Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

Abstract

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.

• Authors: M M Harpold, P R Dobner, R M Evans, F C Bancroft
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 5 Issue 6 Pg. 2039-53 (Jun 1978) ISSN: 0305-1048 [Print] England
• PMID: 353736 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA, Circular
• DNA, Recombinant
• Growth Hormone
• DNA Polymerase I
• DNA-Directed DNA Polymerase

Topics

• Cell Line
• DNA Polymerase I (metabolism)
• DNA, Circular (biosynthesis)
• DNA, Recombinant (metabolism)
• DNA-Directed DNA Polymerase (metabolism)
• Escherichia coli (enzymology)
• Genes
• Growth Hormone (biosynthesis)
• Kinetics
• Nucleic Acid Hybridization
• Plasmids
• Protein Biosynthesis
• Transcription, Genetic