Introduction by molecular cloning of artifactual inverted sequences at the 5' terminus of the sense strand of bovine parathyroid hormone cDNA

C. A. Weaver, D. F. Gordon, B. Kemper

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Abstract

To study the structure and function of the gene for parathyroid hormone, we obtained recombinant plasmids containing bovine parathyroid hormone cDNA. The nucleotide sequence at the 5' terminus (relative to the sense strand) of the cDNA insert in a recombinant plasmid, pPTHi4, was different from that previously reported for the bovine parathyroid hormone cDNA insert of another recombinant plasmid, pPTHm1. The first 50 nucleotides of the pPTHm1 insert were an inverted complement of nucleotides 2-51 of the pPTHi4 insert. The cDNA insert of another plasmid, pPTHi8, contained a sequence identical to nucleotides 2-51 of the pPTHi4 insert but also contained an additional 42 bases at the 5' terminus. The first 41 bases of the pPTHi8 insert were an inverted repeat of an internal sequence of the pPTHi4 insert corresponding to nucleotides 184-224. Restriction endonuclease analysis of pPTHi8 indicated that the internal sequence corresponding to this region was retained. The nucleotide sequence of a restriction fragment hybridized to parathyroid hormone mRNA and extended toward the 5' terminus of the mRNA with reverse transcriptase confirmed that the sequence at the 5' terminus of the pPTHi4 insert was an accurate copy of the parathyroid hormone mRNA sequence. These data suggest that two types of sequence rearrangements may occur during molecular cloning of cDNA: (i) an inversion of a sequence at the 5' terminus, as occurred in pPTHm1, and (ii) an inverted repeat of an internal sequence, as occurred in pPTHi8.

Original languageEnglish (US)
Pages (from-to)4073-4077
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume78
Issue number7 I
DOIs
StatePublished - Jan 1 1981

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