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</mods:affiliation></affiliation></author><author><name sortKey="Secombes, C J" sort="Secombes, C J" uniqKey="Secombes C" first="C. J." last="Secombes">C. J. Secombes</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: c.secombes@abdn.ac.uk</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:B557C68F569F0AD361DAD61798D70DC248EEE113</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1016/S0145-305X(00)00061-6</idno><idno type="url">https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">000186</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title><author><name sortKey="Laing, K J" sort="Laing, K J" uniqKey="Laing K" first="K. J." last="Laing">K. J. Laing</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>1 Equal contribution to the work.</mods:affiliation></affiliation></author><author><name sortKey="Holland, J" sort="Holland, J" uniqKey="Holland J" first="J." last="Holland">J. Holland</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>1 Equal contribution to the work.</mods:affiliation></affiliation></author><author><name sortKey="Bonilla, S" sort="Bonilla, S" uniqKey="Bonilla S" first="S." last="Bonilla">S. Bonilla</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>2 Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</mods:affiliation></affiliation></author><author><name sortKey="Cunningham, C" sort="Cunningham, C" uniqKey="Cunningham C" first="C." last="Cunningham">C. Cunningham</name><affiliation><mods:affiliation>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</mods:affiliation></affiliation></author><author><name sortKey="Secombes, C J" sort="Secombes, C J" uniqKey="Secombes C" first="C. J." last="Secombes">C. J. Secombes</name><affiliation><mods:affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: c.secombes@abdn.ac.uk</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Developmental and Comparative Immunology</title><title level="j" type="abbrev">DCI</title><idno type="ISSN">0145-305X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">25</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="303">303</biblScope><biblScope unit="page" to="312">312</biblScope></imprint><idno type="ISSN">0145-305X</idno></series><idno type="istex">B557C68F569F0AD361DAD61798D70DC248EEE113</idno><idno type="DOI">10.1016/S0145-305X(00)00061-6</idno><idno type="PII">S0145-305X(00)00061-6</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0145-305X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Caspase 6</term><term>Cortisol</term><term>Expression</term><term>LPS, lipopolysaccharide</term><term>ORF, open reading frame</term><term>PCR, polymerase chain reaction</term><term>RT-PCR</term><term>Rainbow trout</term><term>Stress</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>K.J. Laing</name><affiliations><json:string>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</json:string><json:string>1 Equal contribution to the work.</json:string></affiliations></json:item><json:item><name>J. Holland</name><affiliations><json:string>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</json:string><json:string>1 Equal contribution to the work.</json:string></affiliations></json:item><json:item><name>S. Bonilla</name><affiliations><json:string>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</json:string><json:string>2 Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</json:string></affiliations></json:item><json:item><name>C. Cunningham</name><affiliations><json:string>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</json:string></affiliations></json:item><json:item><name>C.J. Secombes</name><affiliations><json:string>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</json:string><json:string>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</json:string><json:string>E-mail: c.secombes@abdn.ac.uk</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Caspase 6</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Rainbow trout</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Expression</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RT-PCR</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Cortisol</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Stress</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>LPS, lipopolysaccharide</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ORF, open reading frame</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PCR, polymerase chain reaction</value></json:item></subject><language><json:string>eng</json:string></language><abstract>The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.