Growth Hormone Gene Sequences Research Articles

Extracellular production of human growth hormone by a head portion of the prepropeptide derived from Bacillus amyloliquefaciens neutral protease in Bacillus subtilis

A hybrid plasmid phGH928 was constructed which contains a human growth hormone gene following a region coding for the promoter and head portion of prepropeptide composed of 48 amino acid residues from the Bacillus amyloliquefaciens neutral protease gene. This plasmid permitted efficient secretion of the hormone in Bacillus subtilis. The N-terminal amino acid sequence analysis suggested that the prepropeptide was deleted during secretion. It showed also that the secreted hormone contained additional amino acid sequences derived from the junction between the prepropeptide coding region and the mature human growth hormone gene sequence. We confirmed that the secreted hormone was biologically active stimulating the growth of rat lymphoma cell.

Relationship between thyroid and glucocorticoid hormone receptor occupancy, growth hormone gene transcription, and mRNA accumulation.

The temporal relationship between the occupancy of the thyroid hormone (3,5,3'-triiodo-L-thyronine (T3)) and glucocorticoid (dexamethasone) receptors and the rate of growth hormone (GH) gene transcription and mRNA accumulation were investigated. The GC line of cultured rat pituitary tumor cells was used in these studies. The rate of GH gene transcription was measured by elongation of in vivo initiated RNA chains in isolated nuclei using radioactive precursors and hybridization of labeled transcripts to immobilized GH gene sequences. T3 receptor occupancy was directly proportional to the rate of GH gene transcription during the initial phase of hormone induction. These results suggest that a single occupied receptor is sufficient to fully activate the gene and that the unoccupied receptor resides close to the gene regulatory site. A decrease in GH gene transcription rapidly followed the initial increase in activity. This decrease occurred in the absence of new protein synthesis and was not accompanied by a proportional decrease in the level of occupied T3 receptor. An analysis of dexamethasone activation of GH gene transcription found maximum stable binding of occupied receptor to nuclei within 5 min of hormone addition, while transcription of the gene did not become fully activated until 30 to 60 min later. Transcription of the gene then declined to a rate 2 to 3 times that observed before addition of dexamethasone. These results suggest that at least one relatively slow molecular step precedes glucocorticoid hormone-receptor enhancement of gene transcription and that a second molecular event later attenuates the response. The simultaneous addition of both hormones produced two peaks of enhanced transcription, each apparently due to the separate effects of the hormones. GH mRNA levels were closely linked to the rate of transcription of the gene under all induction conditions. Comparison of the rates of decay of GH mRNA in cells cultured in the presence or the absence of hormones suggests that GH mRNA is much more stable when T3 is present. The glucocorticoid responses of the gene were consistently obtained only in the presence of T3. These results and the transcriptional synergism of the two hormones suggest a direct interaction of the occupied receptors at their regulatory sites. The transcriptional effects of glucocorticoids were observed only in about half the experiments performed in the absence of T3. When transcriptional activation did not occur, GH mRNA usually still increased 24 h after hormone treatment was begun, but not early in the induction.(ABSTRACT TRUNCATED AT 400 WORDS)

Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line

The mutagenic potential of recombinant DNA in the germ line was investigated in the descendants of mice obtained from eggs injected with the human growth-hormone gene in a pBR322 vector. Six positive animals (including one mosaic) were produced and had 1–20 copies of the donor sequences in unique and often complex integration patterns indicative of a single or an interrupted insertion. All were heterozygotes and transmitted the foreign insert to their progeny, forming six new HUGH strains. Matings between heterozygotes yielded viable healthy homozygotes in four of the strains. However, in the HUGH/3 and HUGH/4 strains, no postnatal homozygotes were found and litter sizes at birth were small. These two independent cases of mutation, both homozygous recessive prenatal lethals, are attributable to disruption of native sequences by alien ones. They constitute the first instances of insertional mutagenesis due to integration of recombinant DNA in the germ line of the mouse. The mutants provide new possibilities for molecular identification of gene functions necessary for normal development.

Distribution of thyroid hormone receptors, glucocorticoid receptors, and growth hormone gene sequences in chromatin from cultured rat pituitary cells.

Chromatin from rat pituitary tumor (GH3) cells was fractionated into transcriptionally active and inactive domains. When the various chromatin fractions were assayed for their content of growth hormone gene sequences, it was found that these sequences were highly enriched in those chromatin fractions most sensitive to micrococcal nuclease. Fraction S1 showed the highest enrichment in growth hormone genes and was impoverished in histones H3 and H4. The distribution of specifically bound thyroid and glucocorticoid receptors in chromatin fractions enriched and depleted in growth hormone gene sequences was also examined. Both thyroid and glucocorticoid receptors are enriched to different extents in transcriptionally active chromatin. Within active chromatin, both types of receptors exist in more than one molecular form.

Human Growth Hormone: Complementary DNA Cloning and Expression in Bacteria

The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.