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V Blasquez

Publications and source records attributed to V Blasquez.

8 recordsLinked to original sources
PubMed1990

Location of nucleosomes in simian virus 40 chromatin.

Over the past decade, the results of numerous indirect mappings analyses have not clarified whether or not nucleosomes occupy preferred positions in simian virus 40 (SV40) chromatin. To address this question more directly, we followed a shotgun cloning approach and determined the nucleotide sequences of over 400 cloned nucleosomal DNA fragments obtained from digestion of SV40 chromatin with micrococcal nuclease. Our results demonstrate and establish that nucleosomes do not occupy unique positions in SV40 minichromosomes and thus indicate the existence of at least several types of chromatin molecules having different nucleosome organization patterns. We developed two types of statistical analysis in order to examine the cloning data in greater detail. One type, overlap analysis, revealed the distribution of the cloned fragments with respect to SV40 DNA. The distribution exhibits an oscillating pattern, dividing the genome into regions of weak or strong nucleosome density. The other analysis determined the distribution of the midpoints of the cloned fragments and revealed potential strong and weak nucleosome location sites, and an early versus late distinction in organization of nucleosomes in SV40 chromatin. The late region appears to contain more strong nucleosome location sites (8) than the early region (4). The strongest nucleosome abuts the late side of the nuclease-hypersensitive region and includes the major transcription initiation site of the late genes. Another strong site precedes this nucleosome and includes sequences implicated in controlling the expression of the SV40 early and late genes. A strong or weak nucleosome location site is not apparent near the early side of the nucleosome-hypersensitive region. Only weak and overlapping nucleosome location sites are found in the region where replication terminates in the SV40 minichromosomes.

Animals↗
PubMed1986

Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes.

We have investigated the average nucleosome spacing in the chromatin from several simian virus 40 virion assembly mutants temperature-sensitive in the major capsid protein VP1. Viral assembly intermediates that accumulate in cells infected with mutants that block virion assembly at the propagation step (tsB) have an average nucleosome repeat length similar to that of wild-type SV40 chromatin, approximately 198(+/- 4) base-pairs. This repeat length is longer than that of the host (BSC-40) cellular chromatin, which has a value of 187(+/- 4) base-pairs. In contrast, SV40 chromatin from cells infected with virus containing a mutation that blocks virion assembly at the initiation step (tsC) has a significantly shorter average repeat length of 177(+/- 4) base-pairs. At the permissive temperature (33 degrees C), tsC chromatin has a nucleosome spacing periodicity essentially the same as that of wild-type SV40 chromatin. In addition to possessing a chromatin structure with nucleosomes that are, on the average, closer together, tsC chromatin contains a nuclease-hypersensitive or open region in nearly all molecules, but apparently the same number of nucleosomes. These findings suggest that nucleosomes are deposited initially on newly replicated SV40 chromatin in such a way as to leave the DNA region containing the origin of replication and transcription enhancers uncovered. Subsequent interaction with capsid proteins appears to increase the average nucleosome spacing and consequently to cover the open region for encapsidation.

Animals↗
PubMed1986

A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.

The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.

Animals↗
PubMed1985

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA.

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.

Capsid↗
PubMed1983

Simian virus 40 morphogenetic pathway. An analysis of assembly-defective tsB201 DNA protein complexes.

Under restrictive conditions, the 220 S SV40 virions are not assembled in tsB201-infected cells. Instead, a new class of SV40 DNA-containing particles is isolated in addition to the 75 S chromatin. This new class of nucleoprotein complex sediments heterogeneously between 100 to 160 S with a peak at 130 S. Under an electron microscope, these complexes appear predominantly as SV40 chromatin associated with a shell-like protein cluster. These structures resemble the wild type assembly intermediates previously observed by Coca-Prados and Hsu (Coca-Prados, N., and Hsu, M.-T. (1979) J. Virol. 31, 199-208). Like the wild type assembly intermediates, the tsB201 DNA-protein complexes are unstable in high salt. In CsCl, they yield a protein species with a density characteristic of empty shells. In 1 M NaCl, they release heterogeneous 55-110 S protein polymers which consist of the capsid proteins VP1, VP2, and VP3. Our results indicate that the tsB201 nucleoproteins consist of capsid proteins, with varying extents of polymerization, held to chromatin by electrostatic bonds. The accumulation of these nucleoproteins is consistent with a simian virus 40 morphogenetic pathway wherein the capsid proteins are added gradually to the 75 S chromatin.

Cell Transformation, Neoplastic↗
PubMed1983

Simian virus 40 chromatin interaction with the capsid proteins.

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.

Capsid↗