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J R Tata

Publications and source records attributed to J R Tata.

At least 73 records · Page 4Linked to original sources
PubMed1984

Sex- and tissue-specific, but hormonally independent, demethylation at the 3'-end of Xenopus vitellogenin gene B1.

We describe three HpaII-specifying sequences in 6 kbp of DNA at the 3'-end of Xenopus vitellogenin gene B1 (two within the coding sequence and one downstream) which remain methylated in all tissues of adult male Xenopus. These sites are however fully demethylated exclusively in adult Xenopus liver parenchymal cells but not in oviduct, kidney or erythrocytes. Developmental studies suggest that this tissue- and sex-specific demethylation may occur gradually after metamorphosis. The methylation of these 3'-sites is independent of transcriptional activation by oestrogen of vitellogenin genes in male or female Xenopus.

Animals↗
PubMed1984

Transient paralysis by heat shock of hormonal regulation of gene expression.

We have investigated the effect of heat shock on primary cultures of male and female Xenopus laevis hepatocytes as a function of estrogen-induced vitellogenin gene expression. Coincident with the induction of heat-shock protein (hsp) synthesis, thermal stress abolishes the estrogen activated transcription and accumulation of vitellogenin mRNA, at the same time causing the destabilization of vitellogenin mRNA accumulated by prior treatment with the hormone. Exposure of the cells to estrogen before heat shock allows an immediate resumption of vitellogenin gene transcription on return to 26 degrees C. Heat shock applied to cells from hormonally naive male Xenopus extends the lag period preceding vitellogenin gene transcription upon return to normal temperatures. This transient and reversible paralysis of estrogen responsiveness is paralleled by reversible changes in the amount of nuclear estrogen receptor in the hepatocytes. Heat shock therefore offers a novel approach in the manipulation and analysis of the early stages of steroid hormonal regulation of gene expression.

Animals↗
PubMed1984

Characterization of nascent and secreted thyroxine-binding globulin in cultured human hepatoma (Hep G2) cells.

Thyroxine-binding globulin (TBG) synthesis by human hepatoma (Hep G2) cells was demonstrated by pulse labeling with [35S]methionine or [3H]mannose and subsequent immunoprecipitation in the medium or cell lysate. Secreted TBG was glycosylated and had the same apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as TBG purified from human serum. Pulse-chase experiments failed to show any precursor form intracellularly. Treatment of cells with the amino acid analogs, canavanine and thialysine, did not cause secretion of large-molecular-weight moieties, in contrast to what was observed in the case of albumin. Thyroxine-binding activity, as assessed by [125I]thyroxine immunoprecipitation with anti-TBG serum, was detectable in the media of oocytes injected with RNA from Hep G2 cells. Translation of this RNA in rabbit reticulocyte lysate, followed by immunoprecipitation with anti-TBG serum, revealed a protein having the same electrophoretic mobility as deglycosylated TBG purified from human serum (Mr approximately 45,000). Since deglycosylated TBG still contains 3% of its weight as carbohydrate, it appears that the translation product contains an additional fragment (signal peptide) of about 1,500 daltons. It is unlikely, however, that TBG is synthesized via a larger-molecular-weight precursor.

Carcinoma, Hepatocellular↗
PubMed1984

Primary culture, cellular stress and differentiated function.

Isolation of specialized cell types for the analysis of tissue-specific gene function often results in loss of the differentiated phenotype. Examples of this type of phenotypic change following tissue disaggregation are reviewed together with possible explanations. Close similarities between the effects of cell isolation with those of other cellular stresses such as heat or anoxia point to common biochemical mechanisms being involved. This suggests that the study of freshly isolated cells will contribute significantly to out understanding of the nature of cellular stress and its consequences for the maintenance of phenotype and induction of tissue specific gene expression.

Animals↗
PubMed1984

The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri).

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 X 10(-2) and 1 X 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.

