Biomedical subjects

Y Aloni

Publications and source records attributed to Y Aloni.

At least 37 records · Page 2Linked to original sources

PubMed1990

A cis downstream element participates in regulation of in vitro transcription initiation from the P38 promoter of minute virus of mice.

We report the use of a HeLa whole cell extract (WCE) runoff transcription system for the study of cis- and trans-acting elements, participating in the regulation of transcription initiation from the P38 promoter of the parvovirus minute virus of mice (MVM). Our initial studies with HeLa WCE indicated that transcription from the P38 promoter is very inefficient, compared with transcription from the P4 promoter. Supplementation of the HeLa WCE with WCE prepared from uninfected Ehrlich ascites cells enhanced transcription from the P38 promoter twofold, indicating a role for a cellular factor in transcription from the P38 promoter. Furthermore, supplementation with WCE prepared from MVM-infected Ehrlich ascites cells enhanced transcription from the P38 promoter about sixfold, indicating a role for a virally encoded or induced factor. Analyses of runoffs produced by transcription of DNA templates digested with various restriction enzymes defined a downstream promoter element (DPE) necessary for efficient transcription initiation from the P38 promoter. This element resides 282 to 647 base pairs 3' to the transcription initiation site, between the NarI site and the HindIII site (2287 to 2652, MVM numbering system). The virally encoded NS1 protein was shown by DNA precipitation to bind directly or indirectly through a cellular factor to the DPE. This interaction is suggested to be involved in the up regulation of the P38 promoter of MVM. Finally, with a DNase I protection assay performed on a fragment containing the DPE, we estimated the sequence involved in the binding of a factor present in uninfected and infected extracts. The correlation between the binding and transcription activation is discussed.

Animals↗

PubMed1989

The block to transcription elongation at the SV40 attenuation site is decreased in vitro by oligomers complementary to segments of the attenuator RNA.

We have previously reported that a mechanism resembling attenuation in prokaryotes regulates simian virus 40 (SV40) late gene expression. We have suggested that modulation of the attenuator RNA secondary structure is an integral element regulating the elongation block at the attenuation site [Hay et al., Cell 29 (1982) 183-193]. In the present study, oligodeoxyribonucleotides (oligos), 13-19 nucleotides long, were used to probe the involvement of the attenuator RNA secondary structure in the control of elongation block at the SV40 attenuation site. These oligos are complementary to segments of the attenuator RNA suggested to play a role in the regulation of attenuation. The oligos were added to an in vitro transcription reaction containing SV40 transcription complexes, and their effect on transcription through the attenuation site was measured. As predicted, the three oligos caused specific decreases in the elongation block at the SV40 attenuation site. These results provide direct evidence for the involvement of RNA secondary structure in the attenuation mechanism in SV40.

Animals↗

PubMed1989

Transcription complexes synthesizing attenuated RNA can serve as a model system for analyzing elongation factors.

In vitro transcription elongation, in HeLa whole cell extract, is partially blocked at the Ad2 and synthetic (8 Ts) attenuation sites when transcription elongation is performed at increasing KCl concentrations (0.2-0.5 M KCl) but not at the standard KCl concentration (50 mM). Furthermore, if briefly initiated transcription complexes are chromatographed on Sephacryl column at increased KCl concentration and the complexes, which are eluted in the void volume, are diluted to 50 mM KCl, they will elongate a substantial fraction of the briefly initiated nascent RNA into attenuated RNA. The addition of a crude preparation of a stimulatory factor to the complexes diluted to 50 mM KCl prevents the elongation blocks. Based on the notion that putative elongation factors dissociate from the transcription complexes at increasing KCl concentrations and in the Sephacryl column they elute after the initiation complexes, we suggest that the column-purified transcription complexes, which have been depleted of elongation factors, can serve as a model system for determining the activities of factors during elongation.

Adenoviruses, Human↗

PubMed1989

RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40.

At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3'-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y. (1982) Cell 29, 183-193). The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements. The hairpin element at the 3'-end of the attenuated RNA is followed by a stretch of Us and resembles a rho-independent terminator in prokaryotes. We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation. Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract. We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2. The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates. The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates.

