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M Yaniv

Publications and source records attributed to M Yaniv.

At least 181 records · Page 10Linked to original sources
PubMed1982

Both early and late control sequences of SV40 and polyoma promote transcription of Escherichia coli gpt gene in transfected cells.

Eucaryotic expression vectors containing two selective markers, the herpes simplex 1 thymidine kinase gene (tk) and the Escherichia coli gpt gene (Eco gpt) coding for a xanthine-guanine phosphoribosyl-transferase ( XGPRT ) were constructed. These plasmids were used to transfect mouse Ltk- cells followed by selection of either tk+ or XGPRT + colonies. The transcription and maturation of the Eco gpt mRNA is dependent on the presence of a eucaryotic promoter sequence at its 5' end and on the presence of a viral intron and a poly(A) addition site at its 3' end ( Mulligan and Berg, 1980). Here, we report that both simian virus 40 (SV40) and polyoma early and late promoters permit the transcription of this gene integrated into the cellular genome. Polyoma DNA fragments lacking the TATA box, or both the TATA and CAAT boxes directing early transcription, efficiently promote Eco gpt expression. Furthermore, a fragment terminating approximately 300 nucleotides upstream of the initiation site of early RNA permits Eco gpt synthesis when the early strand is joined to the Eco gpt-coding strand. The SV40 early promoter is 2- to 3-fold more efficient than the controlling sequence of late transcription. These results strongly suggest that the switch from a predominance of early RNA to that of late RNA occurring after the onset of DNA replication is caused by the increase in the abundance of template and by the concomitant repression of early transcription by the T antigen. The presence of the tk gene in all the plasmids constructed permits the analysis of Eco gpt expression as a non-selected marker in tk+ clones selected for growth in HAT medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions.

Reduction of disulfide bonds involving the major capsid protein with dithiothreitol and removal of the calcium ions by EGTA disrupts the simian virus 40 virions. This process yields normal circular viral minichromosomes containing the four core histones and traces of the capsid proteins at pH values higher than 8.5. However, when carried out at pH 7.5, this procedure yields nucleoprotein complexes that contain both histones and the viral structural proteins. These pH 7.5 complexes appear as circular structures with a mean of 93 +/- 17 beads with a diameter of 7 nm and no visible nucleosomes when observed by electron microscopy. In contrast to the compaction of the viral DNA in minichromosomes, the length of these beaded structures is roughly the same as free DNA. We suggest that VP1, the major capsid protein, can act as a nucleosome unfolding agent in neutral pH and low ionic strength.

Centrifugation, Density Gradient↗
PubMed1982

Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines.

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.

Animals↗
PubMed1982

Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts.

Polyoma virus transforms, upon infection or DNA transfection, nonpermissive Fisher rat fibroblasts. Cloned viral DNA was deleted of sequences around the Bg/I site at nucleotide 86 by Bal31 nuclease treatment and then recloned in Escherichia coli. The extent of deletion for each mutant was then determined by DNA sequencing. Deletions included the early transcription control signals; others stretched into the N-terminal coding sequences of the viral tumor antigens. The transformation efficiency of 16 mutants was tested by transfecting rat fibroblasts. Expression of the T antigens was analyzed by immunofluorescence detection after transfection of rat fibroblasts, mouse secondary embryo cells, and HeLa cells. We found that the absence of the early transcription control sequences (TATA and CAAT boxes) did not significantly alter the transformation capacity of the virus. On the other hand, deletion of the initiator methionine ATG codon or further into the coding sequences did abolish the transformation capacity in some mutants, whereas others maintained a reduced transforming activity, possibly by initiation of translation in a penultimate methionine.

Amino Acid Sequence↗
PubMed1981

Polyoma DNA sequences involved in control of viral gene expression in murine embryonal carcinoma cells.

Polyoma virus can develop lytically in differentiated mouse cells, but is unable to express either early or late functions in undifferentiated early embryonic cells or in embryonal carcinoma (EC) cells of the mouse. However, the EC cells become susceptible during differentiation and then behave as do mouse somatic cells. We have recently described the isolation of a new class of polyoma virus mutant (Py EC) able to develop normally in an EC cell line (PCC4-Aza) that is capable of trigerminal differentiation in vivo but exhibits a very limited differentiation in vitro. The mutants do not grow in another EC cell line (F9) that is 'blocked'--'nullipotent' in vivo, but able to differentiate in vitro when grown in the presence of chemical inducers. Compared with their parental strain, all Py EC genomes analysed so far have an extra 20-50 bases in their HpaII-3 restriction fragment. We now describe polyoma mutants that will grow in EC F9 cells, and the sequence changes in two of them. The region involved in the mutations can be folded into a putative tRNA-like secondary structure which may be involved in the regulation of viral early transcription with respect to the state of differentiation of the host cell.

Animals↗
PubMed1981

[Chromatin structure with no nucleosomes: dissociated SV40 virus].

The mild dissociation of SV40 virus particles at pH 7.5 yields a nucleoprotein complex containing all the capsid proteins and histones of the intact virion. Electron microscopic observations show a bead-on-a-string structure comprising about 70 particles 7 nm in diameter along the viral DNA. No particle of nucleosome size was visible. The compaction of DNA in this complex is lower than 2 and is likely to be close to unity.

Animals↗
PubMed1981

[Biochemical characteristics of a vaccine against hepatitis B].

Hepatitis B Vaccine antigen, purified from HBs positive and HBe negative plasma, is constituted of well defined morphological particles, containing two major polypeptides P22 and P27, and without any trace of viral DNA. These criteria guarantee innocuity and purity of this type of vaccine.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions.

We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction. The intergenic sequence thrA and thrB is only one base pair. The coding region for homoserine kinase is 927 base pairs long. It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB. The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes. The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I.

Amino Acid Sequence↗
PubMed1981

Fine structure of the origin-proximal DNAase I-hypersensitive region in wild-type and EC mutant polyoma.

The chromatin of wild-type polyoma virus displays a unique DNAase I highly sensitive region in situ in the infected nuclei, extending for about 260 nucleotides from the origin of replication to the beginning of the late region. We show that this highly sensitive region is not homogeneous. It displays a well defined pattern of differential sensitivity along its 260 nucleotides, including one protected subregion and two hypersensitive sites, which concern a unique residue or very few nucleotides. All these features were mapped to a precision of +/- 5 bp relative to the DNA nucleotide sequence. In parallel, we studied a PyEC mutant, whose sequence is grossly rear-ranged in this very region. This allows the PyEC to overcome the block in the expression of the early genes in the mouse embryonal carcinoma PCC4 cell line. We show that this mutant, however, displays an identical highly sensitive region with the same fine structure. The mapping of this structure on the mutant nucleotides sequence coincides with that of the wild-type relative to any arbitrary point on the late side of the rearrangement; however, 60% of the mutant molecules display this unique local chromatin structure, instead of 20% for the wild-type ones. Finally, the sequence determinism of this singularity is discussed, as well as its possible role in the control of the early transcription and the establishment of the PyEC phenotype..

Chromatin↗
PubMed1980

Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA.

Electron microscopic examination of SV40 chromatin prepared 44 hr post-infection led to the visualization of a nucleosome-free region (gap) in 15-20% of the minichromosomes. Minichromosomes with and without a gap displayed a mean number of 24 nucleosomes. Measurements carried out on dark field micrographs yielded for the gap a mean length of 249 +/- 13 bp, with a maximum value of 385 bp. The gap was mapped following digestion with three single-cut restriction endonucleases: Bgl l, Bam HI and Eco RI. It was located in the region of the origin of replication in accordance with previous biochemical data. To assess the situ existence of a nucleosome-free region, nuclei from infected cells were digested with DNase I. A highly sensitive region was thus revealed and mapped by secondary digestion with Eco RI. It was located in the same region as the gap, between 0.67 and 0.74 on the physical map. The sensitive region could be detected throughout the late phase of the virus cycle. These findings strongly suggest that a nucleosome-free region exists in the cells. The gap is not likely to be involved in replication, since it is asymmetric with respect to the Bgl I cleavage site, from which replication proceeds symmetrically.

Chromatin↗
PubMed1980

Expression of polyoma early functions in mouse embryonal carcinoma cells depends on sequence rearrangements in the beginning of the late region.

Established mouse cell lines, primary cultures of mouse cells, and differentiated cell lines derived from mouse teratocarcinoma are permissive to polyoma virus. No viral early or late functions are expressed upon infection and penetration of multipotential embryonal cell lines. Polyoma mutants capable of growth on these cells were isolated and their DNA was cloned. Both the linear cloned viral DNA and a hybrid composed of mutant Bam HI (0.58) to Bgl I (0.72) 750 bp fragment (containing the origin of replication) ligated to the complementary wild-type 4.5 kb fragment are able to multiply on PCC4 embryonal carcinoma cells. The nucleotide sequence of two mutants indicated a genomic rearrangement on the late side of the origin, in which a deletion starting at nucleotides 46 (Py 204) and 77 (Py97) and terminating for both in nucleotide 107 was replaced by the duplication of a downstream late sequence starting at nucleotide 138 (Py 204) and 157 Py97) and terminating in nucleotide 220. The fact that the sequence rearrangements permit the expression of early and late functions upon infection suggests that this region participates in the control of early transcription. This control is different in embryonal and differentiated mouse cells.

Animals↗
PubMed1980

Nucleotide sequence of the thrA gene of Escherichia coli.

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1980

Escherichia coli RNA polymerase in vitro mimics simian virus 40 in vivo transcription when the template is viral nucleoprotein.

We have used a low-salt detergent-free extraction procedure on cells infected with simian virus 40 to obtain viral nucleoprotein late after infection. Addition of EScherichia coli RNA polymerase and ribonucleotide triphosphates to the viral minichromosomes permitted transcription of RNA from viral templates. This synthesis was initiated predominantly within a fragment of DNA spanning 0.67 to 0.76 map unit on the genome. The synthesis from this region proceeded primarily along the "late" strand in a clockwise direction. These results were in contrast to the synthesis obtained with naked viral DNA in which initiation occurred on other regions of the genome and from which transcription proceeded counterclockwise along the early strand. These findings indicate that the nucleoprotein template or factors tightly associated with it may be responsible for site(s) and strand selection in transcription of simian virus 40.

DNA, Viral↗
PubMed1980

Molecular cloning, refined physical map and heterogeneity of methylation sites of papilloma virus type 1a DNA.

The entire genome of human papilloma virus type 1a was cloned in Escherichia coli using the plasmid pBR322 as vector. The integrity and the homogeneity of the viral DNA thus obtained was confirmed by restriction endonucleases analysis. Viral DNA isolated from a single wart was partially methylated at only one out of the four HpaII sites, d(C-C-G-G). Recognition sites for Bg/I, Bg/II, PstI and PvuII restriction endonucleases were located on the cloned genome.

Cloning, Molecular↗
PubMed1980

Localization of the binding sites of prokaryotic and eukaryotic RNA polymerases on simian virus 40 DNA.

The binding sites of calf thymus RNA polymerase (B) II, wheat germ RNA polymerase B and of the Escherichia coli RNA polymerase were mapped on the simian virus 40 genome by observation of enzyme-linear DNA complexes by electron microscopy. Three to four major sites and several minor sites are observed for each enzyme; common binding sites for the three enzymes are found in positions 0.17, 0.53 and 0.90 of the viral physical map. Initiation complexes with these enzymes can be stabilized with specific ribodinucleotides and a single ribonucleoside triphosphate. Whereas ApA and ATP greatly enhances the binding of the E. coli enzyme at position 0.17, they stabilize the binding of the eukaryotic enzyme at many sites, some of them located in close proximity of the origin of replication.

Adenosine Monophosphate↗