Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA

David F. Gordon, Byron Kemper

Research output: Contribution to journalArticle

Abstract

DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase. The PTH cDNA contained about 750 bases and was greater tban 95% sensitive to digestion by S1 nuclease. Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA. Sequential incubation of the PTH mRNA with reverse transcriptase and E. coli DNA polymerase I produced near full length double-stranded PTH cDNA. Of the 22 restriction endonucleases tested, double-stranded PTH cDNA could be cleaved with Alu I, Mbo II, Sau 3A, Sst I, and Taq I. The restriction fragments corresponding to the 5′ terminus of the sense strand were identified for the last three enzymes by comparing the size of fragments obtained from PTH cDNA before and after cleavage of the hairpin loop connecting the two strands by S1 nuclease. The restriction map of the cDNA was used to detect clones of bacteria containing recombinant plasmids with near full length PTH cDNA inserts.

Original languageEnglish (US)
Pages (from-to)5669-5683
Number of pages15
JournalNucleic acids research
Volume8
Issue number23
DOIs
StatePublished - Dec 11 1980

ASJC Scopus subject areas

  • Genetics

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