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M Bina

Publications and source records attributed to M Bina.

At least 19 recordsLinked to original sources
PubMed1992

Interactions of HTF4 with E-box motifs in the long terminal repeat of human immunodeficiency virus type 1.

We have identified three consensus E-box motifs in the long terminal repeat of human immunodeficiency virus type 1. One of these E boxes interacts selectively with representative members of the class A group of basic helix-loop-helix proteins, including HTF4, E47, and their heterodimers. Our analyses implicate the helix-loop-helix proteins in regulation of human immunodeficiency virus type 1 gene expression.

Basic Helix-Loop-Helix Proteins↗
PubMed1992

The nucleotide sequence of the human transcription factor HTF4a cDNA.

A partial cDNA has previously been isolated that encodes HTF4, a new member of the basic helix-loop-helix family of DNA binding proteins. We have reconstructed a full cDNA, designated HTF4a cDNA, which spans the entire HTF4 coding region. The 5' end of the reconstructed cDNA includes an open reading frame encoding a 117-residue polypeptide. Three closely spaced ATG codons in the major open reading frame may potentially serve as sites of initiation for HTF4a synthesis, to yield proteins that contain 682, 675, and 657 amino acid residues, respectively. Pairwise dot matrix analysis indicates that there are at least three distinct human genes which encode proteins related to the Drosophila daughterless.

Amino Acid Sequence↗
PubMed1991

In vitro assembly of a positioned nucleosome near the hypersensitive region in simian virus 40 chromatin.

Previous studies have identified a nucleosome near a potential late boundary for the nuclease-hypersensitive region in simian virus 40 chromatin. We have performed in vitro reconstitution analysis to determine whether the underlying DNA sequence encodes for the assembly of this nucleosome and applied hydroxyl radical and DNase I footprinting techniques to examine the structure of the reconstituted nucleosome. Both methods revealed the formation of a precisely positioned nucleosome in vitro, on a fragment spanning the strong in vivo nucleosome location site determined previously in the viral chromatin. The center of the positioned nucleosome maps between nucleotide 384 and 387 on simian virus 40 DNA. The corresponding nucleosome core includes the major-late transcription site (12 base-pairs within the core), the MspI site, and a segment shown previously to adopt a bent structure in the absence of proteins. The hydroxyl radical produces a strikingly well-defined cleavage pattern over the bent DNA incorporated in nucleosomes. The dominant periodicity of DNA in this nucleosome is 10.26 base-pairs per turn. The distribution of the .OH cut sites in the positioned nucleosome provides strong support for models in which the minor grooves of the A/T-rich tracts are oriented toward the histone core while the minor grooves of the G/C-rich sequences are facing outward.

Base Sequence↗
PubMed1991

Sequence of a HeLa cDNA provides the DNA binding domain and carboxy terminus of HE47: a human helix-loop-helix protein related to the enhancer binding factor E47.

In order to identify cDNA encoding regulators of gene expression, a HeLa lambda gt11 expression library was screened with a DNA segment containing multiple copies of a sequence spanning the AP4 site in the simian virus 40 control region. We identified a partial cDNA encoding HE47, a sequence-specific DNA binding protein. The HeLa clone overlaps with a previously reported human B-cell partial cDNA encoding E47. The HeLa cDNA includes the HE47 DNA binding domain, its carboxy terminus, and the 3' untranslated region of its mRNA.

Amino Acid Sequence↗
PubMed1990

Strategy for statistical-mapping of potential regulatory regions in the human genome.

We have explored the feasibility of using crude nuclear extracts and band-shift experiments, to select and clone short human DNA fragments that contain binding sites for sequence-specific nuclear proteins. We show that this strategy is feasible. It provides a means to systematically identify the recognition sequences of proteins found in nuclear extracts prepared from various tissues, in order to compile a protein-binding-sequence catalogue. A complete catalogue would provide the data needed for searching the "raw" human DNA sequences for occurrence of protein-binding sites and thus, to construct a statistical map of potential regulatory regions in the human genome.

Base Sequence↗
PubMed1990

Temperature-mediated regulation and downstream inducible selection for controlling gene expression from the bacteriophage lambda pL promoter.

We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor. Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h. We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E. coli.

Bacteriophage lambda↗
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↗
PubMed1990

Correlation between dinucleotide periodicities and nucleosome positioning on mouse satellite DNA.

