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A Belayew

Publications and source records attributed to A Belayew.

53 records · Page 3Linked to original sources
PubMed1989

Pituitary-specific factor binding to the human prolactin, growth hormone, and placental lactogen genes.

The human genes coding for growth hormone (GH), chorionic somatomammotropin (placental lactogen, CS), and prolactin (Prl) are related evolutionarily but are expressed in phenotypically distinct cell types despite their nucleotide sequence homology. We show here that the promoters of the human Prl and CS genes contain cis-acting sequences that confer pituitary-specific expression in a cell-free transcription assay. Similar data are obtained with the human GH gene, consistent with earlier work by others. Footprinting analysis shows that neighboring sequences in each of these three promoters are protected from deoxyribonuclease I digestion by rat pituitary cell extracts. Footprinting competition experiments and gel retardation assays with synthetic oligonucleotides suggest that a single factor is responsible for the pituitary-specific footprints seen on the human Prl, CS, and GH genes. They also suggest that this factor is identical or closely related to the trans-acting factor GHF-1/Pit-1. Similarities with and differences from the rat GH and Prl genes are discussed.

Animals↗
PubMed1988

Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

Animals↗
PubMed1988

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine.

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.

Animals↗
PubMed1987

Glucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions.

Modulation of gene expression by glucocorticoids involves interaction of these hormones with an intracellular receptor followed by 'transformation' of the hormone-receptor complex into a nuclear binding form. The molecular basis for the antiglucocorticoid action of high-affinity steroid analogues such as RU486 remains controversial. The effects of dexamethasone and RU486 on in vitro and in vivo properties of the receptor were compared using human lymphoblastoid IM-9 cells. In these cells, RU486 fully antagonized the glucocorticoid-specific induction of 5'-nucleotidase activity by dexamethasone. In vitro, however, RU486-bound receptor could be transformed and shown to interact specifically with cloned DNA fragments containing glucocorticoid response elements. These fragments included one from the mouse mammary tumour virus and two from the human growth hormone gene. In vivo, RU486-bound receptor did not behave like dexamethasone-bound receptor. While receptor downregulation, a property of the transformed receptor, was achieved by dexamethasone, this did not occur with RU486. Likewise, RU486 did not affect receptor half-life under conditions when this was shortened by dexamethasone. These seemingly contradictory results can be reconciled by proposing that receptor transformation by agonists involves dissociation of the receptor oligomer to reveal a DNA-binding site that pre-exists on this protein. Although cell-free receptor dissociation and therefore DNA binding can occur even when the receptor is bound to RU486, this steroid maintains receptors in the untransformed state in the intact cell and therefore behaves a glucocorticoid antagonist in vivo.

5'-Nucleotidase↗
PubMed1986

Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions.

Base Sequence↗
PubMed1986

[Localization of transcription regulatory sequences. Application to the genes of the prolactin family].

We are studying nucleotide sequences responsible for the regulation of eukaryotic gene expression. Our test system comprises the human genes coding for prolactin (hPRL), growth hormone (hGH-N) and placental lactogen (hCS-B). We have cloned these genes and are searching within their sequences for in vitro binding sites of the human glucocorticoid receptor on the hGH-N and hCS-B genes; the in vivo activity of such DNA sequences by assaying hybrid gene expression in transfected cells; in vivo "enhancer" activity of different hPRL gene fragments linked to a marker gene and transfected in cultured cells.

Base Sequence↗
PubMed1985

Binding of the human glucocorticoid receptor to defined regions in the human growth hormone and placental lactogen genes.

An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic somatomammotropin, hCS). These genes display 95% sequence homology. Two receptor-binding regions were found in the hGH gene, one of which is located within 290 bp upstream, and one within 251 bp downstream from the transcription initiation site. Two binding regions homologous to those in the hGH gene were found in the hCS gene. The receptor-binding DNA fragment from the structural part of the genes, but not that from their promoter area, contained a sequence homologous to a 15-bp consensus sequence proposed earlier for the glucocorticoid receptor binding site. It is unlikely that the putative difference in glucocorticoid sensitivity between the hGH and hCS genes is accounted for by major differences in glucocorticoid receptor binding pattern.

Base Sequence↗
PubMed1984

Isolation and characterization of the human prolactin gene.

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900-bp sequenced in the 5'-flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Animals↗
PubMed1984

Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes.

