Inheritance and mapping of 11 avirulence genes in Phytophthora sojae

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F-2 Population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F-2 progeny from the two new crosses and two crosses described previously (a total of 212 F-2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4 cM. (C) 2002 Elsevier Science (USA). All rights reserved.

Mould and yeast identification in archival settings: preliminary results on the use of traditional methods and molecular biology options in Portuguese archives

This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media – for the viable fungi – and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents.

Differentiation of Candida species obtained from nosocomial candidemia using RAPD-PCR technique

Thirteen strains of the genus Candida were isolated from catheter, urine and surgical wounds from individual patients of the Santa Casa de Misericórdia, Belo Horizonte, MG, Brazil. Ten strains were characterized as Candida albicans, two as Candida glabrata, and one as Candida parapsilosis. Isolates were evaluated for molecular relatedness by random amplified polymorphic DNA technique using 15 primers. The analysis of the genomic DNA obtained revealed a low intraspecific polymorphism and did not allow for the differentiation between strains of the same species obtained from distinct clinical sources (catheter, urine and surgical wounds). The RAPD profiles generated were able to differentiate among the species of Candida albicans, Candida parapsilosis and Candida glabrata strains isolated in this study.

Molecular and biological diversity of HIV-1 in Brazil

To determine the genomic polymorphism and biological properties present in HIV-1 Brazilian isolates, were analyzed five viral isolates obtained from patients residing in Rio de Janeiro (P1 and P5), São Paulo (P3) and Bahia (P2 and P4) states. For each viral isolate in vitro characteristics such as replication rate, syncytium-inducing capacity and cell death were observed in lymphoblastoid (H9, CEM and peripheral blood mononuclear cells) as well as monocytoid (U937) cells. In addition, the evaluation of the restriction fragment lenght polymorphism of these isolates was also performed using a panel of endonucleases such as Hind III, Bgl II, Sac I, Pst I, Kpn I and Eco RI. One of the isolates (P1), showed the highest phenotypic and genotypic divergence, when compared to others. The results found suggest a HIV heterogeneity in Brazil similar to that already described in other regions of the world.

Maternally inherited Leigh syndrome.

A 6 1/2-year-old girl had developmental regression, and Leigh syndrome was diagnosed. A second girl born to the same mother after heterologous artificial insemination also lost acquired skills and died at 2 1/2 years of age; neuropathologic examination confirmed the diagnosis of Leigh syndrome. Tissues from both children and from the mother had a point mutation at nucleotide 8993 in the adenosinetriphosphatase 6-gene of mitochondrial DNA. This family illustrates that Leigh syndrome can be transmitted by maternal inheritance.

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study.

The identification of genetically homogeneous groups of individuals is a long standing issue in population genetics. A recent Bayesian algorithm implemented in the software STRUCTURE allows the identification of such groups. However, the ability of this algorithm to detect the true number of clusters (K) in a sample of individuals when patterns of dispersal among populations are not homogeneous has not been tested. The goal of this study is to carry out such tests, using various dispersal scenarios from data generated with an individual-based model. We found that in most cases the estimated 'log probability of data' does not provide a correct estimation of the number of clusters, K. However, using an ad hoc statistic DeltaK based on the rate of change in the log probability of data between successive K values, we found that STRUCTURE accurately detects the uppermost hierarchical level of structure for the scenarios we tested. As might be expected, the results are sensitive to the type of genetic marker used (AFLP vs. microsatellite), the number of loci scored, the number of populations sampled, and the number of individuals typed in each sample.

Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela

The mosquito Aedes aegypti is the main vector of dengue in Venezuela. The genetic structure of this vector was investigated in 24 samples collected from eight geographic regions separated by up to 1160 km. We examined the distribution of a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among 1144 Ae. aegypti from eight collections. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Seven haplotypes were detected throughout Venezuela and these were sorted into two clades. Significant differentiation was detected among collections and these were genetically isolated by distance.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

The polymorphic nature of HIV type 1 env V4 affects the patterns of potential N-glycosylation sites in proviral DNA at the intrahost level.

We have previously shown that env V4 from HIV-1 plasma RNA is highly heterogeneous within a single patient, due to indel-associated polymorphism. In this study, we have analyzed the variability of V4 in proviral DNA from unfractionated PBMC and sorted T and non-T cell populations within individual patients. Our data show that the degree of sequence variability and length polymorphism in V4 from HIV provirus is even higher than we previously reported in plasma. The data also show that the sequence of V4 depends largely on the experimental approach chosen. We could observe no clear trend for compartmentalization of V4 variants in specific cell types. Of interest is the fact that some variants that had been found to be predominant in plasma were not detected in any of the cell subsets analyzed. Consistently with our observations in plasma, V3 was found to be relatively conserved at both interpatient and intrapatient level. Our data show that V4 polymorphism involving insertions and deletions in addition to point mutations results in changes in the patterns of sequons in HIV-1 proviral DNA as well as in plasma RNA. These rearrangements may result in the coexistence, within the same individual, of a swarm of different V4 regions, each characterized by a different carbohydrate surface shield. Further studies are needed to investigate the mechanism responsible for the variability observed in V4 and its role in HIV pathogenesis.

The role of the T-cell receptor (TCR) in alpha beta/gamma delta lineage commitment: clues from intracellular TCR staining.

T cells belong to two mutually exclusive lineages expressing either alpha beta or gamma delta T-cell receptors (TCR). Although alpha beta and gamma delta cells are known to share a common precursor the role of TCR rearrangement and specificity in the lineage commitment process is controversial. Instructive lineage commitment models endow the alpha beta or gamma delta TCR with a deterministic role in lineage choice, whereas separate lineage models invoke TCR-independent lineage commitment followed by TCR-dependent selection and maturation of alpha beta and gamma delta cells. Here we review the published data pertaining to the role of the TCR in alpha beta/gamma delta lineage commitment and provide some additional information obtained from recent intracellular TCR staining studies. We conclude that a variant of the separate lineage model is best able to accommodate all of the available experimental results.

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.