Base Sequence↗

PubMed1989

Elements modulating the block of transcription elongation at the adenovirus 2 attenuation site.

We have previously reported that a block of transcription elongation is functioning in vivo and in vitro within the leader sequences of SV40 and the adenovirus 2 major late transcription units and in the regulation of transcription of the P4 promoter of minute virus of mice. In the present study using the HeLa whole cell extract-Sarkosyl system with adenovirus 2 major late promoter as a template we have analyzed several basic parameters that can contribute to our understanding of the mechanism that regulates the elongation block at the adenovirus 2 attenuation site. We show that the elongation block is augmented at elevated temperatures (40-45 degrees C). The elongation block can be reversed by the addition of 0.2 M NaCl to the transcription reaction and the reversibility is temperature-dependent. Furthermore, while at 30-35 degrees C the elongation block is reversible with dilution of the Sarkosyl, at 40-45 degrees C it is only partially reversible. These results may indicate that a factor(s) is involved in the regulation of the elongation block and/or that the conformation of the transcription complex is temperature dependent. Finally, we show that the extent of the elongation block is dependent on the consecutive T residues at the attenuation site and we discuss the involvement of RNA secondary structure in eliciting the elongation block.

Adenoviruses, Human↗

PubMed1989

A block of transcription elongation by RNA polymerase II at synthetic sites in vitro.

We have previously suggested that transcription elongation by RNA polymerase II can be blocked when the nascent RNA is folded into a stem-and-loop structure followed by polyuridines. As an approach to test this suggestion in vitro, several GC-rich deoxyoligonucleotides with dyad symmetries were chemically synthesized and inserted following the adenovirus 2 major late promoter. These constructs were transcribed in vitro using HeLa whole cell extract. The transcripts of the synthetic inserts can potentially form stem-and-loop structures with destabilization energy from 0 to -48 kcal followed by 3, 5, and 8 U residues. The results obtained show that transcription elongation is blocked by these synthetic inserts and that the extent of the elongation block is directly correlated to the stabilities of the potential stem-and-loop structure and the proceeding number of U residues. Three levels of elongation blocks were observed: a brief pause of the polymerase occurs when the RNA could be folded into a secondary structure or when there were 5-6 T residues on the sense DNA strand. An extended pause occurred when the number of T residues on the sense DNA strand was increased to 8. Transcription termination, with a partial release of the attenuated transcript occurred when a stable RNA secondary structure (delta G = -48 kcal) was followed by 8 U residues. The relevancy of these in vitro results to the in vivo mechanism of a transcription elongation block is discussed.

Base Sequence↗

PubMed1989

RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro.

By using the minute virus of mice, we have shown that in vivo and in vitro RNA polymerase II pauses or prematurely terminates transcription at a specific location 142-147 nucleotides downstream from the P4 promoter. The attenuated RNA was found and mapped in vivo in A9 cell late after infection in both the nuclear and cytoplasmic fractions, and the terminal nucleotide was shown to have a 3' OH group. The 3' end of the attenuated RNA is capable of forming a hairpin structure that is followed by a stretch of uridines. To distinguish whether the attenuated RNA is formed as a result of processing, pausing, or termination and to dissect structural elements, factors, or mechanisms that are involved in its formation, we used in vitro systems: isolated nuclei and cell-free extracts from HeLa cells. The results of the in vitro studies show that the attenuated RNA is a result of pausing or termination and not processing. Additionally, a salt-soluble factor and RNA secondary structure were implicated in the process of termination.

Base Sequence↗

PubMed1989

The adenovirus type 2 DNA-binding protein interacts with the major late promoter attenuated RNA.

The adenovirus 72-kilodalton DNA-binding protein (DBP) binds to the attenuated RNA derived from the viral major late promoter. Protection from T1 RNase digestion can be observed when DBP is incubated with attenuated RNA. By using attenuated RNA labeled at one end, the T1 RNase digestion pattern can be mapped to residues located at specific sites in this RNA. Heterologous competitor RNAs do not alter the pattern of DBP protection of a labeled attenuated RNA, as does the identical attenuated RNA. These data indicate some specificity of the interaction between DBP and attenuated RNA. Adenovirus infection of monkey cells results in a more efficient attenuation of RNA initiated at the major late promoter and a reduced level of infectious virus. Adenovirus mutations in DBP relieve this restriction. These DBP mutant proteins do not change their binding properties to the attenuated RNA but suggest a mechanism by which DBP plays a role in the adenovirus host range restriction in monkey cells.

