Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate
acyltransferase (LPA-AT)
enzymes from Limnanthes douglasii. Both
heterologous screening with the
maize pMAT1 clone and in vivo complementation of the
Escherichia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by
heterologous probing, pLAT1, encodes a protein which is most similar to the
maize (
open reading frame in pMAT1) and
yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the
E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a
substrate specificity distinctly different from PlsC and similar to Limnanthes sp.
microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone.
Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+
mRNA from seed,
stem and leaf, demonstrating more widespread expression throughout the plant.
Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small
gene family.
