Transcription of globin genes in reticulocyte chromatin (original) (raw)
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Synthesis of Globin Ribonucleic Acid from Duck-Reticulocyte Chromatin In Vitro
Proceedings of the National Academy of Sciences, 1973
The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo . In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.
Structure of the globin genes in chromatin
Biochemistry, 1975
The distribution of proteins in the neighborhood of the globin genes of duck reticulocyte chromatin has been studied. This chromatin is first shown to be an active template for transcription in vitro of globin messenger-like RNA. The chromatin is then treated with staphylococcal nuclease and the D N A fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). In order to determine the distribution of globin gene sequences in open and covered DNA, these two fractions are annealed to globin cDNA (globin probe). It is found that while all globin gene sequences are represented in covered DNA, a specific portion of the globin gene is missing from open DNA,
Sub-nuclear fractionation II. Intranuclear compartmentation of transcription in vivo and in vitro
Experimental Cell Research, 1974
DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper . Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to a-amanitin, exogenous native and denatured DNA, thermal denaturation at 45, Mg2+ and Mn2 ions, high ionic strength and by the binding of '*C-methyl-y-amanitin. RNA polymerase B (a-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of cc-amanitin subnuclear fractions that had been pre-incubated at 45°C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.
The poly (A)-containing nuclear RNA from sor for fi globin mRNA only. In addition, it was dimethylsulfoxide-induced Friend leukemia shown by electrophoretic analysis that the cells was fractionated by acrylamide gel complex of RNA molecules not resolved by electrophoresis in denaturing conditions and sucrose gradient centrifugatlon (1 IS) com- analyzed for a and fi globin RNA sequences. prises sequences of decreasing size (0.34, The results Indicate that nuclear RNA 0.28, and 0.26 X l0� daltons), which might contains one species of large-size RNA (0.6 be the precursors of a and fi globin x l0� daltons), which is the putative precur- mRNA. I N EUKARYOTES gene transcription is temporally and physically remote from translation. Indeed only a minor proportion of the nuclear transcripts become available for translation in the cytoplasm and long after their production.' Nuclear nonribosomal transcripts (heterogenous RNA, HnRNA) are produced in vast excess over cytoplasmic messenger RN...
The Journal of cell biology, 1980
To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with c...
Partial Purification of the Template-Active Fraction of Chromatin: A Preliminary Report
Proceedings of the National Academy of Sciences, 1974
A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl 2 . The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA·RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.
Experimental Cell Research, 1978
Stimulation of the T3C12 clone of Friend erythroleukemia cells with 1.2% dimethyl sulfoxide (DMSO) results in progressive increase in the concentration of globin mRNA sequences in the total cellular RNA of treated cells, as measured by nucleic acid hybridization employing a globin cDNA probe. The greatest increment in the content of globin RNA occurs between 30 and 40 h after addition of DMSO. Globin cDNA was also used to measure the concentration of globin-specific sequences in the DNA and RNA of transcriptionally active and inactive chromatin fractions prepared from these cells by the DNase II -MgCl, procedure of Gottesfeld et al. [ 161. Essentially equal concentrations of globin sequences are present in the DNA isolated from active and inactive chromatin fractions of cells grown in the presence of 1.2 % DMSO for 50 h (the time of initiation of hemoglobin synthesis). Furthermore, there are no significant differences in the globin gene concentrations between the active chromatin fractions from DMSO-treated and control cultures at either 50 or 120 h after initiation of DMSO treatment. However, chromatin-associated RNA isolated from the active chromatin of cells synthesizing maximum amounts of hemoglobin (120 h) contains a higher concentration of globin sequences than RNA from the active chromatin of control cells. Chromatin fractions from untreated cells also contain a significant amount of RNA which hybridizes to the globin cDNA probe. These observations suggest that both transcriptional and post-transcriptional control mechanisms are involved in hemoglobin gene expression in T3C12 erythroleukemia cells.
Large Globin RNA Molecules and Their Processing
European Journal of Biochemistry, 1981
R N A containing b-globin message sequences larger than 2000 nucleotides could be clearly detected in nuclei of murine erythroid cells using cloned /I-globin cDNA. Under steady-state conditions, when nuclear R N A was separated on denaturing agarose gels and covalently bound to diazobenzyloxymethyl-paper, a 4200-nucleotide and a z 3500-nucleotide band could be seen. The presence of these large molecules could also be visualized under the electron microscope after hybridization to a /I-globin genomic DNA fragment. We suggest that these molecules are precursors to mature mRNAs. In addition to these large molecules, a series of molecules smaller than 2000 nucleotides were seen. These are postulated to be processing intermediates in the maturation of b-globin mRNA.