RESUMO
BACKGROUND: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders in need of innovative 'real-world' outcome measures to evaluate treatment effects. Instrumented gait analysis (IGA) using wearable technology offers a potentially feasible solution to measure "real-world' neurological and motor dysfunction in these groups. METHODS: Children (50% female; 6-16 years) diagnosed with PWS (n = 9) and AS (n = 5) completed 'real-world' IGA assessments using the Physilog®5 wearable. PWS participants completed a laboratory assessment and a 'real-world' long walk. The AS group completed 'real-world' caregiver-assisted assessments. Mean and variability results for stride time, cadence, stance percentage (%) and stride length were extracted and compared across three different data reduction protocols. RESULTS: The wearables approach was found to be feasible, with all participants able to complete at least one assessment. This study also demonstrated significant agreement, using Lin's concordance correlation coefficient (CCC), between laboratory and 'real-world' assessments in the PWS group for mean stride length, mean stance % and stance % CV (n = 7, CCC: 0.782-0.847, P = 0.011-0.009). CONCLUSION: 'Real-world' gait analysis using the Physilog®5 wearable was feasible to efficiently assess neurological and motor dysfunction in children affected with PWS and AS.
Assuntos
Síndrome de Angelman , Síndrome de Prader-Willi , Dispositivos Eletrônicos Vestíveis , Síndrome de Angelman/complicações , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/terapia , Criança , Estudos de Viabilidade , Feminino , Análise da Marcha , Humanos , Imunoglobulina A , MasculinoRESUMO
The biology of three landlocked and a riverine population of Galaxias maculatus were examined in western Victoria, Australia. All systems supported reproducing populations of these fish, including Lake Corangamite which had salinities that on occasion reached 82. Spawning sites in Lake Corangamite were located in adjacent tributaries and not in the main lake as was the case for other populations. The smallest fish were found in the fresh water Lake Purrumbete and the largest in the hypersaline Lake Corangamite. The size at which 50% of the population attained sexual maturity varied across sites, with fish maturing at a smaller size in Lake Purrumbete, followed by the Merri River, Lake Bullen Merri and Lake Corangamite. Condition was higher in the freshwater Lake Purrumbete and there was no relationship between condition and temperature, conductivity, turbidity and pH; but there was a positive relationship between condition and dissolved oxygen. Length frequency analysis suggested that the majority of fishes live for a year.
Assuntos
Osmeriformes/fisiologia , Maturidade Sexual/fisiologia , Análise de Variância , Animais , Feminino , Lagos/química , Masculino , Osmeriformes/anatomia & histologia , Osmeriformes/crescimento & desenvolvimento , Oxigênio/química , Rios/química , Temperatura , VitóriaRESUMO
BACKGROUND: The purpose of this study was to assess the effect of alternative ways of providing information about the risks of twins on couples' perceptions about elective single embryo transfer (eSET). METHODS: Couples undergoing IVF were randomized into three groups. Group 1 received a standard information pack, group 2 an extra information leaflet about twin pregnancy, and group 3 an additional discussion session. The primary outcome measure was acceptability of a hypothetical policy of eSET. Data were collected by means of a questionnaire. RESULTS: eSET was acceptable to 17 (27%), 20 (30%) and 24 (32%) couples in groups 1, 2 and 3, if it meant a slight reduction in pregnancy rates, and to 51 (82%), 55 (83%) and 53 (87%) couples, respectively, if pregnancy rates were unchanged. A fixed charge for all fresh and frozen embryo transfers following a single oocyte retrieval led to acceptability rates of 35 (57%), 36 (55%) and 38 (65%). CONCLUSIONS: Additional information, involving an extra information leaflet and face to face discussion, did not changes couples' attitudes towards eSET. Maintaining existing rates of pregnancy and offering a fixed charge for all embryo transfers resulting from an oocyte recovery may encourage more couples to consider eSET.
