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

Journal Article · Tue Mar 10 00:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00379a023· OSTI ID:6353170

Safford, R; de Silva, J; Lucas, C; Windust, J H.C.; Shedden, J; James, C M; Sidebottom, C M; Slabas, A R; Tombs, M P; Hughes, S G

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 approx. 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.

• Research Organization: Unilever Research, Bedford, England
• OSTI ID: 6353170
• Journal Information: Biochemistry; (United States), Vol. 26:5
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ESTERASES
AMINO ACID SEQUENCE
LABELLING
RECOMBINANT DNA
DNA SEQUENCING
ACETAMIDE
CARBON 14 COMPOUNDS
DNA
DNA-CLONING
ELECTROPHORESIS
IODINE 125
LACTATION
LIQUID COLUMN CHROMATOGRAPHY
MAMMARY GLANDS
METHIONINE
RATS
SULFUR 35
AMIDES
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CHROMATOGRAPHY
CLONING
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
GLANDS
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
RADIOISOTOPES
RODENTS
SEPARATION PROCESSES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques