Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin.

Abstract

Molecular cloning has established the primary structures of two precursors of the human pancreas growth hormone-releasing factor (hpGRF-44), somatocrinin. Both polypeptides contain the sequence of hpGRF-44 flanked by basic processing sites. Furthermore, the precursors include a putative signal sequence and a carboxyl-terminal amidation signal for hpGRF-44. The two forms of mRNA code for pre-pro-GRF-107 and pre-pro-GRF-108. Pre-pro-GRF-108 differs from pre-pro-GRF-107 by the insertion of a serine in the carboxyl-terminal portion of the precursor. In vitro translation of tumor poly(A)+ RNA followed by immunoprecipitation with GRF-specific antiserum and gel electrophoresis showed the molecular weight of preprosomatocrinin to be approximately 13,000, which is in good agreement with the molecular weight deduced from the sequences of the cDNA clones.A 44-amino acid peptide (hpGRF-44, somatocrinint) with high intrinsic growth hormone-releasing activity in vitro and in vivo was recently isolated from a human pancreatic tumor and characterized (1, 2). Preliminary biological (3), immunological (4), and physico-chemical (unpublished data) evidence indicates that the tumor-derived hpGRF-44 is identical to hypothalamic growth hormone-releasing factor. The availability of tumor material, highly enriched in the growth hormone-releasing factor compared with hypothalamic tissue, has permitted the molecular cloning and structural characterization of the mRNA encoding hpGRF-44. MATERIALS AND METHODSPoly(A)+ RNA was obtained from 15 g of tumor tissue (stored in liquid nitrogen or at -80°C) by using the guanidine thiocyanate procedure (5) and chromatography on oligo(dT)-cellulose (6). Double-stranded cDNA was synthesized by using a modification of a published method (ref. 7; unpublished data). A cDNA library was constructed by homopolymer tailing of the cDNA with dGTP, hybridizing it to EcoRV-cut pBR322 that had been tailed with dCTP and transforming Escherichia coli RR1 with the chimeric plasmid (8). The solid-phase phosphotriester method (9) was used for the synthesis of two mixed oligodeoxynucleotide probes (see Fig. 1), which were labeled at their 5' ends with [y-32P]ATP (10). The tetradecamer probe was used for colony screening (11) as follows: hybridizations were carried out in 0.75 M NaCl/0.075 M sodium citrate/0. 1% NaDodSO4/ 0.2% bovine serum albumin/0.2% polyvinylpyrrolidone/0.2% Ficoll/partially hydrolyzed yeast RNA (100 ug/ml) at 300C for 2 hr using 1 pmol of labeled probe per ml. The filters were quickly rinsed in 0.3 M NaCl/0.03 M sodium citrate at room temperature. After a high-stringency wash in 0.6 M NaCl/0.06 M sodium citrate for 60 min at 40'C, the filters were exposed to x-ray film overnight.DNA sequence analysis, Southern and RNA blot analyses, nick-translation, primer extension, and NaDodSO4/polyacrylamide and denaturing agarose gel electrophoresis were carried out according to standard procedures (10,(12)(13)(14)(15)(16)(17)(18). Antibodies to hpGRF were produced in rabbits following immunization with...

Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosomal assignment.

“…The sequence is numbered from the putative mRNA 5' cap site, determined as described in the following section. The sequences of all exons agree precisely with those previously determined from human GRF cDNA clones (7,8). All of the 4 introns begin and end with the consensus sequences G-T and A-G, respectively (19).…”

“…A combination of restriction enzyme mapping and Southern DNA blotting was used to locate regions of the clones that hybridized to the GRF cDNA probe. Exon 1 was localized using a kinase-labeled synthetic oligonucleotide predicted from the sequence of a larger human GRF cDNA (8) to be specific for the 5' nontranslated region of the GRF mRNA (the oligonucleotide corresponds to nucleotides 36-67 of Fig. 3 (11).…”

“…Toward this goal, we, and others, have isolated cDNA recombinant clones encoding human GRF (7,8). Analysis of these cDNA clones indicated that mature GRF is proteolytically processed from a 108-amino acid precursor protein.…”

“…To establish this, we used a synthetic oligonucleotide specific for the 5' nontranslated region, based on the published sequence of another, larger human GRF cDNA clone (ref. 8 Fig. 1B.…”

Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.

“…Double-stranded cDNA was synthesized and cloned in Escherichia coli RR1 as described (10,11). Two oligonucleotide probes were synthesized by the solid-state phosphite method (ref 12; see Fig.…”