Animals↗
PubMed1984

Unequal activation by estrogen of individual Xenopus vitellogenin genes during development.

Using a technique of filter hybridization under very stringent conditions to HindIII fragments of complementary DNA cloned in plasmids, we have measured the accumulation in hepatocytes of mRNA specified by each of the four vitellogenin genes (A1, A2, B1, B2) at different stages of development of Xenopus laevis. The ontogenic competence of embryonic liver to respond to the first exposure to estradiol-17 beta, in terms of activation of transcription of this multigene family, is acquired late in metamorphosis at around Nieuwkoop-Faber stage 58. Upon hormonal induction, the four mRNAs accumulate under non-steady-state conditions at different rates and to different extents at all developmental stages in vivo and in cultured adult hepatocytes. A1 and B1 mRNAs appear more rapidly and accumulate to levels that are five- to eightfold those specified by genes A2 and B2, with higher amounts of B1 than A1 mRNA. A threefold higher absolute rate of synthesis of A1 and B1 mRNAs in hepatocyte cultures, relative to the A2-B2 pair, suggests that hormonal regulation of differential accumulation of vitellogenin mRNA occurs at the transcriptional level. At the early developmental stages (up to stage 61) of acquired competence, there appears to be no fixed pattern of expression, but a pattern of unequal activation of individual genes of the Xenopus vitellogenin multigene family is established thereafter and then retained at all developmental stages of tadpoles, froglets, and in both male and female adults.

Animals↗
PubMed1984

Culture shock. Synthesis of heat-shock-like proteins in fresh primary cell cultures.

The isolation of Xenopus liver, lung and testis cells by collagenase digestion of the tissue, followed by Percoll density gradient centrifugation, was characterized by the preferential synthesis of two proteins whose size and charge were similar to 70 and 85 kD heat-shock proteins. The synthesis of these two heat-shock-like proteins, relative to that of total protein, declined gradually in the first 3-4 days after the cells were plated out for primary culture. In fresh primary cultures of liver parenchymal cells albumin mRNA concentration declined rapidly and plateaued at 3-4 days of culture. Freshly isolated male Xenopus hepatocytes were refractory to induction by estrogen of vitellogenin gene transcription but they reacquired hormonal response during the first 3 days of culture. Both of these differentiated phenotypic functions of the Xenopus hepatocytes were quantitatively associated with the decline in synthesis of hsp-like proteins in freshly prepared primary cell cultures. Freshly isolated or heat-shocked hepatocytes exhibited a rounded shape with little intercellular contacts, whereas during the recovery period of 3 days they acquired a flattened shape with a high degree of intercellular and cell-substratum interaction. These results present the first evidence for the preferential synthesis of heat-shock-like proteins by procedures for establishing primary cell cultures. They emphasize the necessity of monitoring normal and heat-shock protein synthesis and cell morphology before using primary cell cultures for studying normal regulatory and developmental processes.

Albumins↗
PubMed1984

Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures.

We have used primary cell cultures of hepatocytes from male or female Xenopus laevis to study the mechanisms by which estrogen induces vitellogenin gene transcription and how primary exposure to estrogen renders cells more responsive to secondary stimulation. We have characterized the estrogen receptor in hormonally naïve cells and in hepatocytes treated with estrogen under a variety of conditions. Under all conditions the receptor has a Kd congruent to 4 X 10(-10) M. Hormonally naïve male cells contain 300 binding sites whereas female cells or male cells previously exposed to estradiol exhibit 6-7-fold higher levels. In parallel cultures, the absolute rate of vitellogenin gene transcription was determined by hybridization of newly synthesized RNA pulse-labelled with [3H]uridine to cloned Xenopus vitellogenin cDNA. Naïve male cells on primary stimulation with estradiol synthesized vitellogenin mRNA at an average rate of approximately 150 moles/cell/h compared to 1200 moles/cell/h for cells previously exposed to estrogen, thus bearing a close correlation with receptor number. Furthermore, we show that the kinetics of the induced up-regulation of receptor exactly parallel those of the increase in the rate of vitellogenin gene transcription upon secondary hormonal stimulation following various periods of primary exposure to estrogen. Addition of cycloheximide to cell cultures during primary estrogen treatment abolishes both receptor up-regulation and increased rate of vitellogenin gene transcription on secondary stimulation. In addition, primary treatment with the antiestrogen tamoxifen prevents both receptor up-regulation and an enhanced rate of transcription or accumulation of vitellogenin mRNA on secondary hormonal exposure. These results demonstrate that estrogen treatment of male Xenopus hepatocytes results in the rapid up-regulation of its own receptor to female levels via new receptor synthesis, and that receptor number is rate-limiting in vitellogenin gene transcription.

Animals↗
PubMed1983

Simultaneous analysis of conformation and transcription of A and B groups of vitellogenin genes in male and female Xenopus during primary and secondary activation by estrogen.

In male Xenopus, primary estradiol administration results in noncoordinate activation in the liver of the A and B groups of vitellogenin genes, both as judged by transcription and DNase I sensitivity in isolated nuclei, B group genes being activated preferentially in the first 20 hr. Secondary induction in males or "primary" induction in females results in a coordinate and equal transcription of these two groups of genes. The elevated transcriptional activity following primary estrogen stimulation returns to low levels rapidly but the high DNase I sensitivity of these genes persists for 2-3 months. A non-coordinate activation of the A and B groups of vitellogenin genes is however re-established in response to a second administration of estradiol 8 months after primary stimulation of male Xenopus.

Animals↗
PubMed1983

Coordinate and non-coordinate estrogen-induced expression of A and B groups of vitellogenin genes in male and female Xenopus Hepatocytes in culture.

The concentration of mRNA transcribed from A and B groups of vitellogenin genes, induced by estrogen added to primary cultures of hepatocytes from male and female adult Xenopus, was measured by a technique of filter disc hybridization with cloned Xenopus vitellogenin cDNA probes. In cells from naive male Xenopus (i.e. not previously exposed to hormone in vivo or in vitro), the two groups of the multigene family were non-coordinately expressed during the early stages of response to estradiol in vitro. Only B group transcripts could be detected for the first 2-3 h. At later times, or upon successive additions of estradiol to the male cell cultures, both A and B group mRNAs accumulated at the same rate and to the same extent. In female hepatocytes both groups of mRNAs accumulated in parallel at all stages of their response to estrogen, reaching levels 10-fold higher than in naive male cells similarly exposed to the hormone. However, hepatocytes from male Xenopus that had received a single injection of estradiol 5 weeks before the cells were prepared, now exhibited identical rates and extent of accumulation of A and B groups of vitellogenin mRNAs to those observed in female cells. Pulse-labeling of RNA in cultured male Xenopus hepatocytes confirmed that the coordinate and non-coordinate accumulation of mRNAs were largely a function of differential or equal transcription of the A and B groups of vitellogenin genes. A phenomenon analogous to the hormone-induced shift from non-coordinate to coordinate expression of two groups of genes of the same multigene family has not been described previously, and we discuss possible mechanisms underlying the transition.

Animals↗
PubMed1983

Hormonal regulation and expression of vitellogenin multigene family.