The levels of alpha-fetoprotein mRNA in mice are determined by at least two trans-acting, unlinked genes, raf and Rif. raf determines the basal levels of alpha-fetoprotein mRNA in adult mice, while Rif determines its degree of inducibility during liver regeneration. To determine whether these regulatory loci affect other structural genes, we screened a murine fetal liver cDNA library for clones containing mRNA sequences that decrease after birth. One such clone, termed pH19, was identified, and its mRNA was shown to be under the control of both raf and Rif. The single-copy gene for H19 mRNA was localized to chromosome 7, and genetic crosses established that it was unlinked to either raf or Rif. It encodes a 2.5-kilobase mRNA that was identified in those tissues that produce alpha-fetoprotein: visceral endoderm, liver, and fetal gut. The repression of H19 mRNA in neonatal liver occurs several days after the decrease in alpha-fetoprotein mRNA, whereas inductions of both mRNAs during the differentiation of F9 teratocarcinoma cells into visceral endoderm were identical. The tissue-specific expression of H19 mRNA is different from that of alpha-fetoprotein in that H19 mRNA was detected also in both cardiac and skeletal muscle where no alpha-fetoprotein mRNA is produced. Despite the fact that the levels of H19 mRNA decline to 1/10th to 1/20th in cardiac muscle after birth, the adult basal levels are not under the influence of raf. This observation argues that the raf gene is a tissue-specific regulator of mRNA levels.

Animals↗
PubMed1982

Transcriptional control of the murine albumin/alpha-fetoprotein locus during development.

The ontogeny of expression of the alpha-fetoprotein (AFP) and albumin genes was examined in livers from late prenatal to 1-month-postpartum C3H/He mice. A parallel accumulation of both AFP and albumin mRNAs before birth, followed by a selective nonreciprocal decrease in AFP mRNA after birth, was observed. The decrease in AFP mRNA was the result of a decrease in transcription of the AFP gene, as measured by an in vitro nuclear transcription assay. We suggest a model for hepatic expression of the AFP and albumin gene cluster in which transcription of the two genes is activated simultaneously during differentiation and each gene is thereafter modulated independently in committed cells.

Aging↗
PubMed1982

Genetic analysis of alpha-fetoprotein synthesis in mice.

The differential induction of alpha-fetoprotein (AFP) mRNA during liver regeneration in three inbred strains of mice was examined to determine the genetic and molecular bases for the differences in protein production. BALB/cJ, C3H/He, and C57BL/6 mice, previously identified as high, intermediate, and low AFP producers, respectively, were used. Liver AFP mRNA concentrations during normal development and after carbon tetrachloride administration were measured and shown to correlate exactly with the serum protein concentrations. By performing a series of genetic crosses, we identified two unlinked genetic loci that acted independently to affect the inducibility of AFP mRNA. The raf gene, previously identified by Olsson et al. (J. Exp. Med. 145:819-827, 1977), determines the adult basal level of AFP mRNA, and the Rif gene affects its inducibility during regeneration. By using a polymorphic restriction endonuclease site within the albumin-AFP structural gene region, we show that neither regulatory gene is closely linked to the structural genes. In addition, neither gene affects the concentration of albumin mRNA during development or liver regeneration.

Animals↗
PubMed1980

Extraction and translation of collagen mRNA from fetal calf skin.

RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)-rich RNA was separated by oligo(dT)-cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell-free system, unfractionated guanidine-hydrochloride-extracted poly(A)-rich RNA directed the synthesis of two collagenase-sensitive protein bands, while phenol-extracted poly(A)-rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/urea/polyacrylamide gel, these proteins were identified with procollagen alpha 1(I) and procollagen alpha 2. Inhibition of translation by phenol-extracted poly(A)-rich RNA with a sedimentation coefficient lower than 25 S was also observed. Guanidine-hydrochloride-extracted poly(A)-rich RNA from fetal skin directed the synthesis of three distinct collagenase-sensitive proteins in the micrococcal-nuclease-digested rabbit reticulocyte cell-free system; these seemed to correspond to procollagen alpha 1(I), procollagen alpha 2 and procollagen alpha 1 (III).

Animals↗
PubMedDate not supplied

The human genome contains hundreds of genes coding for finger proteins of the Krüppel type.

Our aim was to identify new human proteins with potential DNA binding activity, related to the Krüppel protein which regulates Drosophila segmentation. We screened a human placenta cDNA library and a human genomic DNA library with a synthetic oligonucleotide probe corresponding to the H/C link region that connects finger loops in the multifingered Krüppel protein. We found more than 100 different mRNAs encoding Krüppel multifingered proteins in the human placenta. In the whole human genome, the number of genes encoding such proteins reaches about 300. Sequence analysis of 14 cloned cDNAs indicated that they code for at least nine undescribed human finger proteins. The sequences of the 106 finger repeats present in these nine proteins are highly homologous. Most of the variability lies in a limited number of positions located in their postulated alpha-helical structure, and therefore could be implicated in their DNA-binding specificity.

Amino Acid Sequence↗