No presente estudo, estimou-se a abundância dos transcritos da miostatina foi estimada durante a embriogênese de galinha por análises de RT-PCR competitiva. Níveis basais de mRNA desse gene foram detectados até o estádio HH15, enquanto acúmulos significativos nesses níveis foram observados apenas no estádio HH24, seguido por redução na abundância desses transcritos a partir do estádio HH26. Tais descobertas preliminares proporcionam informações relevantes sobre a ativação do fator de crescimento miostatina durante o desenvolvimento in ovo de aves.

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Important advances have been made in understanding the genetic processes that control skeletal muscle formation. Studies conducted on quails detected a delay in the myogenic program of animals selected for high growth rates. These studies have led to the hypothesis that a delay in myogenesis would allow somitic cells to proliferate longer and consequently increase the number of embryonic myoblasts. To test this hypothesis, recently segmented somites and part of the unsegmented paraxial mesoderm were separated from the neural tube/notochord complex in HH12 chicken embryos. In situ hybridization and competitive RT-PCR revealed that MyoD transcripts, which are responsible for myoblast determination, were absent in somites separated from neural tube/notochord (1.06 and 0.06 10-3 attomol MyoD/1 attomol ß-actin for control and separated somites, respectively; P<0.01). However, reapproximation of these structures allowed MyoD to be expressed in somites. Cellular proliferation was analyzed by immunohistochemical detection of incorporated BrdU, a thymidine analogue. A smaller but not significant (P = 0.27) number of proliferating cells was observed in somites that had been separated from neural tube/notochord (27 and 18 for control and separated somites, respectively). These results confirm the influence of the axial structures on MyoD activation but do not support the hypothesis that in the absence of MyoD transcripts the cellular proliferation would be maintained for a longer period of time.

RESUMO A enterite proliferativa suína (PPE) é uma importante doença descrita em muitos países do mundo. No presente estudo foi utilizada a reação em cadeia pela polimerase (PCR) para detectar o agente Lawsonia intracellularis em amostras de fezes de 1.215 suínos com sinais clínicos de diarréia e idade entre 25 dias e 12 meses. Estas amostras foram colhidas em 207 granjas de suínos de importantes regiões produtoras do Brasil entre janeiro de 1997 e dezembro de 1999. Cento e oitenta e uma amostras de fezes positivas (15%) foram observadas e sessenta e três granjas apresentaram animais positivos (30%). Uma freqüência similar de animais positivos foi observada em algumas faixas etárias: 25 a 42 dias, 70 a 120 dias e 120 a 180 dias, no entanto, suínos com idades variando entre 43 e 70 dias foram significantemente menos positivos para L. intracellularis que entre outras idades. Animais com mais de 180 dias foram significantemente mais afetados por este agente (P < 0,001). Estes resultados reafirmam a importância da PPE nas regiões produtoras de suínos do Brasil. A PCR demonstrou ser um método rápido, sensível e útil para estudos epidemiológicos e diagnóstico da PPE. (PPE mundo (PCR 1215 1 215 1.21 2 meses 20 199 1999 15% 15 (15% 30%. 30 30% . (30%) etárias 4 7 18 entanto L P 0,001. 0001 0,001 0 001 0,001) rápido 121 21 1.2 19 (15 3 (30% 000 0,00 00 1. (1 (30 0,0 ( (3 0,

