Showing papers on "Transcription (biology) published in 1980"

Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping.

Abstract: Publisher Summary This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping. The chapter describes the methodology used to map polyoma virus transcripts on the physical map of the viral genome. The ultimate characterization of viral RNA is the determination of the complete nucleotide sequence of individual RNA species. A rapid procedure for the analysis of labeled viral RNA using an adaptation of the Southern transfer technique (mini-blot hybridization) has also been described. The hybrids formed between polyoma virus RNA and DNA are digested with single-strand-specific endonuclease S1 and the resulting duplexes or their component DNA strands are sized by gel electrophoresis. The 2-D variation of this basic procedure is a general diagonal assay for splicing within RNA molecules. The chapter describes the standard procedures used for the preparation of viral nucleic acids and hybridization probes.

DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract

Abstract: We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.

The ovalbumin gene - sequence of putative control regions

Abstract: We present the sequence of regions of the chicken ovalbumin gene believed to be important in the control of initiation of transcription, splicing, and transcription termination or polyadenylation. Comparison with corresponding areas of other genes reveals some homologous regions which might play a role in these processes.

Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H

Abstract: A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. The plasmid DNA has three principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.

A specific transcription factor that can bind either the 5S RNA gene or 5S RNA.

Abstract: 5S ribosomal RNA specifically inhibits transcription of cloned repeating units of 5S DNA in a nuclear extract of Xenopus oocytes. The inhibition can be explained by the interaction of 5S RNA with a transcription factor that binds specifically to a control region located within the 5S RNA gene. This transcription factor is identical to an abundant cytoplasmic protein that is known to be complexed with 5S RNA in immature Xenopus oocytes. Thus the presence of large amounts of this protein in these cells can account for both the high rate of synthesis and the subsequent storage of 5S RNA to ribosome synthesis.

A herpes simplex virus type 1 function continuously required for early and late virus RNA synthesis.

Abstract: Controlled transcription of animal virus DNAs provides potentially useful models for elucidating the mechanisms which regulate eukaryotic gene expression. The progressive transcription of the herpes simplex virus type 1 (HSV-1) genome has been described previously1–3. Infection of permissive cells with HSV-1 in the presence of the protein synthesis inhibitor cycloheximide resulted in transcription of a restricted set of virus RNAs, referred to as the immediate early RNAs, which map within certain regions of the virus genome only3–5. Removal of cycloheximide led to the transcription of additional virus DNA sequences, which were expressed during the normal replicative cycle both at early and late times post-infection (before and after the onset of virus DNA replication, respectively) and which map throughout the virus genome3. Previously, we have described a temperature-sensitive mutant of HSV-1 Glasgow strain 17, ts K, which accumulated only the immediate early RNAs at the non-permissive temperature (NPT)5. Transfer of ts K-infected cells from NPT to the permissive temperature (PT), even in the absence of de novo protein synthesis, resulted in transcription of the DNA sequences expressed early and late post-infection5. This indicated the persistence at NPT of a non-functional immediate early polypeptide which on transfer to PT regained its function, required for progression from the immediate early to early stage of transcription. Here we demonstrate, by analysing the RNAs made in ts K-infected cells after transfer from PT to the NPT, that this polypeptide's function is required continuously for synthesis of HSV-1 early and late RNAs, thus identifying a control function essential for the expression of early and late HSV genetic information in eukaryotic cells.

Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7

Abstract: Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Pribnow box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.

The Ultrastructural Visualization of Nucleolar and Extranucleolar RNA Synthesis and Distribution

Abstract: Publisher Summary This chapter summarizes current data concerning the ultrastructural localization of transcription sites and the subsequent distribution of newly synthesized ribonucleic acid (RNA) within the cell nucleus. It discusses the roles of ribonucleoprotein (RNP)-containing nuclear structures with respect to the synthesis and processing of nucleolar and extranucleolar RNA. RNA transcription in the nucleolar, as well as extranucleolar, areas takes place predominantly within the regions situated on the border of condensed chromatin. Perichromatin fibrils, considered as the first in situ detected structures containing newly transcribed extranucleolar RNA, originates in these perichromatin regions and the nucleolar fibrillar components are in intimate contact with intranucleolar chromatin areas. DNA replication sites also appear predominantly within this nuclear region. This particular nuclear area consequently plays a key role in the expression of chromatin functions and these findings lead to the conclusion that the layer of chromatin decondensing on the periphery of dense chromatin areas represents the most active chromatin fraction in the nucleus.

Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo

Abstract: Conserved DNA sequence elements of putative regulatory functions were deleted from the prelude region of a sea urchin H2A histone gene. For this, the wild-type H2A gene of the 6-kilobase histone DNA repeat unit was replaced by various mutant H2A genes by cloning. The effects of the manipulation on H2A mRNA synthesis were studied by injection of the mutant DNAs into centrifuged Xenopus oocytes. The unmanipulated H2B gene residing within the same repeat unit provided a suitable internal control for these studies. Deletion of the T-A-T-A-A-A-T-A motif, once thought to be the functional equivalent of the bacterial Pribnow box, did not abolish transcription of the gene; instead, a number of novel mRNA 5' termini were generated. We argue that the T-A-T-A-A-A-T-A motif is a specificity element, a selector of eukaryotic gene transcription. Deletion of the "cap-sequence," 5' pyrimidine-C-A-T-T-C-purine 3' and most of the mRNA leader sequence did not abolish transcription but created yet another mRNA 5' terminus. In contrast to these deletions, which are both down-mutations, deletion of H2A gene-specific conserved DNA sequences upstream from the T-A-T-A-A-A-T-A motif enhanced mRNA synthesis. A hypothesis for the function of these DNA sequences as eukaryotic promoter elements is discussed.

Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter.

Abstract: High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added Nevertheless, oligonucleotide synthesis persists under all conditions tested Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro Production of a long RNA transcript is then essentially an escape from this cycling reaction The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reaction

DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription.

Abstract: A DNA-RNA hybrid oligonucleotide duplex, dC(pA) 5pG:rC(pU)5pG, which contains a (dA:rU) 5 sequence, is at least 200 times less stable at room temperature than the corresponding duplex containing an (rA:dT) 5 sequence, rC(pA)5pG:dC(pT)5pG. This result provides an explanation for the finding that most primary RNA transcripts terminate in several consecutive rU residues, but not rA residues. It strongly supports the idea that instability of the DNA-RNA hybrid at the growing point of transcription plays a role in termination of transcription.

Organization of the recA gene of Escherichia coli

Abstract: The restriction map of a BamHI DNA fragment that contains the recA gene of Escherichia coli has been established and a large portion of the fragment's nucleotide sequence has been determined. The coding region of the recA gene contains 1059 nucleotide residues and encodes a single protein of 353 amino acid residues. The amino acid sequence of the first five residues of the NH2 terminus of the recA protein agrees with the sequence predicted from the DNA sequence except for the absence of formylmethionine in the purified protein. Immediately after the coding sequence, there is a G+C-rich sequence with dyad symmetry followed by an A+T-rich sequence. These could signal termination of transcription. The site of initiation for synthesis in vitro of the recA messenger RNA has been determined by analysis of the 5' nucleotide sequence of [gamma-32P]ATP-labeled transcripts. The promoter region shos a high degree of symmetry and contains sequences commonly found in recognition and binding sites for RNA polymerase.

Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo

Abstract: The control region of a sea urchin H2A histone gene may be functionally dissected into at least three DNA segments, which we have termed modulator, selector, and initiator elements. While the initiator and in particular the selector containing the T-A-T-A-A-A-T-A sequence are specificity elements that dictate the generation of faithful 5' ends to H2A mRNA, the modulators control the rate at which these specificity elements operate [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 1432-1436]. By functional tests of in vitro mutated histone DNA in the Xenopus oocyte we have now discovered that the segment E of the A+T-rich spacer DNA lying at a considerable distance upstream of the conservative T-A-T-A-A-A-T-A sequence is a strong modulator element of H2A gene transcription. Deletion of this element creates a 15- to 20-fold H2A-specific down mutation. Segment E by itself cannot elicit initiation of transcription except in coordination with the prelude sequence of the H2A gene. The nucleotide sequence of the relevant spacer element showing modulator activity has been determined and found to contain a pattern of T and A runs as well as a series of inverted repeats. Additional pre-H2A spacer mutants, including a spacer inversion mutant, have been constructed in vitro, that, when injected into the oocyte nucleus, modulate the expression of the H2A gene by an overall factor as large as 100. Other factors controlling promoter activity are discussed.

