Inhibition of the estrogen-induced synthesis of vitellogenin mRNA in chick liver by tamoxifen

Cloned vitellogenin cDNA (labelled with ³²P) was used as a probe for measuring vitellogenin mRNA sequences in RNA preparations from the liver of chicks treated with estradiol and/or tamoxifen. For the first time it was shown that the antiestrogen tamoxifen inhibits the estradiol-induced synthesis of vitellogenin mRNA in chick liver. This inhibition correlates very well with a reduced capacity of the liver to synthesize vitellogenin. Furthermore, evidence is presented that tamoxifen lacks any agonistic activity in chick liver. Vitellogenin mRNA is not measurable after tamoxifen alone.     

Identification of the mRNA coding for the ACTH-beta-lipotropin precursor in a human ectopic ACTH-producing tumor

The mRNA coding for the ACTH-β-lipotropin precursor from a human ectopic ACTH-producing thymic carcinoid was identified by blot hybridization analysis with the bovine cDNA as a probe. The mRNA from the tumor had the same length (approximately 1,100 nucleotides) as that from the human pituitary. An additional hybridization-positive RNA species of a larger size was found in the tumor. Cell-free translation of the mRNA from the tumor as well as from the pituitary yielded a product with an apparent molecular weight of 38,000 that was reactive both with antibody to ACTH and with antibody to β-endorphin.     

Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks

The regulation of food intake in chickens (Gallus gallus domesticus) represents a complex homeostatic mechanism involving multiple levels of control, and regulation during high ambient temperatures (HT) is poorly understood. In this study, we examined hypothalamic mRNA expression of gonadotropin-inhibitory hormone (GnIH) to understand the effect of HT on an orexigenic neuropeptide. We examined the effects of HT (35 °C ambient temperature for 1, 24 or 48 h) on 14-day old chicks. HT significantly increased rectal temperature and suppressed food intake, and also influenced plasma metabolites. The expression of GnIH precursor mRNA in the diencephalon was significantly increased in chicks at 24-and 48 h of HT when food intake was suppressed significantly, whilst no change was observed for GnIH precursor mRNA and food intake at 1h of HT. In situ hybridization and immunocytochemistry further revealed the cellular localization of chicken GnIH precursor mRNA and its peptide in the paraventricular nucleus (PVN) in the chick hypothalamus. We examined plasma metabolites in chicks exposed to HT for 1 or 48 h and found that triacylglycerol concentration was significantly higher in HT than control chicks at 1h. Total protein, uric acid and calcium were significantly lower in HT chicks than control chicks at 48h. These results indicate that not only a reduction in food intake and alteration in plasma metabolites but also the PVN-specific expression of GnIH, an orexigenic agent, may be induced by HT. The reduced food intake at the same time as GnIH expression was increased during HT suggests that HT-induced GnIH expression may oppose HT-induced feeding suppression, rather than promote it. We suggest that the increased GnIH expression could be a consequence of the reduced food intake, and would not be a direct response to HT.     

Identification of a high molecular weight presumptive precursor to albumin mRNA in the nucleus of rat liver and hepatoma cell line H4AZC2.

Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.     

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluorescent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin.The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules.The fluorescent stain for vittelogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parenchymal cells.The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.     

Identification of the eleocytes as the vitellogenin producing cells in nereids.

Coelomocytes of Nereis diversicolor synthesize and secrete vitellogenin in vitro. Using a monoclonal antibody which specifically recognized vitellogenin, we showed by immunocytochemistry that among the coelomocytes only a subpopulation, called eleocytes, contained vitellogenin. These results assert that eleocytes are the vitellogenin producing cells in nereids.     

Sequence organization of the beta-globin mRNA precursor.

The sequence organization of the beta-globin mRNA precursor has been determined directly by analyzing the resistant fragments from the RNase A digestion of the precursor RNA-globin cDNA hybrid. Three fragments are obtained which proves that the beta-globin mRNA sequence in its precursor is split into three discontinuous segments. The two intervening sequences in the beta-globin gene are therefore transcribed and removed during mRNA maturation.     

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.     

Cell-free synthesis and processing of a large precursor of glutamate dehydrogenase of rat liver

The mitochondrial matrix protein glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using mRNA from free or membrane-bound polysomes from rat liver. Immunoprecipitation of the (³⁵S)methionine labeled translation mixture was performed using rabbit anti-glutamate dehydrogenase serum. Analysis after electrophoresis of the immunoprecipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed that the glutamate dehydrogenase is synthesized ‘in vitro’ as a large precursor. A mitochondrial extract from rat liver processed the precursor synthesized “in vitro” to the mature form.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 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.     

Organization of vitellogenin polysomes, size of the mRNA and polyadenylate fragment.

A heavy polysome fraction containing vitellogenin mRNA was isolated from the liver of oestradiol-treated chicks. As determined by urea-polyacrylamide gel electrophoresis, the molecular weight of vitellogenin mRNA is about 2.5 x 10(6). The mRNA contains a polyadenylate segment of about 220 nucleotides at the 3' end. The remaining 7000 nucleotides are sufficient to code for a polypeptide of Mr about 270000. Combining 'run off' experiments of heavy polysomes in vitro together with radioimmunoprecipitation and polyacrylamide gel electrophoresis of the translation product, we concluded that vitellogenin mRNA is probably monocistronic and the 2.5 x 10(6)-Mr mRNA codes for two polypeptides, Mr 30000 and 240000. The largest polypeptide is, in our cell-free system and liver homogenate, readily cleaved into smaller peptides.