Assuntos
Transferência Embrionária , Aceitação pelo Paciente de Cuidados de Saúde , Educação de Pacientes como Assunto/métodos , Gravidez Múltipla , Gêmeos , Transferência Embrionária/economia , Honorários e Preços , Feminino , Humanos , Gravidez , Taxa de Gravidez , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
All cases S16 years of age with a histological diagnosis of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) presented in Scotland between 1 January 1994 and 31 December 1996 were registered prospectively in the Scotland and Newcastle Lymphoma Group database by a process of total registration. The census population of Scotland in 1996-1997 was 5.1 million. One thousand seven hundred and sixty three patients were registered with NHL and 350 patients with HD. These patients have been followed up for a median of 47 months in the case of NHL and 51 months for HD cases. Actuarial 5-year survival for adult NHL was 35% and for HD, 75%. Outcome for both NHL and HD was particularly poor in the population over 60 years with median survival of 18 months for NHL and 27 months for HD. When analysis of survival was related to degree of material deprivation using the Carstairs score a significantly poorer survival was seen for NHL with increasing deprivation that could not be explained by a different pattern of age or stage at presentation. Deprivation had no impact on incidence or survival in HD. Analysis of impact of caseload of the physician initiating therapy showed no significant difference in 5-year survival.
Assuntos
Doença de Hodgkin/epidemiologia , Linfoma não Hodgkin/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Feminino , Seguimentos , Doença de Hodgkin/mortalidade , Humanos , Incidência , Linfoma não Hodgkin/mortalidade , Masculino , Pessoa de Meia-Idade , Pobreza , Estudos Prospectivos , Sistema de Registros , Escócia/epidemiologia , Fatores Sexuais , Análise de Sobrevida , Resultado do Tratamento , Carga de TrabalhoRESUMO
OBJECTIVES: The aims of this study were to examine emotional distress and infertility-related concerns in male and female members of couples referred to a specialist infertility clinic and to determine changes in these over time. METHODS: A prospective cohort study with a 6-month follow-up. Emotional distress was measured using the Hospital Anxiety and Depression Scale, and concerns by a specially designed questionnaire. RESULTS: The response rate achieved was 38%. At baseline, 25.7% of women and 8.9% of men had scores of greater than 10 on the Hospital Anxiety and Depression Scale (HADS) Anxiety subscale, and 2.7% of women and 1.8% of men had scores of greater than 10 on the HADS Depression subscale. At 6-month follow-up the HADS scores were substantially unchanged. Females reported a significantly greater infertility-related concerns regarding life satisfaction, sexuality, self-blame, self-esteem and avoidance of friends compared with males. CONCLUSIONS: The prevalence of emotional disorder identified was low. There were gender differences in the nature of the specific concerns reported. The degree of distress and concerns did not change significantly over time. There are a minority of patients, mainly females, with clinically significant distress and infertility-related concerns amongst patients attending infertility clinics who deserve psychological attention.
Assuntos
Instituições de Assistência Ambulatorial , Ansiedade/psicologia , Depressão/psicologia , Características da Família , Infertilidade/terapia , Medicina , Encaminhamento e Consulta , Especialização , Adulto , Ansiedade/epidemiologia , Estudos de Coortes , Depressão/epidemiologia , Feminino , Seguimentos , Humanos , Masculino , Prevalência , Estudos Prospectivos , Fatores Sexuais , Inquéritos e QuestionáriosRESUMO
Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Proteínas Fúngicas/genética , Mitose/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endonucleases , Haploidia , Mutação , FenótipoRESUMO
RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.
Assuntos
Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Recombinação Genética , Retroelementos , Saccharomyces cerevisiae/genética , DNA Ligases/metabolismo , DNA Complementar , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , RNA Mensageiro/genética , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiaeRESUMO
An integrated computer aided design/computer aided manufacture system has been used to model the complex geometry of blood vessel anastomoses. Computer models are first constructed with key dimensions derived from radiological images of bypass grafts, and from casts of actual blood vessel anastomoses. Physical models are then fabricated in one of two ways: the surface geometry data can be used to control the movement of a three-axis milling machine; alternatively, the same data can be exported in a form that can be interpreted by a stereolithography apparatus. Both methods produce geometrically defined solid investments that can be used in a multistep casting process that yields high-quality physical models for vascular fluid dynamic studies. This technique is useful for parametric studies.
