In vitro biosynthesis of somatostatin. Evidence for two distinct preprosomatostatin molecules

mRNA isolated from angler fish islets of Langerhans was translated in the wheat germ cell-free protein-synthesizing system and the products identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. As previously shown (Shields, d. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4074), a major polypeptide of 18,000 dalton, designated preprosomatostatin, was immunoprecipitable. Here, evidence is presented for an additional somatostatin-immunoreactive polypeptide of apparent Mr = 19,000. The 19 kilodalton polypeptide was similar, but not identical with the 18 kilodalton preprosomatostatin, as determined by tryptic peptide analysis. Comparison of the tryptic peptides of the 19,000 dalton polypeptide with those of unlabeled somatostatin demonstrated that it contained the authentic somatostatin sequence. Like the 18,000 dalton precursor, the 19,000 dalton polypeptide had the mature somatostatin sequence located at its COOH terminus; it is proposed that this molecule is a minor species of preprosomatostatin.     

Production of EGF-containing polypeptides in Xenopus oocytes microinjected with submaxillary gland mRNA.

The biosynthesis of epidermal growth factor (EGF), a 6045 dalton mitogen produced in the mouse submaxillary gland under androgen regulation, was studied using Xenopus oocytes. Microinjection of total, unfractionated gland mRNA together with [35S]cysteine resulted in the production of a secretory polypeptide of approximately 9000 daltons, specifically immunoprecipitable with anti-EGF antibodies. A minor amount of a similarly immunoreactive 9000 dalton secretory polypeptide was produced from the sucrose gradient 9S fraction of gland mRNA. Other, more intensely labeled polypeptides, a cytoplasmic 125 000 dalton and a secretory 110 000 dalton protein were immunoprecipitated from oocytes injected with the greater than 25S mRNA fraction. The biosynthesis of both can hardly be detected in oocytes injected with unfractionated mRNA. All three polypeptides are produced under androgen regulation and share common immunoreactive properties. Northern blot analysis using a 76 nucleotide synthetic EGF cDNA probe revealed hybridization with a single 28S mRNA species. This, and the apparent interrelation between the three polypeptides, suggest that a gland-specific processing protein, encoded by a 9S mRNA, is required to produce the 9000 dalton pro-EGF from the nascent translation product of EGF mRNA.     

Translational analysis of the killer-associated virus-like particle dsRNA genome of S. cerevisiae: M dsRNA encodes toxin

The M species (medium sized) dsRNA (1.1–1.4 × 10⁶ daltons) isolated from a toxin-producing yeast killer strain (K⁺R⁺) and three related, defective interfering (suppressive) S species dsRNAs of the yeast killer-associated cytoplasmic multicomponent viral-like particle system were analyzed by in vitro translation in a wheat germ cell-free protein synthesis system. Heat-denatured M species dsRNA programmed the synthesis of two major polypeptides, M-P1 (32,000 daltons) and M-P2 (30,000 daltons). M-P1 has been shown by the criteria of proteolytic peptide mapping and cross-antigenicity to contain the 12,000 dalton polypeptide corresponding to the in vivo produced killer toxin, thus establishing that it is the M species dsRNA which carries the toxin gene. An M species dsRNA obtained from a neutral strain (K^?R⁺) also programmed the in vitro synthesis of a polypeptide identical in molecular weight to M-P1, thus indicating that the cytoplasmic determinant of the mutant neutral phenotype is either a simple point mutation in the dsRNA toxin gene or a mutation in a dsRNA gene which is required for functional toxin production. In vitro translation of each of the three different suppressive S dsRNAs resulted in the production of a polypeptide (S-P1) of approximately 8000 daltons instead of the 32,000 dalton M-P1 polypeptide programmed by M dsRNA. This result is consistent with the heteroduplex analysis of these dsRNAs by Fried and Fink (1978), which shows retention of M dsRNA ends, accompanied by large internal deletions in each of the S dsRNAs translated.     

The regulation of somatostatin production in human medullary thyroid carcinoma cells by dexamethasone

There have been few studies of physiological importance on the regulation of somatostatin by hormones. We have studied the effect of the synthetic glucocorticoid dexamethasone on somatostatin production in the human medullary thyroid carcinoma TT cell line, a model for somatostatin production by the parafollicular cell. Dexamethasone inhibited somatostatin production in a dose-related manner with a maximal effect at a concentration of 10(-6) M. TT cells treated with dexamethasone (10(-6) M) showed an almost complete inhibition of somatostatin peptide production by 48 h of treatment. Molecular sizing chromatography demonstrated a decrease in both the probable somatostatin precursor (13,000 dalton) and the fully processed peptide. Analysis of mRNA content by hybridization revealed that dexamethasone also caused a decrease in detectable somatostatin mRNA. The hybridizable somatostatin mRNA decreased to approximately 50% of basal levels within 12 h of treatment. Northern blot hybridization showed a decrease in a single RNA species representing mature somatostatin mRNA. Dose-response experiments revealed inhibition of both peptide and mRNA at concentrations from 1 X 10(-8) to 1 X 10(-5) M dexamethasone. Four days after withdrawal from dexamethasone treatment, peptide and mRNA levels were higher than dexamethasone-treated controls. The sex steroid estradiol had no inhibitory effect on somatostatin production. These results suggest a potential regulator of somatostatin production and provide a system for the study of somatostatin gene regulation.     

Incorporation of the Ca2+ -binding component (g2) into heavy meromyosin and subfragment-1 of pig cardiac myosin.

A new protein component was found in heavy meromyosin and in subfragment-1 (S-1) prepared by chymotrypsin digestion of pig cardiac myosin in the presence of Ca2+. The molecular weight of this protein was estimated as 15,000 dalton. It was able to bind Ca2+ and showed a similar UV absorption spectrum to that of the g2 light chain. Heavy meromyosin and subfragment-1 which contained the 15,000 dalton component incorporated exogenous g2 and the 15,000 dalton component disappeared after such treatment. We concluded that the 15,000 dalton component was produced from g2 by limitted proteolysis. The subfragment-1 was separated into two protein fractions in equal yield by recycling the gel filtration. One contained the 15,000 dalton component and was able to bind Ca2+ while the other did not contain the component and was unable to bind Ca2+. According to analysis by SDS gel electrophoresis, the large polypeptide chain (the f component) of the first S-1 was approximately 5,000 dalton larger than the f component of the second S-1. The polypeptide corresponding to 5,000 dalton was designated polypeptide-C, because it was released from the C terminal of the f component. It seems to be essential for the attachment of the Ca2+-binding light chain g2. The location of g2 in myosin may thus be at the polypeptide-C which links the head to the tail of myosin.     

Cell-free translation of messenger RNA coding for a precursor of human calcitonin

Polyadenylated RNA, extracted from a human medullary thyroid carcinoma, was translated in cell-free systems prepared from wheat germ and reticulocyte lysates. The major product of the translations was a protein of 15,000 M_R which was immunoprecipitated specifically with an antiserum to synthetic human calcitonin. Addition to the translation reactions of microsomal membranes, prepared from canine pancreas, resulted in the partial disappearance of the 15,000 M_R polypeptide and the concomitant appearance of a smaller peptide (11,000 M_R), also immunoprecipitated specifically by antisera to calcitonin. These results indicate that human calcitonin is synthesized in the form of a precursor of 15,000 M_R and suggest that the precursor contains a leader sequence that is cleaved from the polypeptide by enzymes associated with microsomal membranes.     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

The translation products from Cowpea Mosaic Virus (CPMV) RNAs obtained in two different cell-free systems were compared with the viral polypeptides synthesized in CPMV-infected cowpea protoplasts. It was shown that in both the wheat germ system and the rabbit reticulocyte lysate CPMV M component RNA was translated into two polypeptides of 105,000 and 95,000 dalton, which were not detected in CPMV-infected protoplasts. B component RNA however, gave different products depending on the system used. In the reticulocyte system this RNA was translated into a 200,000 dalton polypeptide which was further cleaved to give 170,000 and 32,000 dalton polypeptides. In the wheat germ system this processing step was lacking as only the 200,000 dalton product was formed. Since the 170,000 and 32,000 dalton polypeptides were also found in CPMV-infected protoplasts the two in vitro systems used apparently represent different stages of the expression of the B component RNA, thus providing a tool to study the mechanism of CPMV gene expression in vivo.     

Developmental regulation of yolk protein gene expression in Anastrepha suspensa

A partial cDNA clone for the 48,000 dalton yolk polypeptide gene from Anastrepha suspensa was isolated from a cDNA expression library using a yolk polypeptide antibody probe and hybridization to the Drosophila melanogaster yolk protein 1 gene. The sequenced DNA has greatest homology to the yolk protein genes from Ceratitis capitata, D. Melanogaster, and Calliphora erythrocephala and, similar to these genes, shares amino acid sequence domains with those from lipases. RNA hybridization studies indicated that the yolk protein gene expression is completely female-specific and limited to the ovaries, without apparent regulation by 20-hydroxyecdysone or juvenile hormone. This is in contrast to an earlier study which suggested, based on immunological probes, that a very low level of yolk protein synthesis occurred in fat body and was not sex-specific. Arch. Insect Biochem. Physiol. 36:25–35, 1997.Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Intestinal pre-prosomatostatin. Identification of mRNA coding for a precursor by cell-free translations and hybridization with a cloned islet cDNA

Somatostatin, a tetradecapeptide hormone, is produced in numerous organs including the hypothalamus, pancreatic islets, and the gastrointestinal tract. Recently we identified two separate biosynthetic precursors of somatostatin (Mr = 16,000 and 14,000) among the cell-free translation products encoded by mRNAs prepared from the islets of the anglerfish. The nucleotide sequence of a cloned cDNA encoding the larger of the two pre-prosomatostatins revealed the sequence of the tetradecapeptide somatostatin at the COOH terminus of a polypeptide of 119 amino acids. We now have prepared poly(A)RNA from the intestine of the anglerfish and by immunoprecipitation analyses find a single somatostatin-related translation product that co-migrates during electrophoresis on sodium dodecyl sulfate-polyacrylamide gels with the larger islet pre-prosomatostatin (Mr = 16,000). Analyses of the sizes of the intestinal and islet mRNAs by agarose gel electrophoresis and hybridization with 32P-labeled cDNA containing the coding sequence for the large islet pre-prosomatostatin showed that the complementary RNA in the intestine (600 bases) is 30 nucleotides smaller than that in the islet (620-630 bases). These observations indicate that a gene encoding somatostatin is expressed in the intestine and suggest that the intestinal mRNA is distinct from the two mRNAs encoding the islet somatostatins.     

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

Cell-free synthesis of putative neurophysin precursors from rat and mouse hypothalamic poly (A)-RNA.

Poly(A)-containing RNA has been isolated from rat and mouse hypothalamic tissue and used to direct the synthesis of polypeptides in cell-free systems derived from wheat germ extract and rabbit reticulocyte lysate in the presence of [³⁵S]-L-cysteine and [³H]-L-proline. Translation products were subjected to immunoprecipitation using an antiserum to rat neurophysin proteins. Following purification of the immunoprecipitates by protein A-Sepharose chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide species of molecular weight 17,500 derived from both cell-free systems.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.     

Pancreatic somatostatin-like immunoreactivity suppresses gastric acid secretion in rats.

Somatostatin-like immunoreactivity (SLI) was extracted from the canine pancreas and purified by ion exchange, affinity chromatography and gel filtration. The 1600 dalton fraction, which is physicochemically similar to synthetic somatostatin was infused into the peripheral circulation of anesthetized rats and its effect upon gastric acid secretion was compared with that of synthetic somatostatin. Both synthetic somatostatin and pancreatic SLI in a dose of 7–8 μg/kg/h suppressed pentagastrin-stimulated gastric acid secretion. It is concluded that the highly purified 1600 dalton fraction of canine pancreatic SLI, like synthetic somatostatin, can exert biological activity upon the stomach of rats.     

In vitro translation of mushroom tyrosinase

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.