Protein recovery, separation and purification: selection of optimal techniques using an expert system

The paper discusses the utilization of new techniques ot select processes for protein recovery, separation and purification. It describesa rational approach that uses fundamental databases of proteins molecules to simplify the complex problem of choosing high resolution separation methods for multi component mixtures. It examines the role of modern computer techniques to help solving these questions.

Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library

We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs), i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequencers. ESTs can be used to identify genese onf the basis of their homology whith sequences from other species deposited in DNA or protein databases. Trasncripts with sequences without matches in teh databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.

Contrasting Genomic Variability between Clones from Field Isolates and Laboratory Populations of Schistosoma mansoni

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using randomly amplified polymorphic dNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43% of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analyses of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates

Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1x105 to 4.3x106 conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30°C, compared to 15 and 20°C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Genomic rearrangements in trypanosomatids: an alternative to the "one gene" evolutionary hypotheses?

Most molecular trees of trypanosomatids are based on point mutations within DNA sequences. In contrast, there are very few evolutionary studies considering DNA (re) arrangement as genetic characters. Waiting for the completion of the various parasite genome projects, first information may already be obtained from chromosome size-polymorphism, using the appropriate algorithms for data processing. Three illustrative models are presented here. First, the case of Leishmania (Viannia) braziliensis/L. (V.) peruviana is described. Thanks to a fast evolution rate (due essentially to amplification/deletion of tandemly repeated genes), molecular karyotyping seems particularly appropriate for studying recent evolutionary divergence, including eco-geographical diversification. Secondly, karyotype evolution is considered at the level of whole genus Leishmania. Despite the fast chromosome evolution rate, there is qualitative congruence with MLEE- and RAPD-based evolutionary hypotheses. Significant differences may be observed between major lineages, likely corresponding to major and less frequent rearrangements (fusion/fission, translocation). Thirdly, comparison is made with Trypanosoma cruzi. Again congruence is observed with other hypotheses and major lineages are delineated by significant chromosome rearrangements. The level of karyotype polymorphism within that "species" is similar to the one observed in "genus" Leishmania. The relativity of the species concept among these two groups of parasites is discussed.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Genomic characterization of a Brazilian TT Virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.

Perspectives in the control of infectious diseases by transgenic mosquitoes in the post-genomic era: a review

Arthropod-borne diseases caused by a variety of microorganisms such as dengue virus and malaria parasites afflict billions of people worldwide imposing major economic and social burdens. Despite many efforts, vaccines against diseases transmitted by mosquitoes, with the exception of yellow fever, are not available. Control of such infectious pathogens is mainly performed by vector management and treatment of affected individuals with drugs. However, the numbers of insecticide-resistant insects and drug-resistant parasites are increasing. Therefore, inspired in recent years by a lot of new data produced by genomics and post-genomics research, several scientific groups have been working on different strategies to control infectious arthropod-borne diseases. This review focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to malaria parasites and dengue virus transmission.

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.