Human growth hormone: complementary DNA cloning and expression in bacteria. 1979

in Biotechnology (Reading, Mass.) (1992), 24

A method for isolation of intact, translationally active ribonucleic acid

in DNA (1983), 2(4), 329-35

A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct ... [more ▼]

A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield. [less ▲]

Hormonal regulation of growth hormone mRNA

in DNA (1982), 1(2), 145-53

Cloning of bovine prolactin cDNA and evolutionary implications of its sequence

in DNA (1981), 1(1), 37-50

Prolactin, growth hormone, and chorionic somatomammotropin (placental lactogen) constitute a set of related polypeptides believed to derive from a common evolutionary ancestor protein. We have cloned and ... [more ▼]

Prolactin, growth hormone, and chorionic somatomammotropin (placental lactogen) constitute a set of related polypeptides believed to derive from a common evolutionary ancestor protein. We have cloned and sequenced DNA complementary to the mRNA coding for bovine prolactin. This cDNA contains 702 bases corresponding to 10 amino acids in the leader peptide, all 199 amino acids of the hormone, and 75 nucleotides in the 3' untranslated region of the mRNA. Nucleotide sequence analysis of this cDNA permitted the identification of 10 amino acids in the signal peptide, plus the correction or elucidation of amino acid assignments at 16 sites where aspartic and glutamic acids had not been distinguished from their amides by amino acid sequencing. Codon usage in bovine prolactin mRNA is nonrandom, but, similarly to rat and human prolactins, it does not exhibit the strong preference for G or C in codon third positions seen in bovine, rat, and human growth hormone mRNAs. The translational termination signal in bovine prolactin in UAA, also the same as in rat and human prolactins and differing from the UAG "stop" codon used in bovine, rat, and human growth hormones and human chorionic somatomammotropin. The amino acid and mRNA nucleotide sequences of bovine, rat, and human prolactins and growth hormones were compared by several techniques based on various theories of molecular evolution. The comparison of prolactin to growth hormone is consistent in all three species, suggesting that the genes for these two hormones diverged about 350 million years ago. However, comparisons among the three prolactins or among the three growth hormones to determine the times of evolutionary divergence of the three species generated values that were inconsistent with each other and with the fossil record. Analysis of these discrepancies suggests that the genes for prolactin and growth hormone may now be evolving by different mechanisms. [less ▲]

Cell-free synthesis of acetylcholine receptor polypeptides

in Science (1980), 209(4457), 695-7

Messenger RNA coding for acetylcholine receptor peptides has been identified. This polyadenylate [poly(A)+]RNA from Torpedo californica directs, in a cell-free system, the synthesis of peptides 60,000, 51 ... [more ▼]

Messenger RNA coding for acetylcholine receptor peptides has been identified. This polyadenylate [poly(A)+]RNA from Torpedo californica directs, in a cell-free system, the synthesis of peptides 60,000, 51,000, 49,000 41,000, and 35,000 daltons which account for approximately 2 percent of the total synthesized proteins. The results suggest that several different messenger RNA's code for the receptor subunits. These proteins react specifically to antiserum to native acteylcholine receptor, suggesting that the primary translational product has conformational features similar to the native receptor. Further, the results support the idea that there is post-translational modification of receptor subunits as the molecular weights of the cell-free synthesized proteins differ from those of purified receptor subunits. [less ▲]

Human growth hormone: complementary DNA cloning and expression in bacteria

in Science (1979), 205(4406), 602-7

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 ... [more ▼]

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. [less ▲]

Regulation of growth hormone messenger RNA

in Monographs on Endocrinology (1979), 12

In cultured rat pituitary cells, glucocorticoids regulate growth hormone production by modulating the number of growth hormone messenger RNA molecules. The effect is quite specific, since only a few other ... [more ▼]

In cultured rat pituitary cells, glucocorticoids regulate growth hormone production by modulating the number of growth hormone messenger RNA molecules. The effect is quite specific, since only a few other mRNAs are affected by the hormones. This response is demonstrated by assays involving cell-free mRNA translation and cDNA-RNA hybridization. Furthermore, the inducibility by the glucocorticoids is regulated by at least one other class of hormones, thyroid hormone. Thus, this system serves as a model for studying not only the glucocorticoid regulation of specific mRNA, but also the control of this regulation by other factors in the target tissue. [less ▲]

Hormone genes : Structure, evolution and expression in bacteria

in Molecular Endocrinology (1979)

Synthesis of growth hormone by bacteria

in Nature (1978), 276(5690), 795-8

A hybrid gene was constructed between the beta-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone. This gene is expressed in bacteria and growth hormone sequences are ... [more ▼]

A hybrid gene was constructed between the beta-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone. This gene is expressed in bacteria and growth hormone sequences are detectable by immunological methods. [less ▲]

Thyroid hormonelike actions of 3,3',5'-L-triiodothyronine nad 3,3'-diiodothyronine

in Journal of Clinical Investigation (1977), 60(6), 1230-9

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum ... [more ▼]

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine.Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3).In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2).These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3). [less ▲]