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1.
J Clin Epidemiol ; 54 Suppl 1: S35-43, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11750208

RESUMO

Older individuals (>50 years of age) now comprise over 11% of patients with AIDS in the United States. This percentage is expected to continue to grow, due both to the improved longevity of patients prescribed highly active antiretroviral therapy (HAART) and to new infections among older individuals. This review focuses on the neuropsychiatric and neurological conditions that are most likely to be affected by advancing age-HIV-1-associated cognitive-motor disorder, peripheral neuropathy, progressive multifocal leukoencephalopathy, primary CNS lymphoma, and risk for cerebrovascular accident. Age associations with incidence of these disorders and with treatment foci are specified. Implications for future changes in management are discussed.


Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Doenças do Sistema Nervoso Central/epidemiologia , Transtornos Cognitivos/epidemiologia , HIV-1 , Doenças Neuromusculares/epidemiologia , Doenças do Sistema Nervoso Periférico/epidemiologia , Síndrome da Imunodeficiência Adquirida/epidemiologia , Fatores Etários , Doenças do Sistema Nervoso Central/etiologia , Neoplasias do Sistema Nervoso Central/epidemiologia , Neoplasias do Sistema Nervoso Central/etiologia , Transtornos Cognitivos/etiologia , Progressão da Doença , Humanos , Incidência , Leucoencefalopatia Multifocal Progressiva/epidemiologia , Leucoencefalopatia Multifocal Progressiva/etiologia , Linfoma Relacionado a AIDS/epidemiologia , Linfoma Relacionado a AIDS/etiologia , Linfoma não Hodgkin/epidemiologia , Linfoma não Hodgkin/etiologia , Pessoa de Meia-Idade , Doenças Neuromusculares/etiologia , Doenças do Sistema Nervoso Periférico/etiologia , Fatores de Risco , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologia , Estados Unidos/epidemiologia
2.
Drug Alcohol Depend ; 58(1-2): 153-7, 2000 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-10669066

RESUMO

We quantified HIV-1 RNA load in rinses from needles/syringes (N/S) obtained at shooting galleries in Miami and also analyzed the rinses for antibodies for viral proteins. In rinses from 36 N/S that contained visible blood, 14 (39%) had detectable amounts of HIV-1 RNA. Numbers of copies of HIV-1 RNA ranged from the detection limit (400 copies/ml) to 268,000 copies/ml. We also detected antibodies to HIV-1 polypeptides in 34/36 (94%) of rinses from visibly contaminated N/S using Western blots specific for the HIV-1 proteins. No antibodies were detected in laboratory rinses from six visibly clean needles. The presence of HIV-1 RNA in N/S is an important indication of the risk created by N/S sharing as well as by shared paraphernalia and wash waters by injecting drug users.


Assuntos
HIV-1/isolamento & purificação , Uso Comum de Agulhas e Seringas , RNA Viral/sangue , Abuso de Substâncias por Via Intravenosa , Western Blotting , Florida/epidemiologia , Genes gag , Anticorpos Anti-HIV/sangue , HIV-1/genética , Humanos , Agulhas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Seringas , Carga Viral
3.
CNS Spectr ; 5(4): 31-42, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18277336

RESUMO

Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.

4.
AIDS Res Hum Retroviruses ; 15(9): 811-20, 1999 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-10381169

RESUMO

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.


Assuntos
Encéfalo/virologia , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Fragmentos de Peptídeos/genética , Adulto , Encéfalo/patologia , Evolução Molecular , Feminino , Genes Virais , Infecções por HIV/patologia , HIV-1/classificação , Humanos , Masculino , Filogenia , Análise de Sequência de DNA
7.
J Neuroimmunol ; 83(1-2): 88-101, 1998 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9610677

RESUMO

The epidemiology of cocaine abuse and potential relationships of cocaine withdrawal to human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are discussed. Neuroendocrinological changes in HIV-1 infection of the central nervous system (CNS) are discussed with the relevant impact of cocaine abuse. HIV-1 load in the brain tissue of infected substance users is described along with possible associations with neuropathology and HAD. Finally, the molecular epidemiology and sequence heterogeneity of HIV-1 and their implications for neuropathogenesis are summarized. The complex context of addressing cocaine abuse in the setting of HIV-1 infection appears more tractable when decomposed into its components.


Assuntos
Complexo AIDS Demência/epidemiologia , Cocaína/efeitos adversos , HIV-1 , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Vasoconstritores/efeitos adversos , Complexo AIDS Demência/etiologia , Complexo AIDS Demência/fisiopatologia , Humanos , Neuroimunomodulação/efeitos dos fármacos , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Transtornos Relacionados ao Uso de Opioides/virologia
8.
J Acquir Immune Defic Syndr Hum Retrovirol ; 16(3): 146-52, 1997 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9390565

RESUMO

A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia (HAD) has not yet been established. Knowledge of the neuroanatomic distribution of HIV-1 load in the brain of individuals with HAD and HIV-1 encephalitis may facilitate elucidation of this relation. Nine individuals with AIDS were analyzed postmortem by three independent methods with each assessment performed blinded to the others: 1) a neuropsychiatric review of clinical records for evidence of possible HAD, 2) HIV-1 DNA load determination by quantitative polymerase chain reaction (PCR) across several neuroanatomic regions, and 3) a pathologic examination for diagnosis of HIV-1 encephalitis by immunohistochemical techniques. Of eight AIDS cases with clinical records sufficient for neuropsychiatric review, seven were shown to have evidence for HAD. HIV-1 DNA was detected and quantified in specimens from all of the medial temporal lobe regions analyzed but was not detectable in the frontal lobe at the same level of sensitivity in two of these cases (<1 per 1000 cellular genomes). HIV-1 DNA load in the medial temporal lobe region was significantly larger than that in the frontal lobe. Only four of seven cases with evidence for HAD were also diagnosed with HIV-1 encephalitis.


Assuntos
Complexo AIDS Demência/virologia , Encéfalo/virologia , DNA Viral/análise , HIV-1/genética , Provírus/genética , Carga Viral , Complexo AIDS Demência/patologia , Encéfalo/patologia , Encefalite Viral/virologia , Proteína gp41 do Envelope de HIV/análise , Infecções por HIV/virologia , Humanos , Testes Neuropsicológicos , Reação em Cadeia da Polimerase
9.
J Virol Methods ; 67(2): 177-87, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9300383

RESUMO

The quantitative polymerase chain reaction (PCR) method devised by Fujimura and Bockstahler (1995) was modified for rapid determination of distribution of HIV-1 proviral DNA load in AIDS brains. It was used for analysis of an association with HIV-1 associated dementia and HIV-1 encephalitis (Fujimura et al., 1997). The method has wider applicability for comparative studies of viral DNA load based on PCR amplification. The method is applicable under conditions where target DNA and its PCR-amplified product increase proportionally. An equation was derived to obtain the number of copies of HIV-1 DNA per cellular genome from the amount of PCR amplified product of a tissue specimen DNA. The equalizing constant is the reciprocal of the slope of the amplification of the HIV-1 proviral DNA sequence of the standard cellular DNA included in each experiment. The intercept of the equation is zero.


Assuntos
Encéfalo/virologia , DNA Viral/isolamento & purificação , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Encéfalo/patologia , Criopreservação , Dosagem de Genes , Genoma Viral , Infecções por HIV/virologia , HIV-1/química , HIV-1/isolamento & purificação , Humanos , Provírus/química , Provírus/isolamento & purificação , Reprodutibilidade dos Testes , Carga Viral
10.
Neuroimaging Clin N Am ; 7(3): 561-79, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9376968

RESUMO

HIV-1 infection of brain may be associated with multiple treatment targets, only the most severe of which is represented by HAD. Focusing on earlier treatment targets such as MCMD and cognitive-motor impairment in the absence of any clinical disorder (as well as neuroprotection) may prove to be of greater clinical utility in the treatment and prevention of such impairment than a focus on later-stage cognitive-motor disease, when neuronal cell death is already extensive. This may be especially important now that improvements using the protease inhibitors in triple-drug combination regimens have reduced plasma viral load to unmeasurable levels, while these drugs do not penetrate the CSF well. Currently, peripheral blood markers do not appear to be highly sensitive for central nervous system impairment, and specific CSF laboratory markers have some limited value at present-while requiring a lumbar puncture to obtain. Hence, a role for noninvasive techniques using neuroimaging exists in the clinical management of HIV-1-infected patients. To date, structural imaging techniques have proven limited in value for HIV-1-specific impairment. Several functional techniques (PET, SPECT, and MR spectroscopy) have now provided promising results for the purposes of identifying clinically significant dysfunction, relating such dysfunction to clinical neuropsychiatric symptom status, and for treatment response monitoring. Further studies are needed to examine the extent to which such imaging modalities not only parallel clinically relevant aspects of HIV-1 disease progression, but also match specific types of neuropsychologic performance deficits with potential significance for neuroanatomical localization. It is particularly important to include neurophysiological, neuroimmunological, and virological measures in studies that examine clinical neuropsychiatric status with neuroimaging techniques. In addition, the inclusion of neuropathology data, where possible, is important because demonstration of HIV-1 encephalitis cannot be equated with clinical disorder and because specific HIV-1-associated pathological changes have not yet been proven to be assessed well with neuroimaging techniques (e.g., the extent of microglial cell and macrophage activation). Also, treatment response studies are needed in conjunction with primary antiretroviral therapy regimens specifically aimed at central nervous system penetration (e.g., GW1592, GW141, and nevirapine). The results of such work will provide the data required to determine whether these promising functional neuroimaging techniques will aid in meeting the expected, imminent increase in clinical burden of this frequent complication of HIV-1 infection.


Assuntos
Complexo AIDS Demência/psicologia , Complexo AIDS Demência/diagnóstico , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/fisiopatologia , Complexo AIDS Demência/virologia , Fármacos Anti-HIV/uso terapêutico , Antivirais/uso terapêutico , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Morte Celular , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/imunologia , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Transtornos Cognitivos/virologia , Progressão da Doença , Combinação de Medicamentos , Encefalite Viral/diagnóstico , Encefalite Viral/imunologia , Encefalite Viral/fisiopatologia , Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/líquido cefalorraquidiano , Inibidores da Protease de HIV/uso terapêutico , HIV-1 , Humanos , Ativação de Macrófagos , Espectroscopia de Ressonância Magnética , Microglia/virologia , Transtornos dos Movimentos/diagnóstico , Transtornos dos Movimentos/imunologia , Transtornos dos Movimentos/fisiopatologia , Transtornos dos Movimentos/psicologia , Transtornos dos Movimentos/virologia , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Neuropsicologia , Punção Espinal , Tomografia Computadorizada de Emissão , Tomografia Computadorizada de Emissão de Fóton Único , Viremia/virologia
12.
J Virol Methods ; 55(3): 309-25, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8609197

RESUMO

A PCR method was developed to compare HIV-1 DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-1 provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (10-250 per 10(3) cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.


Assuntos
Encéfalo/virologia , DNA Viral/isolamento & purificação , Infecções por HIV/virologia , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , Síndrome da Imunodeficiência Adquirida/virologia , Actinas/genética , Sequência de Bases , Encéfalo/patologia , Encefalopatias/virologia , Primers do DNA , Estudos de Avaliação como Assunto , Produtos do Gene gag/genética , Humanos , Dados de Sequência Molecular , Provírus/genética
13.
Photochem Photobiol ; 56(1): 35-42, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1387234

RESUMO

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.


Assuntos
DNA/efeitos da radiação , Luz Solar/efeitos adversos , Bacteriófago phi X 174/efeitos da radiação , Dano ao DNA , Humanos , Técnicas In Vitro , Fotoquímica , Doses de Radiação , Água do Mar
14.
Gene ; 97(1): 13-9, 1991 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-1995424

