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Biomedical subjects

J A Sharp

Publications and source records attributed to J A Sharp.

At least 19 recordsLinked to original sources
PubMed2003

Chromatin proteins are determinants of centromere function.

Recent advances in the identification of molecular components of centromeres have demonstrated a crucial role for chromatin proteins in determining both centromere identity and the stability of kinetochore-microtubule attachments. Although we are far from a complete understanding of the establishment and propagation of centromeres, this review seeks to highlight the contribution of histones, histone deposition factors, histone modifying enzymes, and heterochromatin proteins to the assembly of this sophisticated, highly specialized chromatin structure. First, an overview of DNA sequence elements at centromeric regions will be presented. We will then discuss the contribution of chromatin to kinetochore function in budding yeast, and pericentric heterochromatin domains in other eukaryotic systems. We will conclude with discussion of specialized nucleosomes that direct kinetochore assembly and propagation of centromere-defining chromatin domains.

Animals↗
PubMed2001

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing.

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.

Carrier Proteins↗
PubMed2000

Medicine information help lines: a survey of hospital pharmacy-based services in the UK and their conformity with guidelines.

OBJECTIVE: To describe the prevalence and nature of hospital pharmacy-based medicine help lines for consumers in the UK and to compare service provision with published guidelines. BACKGROUND: Since 1992, telephone help lines for patients have proliferated in hospital pharmacies in the UK. There is no common template for such services with variations in target group, number and type of calls, and arrangements for training and audit. Data on these factors will help guide further development of such services. METHODS: All medicine help lines operating from hospital pharmacies in the UK were identified through the national Drug Information Pharmacists network. They were sent a piloted questionnaire covering many aspects of help line operation, including funding, method of advertisement, procedures, target group, number and nature of calls, and audit procedures. RESULTS: Eighty-two help lines were identified in England, Scotland, Wales, and Northern Ireland. Completed responses were received from 69 help lines (84% response rate). The pharmacy drug information center was the help line site in 57% of hospitals; all other help lines were located in the dispensary. In 55% of cases, help lines were open only to patients of the hospital. In the remainder of help lines, calls from the public were answered (although the majority of help lines only advertised to hospital patients). Calls were answered by pharmacists only in 45% of services, and additional staff training had been provided in 43%. Only 48% of services had written procedures or guidelines for operation of the help line. Forty-six percent of the services received fewer than five calls per week, 31% received between five and 10 calls per week, and 22% received 11 or more calls per week. In 59% of the sites, calls took an average of 10 minutes or less to answer; it took 11-15 minutes in 32% of the sites and >15 minutes in 9% of the sites. The most common queries related to adverse effects, dosage and administration, and interactions (including alcohol). Only 33% of help lines had any auditing or monitoring of the service in place. CONCLUSIONS: The increasing use of the telephone to provide services directly to consumers is reflected in the growth of hospital-based medicine help lines in the UK. The telephone route is likely to become more important as patients' needs for information about their medicines increase. However, the rate of calls is low when compared with the number of patients issued prescriptions; further research is needed to investigate the reasons for this low response. There is currently reason for concern because most help lines lack not only professional training in telephone counseling, but also proper documentation, monitoring, and audit procedures.

Drug Information Services↗
PubMed1999

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer.

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB-231, MDA-MB-436, BT549, MCF-7ADR, Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

Animals↗
PubMed1998

Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

Adenosine Triphosphatases↗
PubMed1997

The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator.

Genetic studies have indicated that the facB gene of Aspergillus nidulans is a major regulatory gene involved in acetamide and acetate utilisation. Sequencing of the facB gene revealed that it encodes a protein that contains an N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster for DNA binding, leucine zipper-like heptad repeat motifs and central and C-terminal acidic alpha-helical regions, consistent with a function as a DNA-binding transcriptional activator. The Zn(II)2Cys6 cluster shows strong similarity with those of the Saccharomyces cerevisiae carbon metabolism regulatory proteins CAT8 and SIP4. A significant level of similarity with CAT8 is found throughout the length of the protein, suggesting at least partial functional homology. The facB genes of Aspergillus oryzae and Aspergillus niger were also sequenced and found to be highly conserved. Deletion of the facB gene confirmed that it is required for growth on acetate as a sole carbon source. Functional dissection using deletion and fusion constructs and in vitro mutagenesis indicated that the Zn(II)2Cys6 cluster and the C-terminal end of the protein are required for function.

Acetates↗
PubMed1996

The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters.

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level of amdS expression. Mobility shift studies have identified a factor in A. nidulans nuclear extracts which binds to this CCAAT sequence. In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed an A. nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designated hapC, with extensive homology to HAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, it has been shown that the hapC deletion results in reduced levels of expression of an amdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT binding activity to the amdS or gatA promoters, indicating that hapC may encode a component of the complex binding at this sequence.

Amidohydrolases↗
PubMed1990

Delay and number of food reinforcers: Effects on choice and latencies.

Pigeons were given a choice between two identical-duration situations (terminal links of chain schedules). One terminal link of the choice pair provided two food deliveries, and the other provided five. The exact times of these food deliveries differed between the terminal links and were varied over conditions. A single response during the initial link gave immediate access to the corresponding terminal link. Forced trials, during which only one of the initial-link keys was lighted, were interspersed with choice trials during which both initial-link keys were lighted. Choice tended to favor whichever terminal link was correlated with the higher sum of the immediacies (i.e., the sum of the reciprocals of the delays to each of the reinforcers following the choice, with all delays measured from the choice). Latencies on forced trials and on choice trials also were related (negatively) to the sum of the immediacies. This correlation among response measures (choice and latencies) suggests that both measures are manifestations of the effect of conditioned reinforcement on response tendencies.

Journal Article↗
PubMed1986

The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site.

We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.

Binding Sites↗
PubMed1985

Choline transport across a carbon tetrachloride phase containing a chloroform-methanol extract of brain.

The presence of a chloroform-methanol extract of cat brain in a carbon tetrachloride phase separating two aqueous phases resulted in an increased passage of [3H]choline across the organic phase which was inhibited by the choline transport inhibitor hemicholinium-3 and by high concentrations of non-radioactive choline. In the absence of cat brain extract, [3H]choline passage across carbon tetrachloride was neither inhibited by hemicholinium-3, nor by non-radioactive choline.

Animals↗
PubMed1985

Pausing of RNA polymerase during in vitro transcription through the ilvB and ilvGEDA attenuator regions of Escherichia coli K12.

Synchronized single-round transcriptions, in vitro, from templates encoding the leader RNA of the ilvB and ilvGEDA operons result in the accumulation of a transcript consistent with RNA polymerase pausing after the 1:2 stem that could form in each of the leader RNAs. Addition of L-factor or guanosine 5'-diphosphate,3-diphosphate extended the pause half-life obtained with the ilvB template; addition of L-factor and guanosine 5'-diphosphate,3'-diphosphate together had an additive effect on the pause half-life. L-factor also extended the pause half-life of the pause obtained with the ilvGEDA template; however, addition of guanosine 5'-diphosphate,3'-diphosphate did not. The results obtained are consistent with the model for attenuation proposed by Yanofsky et al. (Yanofsky, C., Das, A., Fisher, R., Kolter, R., and Berlin, V. (1984) UCLA Symposium of Gene Expression (Hamer, D., and Rosenberg, M., eds) pp. 295-310, Alan R. Liss, Inc., New York) in which transcriptional pausing after the synthesis of the 1:2 stem-loop closely couples transcription and translation of the leader region.

Bacterial Proteins↗
PubMed1985

Erythrocyte adenosine transport: effects of Ca2+ channel antagonists and ions.

On the basis of observations of adenosine-Ca2+ competition, we assessed the effects on erythrocyte adenosine transport of Ca2+ channel antagonists, mono- and divalent cations, and Cl- and Cl- transport inhibitors. The Ca2+ channel antagonists, diltiazem and verapamil, competitively inhibited adenosine influx (Ki = 158 +/- 17.4 and 13.5 +/- 1.3 microM at 10 microM adenosine, respectively), despite no apparent effect on transport by Ca2+, Mg2+, Na+, or K+. Verapamil also inhibited uridine efflux (Ki = 1.7 +/- 0.3 microM at 84-100 microM intracellular uridine). The absence of Cl- decreased adenosine influx rates from 0.615 +/- 0.013 to 0.386 +/- 0.008 nmol X s-1 X ml intracellular H2O-1. The Cl- transport inhibitors, diisothiocyanostilbene disulfonate (10 microM), furosemide (1 mM), and NO-3 (145 mM), decreased adenosine influx rates to 0.301 +/- 0.008, 0.325 +/- 0.013, and 0.430 +/- 0.009 nmol X s-1 X ml intracellular H2O-1, respectively. These studies indicate that the Ca2+ channel antagonists inhibit adenosine release and uptake and therefore may modulate adenosine-mediated events. Additionally, they suggest that adenosine and anion transport systems are linked or share common features.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

In vitro and in vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA.

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5' ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.

Chromosome Mapping↗
PubMed1984

Rho-dependent termination and concomitant NTPase activity requires a specific, intact RNA region.

We have investigated the specific DNA and RNA requirements for rho-dependent transcription termination in vitro. As a model, we have used templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t'. Templates containing the trp t' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase activity, and deletion of the trp t' region results in templates that do not induce rho-dependent termination or rho NTPase activity. Addition of ribonuclease T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity. These results demonstrate the requirement for a specific RNA component within the trp t' transcript necessary for NTPase activation and rho-dependent transcription termination. Active transcription is not a prerequisite for rho NTPase activation; trp t' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t' region, activated the rho NTPase when rho was added after inhibition of transcription. As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t' region has few G residues. This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be one determinant of sites specifying rho-dependent termination of transcription. The implications of this are discussed in the light of the lack of significant sequence homologies between rho-dependent transcription termination sites.

Adenosine Triphosphatases↗
PubMed1984

Ultrasonic detection of subdural collections.

Subdural collections in infants are common complications of meningitis, trauma and post surgical procedures. Careful ultrasonic examination via the anterior fontanelle is a reliable method of demonstrating these collections. It has an accuracy comparable to computed tomography and is our first line of investigation.

Female↗
PubMed1983

Each element of the Drosophila tRNAArg gene split promoter directs transcription in Xenopus oocytes.

The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.

Animals↗