Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing.

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.

Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA.

The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.

Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains.

DNA extracted from 32 isolates of Trypanosoma cruzi was subjected to polymerase chain reaction amplification using 4 arbitrary primers resulting in relatively complex DNA profiles that include polymorphic markers known as random amplified polymorphic DNAs (RAPDs). The RAPD profiles of 18 strains belonging to zymodeme 1 (Z1) collected from various regions of South America exhibited a consistant pattern and 59 (59%) of the bands produced were present in all Z1 strains. A similar level of consistency was seen in the number of bands shared between 5 Z2 strains, 4 ZB strains and 2 ZC strains. A phenetic analysis of the 5 most different Z1 strains based on band sharing showed that their interrelationships mirrored their geographical origin. Comparison of the RAPD profiles of strains from different zymodemes showed that less than 7% of bands of strains in one zymodeme are present in strains of another zymodeme. Analysis of band sharing using bands present in all strains of a given zymodeme showed ZB and ZC to be closely related and Z1 and Z2 to form distinct groups.

The random amplification of polymorphic DNA allows the identification of strains and species of schistosome.

The use of arbitrarily selected primers (10-24 nucleotides) and very low stringency annealing conditions (30 degrees C followed by 40 degrees C) for the polymerase chain reaction amplification of 1.0 ng of schistosome DNA resulted in relatively complex patterns of products. Amongst the primers tested some, for example 5'-TCGTAGCCAA, produced patterns that included bands that were polymorphic between strains of Schistosoma mansoni. Other primers, for example 5'-TCACGATGCA, produced apparently identical products using DNA from 5 S. mansoni strains but highly variable patterns when DNA from different schistosome species was used. The results indicate that the random amplification of polymorphic DNA (RAPD) may be an extremely useful approach to the identification of schistosome strains and species.

Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.

A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.

PCR template preparation for capillary DNA sequencing.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.

The use of RAPDs for the analysis of parasites.

There is a lack of sequence information concerning polymorphic loci in parasite genomes. Thus, the use of arbitrary PCR primers under low temperature annealing conditions to generate random amplified polymorphic DNAs (RAPDs) represents an important approach to the study of the structure of parasite populations, their genetic variation as well as improved diagnosis of the diseases they cause. Following the examination of all variables and their effect on the reproducibility of the reaction, we have established a protocol for the analysis of RAPDs that involves amplification at two separate DNA concentrations followed by polyacrylamide gel electrophoresis and silver staining. We find the technique to be sensitive, reproducible, simple and relatively cheap. It has already provided insight into the genetic variation in populations of schistosomes and trypanosomes and is being used to study various other endemic infections. We also use specific primers under low stringency conditions in situations where the objective of the amplification is the detection of a particular sequence and where normal high stringency conditions give a positive/negative answer such as sex determination or diagnosis of blood born infections. Under low stringency conditions, specific amplification products persist but products of low stringency priming are also apparent and serve as a perfect internal control for negative samples.

The use of RAPDs for the study of the genetic diversity of Schistosoma mansoni and Trypanosoma cruzi.

Arbitrary primers have been used for the production of complex, PCR generated DNA profiles in order to undertake a preliminary random amplified polymorphic DNA (RAPD) analysis of strains (and related species) of two parasitic organisms that are responsible for important diseases endemic in Brazil: Schistosoma mansoni that causes schistosomiasis, and Trypanosoma cruzi that causes Chagas' disease. A relatively low level of polymorphism was found in S. mansoni when strains isolated from different regions of Brazil were compared, with less than 10% of bands exhibiting polymorphism. Comparison of different schistosome species, on the other hand, showed them to be distantly related with very few bands shared by even the more closely related species. Trypanosome strains were found to be much more variable. When strains were compared between zymodemes (groups of parasite strains with the same isoenzyme profiles), a maximum of 7% of bands were found to be common whereas among strains in the same zymodeme a clear characteristic pattern was observed. In the zymodeme most thoroughly studied, it was found that 59% of bands were shared. Band sharing analysis showed that the relationships of strains within a zymodeme correlate with their geographical origin and that the relationship between zymodemes correlates closely with that previously determined by isoenzyme analysis. These preliminary data indicate the ready applicability of RAPD analysis to the study of parasites where largely unexplored genetic variations may have an important bearing on the complexity and diversity of diseases.

A phylogenetic analysis of Schistosoma haematobium group species based on randomly amplified polymorphic DNA.

