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

Nucleic Acids Res. 1978 Jun; 5(6): 2039–2053.

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.

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