Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.
Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.
Denis
Laurence
L
Laboratoire mixte CNRS/INRA/Aventis (UMR 1932), Aventis cropscience, 14-20 rue Pierre Baizet, 69263 Lyon CEDEX 9, France.
Grossemy
Marie
M
Douce
Roland
R
Alban
Claude
C
D000483:Alleles; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D014644:Genetic Variation; D007438:Introns; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D018532:Pisum sativum; D011061:Poly A; D011110:Polymorphism, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases