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Biomedical subjects

G R Smith

Publications and source records attributed to G R Smith.

At least 19 recordsLinked to original sources
PubMed1995

Monomeric RecBCD enzyme binds and unwinds DNA.

RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.

Base Sequence↗
PubMed1995

Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. Modulation by ATP and magnesium levels.

RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes single-strand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg2+ ratio. With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3'-side of the Chi octamer (5'-GCTGGTGG-3') and were detected only on the strand bearing that sequence. Three differences were seen with Mg2+ in excess. 1) Chi-dependent 3'-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer. 2) Chi-dependent cuts occurred on the complementary DNA strand. 3) RecBCD enzyme destroyed the 3'-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported. We show here that, with Mg2+ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction.

Adenosine Triphosphate↗
PubMed1995

A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.

Exonuclease I (Exo I) from Schizosaccharomyces pombe, a 5'-->3' double-stranded DNA exonuclease, is induced during meiotic prophase I. The exo1 gene is a member of a family of related DNA repair genes, including RAD2/rad13/xpgc and YKL510/rad2, conserved from yeast to humans. An exo1 mutant displays a mutator phenotype and alters activity of the ade6-M387 marker effect. These results suggest that Exo I acts in a pathway that corrects mismatched base pairs.

Alleles↗
PubMed1995

The initiation and control of homologous recombination in Escherichia coli.

The chromosome of Escherichia coli recombines at low frequency when it is an intact circle but recombines at high frequency when it is broken, for example by X-rays, or when a linear DNA fragment is introduced into the cell during conjugation or transduction. The high recombinogenicity of double-strand (ds) DNA ends is attributable to RecBCD enzyme, which acts on ds DNA ends and is essential for recombination and ds DNA break repair. RecBCD enzyme initiates DNA unwinding at ds DNA ends, and its nuclease activity is controlled by Chi sites (5' G-C-T-G-G-T-G-G 3') in such a way that the enzyme produces a potent single-stranded DNA substrate for homologous pairing by RecA and single-stranded DNA binding proteins. We discuss a unifying model for recombination and ds DNA break repair, based upon the enzymic activities of these and other proteins and upon the behaviour of E. coli mutants altered in these proteins.

Escherichia coli↗
PubMed1995

A trial of the effect of a standardized psychiatric consultation on health outcomes and costs in somatizing patients.

BACKGROUND: Patients who somatize but who do not meet criteria for somatization disorder are common in the community. Virtually no research has been conducted to determine how to treat these patients. METHODS: We conducted a randomized controlled clinical trial of a psychiatric consultation intervention we had previously shown to improve the management of somatization disorder. The study population included 51 physicians treating 56 somatizing patients who had a history of seeking help for six to 12 lifetime unexplained physical symptoms. At the onset of the experiment, physicians randomized to the treatment condition received a consultation letter recommending a specific management approach; physicians randomized to the control/crossover condition received the consultation letter after 12 months. Data on health outcomes and charges were collected every 4 months for 2 years after randomization for 96% of subjects who entered the study. RESULTS: Patients of physicians who received the intervention reported significantly increased physical functioning, an improvement that remained stable during the year after the intervention. The intervention reduced annual medical care charges by $289 (95% confidence interval, $40 to $464) in 1990 constant dollars, which equates to a 32.9% reduction in the annual median cost of their medical care. CONCLUSIONS: Somatizing patients with a lifetime history of six to 12 unexplained physical symptoms reported better physical functioning after their primary care physician was provided appropriate treatment recommendations via a psychiatric consultation. Such a consultation is cost-effective because it reduces subsequent charges for medical care, while improving health outcomes in a chronically impaired population.

Adult↗
PubMed1995

Molecular cloning of the meiosis-induced rec10 gene of Schizosaccharomyces pombe.

The meiotic recombination gene rec10, which encodes a region-specific activator of recombination in Schizosaccharomyces pombe, has been cloned by genetic complementation and its nucleotide sequence determined. The rec10 gene was identified in a 5.6-kb cloned fragment by partial-deletion and insertion experiments. The nucleotide sequence of 3.5 kb of this clone revealed an open reading frame (ORF) encoding a 791 amino-acid polypeptide for the rec10 gene product. During meiosis, thermally induced in a temperature-sensitive pat1-114 mutant, the transcript of rec10 was induced to a maximal level at 2-3 h but was present at much lower levels before and after this time. The transient induction of the rec10 transcript and the rec10 mutant phenotype suggest that the rec10 gene product is involved primarily in the early steps of meiotic recombination localized to chromosome III in S. pombe.

Amino Acid Sequence↗
PubMed1995

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Amino Acid Sequence↗
PubMed1995

DSM-IV field trial: testing a new proposal for somatization disorder.

OBJECTIVE: The purpose of this study was to evaluate for APA a proposed strategy to diagnose somatization disorder for possible inclusion in DSM-IV. METHOD: Five sites--Washington University, University of Kansas, University of Iowa, University of Arkansas, and Mount Sinai Medical Center in New York--participated in a collaborative field trial. Female subjects (N = 353) were recruited from several different services (psychiatry, internal medicine, and family practice) and were evaluated for the presence or absence of the disorder. This assessment was performed with a new instrument constructed by combining all the criteria for somatization disorder from the proposed criteria for DSM-IV, DSM-III, DSM-III-R, Perley-Guze, and proposed criteria for ICD-10. RESULTS: A high level of concordance was found between the proposed diagnostic strategy for DSM-IV and the current criteria (DSM-III-R), as well as the earlier criteria (Perley-Guze and DSM-III). The ICD-10 criteria agreed poorly with all other criteria sets. The level of experience of the rater (expert versus novice) with the earlier (Perley-Guze, DSM-III) and current (DSM-III-R) criteria did not influence the identification of cases by use of DSM-IV criteria. No racial effect was introduced by any of the criteria sets. CONCLUSIONS: The strategy for DSM-IV is an accurate and simpler method of diagnosing somatization disorder that does not require special expertise for proper use.

Adult↗
PubMed1995

Families who somatize.

This investigation extends a previous study of children of people with somatization disorder and children of less severely affected somatizers. We used the revised Diagnostic Interview for Children and Adolescents, revised, and abstracted medical records to compare families in which a child as well as a parent had unexplained somatic complaints; we also identified parental predictors of children's somatization. Children in families with somatizing children had more emergency room use, more suicidal behavior, and more disability. Children in families of somatization disorder adults had 11.7 times as many emergency room visits as less severely affected somatizers and missed 8.8 times as much school. Parental somatization, substance abuse, and antisocial symptoms predicted children's somatization. Physicians in primary care should be aware of the frequency with which somatization occurs in other members of a family when it is identified in one member.

Adolescent↗
PubMed1994

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie.

Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 degrees C for < or = 60 min. Bioassay in rodents showed that none of the regimens produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for < or = 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing < or = 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for < or = 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.

Animals↗
PubMed1994

The production of Clostridium botulinum type A, B and D toxin in rotting carcasses.

Carrion is a major source of botulinal toxin for animals. A type D strain of Clostridium botulinum differed from type A and B strains in producing (1) much higher concentrations of toxin in putrefying mouse carcasses at several different temperatures over a period of 35 days, (2) toxicity that sometimes persisted in mouse carcasses for at least a year, and (3) mouse carcasses with exceptionally high oral toxicity. Fish carcasses were much less favourable than mouse carcasses for type D toxigenesis. The study, together with earlier studies on types C and E, indicated that carrion contaminated with C. botulinum type C or D is likely to be particularly dangerous for animals that may ingest it. This accords with the observation that carrion-transmitted botulism in animals is usually caused by type C or D.

Animals↗
PubMed1992

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.

Chromatography, Affinity↗
PubMed1992

RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.

During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.

Bacteriophage lambda↗
PubMed1992

A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe.

In meiotic cells of the fission yeast Schizosaccharomyces pombe, a DNA exonuclease activity increased approximately 5-fold after premeiotic S-phase and decreased to the initial level before the meiotic divisions. We have purified this activity, designated exonuclease I, to near homogeneity. The activity co-purified with a polypeptide with an apparent molecular weight of 36,000. With a linear double-stranded DNA substrate, exonuclease I degraded only the 5'-ended strand from each end to produce 3'-single-stranded tails. The enzyme also acted on nicked circular DNA with comparable affinity. The meiotic induction of exonuclease I and its mode of action, similar to that of recombination-promoting exonucleases from bacteria, suggest that exonuclease I is involved in meiotic homologous recombination in S. pombe.

Chromatography, Affinity↗
PubMed1992

Chromosomal context dependence of a eukaryotic recombinational hot spot.

The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting.

Chromosomes, Fungal↗
PubMed1992

Transient expression of the coat protein of sugarcane mosaic virus in sugarcane protoplasts and expression in Escherichia coli.

The coat protein (CP) of strain SC of sugarcane mosaic virus (SCMV-SC) was expressed transiently in sugarcane protoplasts after electroporation with one of two plasmids encoding the CP gene. The CP gene was fused with either the cauliflower mosaic virus 35S promoter or the synthetic monocotyledon promoter "Emu". The coat protein gene was also inducibly expressed in Escherichia coli when fused to the trc promoter. The protein expressed in both systems had the same electrophoretic mobility and antigenic specificity as purified SCMV-SC coat protein. Transient expression of the 35S-CP gene in protoplasts could only be demonstrated in Western blots developed with the chemiluminescence enzyme substrate luminol.

Base Sequence↗