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F C Bancroft

Publications and source records attributed to F C Bancroft.

At least 19 recordsLinked to original sources
PubMed1984

The prolactin gene, but not the growth hormone gene, is located on chromosome 9 in the rat.

Experiments from a number of laboratories, employing clonal lines of rat pituitary tumor cells (GH cells), have shown that the cellular levels of the mRNAs for prolactin and growth hormone are subject to regulation by a variety of hormones and other factors. To provide a framework for further studies of the regulation of the expression of these two genes, we have studied their chromosomal locations in the rat, employing our recently developed technique for identifying a unique gene by direct hybridization in situ with a 125I-labeled cDNA plasmid. A rat prolactin cDNA plasmid ( pPRL -1) yielded a signal significantly above that expected on a random basis over chromosomes 9 and 15 from rat FRE cells. Both chromosomes exhibited a subchromosomal clustering of silver grains near the telomere of chromosome 9 and on the short arm of chromosome 15. Control experiments with plasmid pBR322 also yielded a significant signal clustered over the short arm of chromosome 15. Hence, we conclude that chromosome 15 contains sequences homologous to pBR322 and that the prolactin gene is located near the telomere of chromosome 9 in the rat. A rat growth hormone cDNA plasmid (pBR322-GH1) yielded a signal above that expected on a random basis over FRE cell chromosomes 7, 13, and 15, although the labeling was poorer than with prolactin. The signal over chromosome 15 is probably due to sequences homologous to plasmid pBR322. The signals over chromosomes 7 and 13 showed a clear subchromosomal clustering near the centromere on chromosome 7 and near the telomere on the long arm of chromosome 13.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells.

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.

Animals↗
PubMed1983

Epidermal growth factor and thyrotropin-releasing hormone interact synergistically with calcium to regulate prolactin mRNA levels.

The regulation of prolactin RNA sequences by Ca2+, epidermal growth factor (EGF), and thyrotropin-releasing hormone (TRH), singly and in combination, was studied in GH3 cells incubated in a chemically defined medium. Ca2+ increased all large prolactin RNA sequences in the nucleus, thus suggesting a transcriptional site of action. A recently developed cytoplasmic dot hybridization procedure was employed to study regulation of cytoplasmic prolactin mRNA. Ca2+ alone stimulated prolactin mRNA with a lag of between 3 and 9 h and yielded a sizable final increase (14- to 18-fold) of prolactin mRNA. With EGTA present to chelate any residual Ca2+, prolactin mRNA was not detectably increased by TRH, showing that TRH requires Ca2+ to stimulate prolactin mRNA. Under these conditions, prolactin mRNA was only slightly (4-fold) increased by EGF. Either peptide plus an optimal concentration of Ca2+ yielded a 60- to 70-fold increase in prolactin mRNA, showing that both EGF and TRH regulate prolactin mRNA synergistically with Ca2+.

Animals↗
PubMed1982

Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples.

A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numerous small samples of animal cells or tissue is described. The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning. By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis is minimized. Experiments with a clonal line of rat pituitary tumor (GH3) cells showed that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 2.5 x 10(4) cells. The specificity of the technique for prolactin mRNA was shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences. Experiments with cultured rat hemipituitaries showed that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary could be readily detected. This technique is quite simple, requires very small amounts of cells or tissue, and permits the simultaneous analysis of multiple samples. Hence, it should be quite useful for studies with various experimental systems of the regulation of specific mRNA levels.

Animals↗
PubMed1981

Calcium specifically stimulates prolactin synthesis and messenger RNA sequences in GH3 cells.

Addition of CaCl2 (0.4 mM) to GH3 cells incubated in a serum-free, Ca2+-free medium increased relative prolactin synthesis (prolactin synthesis/total protein synthesis) 7- to 150-fold by 3-4 days, but had no effect on relative growth hormone synthesis. Half-maximal and maximal stimulation of relative prolactin synthesis occurred at about 0.07 and 0.2 mM CaCl2, respectively. Neither MgCl2 (0.4 mM) nor KCl 0.4 mM) affected relative prolactin synthesis. 32P-labeled rat prolactin and growth hormone cDNA plasmids were employed to measure by RNA gel blot hybridization changes in cytoplasmic levels of the corresponding mRNA sequences. Both prolactin mRNA sequence levels and translatable prolactin mRNA increased in parallel with the CaCl2-induced increase in relative prolactin synthesis. CaCl2 increased prolactin mRNA sequences by 7- to 200-fold, without affecting growth hormone mRNA sequence levels. Dexamethasone (100 nM) decreased prolactin mRNA sequences in cells incubated with CaCl2, antagonized the CaCl2-induced increases in prolactin mRNA sequences independently of the CaCl2 concentration.

