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G J Cote

Publications and source records attributed to G J Cote.

At least 55 records · Page 3Linked to original sources
PubMed1991

Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA.

The pre-mRNA encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include or exclude the calcitonin-specific exon 4. A minigene containing a viral first exon and exons 4, 5, and 6 from the human CT/CGRP gene was correctly processed in transfected HeLa or F9 teratocarcinoma cells to produce mRNA that included or excluded exon 4, respectively. This processing decision could be reproduced in vitro using nuclear extracts from these two cell lines and an RNA precursor from a similar minigene. Supplementation of extract from HeLa cells with extract from F9 cells resulted in the F9 splicing pattern in which exon 4 was excluded. This model system may be useful for the purification of splicing factors important in the regulation of this splice choice.

Adenoviridae↗
PubMed1990

Transfection of calcitonin gene regulatory elements into a cell culture model of the C cell.

Calcitonin gene expression in the TT cell line can be regulated by phorbol esters, cAMP, glucocorticoids, and 1,25-dihydroxyvitamin D3. To further study the regulation of this gene we have sequenced 1460 bases 5' to the start of calcitonin gene transcription. This DNA sequence contains cis consensus elements for both phorbol ester- and cAMP-responsive elements. To study the role of these elements, calcitonin 5' flanking DNA was coupled to the human growth hormone gene as a reporter and transiently transfected into TT cells, a human thyroid C cell line. Treatment of transfected TT cells stimulated a two- to fivefold increase in reported gene product expression, confirming the existence of functional cAMP- and phorbol ester-dependent enhancers within the calcitonin 5' flanking sequence.

Animals↗
PubMed1990

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

Animals↗
PubMed1990

Calcitonin exon sequences influence alternative RNA processing.

The pre-mRNA encoding calcitonin (CT) and CT gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include exon 4 (which encodes CT) or exclude this exon and splice to exon 5 (which encodes CGRP). We have used a CT-specific in vitro RNA-processing system to identify cis-acting sequences required to prevent splicing to exon 5. Deletion mapping demonstrated the presence of an element within the first 45 nucleotides of the CT-specific exon 4 that was required to suppress splicing to the CGRP-specific exon 5. This element was able to function in a completely heterologous system to suppress splicing when the CGRP exon was replaced with a constitutive viral exon. The element was unable to suppress splicing in the absence of a proximal CT-specific 3' splice site. Our results suggest that CT-specific splicing requires assisted recognition of its 3' splice site.

Calcitonin↗
PubMed1990

Transcription of the human calcitonin gene is mediated by a C cell-specific enhancer containing E-box-like elements.

The calcitonin (CT) gene is expressed normally in thyroidal C-cells and in a restricted population of cells in the central and peripheral nerve system. To define the cis-elements within the 5'-flanking DNA of the human CT gene which mediate this cell-specific expression, we used DNA transfer techniques and a transient transfection approach. We found that a DNA sequence located between -1290 and -820 of the CT 5'-flanking DNA functioned as an enhancer of basal transcription in C-cells (from medullary thyroid carcinoma) but not in rat glioma (C6), hamster insulinoma (HIT), fibroblasts (3T3), or epithelial cells (HeLa and CV1). Further mapping revealed the presence of at least two elements within the enhancer region; an upstream element (USE, located between -1060 and -1030) which could not function independently but its removal caused 70-80% loss of enhancer activity and a downstream element (DSE, located at -1033 to -920) which functioned independently as a cell-specific enhancer but with reduced activity. The binding pattern of nuclear proteins from C-cells to the enhancer elements was studied by an electrophoretic mobility shift assay. A protein-DNA complex was formed with the USE which could be competed, specifically, by an oligonucleotide containing the microE2 motif of the immunoglobulin gene enhancer. A similar complex was formed with the DSE fragment. Nuclear proteins from HeLa cells failed to form complexes with USE. Moreover, the binding pattern of proteins derived from HeLa cells to DSE was different from that of C-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

1,25-Dihydroxyvitamin D3 silences 3',5'-cyclic adenosine monophosphate enhancement of somatostatin gene transcription in human thyroid C cells.

Treatment of the somatostatin (SRIF)-producing TT cell line with 1,25 dihydroxyvitamin D3 (1,25 D3) lowered intracellular SRIF mRNA and peptide concentration. In separate experiments, the cAMP analog 8-(4-chlorophenylthio)-cAMP stimulated a rapid increase in SRIF mRNA content of the TT cells and SRIF peptide secretion. To determine whether 1,25 D3 could inhibit either the transcriptional or secretory effects of cAMP, TT cells were pretreated with 1,25 D3 for 4 days followed by treatment with the cAMP analog. Pretreatment with 1,25 D3 inhibited the cAMP-mediated increase of SRIF mRNA, but had no effect on the secretory response. We conclude that the ability of 1,25 D3 to silence SRIF gene expression is more potent than cAMP enhancer activity but that 1,25 D3 has no effect on that portion of the cAMP-dependent pathway which regulates peptide secretion.