</abstract><qualityIndicators><score>6.409</score><pdfVersion>1.2</pdfVersion><pdfPageSize>544 x 743 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>9</keywordCount><abstractCharCount>1330</abstractCharCount><pdfWordCount>3985</pdfWordCount><pdfCharCount>24804</pdfCharCount><pdfPageCount>10</pdfPageCount><abstractWordCount>202</abstractWordCount></qualityIndicators><title>Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title><pii><json:string>S0145-305X(00)00061-6</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>25</volume><pii><json:string>S0145-305X(00)X0039-0</json:string></pii><pages><last>312</last><first>303</first></pages><issn><json:string>0145-305X</json:string></issn><issue>4</issue><genre></genre><language><json:string>unknown</json:string></language><title>Developmental and Comparative Immunology</title><publicationDate>2001</publicationDate></host><categories><wos><json:string>IMMUNOLOGY</json:string><json:string>ZOOLOGY</json:string></wos></categories><publicationDate>2000</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1016/S0145-305X(00)00061-6</json:string></doi><id>B557C68F569F0AD361DAD61798D70DC248EEE113</id><fulltext><json:item><original>true</original><mimetype>application/pdf</mimetype><extension>pdf</extension><uri>https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/fulltext/pdf</uri></json:item><json:item><original>false</original><mimetype>application/zip</mimetype><extension>zip</extension><uri>https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>ELSEVIER</p></availability><date>2001</date></publicationStmt><notesStmt><note type="content">Fig. 1: Cloning strategy used to obtain the full-length rainbow trout caspase 6 sequence. F, forward primers; R, reverse primers. Products obtained with different primer combinations are shown as bars.</note><note type="content">Fig. 2: Compiled full length trout caspase 6 sequence. Features in bolt include the start and stop codons, putative aspartic acid cut sites, and potential glycosylation sites. A potential integrin binding site is boxed. The linker region is underlined and the propiece is double underlined.</note><note type="content">Fig. 3: Alignment of the predicted trout caspase 6 translation with other known caspase 6 genes. Identical (*) and similar (. or:) residues identified by the clustal program are indicated. Boxed features include the caspase family signature and the active site pentapeptide. Residues involved in the P1 carboxylate binding pocket are in bold, the propiece is in italics, and residues involved in catalysis are in bold and indicated with an arrow.</note><note type="content">Fig. 4: Unrooted phylogenetic tree showing the relationship between the trout caspase 6 amino acid sequence with other known caspase and caspase-related sequences. The tree was constructed by the ‘neighbour-joining’ method using the clustal w and phylip packages, and was bootstrapped 1000 times. C=caspase; Dr/Dros=Drosophila; CE=C. elegans; SF=Spodoptera frugiperda.</note><note type="content">Fig. 5: Analysis of the sites of expression of trout caspase 6. RT-PCR was performed with cDNA from a variety of tissues with trout β-actin primers or trout caspase 6 primers. 1=brain; 2=blood; 3=gill; 4=liver; 5=head kidney; 6=spleen.</note><note type="content">Fig. 6: β-actin and caspase 6 products obtained by RT-PCR, using head kidney leucocyte cDNA as template, with different cycle numbers.</note><note type="content">Fig. 7: Effect of LPS and cortisol addition on expression of caspase 6 and β-actin in cultured head kidney leucocytes. Semi-quantitative RT-PCR was performed with cDNA from head kidney leucocytes cultured with LPS (left) or varying concentrations of cortisol (right), prior to isolation of RNA. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below).</note><note type="content">Fig. 8: Effect of co-incubation with cortisol and LPS on caspase 6 expression, in the absence (left) and presence (right) of RU486. Semi-quantitative RT-PCR was performed with cDNA from head kidney leucocytes pretreated with cortisol in the presence of RU486 for 2h prior to addition of LPS and isolation of RNA 4h later. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below).</note><note type="content">Fig. 9: Semi-quantitative RT-PCR was performed with head kidney cDNA from control trout or trout subjected to a confinement stress for 1, 3 or 6 days. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below), for two representative fish.</note><note type="content">Table 1: Homology of rainbow trout caspase 6 nucleotide and predicted amino acid sequence with other known caspase genes</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title><author><persName><forename type="first">K.J.</forename><surname>Laing</surname></persName><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>1 Equal contribution to the work.</affiliation></author><author><persName><forename type="first">J.</forename><surname>Holland</surname></persName><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>1 Equal contribution to the work.</affiliation></author><author><persName><forename type="first">S.</forename><surname>Bonilla</surname></persName><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>2 Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</affiliation></author><author><persName><forename type="first">C.</forename><surname>Cunningham</surname></persName><affiliation>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</affiliation></author><author><persName><forename type="first">C.J.