Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli ( EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non- human primates belong to the serogroups and/ or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non- human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/ serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non- human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non- human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Search for methylation-sensitive amplification polymorphisms in mutant figs

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools. © FUNPEC-RP.

A biotecnologia nos programas de melhoramento de forrageiras tropicais da Embrapa Gado de Corte.

Neste trabalho procurou-se apresentar e discutir, de forma ampla, o uso da biotecnologia e seu potencial para os programas de melhoramento de forrageiras tropicais realizados na Embrapa Gado de Corte. O uso da biotecnologia nesses programas é uma atividade recente, mas importantes resultados vêm sendo gerados a fim de auxiliar o processo de obtenção de novas cultivares forrageiras. A maioria dos trabalhos apresentados utiliza marcadores Random Amplification of Polymorphic DNA (RAPD) para aplicações em curto prazo: estudos de diversidade em acessos de bancos de germoplasma, identificação de híbridos e estimação da taxa de cruzamento. Aplicações em médio e longo prazos do uso de marcadores, como mapeamento genético e seleção auxiliada por marcadores moleculares (SAMM), ainda necessitam de maiores investimentos, tanto na busca de novos marcadores, quanto no desenvolvimento de populações adequadas para esses estudos. Em 2007, teve início uma nova linha de pesquisa nessa unidade, a prospecção de genes com características econômicas. Genes para a tolerância ao alumínio são o foco dessa pesquisa que pretende explorar a sintenia entre os genomas de gramíneas, visando ao desenvolvimento de cultivares de braquiária mais tolerantes ao alumínio. A Embrapa Gado de Corte vem investindo em pessoal e aquisição de equipamentos para avançar não só na produção de cultivares de forrageiras mais adaptadas às necessidades de um mercado cada vez mais exigente, como também no crescimento institucional do setor de biotecnologia.

The application of randomly amplified polymorphic DNA (RAPD's) to the study of Lasaea rubra (Montagu).

This paper describes the random amplification of polymorphic DNA markers (RAPDs) in Lasaea rubra (Erycinidae: Bivalvia). Present evidence suggests that L. rubra is an asexual species; however, the exact mode of clonal reproduction in this species is still a matter of debate. In this preliminary study, four of the primers used generated polymorphic RAPDs. One primer was able to distinguish between individuals from the same or different crevice population. This same primer also resolved a single band difference between otherwise identical RAPD patterns of a parent and its offspring. No familial differences have been detected in several previous studies using allozyme electrophoresis. This paper suggests that many polymorphic markers could be obtained with this species using the RAPD technique. Population genetic analysis of L. rubra has long been hampered by a dearth of polymorphic markers due to its small size. These findings suggest that this technique has the potential to further the study of population genetics in this asexual species.

A Novel Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Propionibacterium acnes and Characterisation of Type I Cell Surface-Associated Antigens.

We have developed a novel Multilocus Sequence Typing Scheme (MLST) and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and Random Amplification of Polymorphic DNA (RAPD) typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (ST). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 65% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants (SLV), which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore supports an association between acne and P. acnes strains from the type IA cluster and highlights the role of a widely disseminated clonal genotype in this condition. Characterisation of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Molecular genetic profiling of functional genes of pearl oyster, Pinctada fucata

There are a number of genes involved in the regulation of functional process in marine bivalves. In the case of pearl oyster, some of these genes have major role in the immune/defence function and biomineralization process involved in the pearl formation in them. As secondary filter feeders, pearl oysters are exposed to various kinds of stressors like bacteria, viruses, pesticides, industrial wastes, toxic metals and petroleum derivatives, making susceptible to diseases. Environmental changes and ambient stress also affect non-specific immunity, making the organisms vulnerable to infections. These stressors can trigger various cellular responses in the animals in their efforts to counteract the ill effects of the stress on them. These include the expression of defence related genes which encode factors such as antioxidant genes, pattern recognition receptor proteins etc. One of the strategies to combat these problems is to get insight into the disease resistance genes, and use them for disease control and health management. Similarly, although it is known that formation of pearl in molluscs is mediated by specialized proteins which are in turn regulated by specific genes encoding them, there is a paucity of sufficient information on these genes.In view of the above facts, studies on the defence related and pearl forming genes of the pearl oyster assumes importance from the point of view of both sustainable fishery management and aquaculture. At present, there is total lack of sufficient knowledge on the functional genes and their expressions in the Indian pearl oyster Pinctada fucata. Hence this work was taken up to identify and characterize the defence related and pearl forming genes, and study their expression through molecular means, in the Indian pearl oyster Pinctada fucata which are economically important for aquaculture at the southeast coast of India. The present study has successfully carried out the molecular identification, characterization and expression analysis of defence related antioxidant enzyme genes and pattern recognition proteins genes which play vital role in the defence against biotic and abiotic stressors. Antioxidant enzyme genes viz., Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidise (GPX) and glutathione-S-transferase (GST) were studied. Concerted approaches using the various molecular tools like polymerase chain reaction (PCR), random amplification of cDNA ends (RACE), molecular cloning and sequencing have resulted in the identification and characterization of full length sequences (924 bp) of the Cu/Zn SOD, most important antioxidant enzyme gene. BLAST search in NCBI confirmed the identity of the gene as Cu/Zn SOD. The presence of the characteristic amino acid sequences such as copper/zinc binding residues, family signature sequences and signal peptides were found out. Multiple sequence alignment comparison and phylogenetic analysis of the nucleotide and amino acid sequences using bioinformatics tools like BioEdit,MEGA etc revealed that the sequences were found to contain regions of diversity as well as homogeneity. Close evolutionary relationship between P. fucata and other aquatic invertebrates was revealed from the phylogenetic tree constructed using SOD amino acid sequence of P. fucata and other invertebrates as well as vertebrates

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Genetic diversity in Egeria densa and E. najas in Jupia Reservoir, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic variability in natural populations of Zeyheria montana mart. from the Brazilian Cerrado

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

RAPD genomic fingerprinting differentiates Thiobacillus ferrooxidans strains

The PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was optimized and used for assessing genomic variability among eight Thiobacillus ferrooxidans strains. RAPD fingerprints presented variation for the thirty primers used, giving a total of 269 polymorphic bands. Similarity coefficients between the strains were calculated, and UPGMA cluster analysis was used to generate a dendrogram showing relationships among them. Most primers divided T. ferrooxidans strains in two distinct groups - Group 1: S, SSP, V3, AMF and Group 2: CMV, FG-460, I-35, LR. We observed that the T. ferrooxidans strains used in this work have a high degree of genomic diversity and that RAPD is a powerful method to differentiate them.

Tipagem molecular, perfis de sensibilidade e caracterização de transcritos diferencialmente expressos durante a infecção de Cryptococcus neoformans

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Virulence typing of Escherichia coli using microarrays

We describe a microarray based broad-range screening technique for Escherichia coli virulence typing. Gene probes were amplified by PCR from a plasmid bank of characterised E. coli virulence genes and were spotted onto a glass slide to form an array of capture probes. Genomic DNA from E. coli strains which were to be tested for the presence of these virulence gene sequences was labelled with fluorescent cyanine dyes by random amplification and then hybridised against the array of probes. The hybridisation, washing and data analysis conditions were optimised for glass slides, and the applicability of the method for identifying the presence of the virulence genes was determined using reference strains and clinical isolates. It was found to be a sensitive screening method for detecting virulence genes, and a powerful tool for determining the pathotype of E. coli. It will be possible to expand and automate this microarray technique to make it suitable for rapid and reliable diagnostic screening of bacterial isolates.