Please use this identifier to cite or link to this item: https://hdl.handle.net/1959.11/8963
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dc.contributor.authorNgo, Suong Ngoc Thien
dc.contributor.authorMcKinnon, Ross Allanen
dc.contributor.authorStupans, Ievaen
dc.date.accessioned2011-12-01T10:49:00Z-
dc.date.issued2006-
dc.identifier.citationGene, 376(1), p. 123-132en
dc.identifier.issn1879-0038en
dc.identifier.issn0378-1119en
dc.identifier.urihttps://hdl.handle.net/1959.11/8963-
dc.description.abstractIn the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala ('Phascolarctos cinereus'), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327–334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45 ± 0.18 nmol/min/mg protein and 4.79 ± 1.91 nmol/min/nmol CYP (mean ± SD, n = 3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094 ± 0.001 nmol/mg protein (mean ± SD, n = 3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the β-actin mRNA species, much stronger CYP4A mRNA signal was detected for koala liver relative to rat and human. In Southern blot analysis of EcoR 1-digested genomic DNAs, using the same koala CYP4A15 cDNA probe, the size of CYP4A gene fragments observed for the koala and other species were different, suggested a different CYP4A gene organization across species. Collectively, this study provides primary molecular data regarding koala CYP4A15 gene. The possibility that there may be higher CYP4A15 expression in the koala liver could not be excluded.en
dc.languageenen
dc.publisherElsevier BVen
dc.relation.ispartofGeneen
dc.titleCloning and expression of koala ('Phascolarctos cinereus') liver cytochrome P450 CYP4A15en
dc.typeJournal Articleen
dc.identifier.doi10.1016/j.gene.2006.02.020en
dc.subject.keywordsMedical and Health Sciencesen
local.contributor.firstnameSuong Ngoc Thien
local.contributor.firstnameRoss Allanen
local.contributor.firstnameIevaen
local.subject.for2008119999 Medical and Health Sciences not elsewhere classifieden
local.subject.seo2008929999 Health not elsewhere classifieden
local.profile.schoolSchool of Science and Technologyen
local.profile.emailistupans@une.edu.auen
local.output.categoryC1en
local.record.placeauen
local.record.institutionUniversity of New Englanden
local.identifier.epublicationsrecordune-20111130-13452en
local.publisher.placeNetherlandsen
local.format.startpage123en
local.format.endpage132en
local.peerreviewedYesen
local.identifier.volume376en
local.identifier.issue1en
local.contributor.lastnameNgoen
local.contributor.lastnameMcKinnonen
local.contributor.lastnameStupansen
dc.identifier.staffune-id:istupansen
local.profile.roleauthoren
local.profile.roleauthoren
local.profile.roleauthoren
local.identifier.unepublicationidune:9153en
dc.identifier.academiclevelAcademicen
local.title.maintitleCloning and expression of koala ('Phascolarctos cinereus') liver cytochrome P450 CYP4A15en
local.output.categorydescriptionC1 Refereed Article in a Scholarly Journalen
local.search.authorNgo, Suong Ngoc Thien
local.search.authorMcKinnon, Ross Allanen
local.search.authorStupans, Ievaen
local.uneassociationUnknownen
local.year.published2006en
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