Adenoviruses, Human↗

PubMed1989

Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter.

Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuator RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.

Adenoviridae↗

PubMed1988

Transcription termination in animal viruses and cells.

Three experimental systems: isolated nuclei, cell-free reactions and whole cells were used for defining and characterizing cis and trans elements which regulate the block of transcription elongation in animal viruses and cells. In addition we have presented models for transcription termination within and at the end of a gene, which are consistent with the available information on the transcription bubble propagated during transcription elongation and can explain the modes of transcription termination described for various eukaryotic genes.

Adenoviruses, Human↗

PubMed1988

RNA polymerase II allows unwinding and rewinding of the DNA and thus maintains a constant length of the transcription bubble.

Ternary complexes of RNA-DNA-RNA polymerase II, originating from the in vivo transcriptionally active SV40 minichromosomes, can be detected and analyzed by a method previously developed (Choder, M., Bratosin, S., and Aloni, Y. (1984) EMBO J. 3, 2929-2936). Using this method, we compared the electrophoretic mobilities of SV40 ternary complexes with those of SV40 RNA-DNA complexes obtained after the removal of the polymerases. Independent of the in vitro elongation of the nascent RNA, topoisomers of ternary complexes including supercoiled DNA (DNA I) and relaxed DNA (DNA II) electrophoresed on agarose gels as sharp bands. In addition, the polymerase protected 18-22 nucleotides of the nascent RNA from RNase A and T1 digestion. These results demonstrate the constancy of the length of the unwound region in the transcription bubble. In contrast, following the removal of the polymerases topoisomers of RNA-DNA I complexes migrated on agarose gels as a smear between the sites of DNA I and DNA II. RNA with an average length of 120 nucleotides formed hybrid with the DNA and was RNase A- and T1-resistant. The observations that long hybrid formation is prevented as long as the polymerases are present, and the maintenance of the constant length of the transcription bubble during transcription elongation, suggest that during elongation the enzyme that actively unwinds the template allows the synchronous displacement of the nascent transcript and rewinding of the template.

DNA↗

PubMed1988

In vitro transcribed SV40 minichromosomes, as the bulk minichromosomes, have a low level of unconstrained negative supercoils.

The method for detecting the small fraction (1%) of transcriptionally active SV40 minichromosomes, in the presence of the bulk minichromosomes, (14) has been applied to directly analyze the topology of transcribed and non-transcribed minichromosomal DNA. We show here that DNA of both transcribed and non-transcribed minichromosomes have the same number of supercoils which are constrained by nucleosomes. In addition, minichromosomal DNA of both fractions have a low level of unconstrained supercoils (1-2 extra supercoils) which can be relaxed in vitro by topoisomerase I.

Chloroquine↗

PubMed1987

Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal.

Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y. Aloni and N. Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985). In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes. The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract. As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues. Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal. Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl. These results indicate that, at least in isolated nuclei, attenuation can be regulated. The possible involvement of termination factor(s) in the regulation of attenuation is discussed.

Adenoviridae↗

PubMed1986

Efficient and accurate in vitro processing of simian virus 40-associated small RNA.

Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.

Animals↗

PubMed1985

Attenuation of late simian virus 40 mRNA synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems.

Studies were performed to verify the physiological significance of attenuation in the life cycle of simian virus 40 and the role of agnoprotein in this process. For these purposes, nuclei were isolated at various times after infection and incubated in vitro in the presence of [alpha-32P]UTP under the standard conditions which lead to attenuation. Attenuation was evident by the production of a 94-nucleotide attenuator RNA, revealed by gel electrophoresis. In parallel, the synthesis of agnoprotein was studied at various times after infection by labeling the cells for 3 h with [14C]arginine, lysing them, and analyzing the labeled proteins by gel electrophoresis. Both attenuation and the synthesis of agnoprotein were predominant towards the end of the infectious cycle. At earlier times, there was almost no attenuation and no synthesis of agnoprotein. Moreover, there was almost no attenuation even at the latest times after infection in nuclei isolated from cells infected with simian virus 40 deletion mutants that do not synthesize agnoprotein. Finally, analysis by dot blot hybridization showed higher amounts of cytoplasmic viral RNA in cells infected with an agnoprotein gene insertion mutant, delta 79, that does not produce agnoprotein, compared with cells infected with wild-type virus. The present studies indicate that attenuation is temporally regulated and suggest that agnoprotein enhances attenuation in isolated nuclei and that may also enhance it in vivo.

