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J R Tata

Publications and source records attributed to J R Tata.

At least 91 records · Page 5Linked to original sources
PubMed1982

Evolutionary conservation of vitellogenin genes.

Homologous and heterologous hybridizations in solution were performed between sheared genomic DNA and DNA complementary to vitellogenin mRNA of Xenopus, chicken, and migratory locust. The kinetics of hybridization and the thermal stability of the hybrids formed suggested a high degree of conservation of coding sequences of insect, amphibian, and avian vitellogenin genes. These cDNA probes hybridized to calf thymus DNA to a slight, but significant, extent, and not at all to Micrococcus lysodektikus DNA. DNA complementary to Xenopus albumin mRNA did not cross-hybridize significantly with locust or chicken DNA. Further evidence for the evolutionary conservation of vitellogenin genes was obtained from Southern blot analysis of restriction endonuclease-digested genomic DNA from a variety of vertebrate and invertebrate oviparous animals (Xenopus, chicken, migratory and desert locusts, yellow meal worm, carab moth, and Mediterranean fruitfly). When probed with cloned vitellogenin cDNAs from Xenopus and migratory locust, the DNA of these organisms showed varying degrees of homology of parts of the vitellogenin coding sequences. Southern blot analysis also showed that a part of the sequence specified in the cloned Xenopus vitellogenin cDNA was represented as repetitive DNA in the locust genome. However, cloned locust vitellogenin cDNA hybridized to discrete fragments of the restricted vertebrate DNA. These studies demonstrate a remarkably high degree of conservation of insect, amphibian, and avian vitellogenin genes.

Animals↗
PubMed1981

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.

Animals↗
PubMed1981

Vitellogenin gene expression in male Xenopus hepatocytes during primary and secondary stimulation with estrogen in cell cultures.

Primary cultures of male Xenopus liver parenchymal cells that retained their competence to respond to estrogen were used to study the hormone-induced activation of the vitellogenin gene in vitro. The accumulation of vitellogenin mRNA in these cells was monitored by a quantitative diazotized paper disc hybridization procedure with a sensitivity of at least 6 pg of sequences complementary to the probe in total RNA samples of 10 micrograms. A short-term time-course analysis showed that vitellogenin mRNA was detectable within 3 hr of exposure to estrogen during primary stimulation, and that the maximum rate of accumulation was reached at 5--6 hr. A long-term time-course analysis of the accumulation of vitellogenin mRNA showed that it is possible to obtain a primary response, a hormone withdrawal effect and an enhanced secondary response in the same batch of cells in a manner analogous to that observed in vivo. Measurement of hormone concentration dependence showed a response at 10(-9) M estradiol, which continued to increase up to at least 10(-6) M estradiol. This requirement for large doses of estradiol for maximal response can be explained by the rapid metabolism of estradiol by the cultured cells.

Animals↗
PubMed1980

Vitellogenin as a multigene family. Not all Xenopus vitellogenin genes may be in an "expressible" configuration.

DNA-cDNA titrations suggest that the Xenopus genome comprises two albumin genes, whereas there are 12 to 16 vitellogenin-like genes. Both sets of genes are expressed in liver, those coding for albumin constitutively, but vitellogenin only when induced by estrogen in both male and female frogs. Mild micrococcal nuclease digestion of liver nuclei to separate "expressed" from "unexpressed" gene fractions (Tata, J. R., and Baker, B. S. (1978) J. Mol. Biol. 118, 249-272) revealed that the multiple vitellogenin genes are found in two chromosomal states, here termed as "expressible" and "nonexpressible." Both albumin genes were in the chromosomal state associated with expression, but of the 12 to 16 vitellogenin genes, only 4 to 6 are in the potentially expressible state. This distribution pattern is independent of sex and only slightly modified during induction or de-induction of vitellogenesis by estrogenic stimulation or withdrawal.

Animals↗
PubMed1980

Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes.

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

Animals↗
PubMed1980

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity.

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.

Animals↗
PubMed1977

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

Animals↗
PubMed1977

Two fractions of rough endoplasmic reticulum from rat liver. II. Cytoplasmic messenger RNA's which code for albumin and mitochondrial proteins are distributed differently between the two fractions.

Subcellular fractions were obtained from rat liver homogenates under conditions which prevented degradation of polysomes (pH 8.5 and high ionic strength). Rough endoplasmic reticulum (RER) was recovered in high yields from a low-speed nuclear pellet (rapidly sedimenting endoplasmic reticulum, RSER) and from a postmitochondrial supernate (rough microsomes). The polysomal RNA content of these two fractions was very similar. When polyA+-RNA's were translated inthe mRNA-dependent wheat embryo cell-free system, both fractions yielded polypeptide products which had similar electrophoretic patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Activities of messenger RNA's which code for albumin and for polypeptides destined for transport to the inner membrane and matrix of mitochondria (i.e. 'mitoplasts') were assayed by translating in the more active rabbit reticulocyte cell-free system followed by immunoprecipitation of radioactive products and coelectrophoresis with immunoprecipitated marker proteins on SDS-polyacrylamide gels. These tests indicated that albumin mRNA is about equally distributed between the two fractions of RER, or slightly enriched in the RSER fraction when activity is expressed as a percentage of total polypeptide synthesis. Activities of cytoplasmic mRNA's which code for at least some mitoplast proteins could be detected in both fractions, but all were enriched in the rough microsome fraction, not the RSER (two- to threefold when corrected for differences in total polypeptide synthesis in the lysate). Comparisons of mRNA's from free vs. membrane-bound polysomes indicated that most of the albumin mRNA activity (86-91%) and mitoplast protein mRNA activities (75%) were present in the bound fraction. Assuming that RSER and rough microsomes do not derive exclusively from different cells types, the evidence suggests that, compared to albumin and most other membrane-bound mRNA's, cytoplasmic mRNA's coding for mitoplast proteins may be preferentially segregated or compartmentalized within the cell on the microsomal class of RER.

Albumins↗
PubMed1976

Variable stabilities and recoveries of rat-liver RNA polymerases A and B according to growth status of the tissue.

1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed.

Aging↗