</forename><surname>Secombes</surname></persName><email>c.secombes@abdn.ac.uk</email><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</affiliation></author></analytic><monogr><title level="j">Developmental and Comparative Immunology</title><title level="j" type="abbrev">DCI</title><idno type="pISSN">0145-305X</idno><idno type="PII">S0145-305X(00)X0039-0</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">25</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="303">303</biblScope><biblScope unit="page" to="312">312</biblScope></imprint></monogr><idno type="istex">B557C68F569F0AD361DAD61798D70DC248EEE113</idno><idno type="DOI">10.1016/S0145-305X(00)00061-6</idno><idno type="PII">S0145-305X(00)00061-6</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Caspase 6</term></item><item><term>Rainbow trout</term></item><item><term>Expression</term></item><item><term>RT-PCR</term></item><item><term>Cortisol</term></item><item><term>Stress</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>LPS, lipopolysaccharide</term></item><item><term>ORF, open reading frame</term></item><item><term>PCR, polymerase chain reaction</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000-10-20">Registration</change><change when="2000-10-06">Modified</change><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><original>false</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="gr8"></istex:entity><istex:entity SYSTEM="gr9" NDATA="IMAGE" name="gr9"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>DCI</jid><aid>405</aid><ce:pii>S0145-305X(00)00061-6</ce:pii><ce:doi>10.1016/S0145-305X(00)00061-6</ce:doi><ce:copyright type="full-transfer" year="2001">Elsevier Science Ltd</ce:copyright></item-info><head><ce:title>Cloning and sequencing of caspase 6 in rainbow trout, <ce:italic>Oncorhynchus mykiss</ce:italic>, and analysis of its expression under conditions known to induce apoptosis</ce:title><ce:author-group><ce:author><ce:given-name>K.J.</ce:given-name><ce:surname>Laing</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>J.</ce:given-name><ce:surname>Holland</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>S.</ce:given-name><ce:surname>Bonilla</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>C.</ce:given-name><ce:surname>Cunningham</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>C.J.</ce:given-name><ce:surname>Secombes</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>c.secombes@abdn.ac.uk</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</ce:text></ce:correspondence><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para>Equal contribution to the work.</ce:note-para></ce:footnote><ce:footnote id="FN2"><ce:label>2</ce:label><ce:note-para>Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="21" month="1" year="2000"></ce:date-received><ce:date-revised day="6" month="10" year="2000"></ce:date-revised><ce:date-accepted day="20" month="10" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906<ce:hsp sp="0.25"></ce:hsp>bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword" xml:lang="en"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Caspase 6</ce:text></ce:keyword><ce:keyword><ce:text>Rainbow trout</ce:text></ce:keyword><ce:keyword><ce:text>Expression</ce:text></ce:keyword><ce:keyword><ce:text>RT-PCR</ce:text></ce:keyword><ce:keyword><ce:text>Cortisol</ce:text></ce:keyword><ce:keyword><ce:text>Stress</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr" xml:lang="en"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>LPS, lipopolysaccharide</ce:text></ce:keyword><ce:keyword><ce:text>ORF, open reading frame</ce:text></ce:keyword><ce:keyword><ce:text>PCR, polymerase chain reaction</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Cloning and sequencing of caspase 6 in rainbow trout,</title></titleInfo><name type="personal"><namePart type="given">K.J.</namePart><namePart type="family">Laing</namePart><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>1 Equal contribution to the work.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">J.</namePart><namePart type="family">Holland</namePart><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>1 Equal contribution to the work.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">S.</namePart><namePart type="family">Bonilla</namePart><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>2 Present address: Department of Cell Biology, University of Murcia, 30100 Murcia, Spain.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C.</namePart><namePart type="family">Cunningham</namePart><affiliation>SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C.J.</namePart><namePart type="family">Secombes</namePart><affiliation>Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK</affiliation><affiliation>Corresponding author. Tel.: +44-1224-272872; fax: +44-1224-272396</affiliation><affiliation>E-mail: c.secombes@abdn.ac.uk</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><dateValid encoding="w3cdtf">2000-10-20</dateValid><dateModified encoding="w3cdtf">2000-10-06</dateModified><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.</abstract><note type="content">Fig. 1: Cloning strategy used to obtain the full-length rainbow trout caspase 6 sequence. F, forward primers; R, reverse primers. Products obtained with different primer combinations are shown as bars.