ABSTRACT Porcine proliferative enteritis is an important enteric disease that has been reported in many countries around the world. In this study, we used polymerase chain reaction (PCR) to detect Lawsonia intracellularis in fecal samples from 1,215 pigs with clinical signs of diarrhea, ages from 25 days to 12 months. These samples were collected in 207 swine herds from important swine producing areas in Brazil between January 1997 and December 1999. A hundred and eighty-one positive fecal samples (15%) were observed and sixty-three herds showed positive animals (30%). A similar frequency of positive animals was observed in some age groups: 25 to 42 day old, 70 to 120 day old, and 120 to 180 day old. However, pigs in the 43 to 70 day old age group were significantly less positive to L. intracellularis detection than others, and animals older than 180 days were significantly more affected by this agent (P < 0.001). These results assert the importance of PPE in the major swine production regions of Brazil. The PCR showed to be a fast, sensitive and useful method in epidemiological studies. world study (PCR 1215 1 215 1,21 diarrhea 2 months 20 199 1999 eightyone eighty one 15% 15 (15% sixtythree sixty three 30%. 30 30% . (30%) groups 4 7 18 However L others P 0.001. 0001 0.001 0 001 0.001) fast studies 121 21 1,2 19 (15 3 (30% 000 0.00 00 1, (1 (30 0.0 ( (3 0.

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Este estudo foi desenvolvido com o objetivo de avaliar as freqüências gênicas em diferentes gerações de bovinos da raça sintética Canchim (5/8 Charolês, 3/8 Zebu). Uma amostra de 154 animais, representando três classes de gerações de um rebanho das raça Canchim, foi analisada para sete marcadores moleculares. Uma amostra da raça Charolesa (N = 36) foi incluída nas análises para permitir comparações. Observou-se um aumento linear, altamente significativo (P < 0,01), na freqüência do alelo que codifica o aminoácido valina na posição 127 do hormônio de crescimento, ao longo das gerações de Canchim. Este alelo foi observado na amostra da raça Charolesa e não é encontrado em raças zebuínas nacionais. Quatro alelos foram observados para o microssatélite localizado na região 5' não transcrita do gene do fator de crescimento semelhante à insulina do tipo I (IGF-I), com tamanhos variando de 231 a 225 pb. A freqüência do alelo de 225 pb apresentou um aumento significativo (P < 0,05) não linear ao longo das gerações. Este alelo não foi observado na raça Charolesa e é predominante nas raças zebuínas. Para o microssatélite CSFM50, seis alelos foram observados na raça Canchim, tendo sido verificada uma redução linear (P < 0,05) na freqüência do alelo de 168 bp. A hipótese de acasalamento preferencial com relação ao polimorfismo do hormônio de crescimento foi reforçada pelos resultados da análise de estatística F de Wright. O valor estimado de FIS para este loco foi 0,59 (P < 0,01). O aumento simultâneo de um alelo do hormônio de crescimento característico de Bos taurus e de um alelo de IGF-I característico de Bos indicus sugere que a seleção fenotípica tenha favorecido regiões diferentes de ambos os genomas que entraram na formação dessa raça.

This study was developed to evaluate selection effects on gene frequencies in a synthetic beef cattle cross (5/8 Charolais, 3/8 Zebu) named Canchim. A sample of 154 Canchim animals, representing three generation classes, was analyzed for seven molecular markers. Thirty-six Charolais cattle were also studied for comparison. A highly significant variation (P < 0.01), with a linear increase of the allele coding for valine at position 127 of the growth hormone peptide, was observed through generations. This allele was found in the Charolais sample but is not observed in Brazilian Bos indicus breeds. Four alleles were found for a microsatellite in the 5' flanking region of insulin-like growth factor I gene in Canchim. Allele sizes ranged from 231 to 225 bp. There was a significant (P < 0.05) nonlinear increase in the 225-bp allele frequency. This allele was not observed in Charolais. Six alleles were observed for microsatellite CSFM50 in the Canchim population. A significant (P < 0.05) linear reduction of the 168-bp allele was observed over the generations. The hypothesis of preferential mating for growth hormone polymorphism was supported by Wright's F-statistics. Estimated FIS value for growth hormone was 0.59 (P < 0.01). These results suggest that selection influenced three out of seven loci analyzed. The simultaneous increases of a Bos taurus-specific growth hormone allele and a Bos indicus-specific insulin-like growth factor I allele indicate that phenotypic selection favored different regions of both genomes.