Transcription and RNA processing by the DNA tumour viruses.

Abstract: Messenger RNA synthesis by the DNA tumour viruses proceeds by a complex but versatile series of transcription and RNA processing steps. The major mechanistic features of this pathway are probably very similar to those used by the animal cell host itself. The viruses have, however, evolved intricate arrangements of protein coding sequences and sites for RNA initiation, polyadenylation and splicing which allow them to use their genetic information to maximum advantage.

Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes

Abstract: Specific initiation of transcription of cloned conalbumin and ovalbumin genes has been obtained in a cell-free system in the presence of purified calf thymus RNA polymerase B. Comparison with the adenovirus-2 early (E1 A) and major late genes reveals marked variations in transcriptional efficiencies. Specific fragmentation of the conalbumin gene DNA indicates that a short DNA segment 5′ to the initiation site is involved in specific transcription.

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY

Abstract: Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [3H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [3H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH4)2SO4, 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [3H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.

Transcription maps of adenovirus.

Abstract: Publisher Summary This chapter discusses the techniques for the transcription mapping of adenovirus. All true early mRNAs and proteins can be found in adenovirus 2 (Ad2) transformed cell lines. However, only a subset of these early gene products is required to establish a transformed state. With the transition from early to late phase of infection several new mRNAs appear in the cytoplasm that are transcribed from the same regions of the viral DNA as the early mRNAs, but which have novel sizes and sequence content. Two methods have been developed to map spliced Ad2 mRNAs that include (1) the nuclease gel electrophoresis procedure and (2) electron microscopy of RNA-DNA hybrids. The strengths and limitations of these two techniques are reviewed in the chapter. A summary transcription map of Ad2 is also presented.

Evolution of rna viruses

Abstract: These arguments lead to the suggestion that four independent evolutionary lines exist within the general group of RNA viruses. These are positive strand viruses, negative strand viruses, double stranded viruses, and retroviruses. Three of the viral systems may well have shared genes but the double-stranded RNA viruses appear to represent a very different evolutionary line.

In vitro transcription and translation of simian rotavirus SA11 gene products.

Abstract: Rotavirus gene products were examined, with the simian rotavirus SA11 as a model. The endogenous viral RNA-dependent RNA polymerase associated with single-shelled virus particles or with activated double-shelled particles was used to synthesize viral RNA transcripts. Sedimentation velocity sucrose gradient analysis of the RNA transcripts revealed four peaks at 9S, 12S, 14S, and 18S, whereas agarose gel electrophoresis under partially denaturing conditions revealed eight groups of RNA species ranging in molecular weight from 2 x 10(5) to 1.2 x 10(6). The transcripts synthesized in vitro were active in an mRNA-dependent cell-free translation system derived from rabbit reticulocytes. The transcripts directed the synthesis of 11 polypeptides that had molecular weights ranging from 125,000 to 20,000 when analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The products of in vitro translation were compared with polypeptides from purified virus and those synthesized in infected cells. Several of the polypeptides synthesized in vitro were designated as structural polypeptides by comparing the molecular weights determined by polyacrylamide gel electrophoresis analysis or by precipitation with hyperimmune serum prepared against purified virus. Three of the viral structural polypeptides (VP4, -5, and -5a) were not synthesized in vitro as primary gene products, demonstrating that processing must occur for the production of some structural polypeptides. Other in vitro-synthesized polypeptides were tentatively identified as either precursors to the viral glycoproteins or nonstructural polypeptides.