</note><note type="content">Fig. 2: Compiled full length trout caspase 6 sequence. Features in bolt include the start and stop codons, putative aspartic acid cut sites, and potential glycosylation sites. A potential integrin binding site is boxed. The linker region is underlined and the propiece is double underlined.</note><note type="content">Fig. 3: Alignment of the predicted trout caspase 6 translation with other known caspase 6 genes. Identical (*) and similar (. or:) residues identified by the clustal program are indicated. Boxed features include the caspase family signature and the active site pentapeptide. Residues involved in the P1 carboxylate binding pocket are in bold, the propiece is in italics, and residues involved in catalysis are in bold and indicated with an arrow.</note><note type="content">Fig. 4: Unrooted phylogenetic tree showing the relationship between the trout caspase 6 amino acid sequence with other known caspase and caspase-related sequences. The tree was constructed by the ‘neighbour-joining’ method using the clustal w and phylip packages, and was bootstrapped 1000 times. C=caspase; Dr/Dros=Drosophila; CE=C. elegans; SF=Spodoptera frugiperda.</note><note type="content">Fig. 5: Analysis of the sites of expression of trout caspase 6. RT-PCR was performed with cDNA from a variety of tissues with trout β-actin primers or trout caspase 6 primers. 1=brain; 2=blood; 3=gill; 4=liver; 5=head kidney; 6=spleen.</note><note type="content">Fig. 6: β-actin and caspase 6 products obtained by RT-PCR, using head kidney leucocyte cDNA as template, with different cycle numbers.</note><note type="content">Fig. 7: Effect of LPS and cortisol addition on expression of caspase 6 and β-actin in cultured head kidney leucocytes. Semi-quantitative RT-PCR was performed with cDNA from head kidney leucocytes cultured with LPS (left) or varying concentrations of cortisol (right), prior to isolation of RNA. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below).</note><note type="content">Fig. 8: Effect of co-incubation with cortisol and LPS on caspase 6 expression, in the absence (left) and presence (right) of RU486. Semi-quantitative RT-PCR was performed with cDNA from head kidney leucocytes pretreated with cortisol in the presence of RU486 for 2h prior to addition of LPS and isolation of RNA 4h later. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below).</note><note type="content">Fig. 9: Semi-quantitative RT-PCR was performed with head kidney cDNA from control trout or trout subjected to a confinement stress for 1, 3 or 6 days. Data are presented as caspase 6 PCR products (above) and after normalising against products obtained after PCR of the same samples with β-actin primers (below), for two representative fish.</note><note type="content">Table 1: Homology of rainbow trout caspase 6 nucleotide and predicted amino acid sequence with other known caspase genes</note><subject lang="en"><genre>Keywords</genre><topic>Caspase 6</topic><topic>Rainbow trout</topic><topic>Expression</topic><topic>RT-PCR</topic><topic>Cortisol</topic><topic>Stress</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>LPS, lipopolysaccharide</topic><topic>ORF, open reading frame</topic><topic>PCR, polymerase chain reaction</topic></subject><relatedItem type="host"><titleInfo><title>Developmental and Comparative Immunology</title></titleInfo><titleInfo type="abbreviated"><title>DCI</title></titleInfo><genre type="Journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">200105</dateIssued></originInfo><identifier type="ISSN">0145-305X</identifier><identifier type="PII">S0145-305X(00)X0039-0</identifier><part><date>200105</date><detail type="volume"><number>25</number><caption>vol.</caption></detail><detail type="issue"><number>4</number><caption>no.</caption></detail><extent unit="issue pages"><start>269</start><end>352</end></extent><extent unit="pages"><start>303</start><end>312</end></extent></part></relatedItem><identifier type="istex">B557C68F569F0AD361DAD61798D70DC248EEE113</identifier><identifier type="DOI">10.1016/S0145-305X(00)00061-6</identifier><identifier type="PII">S0145-305X(00)00061-6</identifier><accessCondition type="use and reproduction" contentType="">© 2001Elsevier Science Ltd</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Elsevier Science Ltd, ©2001</recordOrigin></recordInfo></mods></metadata><enrichments><istex:catWosTEI uri="https://api.istex.fr/document/B557C68F569F0AD361DAD61798D70DC248EEE113/enrichments/catWos"><teiHeader><profileDesc><textClass><classCode scheme="WOS">IMMUNOLOGY</classCode><classCode scheme="WOS">ZOOLOGY</classCode></textClass></profileDesc></teiHeader></istex:catWosTEI></enrichments><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Musique/explor/MonteverdiV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000186 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 000186 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Musique
|area= MonteverdiV1
|flux= Istex
|étape= Corpus
|type= RBID
|clé= ISTEX:B557C68F569F0AD361DAD61798D70DC248EEE113
|texte= Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss , and analysis of its expression under conditions known to induce apoptosis
}}
| This area was generated with Dilib version V0.6.21. |  |