Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 145 records · Page 8Linked to original sources

PubMed1993

Kinetic analysis of template.primer interactions with recombinant forms of HIV-1 reverse transcriptase.

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1993

Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Short gap-filling synthesis by DNA polymerase beta is processive.

Mammalian DNA polymerase beta (beta-pol) is considered to be part of the DNA repair machinery. However, there have been conceptual problems with this idea because beta-pol does not completely fill some gapped substrates, and unlike other DNA polymerases, beta-pol adds just one dNMP for each cycle of binding to the DNA substrate, incorporation, and product release (distributive synthesis). To examine the questions of complete gap filling and gap recognition by beta-pol, we designed template-primer substrates with a range of gap sizes from 1 to 53 template residues between the 3'-OH primer and a downstream polynucleotide. M13mp18(+) single-stranded DNA was used as template, and synthetic deoxyoligonucleotides were used as primers and downstream polynucleotides. We find that beta-pol can completely fill gaps and that the gap-filling activity on substrates with gaps of up to 6 nucleotides is highly processive rather than distributive; processive synthesis to fill a gap strictly required 5'-phosphate on the 5'-moiety of the gap. Similar specificity for filling short gaps was not observed with other DNA polymerases. These results on substrate specificity and the mechanism of beta-pol suggest distinct requirements for the role of beta-pol in vivo.

Animals↗

PubMed1993

Identification of a nuclear protein binding element within the rat brain protein kinase C gamma promoter that is related to the developmental control of this gene.

Protein kinase C gamma (PKC gamma) is a brain-specific isozyme expressed at a high level in the adult but not in the fetal or newborn rat. At least seventeen nuclear protein binding sites within the 5'-flanking region extending from -1612 to +243 had been identified by DNase I footprinting analysis and gel mobility shift assays. Among them, one site, GAATTAATAGG, at -669 to -679 is protected from DNase I digestion by nuclear protein from newborn but not from the adult rat brain. The levels of this binding protein, as determined by gel mobility shift assay, were found inversely related to the levels of PKC gamma in rat brain at different stages of development. These results suggest that this particular binding site may participate in the developmental regulation of PKC gamma gene.

Animals↗

PubMed1993

Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem.

During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult. At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds. These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related. Mechanisms producing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. Several general trends emerged from the study. First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T. Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end. A and C as template bases in the template-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.

Base Sequence↗

PubMed1993

Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots.

We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences. We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J. D., Wilson, S. H., and Kunkel, T. A. (1989) J. Biol. Chem. 264, 16948-16956). Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors. The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability. The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots. The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides. Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run. These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.

Base Sequence↗

PubMed1993

Mammalian heterogeneous ribonucleoprotein A1 and its constituent domains. Nucleic acid interaction, structural stability and self-association.

With a view toward further understanding the structure-function relationships of the eukaryotic heterogeneous ribonucleoprotein (hnRNP) A1, and in particular its multiplicity of nucleic acid-interactive domains, we have studied the nucleic acid binding properties of the globular N-domain (UP1) and sequence-repetitive, flexible C-domain, the thermal denaturation of UP1 and the concomitant effects of binding polynucleotide, and the self-associative properties of the full-length protein. Utilizing protein tryptophan fluorescence as a probe, polynucleotide binding was shown to stabilize UP1 against thermal unfolding. The denaturation profile of UP1-poly(thymidylic acid) complexes was biphasic, suggesting that unfolding of the two subdomains of UP1 can occur independently. This is in agreement with a previously proposed structure in which only one of the two UP1 subdomains binds the nucleic acid. The subdomains of UP1 can be prepared by controlled proteolysis of A1, further indicating that these two globular segments within A1 are connected by an exposed, flexible linkage. Circular dichroism measurements on UP1 confirm previous data that this portion of A1 binds single-stranded nucleic acids non-co-operatively. UP1 clearly shows a preference for single-stranded nucleic acids with a 2'-OH, since its affinity for poly(U) is three times higher than for poly(dU), and five times higher than its affinity for poly(2'-OCH3U). The nucleic acid-interactive properties of the C-domain were further examined by preparing a synthetic peptide polymer (M(r) approximately 12,000) containing about seven repeats of a 16-residue sequence, GNFGGGRGGNYGGSRG, which in turn comprises two copies of the C-terminal consensus, GN(F/Y)GG(G/S)RG. The polymer of this sequence exhibited significant affinity for the fluorescent polyribonucleotide, poly(ethenoadenylic acid), binding stoichiometrically at < or = 0.2 M-Na+. Complex formation was accompanied by an increase in aggregate formation, as indicated by the appearance of scattering. For purposes of comparison, the data were analyzed via the linear co-operative model of McGhee and von Hippel, though this model may not be fully descriptive of the protein-nucleic acid complex(es) formed in this case. In contrast to the non-co-operative binding mode of the UP1 domain, the C-polymer exhibited moderate co-operativity, comparable to that seen with full-length A1. Although addition of sufficient NaCl reversed the interaction, a sigmoidal binding isotherm could still be observed (with sufficient added polymer) at 0.8 M-NaCl. This suggests that non-electrostatic interactions contribute significantly to the free energy of binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli.

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.

Amino Acid Sequence↗

PubMed1993

Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system.

We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system. This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur. After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates. RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa. Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain. Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E. coli-derived RT. Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.

Animals↗

PubMed1993

Unemployment and health: a review.

Unemployment has an adverse effect on health. This effect is still demonstrable when social class, poverty, age and pre-existing morbidity are adjusted for. Unemployed men and their families have increased mortality experience, particularly from suicide and lung cancer. Unemployed men also have a reduction in psychological well-being with a greater incidence of parasuicide, depression and anxiety. Unemployed men are more likely to use general practitioner and hospital services and receive more prescribed medicines. Smoking and alcohol consumption are often increased after the onset of unemployment. Women are less affected by enforced unemployment, but families are put at greater risk of physical illness, psychological stress and family breakdown. Maintaining financial security, providing proactive health care and retraining for re-employment can all reduce the impact of unemployment on health.

Family Health↗

PubMed1992

DNA damage response of cloned DNA beta-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A.

DNA beta-polymerase (beta-pol), one of the recognized DNA polymerizing enzymes in vertebrates, has a role in 'very short patch' gap-filling synthesis during nucleotide excision DNA repair. In human and mouse, the enzyme is encoded by a single-copy gene located on the short arm of chromosome 8 near the centromere. In a series of studies, we have found that the cloned human beta-pol promoter is regulated by signals acting through the single ATF/CRE palindrome in the core promoter. These signals include transactivation by: adenovirus E1a/E1b proteins; activated p21ras; and in CHO cells, treatment with the DNA damaging agent MNNG. Hence, several types of stimulatory signals are mediated through the single ATF/CRE site, including DNA damage induction. To understand the mechanism of beta-pol promoter activation by MNNG in CHO cells, we asked whether induction of the cAMP/protein kinase A pathway can increase transcription of the cloned promoter in this system. Agents that increase cellular cAMP levels (8-BrcAMP; forskolin and IBMx) activated the beta-pol promoter fusion gene in transient expression experiments, and a mutation in the ATF/CRE palindrome blocked this response. Thus, the ATF/CRE site appears to be cAMP responsive in the CHO cell system. We found that the activation of the cloned beta-pol promoter by MNNG does not occur with two mutant CHO cell lines that are deficient in protein kinase A activity. Further, simultaneous treatment of wild-type CHO cells, with MNNG and to elevate cAMP, failed to result in an additive effect for activation of the beta-pol promoter. Thus, these effectors may act through a common pathway. These results suggest that the activation of the cloned beta-pol promoter in CHO cells following MNNG treatment is mediated through the cAMP/protein kinase A signal transduction pathway.

Animals↗

PubMed1992

Mammalian DNA polymerase beta: characterization of a 16-kDa transdomain fragment containing the nucleic acid-binding activities of the native enzyme.

The 39-kDa DNA polymerase beta (beta-Pol) molecule can be readily converted into two constituent domains by mild proteolysis; these domains are represented in an 8-kDa N-terminal fragment and a 31-kDa C-terminal fragment [Kumar et al. (1990a) J. Biol. Chem. 265, 2124-2131]. Intact beta-Pol is a sequence-nonspecific nucleic acid-interactive protein that binds both double-stranded (ds) and single-stranded (ss) polynucleotides. These two activities appear to be contributed by separate portions of the enzyme, since the 31-kDa domain binds ds DNA but not ss DNA, and conversely, the 8-kDa domain binds ss DNA but not ds DNA [Casas-Finet et al. (1991) J. Biol. Chem. 266, 19618-19625]. Truncation of the 31-kDa domain at the N-terminus with chymotrypsin, to produce a 27-kDa fragment (residues 140-334), eliminated all DNA-binding activity. This suggested that the ds DNA-binding capacity of the 31-kDa domain may be carried in the N-terminal segment of the 31-kDa domain. We used CNBr to prepare a 16-kDa fragment (residues 18-154) that spans the ss DNA-binding region of the 8-kDa domain along with the N-terminal portion of the 31-kDa domain. The purified 16-kDa fragment was found to have both ss and ds polynucleotide-binding capacity. Thermodynamic binding properties for these activities are similar to those of the intact enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

DNA polymerase beta and DNA synthesis in Xenopus oocytes and in a nuclear extract.

The identities of the DNA polymerases required for conversion of single-strand (ss) M13 DNA to double-strand (ds) M13 DNA were examined both in injected Xenopus laevis oocytes and in an oocyte nuclear extract. Inhibitors and antibodies specific to DNA polymerases alpha and beta were used. In nuclear extracts, inhibition by the antibody to polymerase beta could be reversed by purified polymerase beta. The polymerase beta inhibitors, dideoxythymidine triphosphate (ddTTP) and dideoxycytidine triphosphate (ddCTP), also blocked DNA synthesis and indicated that polymerase beta is involved in the conversion of ssDNA to dsDNA. These results also may have particular significance for emerging evidence of an ssDNA replication mode in eukaryotic cells.

Animals↗

PubMed1992

Selective photochemical modification by trichloroethanol of tryptophan residues in proteins with a high tyrosine-to-tryptophan ratio.

We present an improved procedure for the selective modification of tryptophan residues in proteins. A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol. This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct. Except for tyrosine, no reaction with other amino acid side chains are known. Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses. Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density. Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved. Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes.

Animals↗

PubMed1992

An audit of cervical cancer deaths in Nottingham.

Death certificates were reviewed and 57 women were identified whose primary cause of death was cervical cancer. Their cervical smear records were reviewed from laboratory files. Only nine had participated in the cervical cancer screening programme before the diagnosis of cervical cancer was made. One woman had an abnormal smear as a result of cervical screening but failed to attend for follow-up. The problem of false negative smears is also addressed and the value of review of negative smears for the purposes of cytology audit is emphasized.

Adult↗

PubMed1992

Inhibitors of HIV-1 reverse transcriptase and fidelity of in vitro DNA replication.

Mechanisms of the effects of the dTTP analogues 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) and 3'-amino-3'-deoxythymidine 5'-triphosphate (NH2 TTP) upon the HIV-1 reverse transcriptase (RT) are discussed. These compounds block the RT in vitro and do so by different kinetic mechanisms. Infidelity of replication is a hallmark of the HIV-1 RT, and replication errors by the enzyme on RNA and DNA templates are discussed. The enzyme's infidelity has ramifications for inhibition: On the one hand, the propensity to produce mutations enhances the ability of the virus to escape inhibitors whereas on the other hand, the infidelity of the reverse transcriptase may allow the development of imaginative inhibitor strategies.

DNA Replication↗

PubMed1992

Teenage conception and contraception in the English regions.

Nationally available data on teenage fertility, family planning care and mortality were analysed to determine the relationship between teenage conception, availability of abortion and family planning care, and an indicator of socioeconomic disadvantage--the Standardized Mortality Ratio (SMR). In the 14 regions of England the strongest correlate of teenage conception and of the proportion of teenage conceptions aborted was female all-causes SMR. High levels of provision of NHS abortion services and uptake of family planning clinic care did not significantly reduce teenage fertility. Provision of traditional family planning services obviously plays an important role in preventing teenage pregnancy, but innovation in this service coupled with a concerted effort to reduce social disadvantage might have a greater impact on teenage fertility in England.

Abortion, Induced↗

PubMed1991

Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding.

Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a approximately 51,000 Mr polypeptide and a approximately 66,000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15,000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986). We prepared individual bacterial-recombinant RTs as the approximately 66,000 Mr polypeptide (p66) or as the approximately 51,000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments. Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 x 10(4) M-1. p51 failed to dimerize and behaved as a monomer under these conditions. Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 10(5) M-1. These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns. Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay. Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component. C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66. The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of p66 appears to be required also, as p51 alone did not dimerize.

Blotting, Western↗