Previous studies demonstrated 16 well-defined nucleosome locations (A-P) on a tandemly repeated prototype 234 base pair (bp) mouse satellite repeat unit. We have aligned the A-P fragments to search for DNA sequence elements that might contribute to nucleosome placement at these positions. Our results demonstrate a strikingly regular, uninterrupted, periodic pattern for the AA dinucleotide occurrences along the entire length of the aligned fragments. The periodicity of the AA occurrences is about 9.7 bp. The pattern exhibits a local minimum at position 74, near the nucleosome dyad axis of symmetry. Other dinucleotides--including AC: GT, CA: TG, and CC: GG--are also placed periodically, but their patterns of occurrence are less regular and less frequent than AA. The calculated spacings between consecutive preferred nucleosome locations on mouse satellite DNA are nearly identical, corresponding to multiples of 9.7 bp. The correlation between the periodicity of dinucleotide occurrences and the average spacing of nucleosome positions suggests that the preferred nucleosome locations recur at intervals that may correspond to the DNA helical repeat in the mouse satellite nucleosomes, and that the histone octamers sample (or slip along) the duplex in steps of 9.7 bp during nucleosome formation on mouse satellite DNA.

Animals↗
PubMed1989

Locations of nucleosomes on the regulatory region of simian virus 40 chromatin.

We have asked where the nucleosomes are located with respect to the replication origin and regulatory region of simian virus 40 DNA, what would be the possible functional consequences of the identified locations, and to what extent these locations correlate with the current views on mechanisms involved in establishing nucleosome-free regions in chromatin. To identify the precise location of nucleosomes, we have shot-gun cloned and sequenced nucleosomal DNA obtained from micrococcal nuclease digestion of wt776 chromatin prepared late in infection. Our results indicate that nucleosomes do not occupy unique positions over the replication origin or the elements involved in transcriptional control. However, it appears that the nucleosome distribution is not random, since several nucleosomes are represented by two or more independently generated clones. Two nearly identical cloned fragments map over the replication origin; five include 1.5 copies of the 72 base-pair enhancer sequences; and eight map to a region that spans a DNA bending locus and the major transcription initiation site of the late genes. The complex nucleosome distribution pattern observed in our direct analysis suggests that disparate nucleosome-free regions may be involved in controlling replication, and selective expression of the viral early or late genes.

Cell Line↗
PubMed1989

The capsid of small papova viruses contains 72 pentameric capsomeres: direct evidence from cryo-electron-microscopy of simian virus 40.

The three-dimensional structure of the simian virus 40 capsid is remarkably similar to the structure of the polyoma empty capsid. This similarity is apparent despite striking differences in the methods used to determine the two structures: image analysis of electron micrographs of frozen-hydrated samples (SV40 virions) and an unconventional x-ray crystallographic analysis (polyoma empty capsids). Both methods have clearly resolved the 72 prominent capsomere units which comprise the T = 7d icosahedral capsid surface lattice. The 12 pentavalent and 60 hexavalent capsomeres consist of pentameric substructures. A pentameric morphology for hexavalent capsomeres clearly shows that the conserved bonding specificity expected from the quasi-equivalence theory is not present in either SV40 or polyoma capsids. Determination of the SV40 structure from cryo-electron microscopy supports the correctness of the polyoma structure solved crystallographically and establishes a strong complementarity of the two techniques. Similarity between the SV40 virion and the empty polyoma capsid indicates that the capsid is not detectably altered by the loss of the nucleohistone core. The unexpected pentameric substructure of the hexavalent capsomeres and the arrangement of the 72 pentamers in the SV40 and polyoma capsid lattices may be characteristic features of all members of the papova virus family, including the papilloma viruses such as human wart and rabbit papilloma.

Capsid↗
PubMed1989

Evaluation and optimization of variables in immunofixation electrophoresis for the detection of IgG paraproteins.

We examined different antisera and varying serum dilutions for their effects on the sensitivity of immunofixation electrophoresis (IFE) for detecting and characterizing serum IgG paraproteins. Different antisera reacted unequally with a single paraprotein and conversely, a single antiserum reacted unequally with several different paraproteins. The dilution of serum was extremely important; the adoption of a single dilution for all sera could result in failure to detect low-concentration or poorly immunoreactive paraproteins. While the application of a standardized amount of paraprotein is recommended, it must be emphasized that no single antiserum can be relied on to provide unequivocal detection of all paraproteins of that particular isotype.

Blood Protein Electrophoresis↗
PubMed1989

Photochemically induced binding of Rh(phen)2Cl2+ to DNA.

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.

Animals↗
PubMed1988

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli.

The complete sequences of the SV40 agnogene (LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 micrograms/ml in the case of NS1A-LP1 to approx. 5 micrograms/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with Mrs expected for isolated NS1A and LP1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.

Amino Acid Sequence↗
PubMed1988

Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatin core.

