Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

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Abstract
Poly(A) + RNA from pregnant rat mammary glands was size-fractionated sucrose gradient centrigugation, 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 sequences data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from .apprx. 1100 to 1550 base pairs (bp). A 1,550-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 sequences of the clone to the amino acid sequences of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1,500 nucleotides, and it is therefore concluded that the 1,550-bp insert (including G .cntdot. 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.