Randomly amplified polymorphic DNA (RAPD) profiles were produced using four oligonucleotide primers with genomic DNA from 15 isolates of schistosome. Both inter- and intraspecific variation were noted. Intraspecific variation was greater for two species of the S. haematobium group (S. haematobium and S. intercalatum) than for S. mansoni. The inferred phylogeny placed S. curassoni and S. bovis as sister groups to S. mansoni-S. rodhaini group. S. mattheei and S. leiperi formed a separate lineage. The results confirm that RAPD profiles may be used for both strain and species differentiation and for the generation of phylogenetic trees.

Populational structure of Schistosoma mansoni assessed by DNA microsatellites.

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Dot-dye-immunoassay and dot-ELISA for the serological differentiation of acute and chronic schistosomiasis mansoni using keyhole limpet haemocyanin as antigen.

Two immunoassays, dot enzyme-linked immunosorbent assay (dot-ELISA) and dot-dye immunoassay (dot-DIA), using soluble egg antigen and keyhole limpet haemocyanin as antigens, were evaluated for the serological differentiation of 25 acute and 37 chronic patients infected with Schistosoma mansoni and 20 non-infected individuals, in comparison with ELISA. Efficiency was 92.7%, 90.0% for ELISA, dot-ELISA and dot-DIA, respectively. Dipstick dot-ELISA and dot-DIA are described and shown to be reliable cheap and simple methods for the serological differentiation of acute and chronic schistosomiasis.

A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls.

Several different PCR protocols for the detection of Bovine herpesvirus-1 (BHV-1) in bovine semen, are available in the literature. Most of them are rather laborious and the majority were performed on laboratory samples, artificially contaminated semen or semen provided from experimentally inoculated animals. Furthermore, to obtain higher levels of sensitivity, additional dot-blot procedures are frequently necessary. We describe the detection of BHV-1 in bovine semen and the supernatant of cell cultures with titres of 0.001 TCID50/50 microliter by a nested PCR assay, with no further hybridization procedures. The high sensitivity was achieved by filtering the semen samples on chromatography columns before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels. Specificity of the amplified fragments was confirmed by RFLP and sequence analysis of the PCR products. This nested PCR procedure was performed in parallel with viral isolation (VI) on 101 semen samples provided from naturally infected bulls housed at an artificial insemination centre. The nested PCR was shown to be more sensitive, faster and easier to perform than the standard VI test. To our knowledge, it is the most sensitive PCR test for BHV-1 detection in bovine semen and could be easily used for routine diagnosis.

Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12.

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Partially degraded DNA of parasitological interest serves as an adequate template for the production of random amplified polymorphic DNAs (RAPDs).

Genomic DNA was extracted from Schistosoma mansoni adult worms and deliberately degraded by sonication. Samples with varying average molecular weight were subjected to RAPD (randomly amplified polymorphic DNA) analysis using the primer 3307 (5'-AGTGCTACGT-3') and other primers. Reproducible and complex DNA banding patterns were obtained, irrespective of the extent of DNA degradation. The same amplification protocol was employed with naturally degraded Biomphalaria glabrata genomic DNA and the primer 3302 (5'-CTGATGCTAC-3'). Again, reproducible RAPD patterns resulted. The experiment shows that the partially degraded DNA samples can be safely compared in RAPD analysis without artifactual bands compromising the accuracy of genetic analysis. Thus RAPD analysis permits complex and reproducible DNA fingerprinting from degraded samples of parasitological interest.

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

[Susceptibility to chemotherapeutic agents of Schistosoma mansoni isolates from patients treated with oxamniquine and praziquantel and not cured]. / Suscetibilidade aos agentes quimioterápicos de isolados de Schistosoma mansoni oriundos de pacientes tratados com oxamniquine e praziquantel e não curados.

Ten inhabitants of Itaquara, Bahia, Brazil treated with oxamniquine and subsequently praziquantel were not cured. Schistosoma mansoni isolates derived from these patients were studied. Snails were infected with miracidia derived from the feces of these patients and the cercariae produced used to infect albino mice. The animals were then treated with a single oral dose of oxamniquine (25, 50 and 100mg/kg) or praziquantel (100, 200 and 400 mg/kg). The response to chemotherapy was significantly different in some of the isolates although it was not possible to characterize any of them as resistant. In addition, DNA analysis of the isolates by means of "Random Amplified Polymorphic DNA" indicated a low degree of variability as compared with a laboratory strain, LE. Thus, it was not possible to characterize these organisms at a genetic level as a distinct strain.

Comparative study of the accuracy of different technique for the laboratory diagnosis of schistosomiasis mansoni in areas of low endemicity in Barra Mansa City, Rio de Janeiro State, Brazil

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82­95.5%), differed significantly from COPT in positivity , and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

Genomics and proteomics approaches to the study of cancer-stroma interactions.

BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.