Animals↗
PubMed1981

Thyroid or glucocorticoid hormone induces pre-growth-hormone mRNA and its probable nuclear precursor in rat pituitary cells.

Thyroid or glucocorticoid hormone increases the synthesis of growth hormone (GH) by clonal lines of rat pituitary tumor cells. To investigate whether these increases arise from increased accumulation of GH-specific RNA sequences in the cytoplasm and nuclei of these cells, we adapted two existing procedures so that a 32P-labeled hybrid plasmid containing a cDNA sequence could be used to quantitate relative concentrations of the corresponding mRNA. One method (RNA gel blot hybridization) used electrophoresis of RNA, transfer to nitrocellulose paper, and hybridization to 32P-labeled plasmid. The other (RNA dot hybridization) used covalent attachment of RNA to activated cellulose paper squares and hybridization to 32P-labeled plasmid. As probe, we used a hybrid plasmid (pBR322-GH1) which we show by restriction analysis to contain a DNA sequence coding for rat GH. The results were comparable from both techniques and showed that incubation of GH3 cells with a thyroid hormone (triiodothyronine), a glucocorticoid hormone (dexamethasone), or both hormones caused an increase of cytoplasmic pre-GH mRNA sequences of about 4-, 22-, and 13-fold, respectively. Results obtained with the RNA gel blot hybridization method showed that hormonal stimulation leads to the induction of a single 1.0-kilobase species of pre-GH mRNA in the cytoplasm and of 2.7- and 1.0- kilobase species of GH-specific RNA in the nucleus.

Animals↗
PubMed1981

Localization of a unique gene by direct hybridization in situ.

Previous determination of the chromosomal location of unique genes have required that the chromosomes of interest be fractionated, either by species-specific chromosome loss from interspecies hybrids, or by physical fractionation procedures. We have developed a general technique for the localization of a unique gene, which requires no prior chromosome fractionation. The technique involves the use of a labeled hybrid cDNA plasmid for direct hybridization in situ to metaphase cells from the organism under investigation. As a model system for development of this technique, we have employed a human alpha-globin cDNA plasmid (JW101) to localize the corresponding gene cluster. To obtain a sufficiently large autoradiographic signal, we have both labeled this plasmid with 125I to a high specific activity (10(9) dpm/micrograms) and taken advantage of the ability of a double-stranded probe to form networks. To obtain sufficient hybridization specificity, various experimental procedures were used, the most important of which was the choice from among a range of probe concentrations of the highest that does not yield excessive background hybridization. We have shown that, with an autoradiographic exposure time of only 12 days, use of this technique correctly localized the human alpha-globin gene cluster to chromosome 16. This technique should be generally applicable to the localization of any gene for which a corresponding cDNA hybrid plasmid is available.

Chromosome Mapping↗
PubMed1979

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.

Animals↗
PubMed1979

Rat growth hormone gene: intervening sequences separate the mRNA regions.

Rat genomic DNA was digested with various restriction endonucleases, separated by gel electrophoresis and hybridized to 125 I-labeled mRNA for the precursor protein to growth hormone. Restriction sites were found within the genomic DNA that were not found in the previously reported DNA sequence corresponding to the mRNA (19). It appears that the gene for growth hormone contains intervening sequences separating the DNA regions that specify mRNA sequences.

Animals↗
PubMed1979

Interferon priming. Effects on interferon messenger RNA.

The effects of priming mouse cells with interferon on the production of interferon and its mRNA were investigated. Interferon-treated (primed) mouse L929 cells produce 3 to 10 times more interferon than do nonprimed cells following induction with Newcastle disease virus. Interferon appears 2 to 4 h sooner in the primed cultures than in nonprimed cultures and interferon production by primed cells becomes resistant to inhibition by actinomycin D about 4 h sooner than interferon production in nonprimed cells. Interferon mRNA is detected in primed-induced cells about 2 h earlier than in nonprimed-induced cells. It reaches peak levels about 2 to 4 earlier in primed cells, but it also disappears sooner in primed cells. The total amounts of interferon mRNA isolated from primed-induced cells and nonprimed-induced cells were indistinguishable, by the methods utilized. Therefore, although primed cells can produce significantly more interferon and make interferon mRNA sooner than nonprimed cells, the total amount of interferon mRNA produced is apparently not increased, nor is its half-life prolonged in primed cells. Thus, enhanced interferon production in primed cells may result from enhanced efficiency of translation of interferon mRNA in the primed cells.

Animals↗
PubMed1978

Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.

Cell Line↗
PubMed1977

Glucocorticoid induction of growth hormone synthesis in a strain of rat pituitary cells.

Conditions were determined for measuring growth hormone synthesis by a clonal strain of rat pituitary cells grown in suspension culture. Incubation of the cells with [3H]leucine in either continuous labeling or pulse-chase experiments showed that secretion of newly synthesized growth hormone commences only after a lag of about 15 min. The pulse-chase experiments also demonstrated that there is no detectable degradation by the cells of growth hormone. Thus growth hormone synthesis could be measured, in the absence of complications arising either from secretion or degradation of growth hormone, by incubating the cells with [3H]leucine for 10 min. Exposure of cells grown under the usual culture conditions to dexamethasone (a synthetic glucocorticoid) led to an average stimulation of specific growth hormone synthesis (growth hormone synthesis/total cytoplasmic protein synthesis) of only 2.6-fold. However, two other growth conditions were found in which dexamethasone routinely yielded a 5- to 15-fold stimulation of specific growth hormone synthesis. One of these conditions, involving substitution of 10% fetal calf serum for the normal serum supplement, was employed in subsequent experiments. A stimulation of specific growth hormone synthesis could be observed at 10(-9) M dexamethasone, and the maximum stimulation was observed at dexamethasone concentrations of about 10(-8) to 10(-7) M. There was a lag of about 6 h before a stimulation by dexamethasone of specific growth hormone synthesis was detected. Thereafter, the stimulation increased in a nearly linear fashion until maximum stimulation was reached at about 48 h.

Animals↗
PubMed1977

Pregrowth hormone messenger RNA: glucocorticoid induction and identification in rat pituitary cells.

We have investigated whether the large (5- to 15-fold) stimulation of synthesis of growth hormone (somatotropin) observed upon exposure of a strain of rat pituitary (GH3) cells to dexamethasone (a synthetic glucocorticoid) arises from a stimulation of pregrowth hormone mRNA. A good agreement was observed between the stimulation of growth hormone synthesis and the stimulation of cytoplasmic levels of pregrowth hormone mRNA, assayed by translation in wheat germ extracts. Electrophoresis on formamide-acrylamide gels of oligo(dT)-treated membrane fraction RNA revealed a band that was induced by dexamethasone to about the same extent as was translatable pregrowth hormone mRNA. Elution of the band and translation in wheat germ extracts showed directly that it contained pregrowth hormone mRNA. These results suggest that the dexamethasone induction of growth hormone synthesis in GH3 cells arises from an accumulation of pregrowth hormone mRNA in the cytoplasm of the cells. The pregrowth hormone mRNA band observed by gel electrophoresis had an estimated molecular weight of 3.6 X 10(5), suggesting the existence in this molecule of an untranslated region at least 200 nucleotides in length.

Cell Line↗
PubMed1976

Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone.

Membrane fraction RNA isolated from rat pituitary tumor (GC) cells has been translated in a wheat germ extract. A product was synthesized which was immunologically related to growth hormone, but which migrated more slowly than growth hormone upon sodium dodecyl sulfate-acrylamide gel electrophoresis. The mobility of the cell-free product on gels of this type was unchanged by treatment with either KOH or RNase. The mobilities during paper electrophoresis of the methionine-containing tryptic peptides obtained from the cell-free product were identical to those obtained from growth hormone synthesized and secreted by the GC cells. Molecular weights for growth hormone and the cell-free product of 19,500 and 24,000, respectively, were determined by gel electrophoresis of these proteins together with marker proteins of known molecular weights. No protein with the properties of the cell-free product was detected after a 2 min incubation of the GC cells with [35S]methionine. However, treatment of the GC cells, with a protease inhibitor, L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK), led to the appearance of a new polypeptide, immunologically related to growth hormone, and with a mobility on gels identical to that of the cell-free product. These results strongly imply that the cell-free product represents a growth hormone precursor (pregrowth hormone) which is rapidly converted to growth hormone in pituitary cells.

Cell Line↗