RESUMO

T5 DNA polymerase (T5Pol), an essential enzyme for bacteriophage T5 DNA replication, is unusual because of its high processivity and strand-displacing ability. These two properties in a single polypeptide make T5Pol an ideal candidate for structural and functional analysis. Therefore, the structural gene encoding the DNA polymerase of bacteriophage T5 (T5pol) has been cloned and overexpressed in Escherichia coli. Elimination of sequences upstream from the 5' end of the T5pol by exonuclease III digestion was necessary to obtain stable clones containing a full-length structural gene. Determination of the nucleotide (nt) sequence of the region deleted during clone construction revealed the presence of a promoter sequence having extensive homology with known T5 phage 'early' promoters. By primer extension of mRNA isolated from T5 phage-infected cells, two successive G residues located 6 and 7 nt downstream from the -10 region of this promoter were identified as the initiating nt at the 5' end of T5pol mRNA. T5Pol produced in E. coli from the cloned gene under control of a tac or phage lambda pL promoter represented as much as 40% of total cell protein. The majority of the T5Pol present in extracts of E. coli was insoluble. The amount of active enzyme present was estimated to be a maximum of tenfold higher than that found in extracts of T5 phage-infected cells.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Expressão Gênica , Genes Virais , Fagos T/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Vetores Genéticos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Fagos T/enzimologia , Tiorredoxinas/farmacologia , Transcrição Gênica
15.
Anal Biochem ; 153(2): 299-304, 1986 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3010761

RESUMO

Chromatography of tRNA and DNA fragments on columns of reverse-phase 5 (RPC-5) exchange material has been widely employed for analytical and preparative studies. The Plaskon bead that formed the solid support on which a quaternary amine was absorbed is no longer commercially available. A Voltalef bead is available and provides similar, though not identical, chromatograms for Asp-tRNA, Ser-tRNA, and certain DNA fragments. Procedures are described for preparation of the column packing and for long-term operation of the column.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , DNA/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II , RNA de Transferência/isolamento & purificação , Enzimas de Restrição do DNA , DNA Viral/isolamento & purificação , Aminoacil-RNA de Transferência/isolamento & purificação
16.
J Virol ; 56(1): 245-9, 1985 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3897573

RESUMO

We identified by immunobinding assay the polypeptides synthesized as the result of amber mutations in the DNA polymerase gene of bacteriophage T5. Comparison of the size of such polypeptides revealed the order of mutagenic loci of these mutations and the direction of transcription of the gene. Extracts of cells infected with wild-type T5 and with five amber mutants of the polymerase gene (D7, D8, D9, am1, and am6) were prepared, and the proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After transfer of the proteins to a nitrocellulose sheet, a radioimmunolabeling technique was used to identify the T5 DNA polymerase and its amber mutant polypeptides. Based on the relative sizes of the polypeptides, the transcription of the T5 DNA polymerase gene was determined to proceed in the order D7, D8, am1, D9, and am6. The molecular weights of the DNA polymerase polypeptides coded by D8, am1, D9, am6, and T5+ were 23,000, 45,000, 75,000, 83,000, and 96,000, respectively. The D7-coded polypeptide was not detectable. These data suggest that the carboxyl-terminal region of the enzyme is essential for the polymerase function.


Assuntos
Bacteriófagos/enzimologia , DNA Polimerase Dirigida por DNA/genética , Especificidade de Anticorpos , Bacteriófagos/genética , Mapeamento Cromossômico , DNA Viral/genética , DNA Polimerase Dirigida por DNA/imunologia , Técnicas de Imunoadsorção , Peso Molecular , Mutação , Transcrição Gênica
17.
J Virol ; 53(2): 495-500, 1985 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2982033

RESUMO

Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Genes Virais , Genes , Fagos T/genética , Enzimas de Restrição do DNA , Técnicas Genéticas , Mutação , Fagos T/enzimologia
18.
J Virol ; 46(3): 778-87, 1983 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6304341

RESUMO

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.


Assuntos
DNA Helicases/metabolismo , DNA Viral/metabolismo , Inibidores da Síntese de Ácido Nucleico , Fagos T/enzimologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Hidrólise , Fagos T/genética , Moldes Genéticos
19.
J Biol Chem ; 257(24): 14811-6, 1982 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-6294081

RESUMO

Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.


Assuntos
DNA Helicases/genética , DNA Viral/genética , Genes Virais , Genes , Fagos T/genética , Centrifugação com Gradiente de Concentração , DNA Helicases/isolamento & purificação , DNA Viral/isolamento & purificação , Proteínas de Ligação a DNA , Escherichia coli/genética , Metrizamida
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