Assuntos
Artérias/anatomia & histologia , Desenho Assistido por Computador , Modelos Cardiovasculares , Anastomose Cirúrgica , Prótese Vascular , Hemodinâmica , Imageamento por Ressonância Magnética , Desenho de Prótese , Reprodutibilidade dos Testes , SoftwareRESUMO
The influence of surfaces on the reactions of platelets in whole blood under laminar flow was investigated in a cone and plate viscometer. Citrated whole blood was exposed to steel, PMMA and PMMA modified with PEO at low (500 s(-1)) and high (4000 s(-1)) wall shear rates at room temperature for a period of 100 s. Treated blood samples were fixed with paraformaldehyde, stained with a monoclonal antibody for CD41 (platelet GPIIb/IIIa) conjugated with phycoerythrin and analyzed by flow cytometry. The reactions of platelets (microparticle generation and formation of platelet-platelet, platelet-red blood cell and red blood cell-microparticle aggregates) to these environments were quantified. Additionally, the size of platelet-platelet aggregates was assessed. The percentage platelet aggregation and numbers of microparticles generated were independent of surface type at any shear rate. The composition of the aggregates formed was influenced by the surface: at low and high shear rates PMMA caused the generation of platelet-platelet aggregates of the greatest size. The numbers of red blood cell-platelet and red blood cell-microparticle aggregates also varied depending on the surface. Fewer red blood cell-platelet aggregates were formed at higher shear rates, whereas the reverse was true for red blood cell-microparticle aggregates. It is concluded that these variations may help to explain the differential effects of surfaces to the induction of distant thrombotic events: microparticles may be protected from loss from the blood stream by their association with red blood cells at high shear rates.
RESUMO
The role of temperature and shear rate in the activation status of aggregating platelets and platelet microparticles (MPs) was investigated in a modified concentric-cylinder rotational viscometer. Whole blood anticoagulated with citrate was exposed to a range of shear rates typical of cardiopulmonary bypass circuits (0, 1000, 2000 and 4000 s(-1)) over four temperatures spanning hypothermic to mildly hyperthermic conditions (24, 30, 37 and 42 degrees C) for short durations (100 s). Aliquots of blood were double-stained for CD41 (platelet GPIIb/IIIa) and CD62 (P-selectin). Platelets, platelet aggregates, MPs and red blood cell-platelet and -MP aggregates were identified by flow cytometry by acquiring only CD41-positive particles and differentiating on a plot of CD41 versus forward light scatter. The activation status of each particle was quantified by measuring CD62 expression (alpha-granule release). A degree of correlation between the shedding of MPs and the formation of platelet-platelet aggregates was observed for the data as a whole (r=0.85 for p<0.01), although this trend was not observed for a shear rate of 4000 s(-1). The mean expression of CD62 on both platelets and MPs was maintained at a very low level for all temperature and shear rate combinations. There was, however, a number of very highly activated MPs associated with red blood cells at high shear rates.
RESUMO
Samples of whole blood were obtained from male volunteers and exposed to combinations of shear rates and temperatures representative of cardiopulmonary bypass (CPB) in a modified computer-controlled concentric cylinder rotational viscometer for a period of 100 s. Blood sampled from the chamber was fixed in paraformaldehyde, stained with CD41 and analysed by flow cytometry. Only platelet-positive particles were acquired, each individual cell, or aggregate of cells, identified by analysis of its fluorescence and forward light scatter characteristics. Little platelet aggregation was observed at shear rates of less than 4000 s(-1) for temperatures of greater than 24 degrees C, but large numbers of aggregates were formed at all temperatures at 4000 s(-1) (p<0.05), with more aggregates forming at 24 and 30 degrees C than at 37 and 42 degrees C (p<0.05). We conclude that the process of aggregation is dependent on both temperature and shear rate. We note that a large number of platelets become involved in aggregates under conditions of temperature and shear-rate typical of CPB.
RESUMO
Patients with intermittent claudication have been reported to have disturbances in blood rheology and haemostasis. Whether these disturbances are a result of, or largely independent of, smoking history and arterial narrowing has not yet been established. The levels of whole blood and plasma viscosity, haematocrit, von Willebrand factor antigen, fibrin D-dimer antigen and urinary fibrinopeptide A antigen were compared in 617 claudicants and 722 controls from two epidemiological studies in Edinburgh. After adjustment for age and sex, all factors, except whole blood viscosity and haematocrit, were significantly higher in the claudicants compared to controls (P < or = 0.001). The risk of intermittent claudication was significantly raised for unit change in each factor, except for whole blood viscosity and haematocrit. Adjustment for lifetime smoking had little effect on the odds ratios. After further adjustment for the ankle brachial pressure index (as a measure of the extent of peripheral arterial disease), haematocrit, von Willebrand factor and urinary fibrinopeptide A showed a significant independent relationship with the risk of intermittent claudication. We conclude that the association between selected rheological and haemostatic factors and leg ischaemia is largely independent of both smoking history and the extent of arterial narrowing, and may be directly related to microvascular ischaemia.
Assuntos
Fatores de Coagulação Sanguínea/análise , Hemorreologia , Hemostasia/fisiologia , Claudicação Intermitente/sangue , Doenças Vasculares Periféricas/complicações , Fumar/sangue , Idoso , Viscosidade Sanguínea , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinopeptídeo A/urina , Hematócrito , Humanos , Claudicação Intermitente/complicações , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fator de von Willebrand/análiseRESUMO
The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.
Assuntos
Genes Fúngicos/genética , Mitose/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Inversão Cromossômica , Troca Genética , Dano ao DNA , Epistasia Genética , Raios gama/efeitos adversos , Conversão Gênica , Modelos Genéticos , Tolerância a Radiação/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 x 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 has functions in addition to those of the Rad51/Rad52 protein complex.
Assuntos
Cromossomos Fúngicos , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Mitose/genética , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genética , Troca Genética , DNA Fúngico , Conversão Gênica , Rad51 Recombinase , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiaeRESUMO
Haemostatic and rheological factors may predict cardiovascular disease. We studied patients with intermittent claudication to see if the progression of peripheral arterial disease and the risks of coronary events could be predicted by baseline packed cell volume, plasma fibrinogen, blood and plasma viscosites, von Willebrand factor antigen, cross-linked fibrin degradation products (XLFDP), urinary fibrinopeptide A, and plasma leucocyte elastase. In 617 patients with claudication followed up for one year, baseline XLFDP was related most strongly to coronary events, relative risk 4.4 (95% CI 1.3-19.0) between top and bottom quintiles. Plasma fibrinogen was the strongest independent predictor of death from coronary disease. XLFDP was the only factor, in addition to age and cigarette smoking, that was independently associated (p = 0.008) with deterioration in peripheral arterial disease. We conclude that, in patients with peripheral arterial disease, plasma concentration of XLFDP, a measure of ongoing fibrin formation and degradation, is a strong predictor of both disease progression and future coronary risk. These results accord with the hypothesis that fibrin formation contributes to progression of coronary and peripheral atherosclerosis.