Calcitriol↗
PubMed1989

Transcriptional regulation of the human calcitonin gene: a progress report.

We have applied DNA transfer techniques to study the transcriptional regulation of the calcitonin (CT) gene in a C-cell line (TT) derived from a human medullary thyroid carcinoma. TT cells were transfected with a fusion gene containing the CT gene promoter and 5'-flanking DNA attached to the promoter-less growth hormone gene (reporter). We quantitated the reporter gene product to monitor transcriptional activation by the CT promoter and deletion mutants of the 5'-flanking DNA. We found that the proximal CT promoter which includes the DNA sequence from +1 to -129 bp upstream from the CT transcription start site did not induce transcription in C-cells or in NIH 3T3 cells. The attachment of additional 5'-flanking DNA, extending up to -1460 bp enhanced transcription up to twelvefold in TT cells but had no effect on transcription in 3T3 cells. Deletion of a sequence located at -1290 to -820 bp on the CT 5'-flanking DNA abolished the transcription of the reporter gene. Attachment of the DNA sequence located between -1333 to -731 to the fusion gene, containing the CT promoter (+1 to -129) and the reporter gene, restored transcription of the reporter gene in TT cells. We conclude that an enhancer of CT transcription, which is active in C-cells but not in 3T3 cells, is located between -1290 and -820 of the CT 5'-flanking DNA.

Calcitonin↗
PubMed1989

Calcitonin production: an examination of the regulatory effects of extracellular calcium and 1,25-dihydroxyvitamin D3.

Acute elevations of the serum calcium concentration stimulate release of preformed calcitonin. Whether chronic alterations of the serum calcium of 1,25 dihydroxyvitamin D3 concentrations cause an effect on synthesis or secretion of this major calcium-regulating hormone has not been determined with certainty. Recent studies, including those presented in this manuscript, indicate that 1,25 dihydroxyvitamin D3 and, perhaps, increases of the serum calcium concentration inhibit transcription of the calcitonin gene resulting in decreased production of calcitonin. Inhibition of calcitonin production in the hypercalcemic or vitamin D toxic patient may play a role in the pathophysiology of these conditions.

Calcitonin↗
PubMed1987

The effect of 1,25-dihydroxyvitamin D3 treatment on calcitonin and calcitonin gene-related peptide mRNA levels in cultured human thyroid C-cells.

We utilized the TT cell, a human C-cell line derived from a medullary thyroid carcinoma, to study the effects of 1,25-dihydroxyvitamin D3 on cell growth and expression of the calcitonin gene. The growth rate of cells treated for 8 days with 1,25-dihydroxyvitamin D3 did not differ significantly from control or 24,25-dihydroxyvitamin D3 treated cells. Total RNA was isolated, and calcitonin and calcitonin gene-related peptide (CGRP) levels were measured by hybridization. 1,25 D3 lowered calcitonin and CGRP mRNA levels in a time- and dose-dependent fashion; 24,25 D3 had no effect. Northern blots revealed a decrease in the mature mRNA as well as the common precursor forms, indicating a transcriptional effect of 1,25 D3.

24,25-Dihydroxyvitamin D 3↗
PubMed1987

The expressions of oncogenes and liver-specific genes in Morris hepatomas.

The expression of three liver-specific genes and four oncogenes was studied in the Morris hepatomas 8994, 7288c, 7777, 5123tc, and 7800. Total RNA isolated from these tumors was probed with cDNA's for alpha-fetoprotein (AFP), albumin, tyrosine aminotransferase (TAT), and the oncogenes Ha-ras, Ki-ras, myc and src. When compared to mRNA's levels expressed in normal adult liver, we found AFP levels elevated in AFP-producing tumors, albumin and TAT mRNA levels depressed in all tumors, except TAT is elevated in 5123tc and the oncogenes with the exception of src elevated in all tumors. These results argue against a coordinated expression of these genes as a result of transformation, but suggest that oncogene expression is related to tumorigenesis or proliferation.

Animals↗
PubMed1987

A specific alpha-fetoprotein gene binding protein in alpha-fetoprotein producing rat hepatomas.

A specific alpha-fetoprotein (AFP) gene binding nuclear protein (Mol. Wt. 149,000) was determined in Morris hepatoma 7777 cells by the protein blotting technique. This protein is not present in normal adult rat liver and non-AFP producing Morris hepatoma 5123tc. Neoplasia induced in rats fed the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzine enhanced AFP gene activity and re-expressed specific AFP gene binding nuclear protein. The precise role of this protein in AFP gene regulation remains to be determined.

Animals↗
PubMed1987

Hepatoma-associated nonhistone chromosomal proteins are present in active chromatin.

Recently a group of nonhistone proteins with molecular weights ranging from 180-200 K were discovered which are associated with rat hepatoma chromatin specifically (Burkhardt et al., Biochim. Biophys. Acta 781, 165-172, 1984). These hepatoma-associated nonhistone proteins appeared and increased in rats treated with a hepatocarcinogen. Two approaches were used in this study to investigate whether the hepatoma-associated nonhistone chromosomal proteins are present in actively transcribed regions. We found that the limited DNase I digestion of Morris hepatoma 7777 chromatin released antigenic proteins not detected in normal liver chromatin digests. The association of antigenic nonhistone proteins with nuclear matrices was also studied. Using immunoblot analysis of nuclear matrices and total chromatin, the antigenic nonhistone chromosomal proteins were determined. Hepatoma-associated nonhistone protein antigens were extensively concentrated in the nuclear matrices. In the present study, the transcriptionally-active alpha-fetoprotein gene and the nontranscribed beta-globin gene were used as gene markers to determine the transcriptionally active chromatin region. Data presented in this paper indicate that hepatoma-associated NHPs are localized in active chromatin.

Animals↗
PubMed1987

Studies of short-term secretion of peptides produced by alternative RNA processing.

Alternative RNA processing of the calcitonin gene primary transcript results in production of two peptides, calcitonin and calcitonin gene-related peptide (CGRP). We have used the TT cell line, which produces both peptides, to ascertain whether secretion of peptides produced by alternative RNA processing is under identical regulatory control. Short-term treatment of TT cells with phorbol esters and cAMP analogs caused a rapid and parallel release of both calcitonin and CGRP. The measurement of calcitonin and CGRP mRNA levels during treatment revealed that new RNA synthesis was not required for secretion. Four potential regulators of phorbol ester-mediated and five of cAMP-mediated secretion were identified by incorporation of radioactive phosphate into protein as analysed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. From these results we conclude that calcitonin and CGRP secretion in this human C cell model is not differentially affected by alternative RNA processing for the phorbol ester-, and cAMP-dependent secretory pathways.

Calcitonin↗
PubMed1986

Dexamethasone differentially affects the levels of calcitonin and calcitonin gene-related peptide mRNAs expressed in a human medullary thyroid carcinoma cell line.

The TT cells are a continuous line of human C-cells derived from a medullary thyroid carcinoma. These cells produce large quantities of calcitonin and calcitonin gene-related peptide (CGRP) by the differential splicing of a single calcitonin gene transcript. We have used specific cDNA probes for calcitonin and CGRP to study the regulation of the calcitonin gene by dexamethasone, a synthetic glucocorticoid. Northern blot analysis of total cellular RNA isolated from the TT cells showed hybridization of the calcitonin probe to 3600- and 1000-base RNA species. The CGRP probe hybridized to 3600- and 1050-base RNA species. Dexamethasone treatment (10(-9) to 10(-5) M) of TT cells (for 6 days) caused a dosage-dependent increase in calcitonin mRNA levels and a decrease in CGRP mRNA levels. These findings were confirmed in time course studies where dexamethasone treatment (10(-6) M) caused a 2-13-fold increase in calcitonin mRNA and a 40-60% decrease in CGRP mRNA between 4 and 6 days of treatment; the effect was reversible after dexamethasone withdrawal. After excluding an effect of dexamethasone on calcitonin and CGRP mRNA stability, we have concluded that dexamethasone affects the splicing mechanism to favor production of calcitonin mRNA over CGRP mRNA.

Calcitonin↗
PubMed1986

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.

Cell Line↗
PubMed1985

alpha-Fetoprotein gene DNA-binding proteins.

The technique of protein blotting was used to study nuclear protein interaction with the alpha-fetoprotein (AFP) gene. The DNA-binding specificity was optimized by varying the amount of competitor DNA and the ionic strength. The specific binding of AFP gene DNA was observed for a set of Morris hepatoma 7777 nuclear proteins. Similar specificity was not seen for these same proteins in liver. In normal rat livers, however, two unique proteins were observed which displayed specific binding. The speculated involvement of these proteins in AFP gene regulation is discussed.

Animals↗
PubMed1985

Dexamethasone inhibits alpha-fetoprotein gene transcription in neonatal rat liver and isolated nuclei.

The effect of dexamethasone on rat alpha-fetoprotein (AFP) expression has been further examined. Quantitation of serum AFP levels from newborns treated with dexamethasone showed a dose-response relationship between the quantity of dexamethasone administered and the reduction in AFP serum level. RNA blots, utilizing cloned AFP cDNA as probe, showed a marked reduction in AFP mRNA in dexamethasone treated livers. The extent of AFP mRNA depletion was correlated with dexamethasone dosage. The effect of dexamethasone on AFP mRNA concentration was relatively rapid; a substantial reduction occurred 12 hours after a single injection. The effect of dexamethasone appeared to be irreversible as hormone withdrawal did not cause AFP mRNA levels to rise. One putative AFP nuclear RNA precursor was identified which rapidly disappeared following dexamethasone treatment. AFP mRNA synthesis was also diminished in nuclei transcribed in vitro. The direct inhibitory effect of glucocorticoid hormone on AFP gene transcription was demonstrated in a reconstituted cell-free nuclear system.

Animals↗