Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones.

Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3' non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.

The isolation and sequence analysis of two seed-expressed acyl carrier protein genes from Brassica napus.

Genomic Southern blot analysis of Brassica napus DNA indicates that seed-expressed acyl carrier protein (ACP) is encoded by a multigene family of some 35 genes/haploid genome. Two genomic clones encoding B. napus ACP have been isolated and sequenced. The coding sequences of the 2 respective genes were found to be perfectly homologous to 2 distinct B. napus seed-expressed cDNAs and therefore represent seed-expressed forms of ACP. The 2 genomic ACP sequences share 94% homology within their coding sequences. Both genes are interrupted by 3 intervening sequences whose position within the 2 coding sequences is conserved. RNase protection studies were used to map the transcription start site of one of the genes and to provide further evidence that the gene is seed-expressed. The expression of a sub-group of the ACP gene family was found to be developmentally regulated in concert with the storage lipid synthetic phase of seed development. The coding sequence of both B. napus genes are highly homologous (96% and 93% respectively) to a Brassica campestris ACP cDNA sequence, suggesting that they may have evolved from this ancestral gene.

Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC.

The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon. It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator. Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor. The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones. We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function. We have designated these operons kcrA, B and C for KorC-regulated. Putative kilC is designated kcrC. The homology between the expression signals of these operons suggests that they have arisen by duplication. This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons.