cDNA cloning of the bovine low density lipoprotein receptor: Feedback regulation of a receptor mRNA

D. W. Russell, T. Yamamoto, W. J. Schneider, Clive A. Slaughter, M. S. Brown, J. L. Goldstein

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Abstract

The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of ≃5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of ≃5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns.

Original languageEnglish (US)
Pages (from-to)7501-7505
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume80
Issue number24 I
DOIs
StatePublished - Jan 1 1983

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LDL Receptors
Organism Cloning
Complementary DNA
Messenger RNA
Cyanogen Bromide
Amino Acid Sequence
Plasmids
Proteins
Polyribosomes
Sterols
Adrenal Glands
Southern Blotting
Endocytosis
Gene Library
Oligonucleotides
Introns
Libraries
Culture Media
Squamous Cell Carcinoma
Membrane Proteins

ASJC Scopus subject areas

  • General

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cDNA cloning of the bovine low density lipoprotein receptor : Feedback regulation of a receptor mRNA. / Russell, D. W.; Yamamoto, T.; Schneider, W. J.; Slaughter, Clive A.; Brown, M. S.; Goldstein, J. L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, No. 24 I, 01.01.1983, p. 7501-7505.

Research output: Contribution to journalArticle

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