A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies.
A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.
Nishida
I
I
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan. nishida@biol.s.u-tokyo.ac.jp
Sugiura
M
M
Enju
A
A
Nakamura
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology*
D018076:DNA, Complementary; D007527:Isoenzymes; D005992:Glycerol-3-Phosphate O-Acyltransferase