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W Ross

Publications and source records attributed to W Ross.

At least 19 recordsLinked to original sources
PubMed2001

Fine structure of E. coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove.

The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding. On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.

Amino Acid Motifs↗
PubMed2001

Promoter recognition and discrimination by EsigmaS RNA polymerase.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sigmaS (EsigmaS), and it is known that there is some overlap between the promoters recognized by EsigmaS and by the major E. coli holoenzyme (Esigma70), the sequence elements responsible for promoter recognition by EsigmaS are not well understood. To define the DNA sequences recognized best by EsigmaS in vitro, we started with random DNA and enriched for EsigmaS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EsigmaS contained the known consensus elements (-10 and -35 hexamers) for recognition by Esigma70. Using genetic and biochemical approaches, we show that EsigmaS and Esigma70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EsigmaS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Esigma70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EsigmaS versus Esigma70 thus presents an alternative solution to the problem of promoter selectivity.

Bacterial Proteins↗
PubMed2001

Daytime sleepiness is associated with 3-year incident dementia and cognitive decline in older Japanese-American men.

OBJECTIVES: To assess the longitudinal association between sleep disturbances (insomnia and daytime sleepiness) and incidence of dementia and cognitive decline in older men. DESIGN: Community-based longitudinal cohort study. SETTING: The Honolulu-Asia Aging Study of dementia that is linked to the Honolulu Heart Program's fourth examination, conducted 1991-1993, and the 3-year follow-up fifth examination, conducted 1994-1996. PARTICIPANTS: Two thousand three hundred forty-six Japanese-American men age 71 to 93 years who screened negative for prevalent dementia at baseline and were screened again for dementia incidence in a 3-year follow-up examination. MEASUREMENTS: Baseline self-reports of trouble falling asleep or early morning awakening (insomnia) and being sleepy during the day (daytime sleepiness); Cognitive Abilities Screening Instrument (CASI) scores from baseline and followup; clinical diagnosis of incident dementia; and other baseline measures including age, years of education, body mass index, depressive symptoms, and history of hypertension, heart disease, diabetes mellitus, asthma, and use of benzodiazepines. RESULTS: After adjusting for age and other factors, persons reporting excessive daytime sleepiness at baseline (8%) were twice as likely to be diagnosed with incident dementia than were those not reporting daytime sleepiness (odds ratio (OR)=2.19, 95% confidence interval (CI)=1.37-3.50) and about 40% more likely to have >or=9 point drop in their CASI score between examinations (OR=1.44, 95% CI=1.01-2.08). In contrast, insomnia was not associated with cognitive decline or incidence of dementia. CONCLUSIONS: Daytime sleepiness in older adults may be an early indicator of decline in cognitive functioning and onset of dementia.

Aged↗
PubMed2001

Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli.

The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

Base Sequence↗
PubMed2001

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Base Sequence↗
PubMed2001

Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous Simian/human immunodeficiency virus infection in pigtailed macaques.

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV(DH12)) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV(DH12) strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV(DH12) challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

AIDS Vaccines↗
PubMed2001

Missing a therapeutic window of opportunity: an audit of patients attending a tertiary teaching hospital with potentially osteoporotic hip and wrist fractures.

OBJECTIVE: To establish the rate of and the predictors for performance of a bone mineral density (BMD) test and the treatment of osteoporosis in an at-risk cohort of patients attending a tertiary teaching hospital with fracture possibly related to osteoporosis. METHODS: A list of all patients between ages 40 and 85 who had been admitted to a tertiary teaching hospital in the last 18 mo with hip fracture or seen in the accident and emergency department with a wrist fracture over the last 30 mo was obtained from computer records; those patients were invited to participate in the audit. In a followup telephone questionnaire, they were queried about potential risk factors for osteoporosis and subsequent fracture, the performance of a BMD test, any information received on osteoporosis and the source of this information, and the prescription of any treatment for osteoporosis. RESULTS: In total, 218 patients were included in the audit from a potential 374 eligible patients. The majority were female (78%), with hip and wrist fractures in 42% and 58%, respectively; 32% subsequently had BMD measured and 39% were offered treatment for osteoporosis. Ninety-four percent of patients had heard of osteoporosis, with the major source of information being the media (83%) and friends (23%), with little information from the medical profession (34%). The major predictors for a patient to have a subsequent BMD test were female sex (OR 3.4, 95% CI 1.3-9.9), history of a previous fracture after the age of 50 (OR 2.3, 95% CI 1.0-5.6), family history of osteoporosis (OR 3.5, 95% CI 1.3-9.5), or the use of concurrent medications with a potential to cause osteoporosis (OR 2.5, 95% CI 1.1-5.8). The main predictors of treatment for osteoporosis being offered were age (risk increased by 1.04 for every year of life), abnormal result on the BMD test (OR 19, 95% CI 6-60), history of fracture after the age of 50 (OR 2.6, 95% CI 1.1-6.7), and a history of fracture with minimal trauma (OR 2.6, 95% CI 1.1-4.2). There was a range of treatments offered, with calcium supplementation alone accounting for 60% of treatments. CONCLUSION: Osteoporosis was overlooked by medical practitioners responsible for the care of this at-risk patient cohort, with little evidence of the medical profession offering information, further investigation, or treatment of patients who presented with a probable osteoporotic fracture of the hip or wrist. This suggests that greater education of the accident and emergency and orthopedic medical staff as well as the general public is required concerning this opportunity to investigate and treat symptomatic osteoporosis.

Adult↗
PubMed2000

UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition.

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the sigma subunit, but also between a DNA element upstream of the core promoter and the alpha subunit. DNA binding by alpha can increase transcription dramatically. Here we review the current state of our understanding of the alpha interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).

Bacteria↗
PubMed2000

More than skin deep.

Explore the source record for details and available documents.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters.

We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.

Carrier Proteins↗
PubMed1999

Mutatect: a mouse tumour model for detecting radiation-induced mutations in vivo.

A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.

Animals↗
PubMed1999

Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit.

We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.

Consensus Sequence↗
PubMed1999

Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys.

Virus-specific antibodies protect individuals against a wide variety of viral infections. To assess whether human immunodeficiency virus type 1 (HIV-1) envelope-specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV-1 isolates, and used this for passive immunization of pig-tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian-human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV-1DH12, a dual-tropic primary virus isolate. Here we show that anti-SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody-mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV-challenged macaques was in the range of 1:5-1:8. The HIV-1-specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non-neutralizing anti-HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Animals↗
PubMed1999

Characterization of binding of adenylate cyclase toxin to target cells by flow cytometry.

Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K(+) and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K(+) efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 micrograms/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37 degrees C; however, the total binding at 0 degrees C is less than that at 37 degrees C. In human erythrocytes, AC toxin does not cause an increase in K(+) efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K(+) efflux, and hemolysis produced by AC toxin.

Adenylate Cyclase Toxin↗
PubMed1999

Activation of Escherichia coli leuV transcription by FIS.

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.

Base Sequence↗
PubMed1998

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit.

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the alpha subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP alphaCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Base Sequence↗
PubMed1998

Identification of an UP element consensus sequence for bacterial promoters.

The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: -59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the alpha C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the alpha-DNA interaction. Based on the evolutionary conservation of the residues in alpha responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.

Base Sequence↗