Carlos Souza do Nascimento

Differential expression of genes in resistant versus susceptible Gyr x Holstein cattle challenged with the tick Rhipicephalus (Boophilus) microplus.

The bovine tick Rhipicephalus (Boophilus) microplus causes major losses in cattle production systems in tropical regions. Bos indicus breeds are more resistant to ticks than B. taurus breeds. Resistance genes could be an alternative to control this parasite. We examined the pattern of gene expression of three calcium-binding-protein genes: translationally controlled tumor protein 1 (TPT1), allergen Bos d3 (S100A7), calcium channel protein transient receptor potential vanilloid 6 (TRPV6), and the cysteine proteinase inhibitor gene (CST6). These genes were selected from cDNA libraries prepared from skin biopsies taken from resistant and susceptible Gyr x Holstein F₂ animals. These biopsies were also used to study the expression level of these genes through real-time PCR analysis. The relative expression levels of the S100A7, TPT1, TRPV6, and CST6 genes were 2.01 ± 0.6, 1.32 ± 0.9, 1.53 ± 1.2, and 2.03 ± 0.7 times higher in the susceptible group, respectively. Skin lesion tissue from the susceptible animals showed significantly more mRNA transcripts of these genes in comparison with the resistant animals (P = 0.001). However, this hypersensitivity does not seem to protect the susceptible animals against tick infestation.

Mapping of quantitative trait loci for performance on chromosome 4 in pigs.

Uma populacao F2 de suinos obtida a partir do intercruzamento da geracao F1, proveniente do acasalamento divergente de dois machos da raca naturalizada brasileira Piau com 18 femeas comerciais (Landrace × Large White × Pietrain) e genotipada para cinco marcadores tipo microssatelite foi utilizada com o objetivo de mapear locos de caracteristicas quantitativas associados a caracteristicas de desempenho no cromossomo 4. As caracteristicas avaliadas foram: numero de tetas, peso ao nascimento, peso aos 21, 42, 63, 77 e 105 dias de idade, peso ao abate, consumo de racao, conversao alimentar e ganho de peso medio diario dos 77 aos 105 dias de idade e idade ao abate. Utilizou-se o metodo de regressao por intervalo de mapeamento com analises realizadas por meio do programa QTL EXPRESS. Verificou-se a presenca de apenas um QTL significativo (para peso aos 77 dias). A utilizacao deste QTL na Selecao Assistida por Marcadores deve ser feita depois que a posicao desse QTL for refinada, com possivel identificacao da mutacao causal e estimacao de seus efeitos.

Follicular dynamics and gene expression in granulosa cells, corpora lutea and oocytes from gilts of breeds with low and high ovulation rates.

Follicular dynamics and the expression of candidate genes using real-time polymerase chain reaction (PCR) were compared during the oestrous cycle of pig breeds with high (commercial line; n=24) and low (local Brazilian Piau; n=21) ovulation rates and prolificacy. Gilts were killed on Days 0, 4, 10 and 18 of the oestrous cycle and visible ovarian follicles were classified by follicular diameter. Recovered cumulus-oocyte complexes were classified as normal or atretic and frozen in liquid nitrogen until RNA extraction. Low ovulation rates and/or prolificacy in Piau gilts was associated with a different pattern of follicle development, with lower numbers of small follicles on Day 18, fewer large follicles on Days 0 and 18 (P≤0.05) and a higher proportion of atretic follicles on Days 0 and 18 (P≤0.05). Compared with commercial line gilts, less-prolific Piau gilts exhibited higher expression of apoptotic genes during luteolysis (CASP3 and FASL; P≤0.05), decreased expression of TGFBR2 and BAX mRNA in the corpus luteum (P≤0.05), higher expression of apoptotic genes (FAS, BCL2 and CASP8; P≤0.05) in granulosa cells and a greater abundance (P≤0.05) of genes controlling oocyte-secreted factors (GDF9, BMP15 and BMP6), suggesting underlying mechanisms controlling differences in follicular development, ovulation rate and inherent prolificacy in this pig breed.

Transcript profiling of expressed sequence tags from semimembranosus muscle of commercial and naturalized pig breeds.

In general, genetic differences across different breeds of pig lead to variation in mature body size and slaughter age. The Commercial breeds Duroc and Large White and the local Brazilian breed Piau are ostensibly distinct in terms of growth and muscularity, commercial breeds are much leaner while local breeds grow much slower and are fat type pigs. However, the genetic factors that underlie such distinctions remain unclear. We used expressed sequence tags (ESTs) to characterize and compare transcript profiles in the semimembranosus muscle of these pig breeds. Our aim was to identify differences in breed-related gene expression that might influence growth performance and meat quality. We constructed three non-normalized cDNA libraries from semimembranosus muscle, using two samples from each one, of these three breeds; 6902 high-quality ESTs were obtained. Cluster analysis was performed and these sequences were clustered into 3670 unique sequences; 24.7% of the sequences were categorized as contigs and 75.3% of the sequences were singletons. Based on homology searches against the SwissProt protein database, we were able to assign a putative protein identity to only 1050 unique sequences. Among these, 58.5% were full-length protein sequences and 17.2% were pig-specific sequences. Muscle structural and cytoskeletal proteins, such as actin, and myosin, were the most abundant transcripts (16.7%) followed by those related to mitochondrial function (12.9%), and ribosomal proteins (12.4%). Furthermore, ESTs generated in this study provide a rich source for identification of novel genes and for the comparative analysis of gene expression patterns in divergent pig breeds.

Expressed sequenced tags profiling of resistant and susceptible Gyr x Holstein cattle infested with the tick Rhipicephalus (Boophilus) microplus

Tick resistance in cattle is mainly found in zebu (Bos indicus) animals, although it is also present in some taurine (B. taurus) breeds. In order to characterize functional genes involved in tick resistance/susceptibility in cattle, two cDNA libraries were generated using skin tissues of selected Holstein x Gyr animals. A total of 2700 high-quality reads from both resistant and susceptible cDNA were assembled into 458 sequences (contigs) and 834 singletons, with a mean size of 447.7 nucleotides. Assignment of homologous proteins by BLASTX revealed 790 (61.1%) and 300 (23.2%) hits in resistant and susceptible cDNA, respectively; 121 of these hits matched bovine proteins. A total of 502 (38.9%) unique sequences were found to have no significant homology with known sequences and were classified as novel sequences. In general, the most abundant sequences consisted of those coding for hypothetical proteins whose function had not yet been determined, in addition to ribosomal proteins, binding proteins and structural proteins, such as keratin and collagen. The most abundant protein found was collagen type III alpha, although ribosomal proteins accounted for half of the 40 most frequent hits. In addition, five matches within the top 40 best hits corresponded to immune response proteins. These sequences could be used for future studies on functional genomics of cattle tick resistance as well as for genomic sequencing projects.

Treatment of long-term stored DNA—Comparison between different methods to obtain high-quality material

Long‐term stored DNA can be sometimes the only source of genetic material of an organism that does not exist anymore, but a research interest still persists. However, there is a lack of information about useful methods to improve quality from such type of material. In this study, we compared four different protocols using DNA samples collected in 1998. Fresh DNA was also tested aiming to check the differences between these two material types. Sixteen samples of each DNA type treated with phenol‐chloroform with PEG 5.0%, silica‐gel membrane spin column, PEG 7.5%, and glass‐fiber matrix spin column were submitted to spectrophotometer measurements, electrophoresis, PCR, and RFLP‐PCR to assess the best method concerning yield, quality, and purity. Based on the results, purification with PEG 7.5% was considered the best method to treat aged DNA samples. In addition to the efficiency, this protocol has low cost. Analyzing the data, we also conclude that long‐term stored DNA may be considered a reliable and potential resource for future molecular studies.