Assuntos
Doença das Coronárias/etiologia , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Claudicação Intermitente/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Pressão Sanguínea , Viscosidade Sanguínea , Feminino , Fibrinogênio/análise , Fibrinopeptídeo A/urina , Hematócrito , Humanos , Claudicação Intermitente/fisiopatologia , Leucócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/sangue , Fatores de Risco , Fator de von Willebrand/análiseRESUMO
In a previous study, meiotic recombination events were monitored in the 22-kb LEU2 to CEN3 region of chromosome III of Saccharomyces cerevisiae. One region (the hotspot) was shown to have an enhanced level of both gene conversion events and reciprocal crossovers, whereas a second region (the coldspot) was shown to have a depressed level of both types of recombination events. In this study we have analyzed the effects of a replication origin, ARS307, located about 2 kb centromere proximal to the hotspot region, on the distribution of meiotic recombination events. We find that a deletion of this origin results in a reduction of both gene conversions and reciprocal crossovers in the hotspot region, and that a 200-bp fragment of this ARS element can stimulate both types of recombination events when relocated to the coldspot region. Although the magnitude of stimulation of these events is similar in both orientations, whether the ARS is functional or not, the distribution of events is dependent upon the orientation of the element.
Assuntos
Meiose/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Troca Genética , Replicação do DNA/genética , Conversão Gênica , Genes Fúngicos , Mutação , Saccharomyces cerevisiae/citologia , Deleção de SequênciaRESUMO
A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.
Assuntos
HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Bases , DNA Viral/biossíntese , Transcriptase Reversa do HIV , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , Mutação , Purinas/metabolismo , Especificidade por SubstratoRESUMO
At least one of the procyclic acidic repetitive protein (PARP or procyclin) loci of Trypanosoma brucei is a small (5- to 6-kilobase) polycistronic transcription unit which is transcribed in an alpha-amanitin-resistant manner. Its single promoter, as mapped by run-on transcription analysis and UV inactivation of transcription, is located immediately upstream of the first alpha-PARP gene. Transcription termination occurs in a region approximately 3 kilobases downstream of the beta-PARP gene. The location of the promoter was confirmed by its ability to direct transcription of the bacterial chloramphenicol acetyltransferase gene in insect-form (procyclic) T. brucei. The putative PARP promoter is located in the region between the 3' splice acceptor site (nucleotide position 0) and nucleotide position -196 upstream of the alpha-PARP genes. Regulatory regions influencing the levels of PARP expression may be located further upstream. We conclude that a single promoter, which is located very close to the 3' splice acceptor site of the alpha-PARP genes, directs the transcription of a small, polycistronic, and alpha-amanitin-resistant transcription unit.
Assuntos
Amanitinas/farmacologia , Genes , Glicoproteínas de Membrana , Regiões Promotoras Genéticas , Proteínas de Protozoários , Transcrição Gênica , Transfecção , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Antígenos de Protozoários/genética , Sequência de Bases , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , DNA/genética , Relação Dose-Resposta à Radiação , Resistência a Medicamentos/genética , Dados de Sequência Molecular , RNA/genética , RNA/isolamento & purificação , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
The reverse transcriptase-associated RNase H activity is responsible for producing the plus-strand RNA primer during reverse transcription. The major plus-strand initiation site is located within a highly conserved polypurine tract (PPT), and initiation of DNA replication at this site is necessary for proper formation of the viral long terminal repeats (LTRs). We present here a compilation of PPT sequences from an evolutionarily diverse group of retroviruses and retrotransposons, which reveals that there is a high degree of sequence conservation at this site. Furthermore, we found previously that secondary plus-strand origins, identified in vitro, also show strong similarity to the PPT. Taken together, these data suggest that RNase H recognizes a specific sequence at the PPT as a signal to cleave the RNA at a precise location, producing a primer for the initiation of plus-DNA strands. We have analyzed the RNase H recognition sequence by producing a large number of single and double mutations within the PPT. Our findings suggest that no single residue in the +5 to -6 region (where the cleavage occurs between -1 and +1) is essential; mutations at these positions introduced heterogeneity at the cleavage site, but cleavage is still predominantly at the correct location. Furthermore, base-pairing is not required at the +1 position of the RNase H cleavage site, but a mismatched base-pair at the -1 position causes imprecision in the cleavage reaction. Interestingly, the A residue at position -7 seems to be critical in positioning the RNase H enzyme for correct cleavage. The preference of the enzyme for cleaving between G and A residues may play a minor role in determining the specificity.