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Y Aloni

Publications and source records attributed to Y Aloni.

At least 55 records · Page 3Linked to original sources
PubMed1984

Control of late simian virus 40 transcription by the attenuation mechanism and transcriptionally active ternary complexes are associated with the nuclear matrix.

Isolated nuclei derived from simian virus 40 (SV40)-infected cells and incubated with [alpha-32P]UTP can elongate the in vivo preinitiated SV40 late RNA, synthesizing a viral RNA species 94 nucleotides long (attenuator RNA) as well as longer RNA molecules. In contrast to newly synthesized SV40 RNA, the attenuator RNA is not associated with the nuclear matrix. Pretreating the cells with 5,6-dichloro-1-beta-ribofuranosylbenzimidazole before the incubation of isolated nuclei in vitro, enhances the accumulation of the attenuator RNA, but again it is removed from nuclei by DNase and high salt. In contrast, pretreating the cells with proflavine, an intercalating drug that interferes with RNA secondary structure, prevents the accumulation of the attenuator RNA and increases the amount of the long RNA molecules. These RNA molecules become associated with the nuclear matrix. Isolated nuclear matrices from SV40-infected cells are highly enriched in transcriptionally active ternary complexes. Thus, isolated nuclear matrices that contain from 2 to 6% of SV40 DNA are capable of synthesizing at least 35% of the viral RNA synthesized in isolated nuclei after 2 to 15 minutes incubation with [alpha-32P]UTP. The RNA synthesized in vitro on purified nuclear matrices and isolated nuclei is derived from the same regions of the viral genome, suggesting that there is an association between transcribed DNA sequences and the nuclear matrix. The results suggest a major role for the nuclear matrix in controlling SV40 gene expression.

Base Sequence↗
PubMed1984

SV40 deletion mutant (d1861) with agnoprotein shortened by four amino acids.

d1861 is an SV40 deletion mutant which was thought to lack the agnoprotein coding region and was used to verify the role of agnoprotein in the life cycle of SV40. In the present study the region flanking the deletion was sequenced and, in contrast to the available information, it was found that d1861 lacks 12 nt in phase, downstream from the AUG start codon of agnoprotein (residues 347-358). Using the runoff protocol with viral transcriptional complexes (VTC), that in vitro elongate the in vivo preinitiated nascent RNA, it was found that in vivo the major initiation site for late transcription is at residue 325, the same as in wild type (WT). In comparison with WT, d1861 encodes information for agnoprotein shortened by four amino acids and it has been identified in d1861 infected cells. However, pulse-chase experiments indicated that the rate of synthesis of d1861 agnoprotein is slower than that of WT agnoprotein and that it has a turnover rate of 1 hr as compared to 3 hr of WT agnoprotein. The reduced rate of synthesis of d1861 agnoprotein can be explained by nuclease S1 analyses in which the major leader of d1861 16 S RNA, that encodes the agnoprotein, appeared in significantly lower amounts as compared to the major leader of WT 16 S RNA. Furthermore, analysis of the potential secondary structures at the 5' end of the leader of d1861 16 S RNA has revealed stable structures in which the start codon of agnoprotein is sequestered in a stem. The involvement of RNA secondary structures in regulating the synthesis of agnoprotein is discussed.

Amino Acid Sequence↗
PubMed1984

Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation.

To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [alpha-32P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 +/- 5 and 2005 +/- 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [alpha-32P]UTP or [alpha-32P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 +/- 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142-nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase.

Base Sequence↗
PubMed1984

Mapping in vivo initiation sites of RNA transcription and determining their relative use.

Runoff transcripts were generated on viral transcriptional complexes cleaved with restriction enzymes and incubated in vitro with [alpha-32P]UTP under pulse-chase conditions. As viral transcriptional complexes in vitro elongated the nascent RNA preinitiated in vivo, size analysis by gel electrophoresis of the runoff transcripts allowed identification of the in vivo initiation sites. Moreover, scanning the intensities of the runoff bands as they appeared in the autoradiogram of the gel allowed determination of the relative use of these sites. A model system in which the initiation sites of simian virus 40 late RNA were identified and their relative use determined is presented.

Animals↗
PubMed1983

Solubilization of the UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase (cellulose synthase) from Acetobacter xylinum. A comparison of regulatory properties with those of the membrane-bound form of the enzyme.

A procedure has been developed for the effective solubilization of UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase (cellulose synthase) by treatment of membranes from the bacterium Acetobacter xylinum with digitonin. Low concentrations of digitonin (0.1%, w/v) cause stimulation of the enzyme activity in membranes; treatment with higher concentrations of digitonin (1-10%) results in solubilization of up to 70% of the digitonin-stimulated activity. The digitonin-solubilized enzyme displays regulatory properties quite similar to those of the membrane-bound form of the enzyme, showing specific activation by GTP. GTP activation requires the presence of a protein factor which can be separated from the enzyme by washing the membranes prior to enzyme solubilization. Association of this protein factor with the membrane-bound enzyme is promoted by polyethylene glycol or by Ca2+; however, these compounds are ineffective in enhancing enzyme-factor association for the enzyme in the solubilized state. The observation that Ca2+ promotes enzyme-factor association in the membranes suggests that this cation, in addition to GTP, may play a role in the regulation of cellulose synthesis in vivo in A. xylinum.

Arabidopsis Proteins↗
PubMed1983

Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region.

Viral transcription complexes were isolated from SV40-infected cells and incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the promoter-proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243-336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259-336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13-16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC-rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.

5' Untranslated Regions↗
PubMed1983

In vitro premature termination in SV40 late transcription.

