Association and in Silico Assignment of Sequences from Turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

Characterisation of cDNAs for ribulose bisphosphate carboxylase small subunit genes in different species of Avena (Poaceae)

ABSTRACT

We report the cloning and sequencing of 14 rbcS cDNAs in six species of Avena (Poaceae) with different genome and ploidy levels. The nucleotide sequences 504 bp long were aligned with the published sequences of cultivated hexaploid oat, wheat, barley, rye, rice and corn and subjected to cladistic and phenetic analyses. The parsimonious analysis generated a tree with a topology very similar to the phenogram generated by the Neighbor-Joining analysis based on the Jukes and Cantor distances. Within the monophyletic assemblage of the tribe Aveneae, consistent clades composed of rbcS clones belonging to different species are recognized. It is suggested that they correspond to orthologous genes belonging to different subfamilies, and that the “within-species?d homogenisation may have occurred at a slow rate with respect to species evolution. In the monophyletic group of Pooideae, the topologies place barley rbcS sequences closer to wheat and rye than to oat sequences. This grouping agrees with most taxonomic and phylogenetic views.     

Detection of Fusarium wilt pathogens of Psidium guajava L. in soil using culture independent PCR (ciPCR)

Traditional culturing methods take a long time for identification of pathogenic isolates. A protocol has been developed for the detection of Fusarium from soil samples in the early stage of infection. Seventeen soil samples from different locations were collected before the onset of rains to find out the presence of Fusarium spp. population present in the soil of guava orchards and to correlate its presence with incidence of wilt. A PCR based method was developed for the molecular characterization of Fusarium using Fusarium spp. specific primer. DNA extracted by this method was free from protein and other contaminations and the yield was sufficient for PCR amplification. The primer developed in this study was amplifying ∼230 bp in all infected samples while not in healthy soil. The specificity and sensitivity of primer were tested on several Fusarium spp. and found that this primer was amplifying 10⁻⁶ dilution of the fungal DNA. The present study facilitates the rapid detection of Fusarium spp. from infected soil samples of guava collected from different agroclimatic regions in India. A rapid detection method for pathogens and a diagnostic assay for disease would facilitate an early detection of pathogen and lead to more effective control strategies.     

Genome analysis with distance to the nearest dissimilar nucleotide

DNA may be represented by sequences of four symbols, but it is often useful to convert those symbols into real or complex numbers for further analysis. Several mapping schemes have been used in the past, but most of them seem to be unrelated to any intrinsic characteristic of DNA. The objective of this work was to study a mapping scheme that is directly related to DNA characteristics, and that could be useful in discriminating between different species.Recently, we have proposed a methodology based on the inter-nucleotide distance, which proved to contribute to the discrimination among species. In this paper, we introduce a new distance, the distance to the nearest dissimilar nucleotide, which is the distance of a nucleotide to first occurrence of a different nucleotide. This distance is related to the repetition structure of single nucleotides. Using the information resulting from the concatenation of the distance to the nearest dissimilar and the inter-nucleotide distance, we found that this new distance brings additional discriminative capabilities. This suggests that the distance to the nearest dissimilar nucleotide might contribute with useful information about the evolution of the species.     

Molecular cytogenetic analysis of Haemulidae fish (Perciformes): Evidence of evolutionary conservation

Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric chromosomes (FN = 48), and apparently similar chromosomal microstructure, especially in genus Haemulon, representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Ag-NOR, DAPI/CMA₃ staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n = 48 acrocentric chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA₃⁺/DAPI^-regions in the karyotype. Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes.     

Multilocus variable number tandem repeat analysis (MLVA) of Streptococcus pyogenes

We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson's Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28.We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE.     

Environmental Vibrio parahaemolyticus DNA signatures validation

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.     

Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function

Knowledge of the detailed organization of nucleosomes across genomes and the mechanisms of nucleosome positioning is critical for the understanding of gene regulation and expression. In the present work, the bias of 4-mer frequency in nucleosome and linker sequences of the S. cerevisiae genome was analyzed statistically. A novel position-correlation scoring function algorithm based on the bias of 4-mer frequency in linker sequences was presented to distinguish nucleosome vs linker sequences. Five-fold cross-validation demonstrated that the algorithm achieved a good performance with mean area under the receiver operator characteristics curve of 0.981. Next, the algorithm was used to predict nucleosome occupancy throughout the S. cerevisiae genome and relatively high correlation coefficients with experiment maps of nucleosome positioning were obtained. Besides, the distinct nucleosome depleted regions in the vicinity of regulatory sites were confirmed. The results suggest that intrinsic DNA sequence preferences in linker regions have a significant impact on the nucleosome occupancy.     

A career pathway in protein folding: from model peptides to postreductionist protein science

This review discusses the inherent challenge of linking "reductionist" approaches to decipher the information encoded in protein sequences with burgeoning efforts to explore protein folding in native environments-"postreductionist" approaches. Because the invitation to write this article came as a result of my selection to receive the 2010 Dorothy Hodgkin Award of the Protein Society, I use examples from my own work to illustrate the evolution from the reductionist to the postreductionist perspective. I am incredibly honored to receive the Hodgkin Award, but I want to emphasize that it is the combined effort, creativity, and talent of many students, postdoctoral fellows, and collaborators over several years that has led to any accomplishments on which this selection is based. Moreover, I do not claim to have unique insight into the topics discussed here; but this writing opportunity allows me to illustrate some threads in the evolution of protein folding research with my own experiences and to point out to those embarking on careers how the twists and turns in anyone's scientific path are influenced and enriched by the scientific context of our research. The path my own career has taken thus far has been shaped by the timing of discoveries in the field of protein science; together with our contemporaries, we become part of a knowledge evolution. In my own case, this has been an epoch of great discovery in protein folding and I feel very fortunate to have participated in it.