Molecular cloning of DNA complementary to bovine growth hormone mRNA

in Journal of Biological Chemistry (1980), 255(16), 7521-4

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x ... [more ▼]

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%. [less ▲]

Nucleotide sequence of part of the gene for human chorionic somatomammotropin: purification of DNA complementary to predominant mRNA species

in Cell (1977), 12(1), 157-65

A novel purification procedure for DNA complementary to individual mRNA species has been developed by using restriction endonuclease cleavage of cDNA transcribed from a complex mixture of mRNAs. This ... [more ▼]

A novel purification procedure for DNA complementary to individual mRNA species has been developed by using restriction endonuclease cleavage of cDNA transcribed from a complex mixture of mRNAs. This procedure has allowed us to isolate and analyze DNA fragments complementary to the mRNA coding for the human peptide hormone chorionic somatomammotropin (HCS). The mRNA for this hormone is a major constituent of placental polyadenylated RNA as shown by in vitro translation of placental RNA and by nucleic acid hybridization using HCS cDNA as a specific probe. The purification of HCS cDNA was achieved by Hae III and Hha I restriction endonuclease cleavage of single-stranded cDNA synthesized in vitro from total polyadenylated placental RNA. Polyacrylamide gel electrophoresis of the products allowed detection and purification of discrete DNA fragments. A comparison of the nucleotide sequence of these fragments with that predicted from the amino acid sequence of HCS demonstrated that the fragments are transcripts of HCS mRNA, containing most of the translated and 3′ untranslated regions. The latter region is characterized by a UAG termination codon immediately adjacent to the translated region (a second in phase UAG occurs 9 nucleotides away) and a palindromic sequence (GUGACCCCUCCCCAGUG) centered 27 nucleotides from the termination codon. The purification scheme outlined for HCS cCNA should be applicable to DNA sequences complementary to mRNA species which represent at least 2% of any polyadenylated RNA preparation. This was demonstrated by restriction endonuclease cleavage of cDNA synthesized from a mixture of purified rabbit globin and total polyadenylated human placental RNAs. [less ▲]

Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones

in Proceedings of the National Academy of Sciences of the United States of America (1977), 74(5), 1816-20

Thyroid and glucocorticoid hormones stimulate growth hormone synthesis in cultured rat pituitary cells (GC). We have compared changes in growth hormone production and mRNA in these cells. Triiodothyronine ... [more ▼]

Thyroid and glucocorticoid hormones stimulate growth hormone synthesis in cultured rat pituitary cells (GC). We have compared changes in growth hormone production and mRNA in these cells. Triiodothyronine (10 nM) and dexamethasone (1 micron) stimulated increases in growth hormone production by 2.5- and 3.8-fold, respectively. There were corresponding increases in the capacity of RNA from hormone-treated cells to direct synthesis of pregrowth hormone in a wheat germ cell-free translation system, suggesting hormone-regulated increases in growth hormone mRNA. Hormone-induced changes in mRNA were also demonstrated by determining the kinetics of hybridization of a cDNA probe prepared from RNA enriched (about 70%) for growth hormone translational activity with RNA from control and hormone-treated cells. These results suggest that thyroid and glucocorticoid hormones can regulate growth hormone production by influencing the levels of its mRNA. [less ▲]