Complete genomic sequence of the Australian south-west genotype of Sindbis virus: Comparisons with other Sindbis strains and identification of a unique deletion in the 3 '-untranslated region

Our previous studies have shown that two distinct genotypes of Sindbis (SIN) virus occur in Australia. One of these, the Oriental/Australian type, circulates throughout most of the Australian continent, whereas the recently identified south-west (SW) genetic type appears to be restricted to a distinct geographic region located in the temperate south-west of Australia. We have now determined the complete nucleotide and translated amino acid sequences of a SW isolate of SIN virus (SW6562) and performed comparative analyses with other SIN viruses at the genomic level. The genome of SW6562 is 11,569 nucleotides in length, excluding the cap nucleotide and poly (A) tail. Overall this virus differs from the prototype SIN virus (strain AR339) by 23% in nucleotide sequence and 12.5% in amino acid sequence. Partial sequences of four regions of the genome of four SW isolates were determined and compared with the corresponding sequences from a number of SIN isolates from different regions of the World. These regions are the non-structural protein (nsP3), the E2 gene, the capsid gene, and the repeated sequence elements (RSE) of the 3'UTR. These comparisons revealed that the SW SIN viruses were more closely related to South African and European strains than to other Australian isolates of SIN virus. Thus the SW genotype of SIN virus may have been introduced into this region of Australia by viremic humans or migratory birds and subsequently evolved independently in the region. The sequence data also revealed that the SW genotype contains a unique deletion in the RSE of the 3'UTR region of the genome. Previous studies have shown that deletions in this region of the SIN genome can have significant effects on virus replication in mosquito and avian cells, which may explain the restricted distribution of this genotype of SIN virus.

Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Identification of a 0.4 Kb deletion region in 10q26 associated with endometrial carcinoma

We have identified an allelic deletion common region in the q26 region of chromosome 10 in endometrial carcinomas, which has been reported previously as a potential target of genetic alterations related to this neoplasia. An allelotyping analysis of 19 pairs of tumoral and non-tumoral samples was accomplished using seven microsatellite polymorphic markers mapping in the 10q26 chromosomal region. Loss of heterozygosity for one or more loci was detected in 29% of the endometrial carcinoma samples. The observed pattern of loss enabled the identification of a 3.5 Mb common deleted region located between the D10S587 and D10S186 markers. An additional result from an endometrial sample with evidence of a RER phenotype may suggest a more centromeric region of loss within the above-mentioned interval. This 401.84 Kb interval flanked by the D10S587 and D10S216 markers may be a plausible location for a putative suppressor gene involved in early stage endometrial carcinogenesis.

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16D), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16D mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H?-ATPase (VATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1D or vma2D) or membrane domain (vph1D or vma3D) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.

Juvenile Parkinson disease caused by parkin mutations: large deletions and pathogenic mechanisms

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Study of antiretroviral mutants in HIV patients with treatment failures and the effect of risk factors in the virological failures

INTRODUCTION: Information about HIV phenotypes of resistant to available ART and the influence of different risk factors on virological failures (VF) in Costa Rican HIV positive patients prior or during HAART is unknown. MATERIALS AND METHODS: Eighty nine samples, 72 VF and 17 basal (before treatment) were analyzed by examining resistant mutants in reverse transcriptase (RT) and protease (PT) regions using Trugene or LIPA genotyping kits. Sixty eight control patients were selected and relevant information was collected in a questionnaire. RESULTS: Poor adherence, presence of resistant mutations and number of treatment's changes were the only significant factors found (p = 0.006, 0.04 and 0.01 respectively). From 66 sequenced samples, 78%, 50% and 50% showed resistance to NRTI (nucleoside reverse transcriptase inhibitors), NNRT (non-nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors), respectively. The most frequent mutations were M41L, M184V, and T215FY in RT and L62PI, L10FIRV and M36I in PT. DISCUSSION: The most important factor related to treatment response in this study was adherence to treatment. Mutations in RT were related to the treatment failure while the ones found in PT were secondary mutations which have been previously described to influence the selection of primary resistance mutations in these regions. The study reveals the urgency to detect resistant mutations in VF to be considered by physicians for selection of treatment schedule, to analyze basal HIV patients for monitoring of the spread of resistant mutations and the importance to reinforce the adherence in the patients for overall treatment outcome.

ABCC Subfamily Vacuolar Transporters are Involved in Pb (Lead) Detoxification in Saccharomyces cerevisiae

The present work has as objective to contribute for the elucidation of the mechanism associated with Pb detoxification, using the yeast Saccharomyces cerevisiae as a model organism. The deletion of GTT1 or GTT2 genes, coding for functional glutathione transferases (GST) enzymes in S. cerevisiae, caused an increased susceptibility to high Pb concentrations (500-1000 μmol L(-1)). These results suggest that the formation of glutathione-Pb conjugate (GS-Pb), dependent of GSTs, is important in Pb detoxification. The involvement of ATP-binding cassette (ABC) vacuolar transporters, belonging to class C subfamily (ABCC) in vacuolar compartmentalization of Pb, was evaluated. For this purpose, mutant strains disrupted in YCF1, VMR1, YBT1 or BPT 1 genes were used. All mutants tested, without vacuolar ABCC transporters, presented an increased sensitivity to 500-1000 μmol L(-1) Pb comparative to wild-type strain. Taken together, the obtained results suggest that Pb detoxification, by vacuolar compartmentalization, can occur as a result of the concerted action of GSTs and vacuolar ABCC transporters. Pb is conjugated with glutathione, catalysed by glutathione transferases and followed to the transport of GS-Pb conjugate to the vacuole by ABCC transporters.

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

The PROP1 2-Base Pair Deletion Is a Common Cause of Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301–302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301–302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301–302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301–302delAG deletion suggests that rather than being inherited from a common founder, the 301–302delAG may be a recurring mutation.

The PROP1 2-Base Pair Deletion Is a Common Cause of Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301-302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301-302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301-302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301-302delAG deletion suggests that rather than being inherited from a common founder, the 301-302delAG may be a recurring mutation.

Biological and Biochemical Characterization of Mutants of the RNase II family of enzymes

Dissertação para a obtenção de grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa.