Emmanuel Dias Neto

Whole genome linkage disequilibrium maps in cattle

BackgroundBovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides background information concerning the extent of long range linkage disequilibrium in cattle.ResultsLinkage disequilibrium was assessed using r2 among all pairs of syntenic markers within eight breeds of cattle from the Bos taurus and Bos indicus subspecies. Bos taurus breeds included Angus, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black and Limousin while Bos indicus breeds included Brahman and Nelore. Approximately 2670 markers spanning the entire bovine autosomal genome were used to estimate pairwise r2 values. We found that the extent of linkage disequilibrium is no more than 0.5 Mb in these eight breeds of cattle.ConclusionLinkage disequilibrium in cattle has previously been reported to extend several tens of centimorgans. Our results, based on a much larger sample of marker loci and across eight breeds of cattle indicate that in cattle linkage disequilibrium persists over much more limited distances. Our findings suggest that 30,000–50,000 loci will be needed to conduct whole genome association studies in cattle.

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 generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define ≈23,500 genes, of which only ≈1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

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.

An assessment of population structure in eight breeds of cattle using a whole genome SNP panel

BackgroundAnalyses of population structure and breed diversity have provided insight into the origin and evolution of cattle. Previously, these studies have used a low density of microsatellite markers, however, with the large number of single nucleotide polymorphism markers that are now available, it is possible to perform genome wide population genetic analyses in cattle. In this study, we used a high-density panel of SNP markers to examine population structure and diversity among eight cattle breeds sampled from Bos indicus and Bos taurus.ResultsTwo thousand six hundred and forty one single nucleotide polymorphisms (SNPs) spanning all of the bovine autosomal genome were genotyped in Angus, Brahman, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black, Limousin and Nelore cattle. Population structure was examined using the linkage model in the program STRUCTURE and Fst estimates were used to construct a neighbor-joining tree to represent the phylogenetic relationship among these breeds.ConclusionThe whole-genome SNP panel identified several levels of population substructure in the set of examined cattle breeds. The greatest level of genetic differentiation was detected between the Bos taurus and Bos indicus breeds. When the Bos indicus breeds were excluded from the analysis, genetic differences among beef versus dairy and European versus Asian breeds were detected among the Bos taurus breeds. Exploration of the number of SNP loci required to differentiate between breeds showed that for 100 SNP loci, individuals could only be correctly clustered into breeds 50% of the time, thus a large number of SNP markers are required to replace the 30 microsatellite markers that are currently commonly used in genetic diversity studies.

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

ABSTRACT. Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co‐infections with Trypanosoma cruzi, demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.

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.

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.

Association of a new polymorphism in ALOX12 gene with bipolar disorder

Abstract. Bipolar disorder (BPD) is characterised by episodes of excitement interspersed with periods of depression. The role of genetic factors in BPD is indicated by studies in monozygotic twins showing 40–70 % of concordance. Studies using genetic markers showed linkage of genes for affective disorders in different chromosome regions, emphasising the polygenic and multifactorial traits. The main goal of our research is to search non-synonymous SNPs (those that result in modifications in protein sequence) in genes that can be associated with psychiatric diseases as suggested by genomic mapping and/or by physiological function of the protein. Using DNA sequencing we could confirm a new non-synonymous SNP in the conservative domain of the ALOX12 gene (17p13.1), suggested by EST alignment. This SNP is an alteration from G to A that leads to a change of an arginine (A) to a glutamine in one of the most important domains of the protein. This SNP was evaluated by DNA sequencing in 182 patients with BPD and 160 control individuals. An increased presence of allele A among patients (60 % in controls and 73.1 % in cases; χ2 = 6.581, P = 0.010; OR = 1.8095, 95 % CI = 1.1477–2.853) was found, suggesting an association of this polymorphism with the BPD in this Brazilian sample.

Alterações genéticas na doença de Alzheimer

Com o aumento da expectativa de vida, visto hoje em todo o planeta, um maior numero de individuos alcanca uma idade avancada em que a manifestacao de doencas neurodegenerativas e mais frequente. Entre essas, a doenca de Alzheimer (DA) e a causa mais frequente de demencia. Os achados mais marcantes na DA, em cerebros de pacientes acometidos pela doenca, sao as placas senis, os emaranhados neurofibrilares e a extensa perda neuronal. No entanto, existe uma carencia generalizada de marcadores biologicos preditivos ou com valor diagnostico para a DA. Estudos de genetica molecular permitiram identificar quatro genes consistentemente associados com o maior risco de desenvolvimento da doenca: APP, apoE, PSEN1 e PSEN2. No entanto, inumeros estudos apontam para papel importante de outros genes, fortalecendo a hipotese de uma doenca poligenica e multifatorial. Neste sentido, novas abordagens de estudo tem um futuro promissor, podendo indicar uma vasta populacao de genes ou alteracoes moleculares que possam explicar o surgimento da doenca, vindo a fornecer as bases para a compreensao da DA e tambem para o delineamento de novas e mais eficazes abordagens de tratamento ou prevencao da doenca.