Animals↗

PubMed1985

Attenuation may regulate gene expression in animal viruses and cells.

In eukaryotes, an abundant population of promoter-proximal RNA chains have been observed and studied, mainly in whole nuclear RNA, in denovirus type 2, and in SV40. On the basis of these results it has been suggested that a premature termination process resembling attenuation in prokaryotes occurs in eukaryotes. Moreover, these studies have shown that the adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) enhances premature termination, but its mode of action is not understood. The determination of the nucleotide sequences of SV40 and other viruses and cellular genes provide means for elucidating the nucleotide sequences involved in the attenuation mechanism. A model has recently been described in which attenuation and mRNA modulation in a feedback control system quantitatively regulate SV40 gene expression. The suggested mechanism described in this model opens up approaches to the investigation of attenuation and mRNA modulation as a possible mechanism whereby eukaryotes may regulate transcription in a variety of different circumstances.

Animals↗

PubMed1984

A direct analysis of transcribed minichromosomes: all transcribed SV40 minichromosomes have a nuclease-hypersensitive region within a nucleosome-free domain.

A direct quantitative method was developed for detecting a small fraction of minichromosomes active in transcription and for analyzing their chromatin structure. For this, SV40 minichromosomes were incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the in vivo pre-initiated nascent RNA and thus labeling of the in vivo transcriptionally active molecules. Subsequently the minichromosomes were treated with Sarkosyl to remove most of the proteins bound to the viral DNA while leaving the 32P-labeled RNA-DNA-polymerase ternary complexes intact. Following agarose gel electrophoresis, the bulk SV40 DNA was identified by staining with ethidium bromide and the labeled ternary complexes by autoradiography, although they are not well separated in the gel. When the viral minichromosomes were cleaved with certain restriction enzymes or mildly with DNase I prior to the in vitro labeling and Sarkosyl treatment, analysis by gel electrophoresis showed that all transcriptionally active minichromosomes were cleaved, whereas a large fraction of the bulk minichromosomes was resistant to cleavage. In all molecules the cleavage occurred within the transcriptional regulatory region. In addition, analysis by electron microscopy revealed that SV40 minichromosomes having a nucleosome-free region (gap) are also more sensitive to the initial cleavage by both DNase I and Bg/I restriction enzyme than non-gapped molecules and that the initial DNase I cleavage occurs within the gap. Thus, at least in SV40, the nuclease-hypersensitive region is positively correlated with the existence of a gap.

Animals↗

PubMed1984

Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B.

Nuclei which were isolated from SV40 infected cells with a hypotonic detergent-free buffer were used to establish in vitro conditions which lead to transcription-termination at the attenuation site of SV40. This system allowed us to identify regulatory elements involved in transcription-termination by RNA polymerase B transcribing SV40. Transcription-termination at the attenuation site was found to be ionic strength dependent. Efficient termination occurred at low (100 mM NaCl) but not at high (100 mM (NH4)2 SO4 or 300 mM NaCl) ionic strength. When nuclei were prewashed with 300 mM NaCl, the efficiency of transcription-termination was low even when transcription was carried out at low ionic strength (100 mM NaCl). Efficient transcription-termination in the high salt prewashed nuclei was reconstituted by complementation with a high salt (300 mM NaCl) soluble factor extracted from nuclei of uninfected cells. In addition, the efficiency of transcription-termination was significantly reduced when ITP replaced GTP in the transcription reaction mixture. Our data indicate that a nuclear factor and RNA secondary structure are essential regulatory elements involved in transcription-termination by RNA polymerase B.

Animals↗