Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 5 1 -amino-acid N-terminal extension.Immunocytochemical studies showed ACP to be localised solely within the plastids of B. nupus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.De novo synthesis of fatty acids is catalysed by fatty acid synthetase which consists of seven or eight catalytic domains. In animals [l] and yeast [2] the domains are present on one or two multifunctional polypeptide chains (type I fatty acid synthetase), which are localised within the cytoplasm. In contrast, in plants [3] the fatty acid synthetase domains exist as discreet, monofunctional activities (type 11) which are organellar in location.Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds. As a first step towards that objective we report here the molecular cloning of cDNA encoding seed-expressed acylcarrier protein (ACP) from Brassica napus (oil seed rape).ACP is a key component of the plant Fatty acid biosynthetic machinery, serving both as a component of fatty acid synthetase and also as an acyl donor in desaturation and acyltransfer reactions [6]. Recent studies have shown two major ACP isoforms to be expressed in leaf tissue [7, 81, but apparently only one major isoform in seeds [8]. To date, characterisation of plant ACP has been largely confined to the leafexpressed forms. Thus, spinach [S] and barley [7] isoforms have been purified and N-terminal analysis suggests that, in both species, the isoforms are products of distinct genes. Using ACP as a representative marker protein, the site of fatty acid biosynthesis in leaves has been identified as the chloroplast [9]. In developing soybean seeds, ACP levels increase in close correlation with storage lipid synthesis [lo] suggestive of a regulatory role for ACP in this process. Despite this important role, ACP has not previously been localised within, or purified from, a seed source.This paper provides the first insight into the origin, structure and expression of gene...

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Poly(A)+ RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A)+ RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approximately 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G X C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.

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