Yolk proteins are the most abundant egg proteins in oviparous animals. They are deposited during oocyte maturation for use after fertilization and are synthesized in the liver or fat body as a common precursor termed vitellogenin. Hybridization with cloned DNA complementary to vitellogenin messenger RNA has revealed a surprisingly high degree of evolutionary conservation of sequence of vitellogenin genes among insects, amphibians and birds. The synthesis of vitellogenin in vertebrates is directly under the control of oestrogen at the level of gene transcription. In the frog, Xenopus, vitellogenin genes occur as a multigene family, four of which are actively expressed and are grouped as A and B genes. This multiplicity offers a useful system for investigating the possible selective hormonal regulation of expression of individual members of multigene families. When X. laevis vitellogenin genes were activated by oestrogen in the liver of whole animals or in cultures of parenchymal cells, the two groups of expressed genes were not induced in an identical manner in cells from male and female animals. The activation of A and B groups of genes was non-coordinate in male hepatocytes and coordinate in female cells. Prior exposure of male hepatocytes to oestradiol in vivo or in culture caused the pattern of expression to shift to that in female cells. Since the X. laevis oocyte itself does not synthesize vitellogenin in response to oestrogen, an attempt was made to activate its dormant vitellogenin genes by transferring oestrogen-binding proteins from the liver. Preliminary results show that the microinjection into the oocyte of a preparation containing liver receptor-hormone complex led to the synthesis of vitellogenin by the oocyte. Extension of these experiments will not only enable a more precise analysis of the activation of the vitellogenin multigene family to be made but will also provide direct functional evidence for the role played by steroid hormone receptors in regulating gene expression.

Animals↗
PubMed1983

Rapid estrogen metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures.

Male hepatocytes metabolized estradiol-17 beta, 17 alpha-ethinylestradiol and mestranol extremely rapidly (t 1/2 = 40, 60 and 300 min, respectively), whereas these were more stable in cultures of female hepatocytes (t 1/2 = 120, 150 and 640 min, respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10(-6) M estradiol to male hepatocyte cultures; mestranol, but not 17 alpha-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5-15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17 alpha-ethinylestradiol and diethylstilbestrol. Ovariectomy, 60 days prior to cell culture, did not alter the metabolism of estradiol or the vitellogenic response of female hepatocytes. On the other hand, a single administration of estradiol in vivo to male Xenopus caused a long-lasting shift (at least 16 weeks) to the female pattern of its metabolism, although the enhanced inducibility of vitellogenin genes was partially reversed between 4 and 16 weeks after hormonal treatment. The addition of fresh estradiol every 4 h to male hepatocyte cultures to compensate for its rapid metabolism resulted in a continuous and sustained accumulation of vitellogenin mRNA at rates comparable to those attained in vivo. Our findings explain the requirement for high levels of estrogen to activate vitellogenin genes and establish Xenopus hepatocyte cultures as a reproducible system for analysing the expression of this multigene family.

Animals↗
PubMed1982

Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.

Animals↗
PubMed1982

Differential sensitization to deoxyribonuclease I of Xenopus vitellogenin and albumin genes during primary and secondary induction of vitellogenesis by oestradiol.

The sensitivity to DNAase (deoxyribonuclease) I (which preferentially digests transcribed sequences) of vitellogenin and albumin genes in liver and erythrocytes of male Xenopus after primary and secondary induction of vitellogenesis by oestrogen was measured by hybridization to cDNA (complementary DNA) of the residual DNA after enzymic digestion of isolated nuclei. Vitellogenin sequences were rendered selectively more sensitive to limited DNAase-I digestion (15-20% of DNA rendered acid-soluble) during primary hormonal activation (5 days) of vitellogenin genes in liver, but not erythrocyte, nuclei. Hormone withdrawal (25 days after first injection) did not result in reversion to a pre-activation gene configuration, nor did secondary hormonal stimulation (5 days after second and 25 days after first injection) augment the sensitivity of the genes to digestion by the nuclease. Similar hormone treatment did not affect the sensitivity of the constitutively expressed albumin genes in liver nuclei, nor their insensitivity in erythrocyte nuclei. Under the same conditions, globin genes remained indigestible in liver nuclei. It is concluded that primary induction of vitellogenesis in male Xenopus liver is accompanied by relatively long-lasting (3-4 weeks) change in the configuration of vitellogenin genes in hepatic nuclei which is not reversed or further modified during short-term oestrogen withdrawal or upon secondary stimulation.

Albumins↗