Nuclear extracts and viral transcribing minichromosomes were prepared from SV40-infected cells and incubated in vitro with [alpha-32P]UTP under conditions which allow the elongation of preinitiated RNA chains. Sucrose gradient lysis of the transcription mixtures revealed two populations of SV40-specific RNA: elongating chains that remain associated with the viral minichromosomes, and, at the top of the gradient, small free RNA detached from the template and hybridizing exclusively to the promoter-proximal region of SV40 DNA. This free RNA was shown by polyacrylamide gel electrophoresis to comprise essentially a 94 nucleotide species, which could, however, at high UTP concentration, be elongated a further few nucleotides before terminating. These results thus show that the actively transcribing minichromosomes provide a sytem in which the attenuated RNA can be released from the template. Moreover, this is the first demonstration of specific in vitro termination of polymerase B transcription. The conditions which lead to transcription termination are discussed.

Animals↗
PubMed1983

Attenuation and modulation of mRNA secondary structure in a feedback control system regulating SV40 gene expression.

Alternative secondary structures can be predicted for the initial 94 nucleotides synthesized from the major transcription initiation site of SV40 late RNA: a transcription-termination conformation results in the production of aborted RNA and a readthrough conformation leads to the synthesis of the primary SV40 late RNA. In the cytoplasm similar alternative conformations can be predicted for the initial nucleotides at the 5' ends o both the major 16S and 19S late mRNAs. In one of these alternative conformations the AUG initiation codon of the leader protein (agnoprotein) is sequestered and not available for ribosome binding. In the alternative conformation the same AUG is accessible for ribosome binding. We suggest that these mutually exclusive conformations are fundamental elements in a transcription and translation feedback control mechanism regulating the synthesis of 16S and 19S mRNA in the nucleus and the translation of their encoded proteins in the cytoplasm.

Feedback↗
PubMed1983

Processing of SV40 RNA is associated with the nuclear matrix and is not followed by the accumulation of low-molecular-weight RNA products.

Nuclear matrices from SV40-infected cells were prepared by treating purified nuclei with DNase and salt to extract DNA and histones. After a 10-min pulse with [5,6(-3)H]uridine over 85% of the viral RNA was found in association with the nuclear matrix. Following a 3-hr chase with glucosamine and unlabeled uridine, 2-4 S nuclear viral components accumulate, but they are not associated with the nuclear matrix. The 2-4 S components had been characterized previously as viral RNA processing products (N. H. Chiu, M. F. Radonovich, M. M. Thoren, and N. P. Salzman, J. Virol. 28, 590-599, 1978). The results of the present study identify the 2-4 S components as DNA rather than RNA, thus indicating that the synthesis and the majority of the processing products of the viral RNA are associated with the nuclear matrix.

Animals↗
PubMed1982

SV40 virions and viral RNA metabolism are associated with cellular substructures.

Nuclear matrices were prepared by DNase and high salt extraction of SV40-infected epithelial monkey cells. The matrices retain the majority of SV40 virions. This conclusion is based on electron microscopic observations of the occurrence of encapsidated viral DNA that is resistant to DNase digestion and on the analysis of viral proteins by gel electrophoresis. Pulse labeled SV40 RNA is also associated with the nuclear matrix (less than 15% of the viral RNA is removed by DNase and high salt). Pulse-chase experiments revealed that processing of SV40 RNA takes place on the nuclear matrix and the processed molecules are directly transported to the cytoplasm where they are associated with the cytoskeleton. These results suggest a central role for the nuclear and cytoplasmic substructures in virus assembly and in the biogenesis of viral RNA.

Animals↗
PubMed1982

Attenuation in the control of SV40 gene expression.

Nuclei and viral transcriptional complexes were prepared from cells infected with simian virus 40 and incubated in vitro in the presence of alpha- 32P-UTP. The in vitro elongated viral RNA appeared with a peak of 5S in sucrose gradients and hybridized preferentially to a promoter-proximal region of SV40 DNA. Treatment of infected cells with proflavine led to transcription of elongated RNA, while treatment of cells with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a drug known to enhance premature termination, augmented accumulation of the promoter-proximal RNA. The in vitro elongated RNA produced a major band of 93-95 nucleotides in length in acrylamide gel. This RNA was found to map between the major initiation site at nucleotide 243 and nucleotides 335-337. The significance of these observations with respect to the transcription termination signal and the control of SV40 gene expression is discussed.

Animals↗
PubMed1982

Achievement of high rates of in vitro synthesis of 1,4-beta-D-glucan: activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor.

Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 2.4.1.12) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.

Bacterial Proteins↗
PubMed1982

Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Sedimentation analysis of pulse-labeled RNA synthesized in nuclei isolated from simian virus 40-infected cells revealed an abundance of short cellular and viral RNAs. The relative amount of the short chains is increased in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The short viral RNAs were purified by hybridization to and elution from simian virus 40 DNA on filters, and their sizes were determined by gel electrophoresis. A major band of 93- to 95-nucleotide-long RNA was observed along with additional minor bands. Identical bands were revealed when the viral RNA was purified from nuclei of cells pretreated with DRB. The major band was identified as an aborted transcript of a RNA that initiated at the major initiation site (nucleotide 243). We have found that the DNA region where the RNA stops is A+T rich and is immediately preceded by a G+C-rich region that exhibits dyad symmetry, resembling the termination signal in prokaryotes. These observations show that RNA polymerase II responds to the same termination signal as the prokaryotic enzyme and suggest that a mechanism of attenuation regulates simian virus 40 late transcription.

DNA, Viral↗
PubMed1982

Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure.

Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.

Chromosomes↗