The three-dimensional structure of the capsid and the nucleohistone core of simian virus 40 (SV40) has been reconstructed by image analysis of electron micrographs of frozen hydrated samples. The 72 prominent capsomere units that comprise the T = 7d icosahedral surface lattice of the capsid are clearly resolved. Both the pentavalent and hexavalent capsomeres appear with pentameric substructure, indicating that bonding specificity in the shell is not quasi-equivalent. There is a remarkable similarity between the structure of the SV40 virion capsid and the structure reported for the polyoma empty capsid. This result establishes that (i) the unexpected pentameric substructure of the hexavalent capsomeres is also present in virions and (ii) the arrangement of the 72 pentamers in the capsid lattice may be a characteristic feature of the entire papova family of viruses. The center of the SV40 reconstruction reveals electron density corresponding to the nucleohistone core. This density is smeared, suggesting that the minichromosome is not organized with icosahedral symmetry matching the capsid symmetry. The visualization of the virion chromatin provides a basis for invoking new models for the higher order structure of the encapsidated minichromosome.

Capsid↗
PubMed1988

Analysis of temperature-sensitive mutations in the simian virus 40 gene encoding virion protein 1.

Temperature-sensitive (ts) assembly mutants of the tumorigenic virus simian virus 40 (SV40) fail to follow the normal pathway of virion morphogenesis at 40 degrees C. The mutations were previously mapped to the gene coding for the major virion protein VP1 and fall into three groups: tsB, tsBC, and tsC. We have determined the tsB/C mutations by DNA sequence analysis and deduced the corresponding amino acid substitutions. We find that the mutations are global and span 68% of the VP1 gene. They result predominantly in single amino acid substitutions. The B mutations are localized between nucleotides 1667 and 2091, spanning the VP1 amino acid residues 54-195. With the exception of one mutation in tsC260, the C group mutations occur between the nucleotides 2141 and 2262, spanning VP1 residues 212-252. The tsBC substitutions are not localized within a distinct region. We present a model for the VP1 structure. The model correlates the distribution of ts assembly mutations in the SV40 VP1 gene with the VP1 functional domains, deduced form the phenotypes exhibited by the assembly mutants, and the VP1 structural domains, deduced recently from the cryoelectron microscopic studies of the SV40 virions. We summarize the behavior of the SV40 ts mutants and discuss the possible relationship between the ts phenotype and amino acid substitutions.

Amino Acid Sequence↗
PubMed1988

Sensitivity of immunofixation electrophoresis for detecting IgM paraproteins in serum.

Detection and characterization of small IgM paraproteins by immunoelectrophoresis are often difficult because of (a) slow diffusion of the macromolecular immunoglobulin, and (b) the obscuring of the light chain, especially kappa, by the light chains of background polyclonal immunoglobulin (the "umbrella effect"). Various methods of sample pretreatment have been tried in an attempt to overcome this problem. Using immunofixation electrophoresis (IFE), we were able to detect and characterize IgM paraproteins in serum at concentrations as low as 0.25 g/L, even when the polyclonal immunoglobulin background was increased. With routine IFE, our laboratory identified 58 patients with IgM paraproteinemia in a four-year period. In 36, the paraproteins were present at less than 5 g/L; 10 of these were detectable only by IFE.

Humans↗
PubMed1987

The flexibility and topology of simian virus 40 DNA in minichromosomes.

The linking number of DNA in minichromosomes increases by 2 turns during SV40 assembly. Changes in temperature also influence the average linking number of the total intracellular forms of SV40 DNA. When the isolated minichromosomes assembled in vivo are incubated with topoisomerase I at 33 degrees C in vitro, the linking number of SV40 DNA decreases. This decrease is about: -1.1 turns for minichromosomes with an average nucleosome spacing of 198 base pairs (bp), wt776; and -0.6 turns for minichromosomes containing a shorter average nucleosome repeat (177 bp), tsC219. The difference between the average linking number of naked SV40 DNA relaxed with topoI at 33 degrees C and minichromosomes relaxed with the enzyme at the same temperature indicates that SV40 chromatin contains on the average 26 nucleosomes. However, the results of studies obtained both on DNA flexibility in chromatin and in naked DNA, and on the shape of the topoisomer distribution curves, indicate that all of the minichromosomes, regardless of their overall structure, do not contain the same number of nucleosomes; this heterogeneity may be as large as 8 nucleosomes. We find no apparent correlation between the amount of minichromosomes containing unconstrained torsional stress and the abundance of the molecules with a structure characteristic of transcriptionally active chromatin.

Chromatin↗