Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.
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.
D002460:Cell Line; D004256:DNA Polymerase I / Q000378:metabolism*; D004270:DNA, Circular / Q000096:biosynthesis; D004274:DNA, Recombinant / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D013006:Growth Hormone / Q000096:biosynthesis*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D004270:DNA, Circular; D004274:DNA, Recombinant; D013006:Growth Hormone; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase