Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5,...

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Bibliographic Details
Published in:Biochemical journal 1982-01, Vol.201 (1), p.81-90
Main Authors: Suard, Y M, Tosi, M, Kraehenbuhl, J P
Format: Article
Language:English
Subjects:
alpha-casein
Animals
Caseins - analysis
Caseins - genetics
cloning
Cloning, Molecular
DNA - genetics
DNA, Recombinant
Female
Gene Expression Regulation
genes
Kappa -caaein
Lactalbumin - analysis
Lactalbumin - genetics
Lactation
mammary gland
Mammary Glands, Animal - analysis
mapping
Poly A - genetics
Poly A - isolation & purification
Pregnancy
Protein Biosynthesis
proteins
purification
Rabbits
restriction endonuclease
RNA, Messenger - genetics
RNA, Messenger - isolation & purification
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Summary:Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2010081