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Eight novel microsatellite markers were isolated from the woolly mouse opossum from the Amazon Forest in Peru, Micoureus demerarae, using a partial genomic DNA library and an enrichment protocol. These loci were polymorphic in M. demerarae and Micoureus paraguayanus populations from the Atlantic Forest in Brazil with the number of alleles ranging from two to 23. Those eight loci plus another five already described for M. paraguayanus will allow for the evaluation of genetic diversity of populations from the ‘Rio Doce’ Park, one of the last Atlantic Forest fragments in Minas Gerais state, Brazil. [ABSTRACT FROM AUTHOR]

A new, quick, and inexpensive method for detecting the bovine acyl-CoA:diacylglycerol acyltransferase1 (DGAT1) polymorphism (K232A) through tetra-primer amplification refractory mutation system by PCR (ARMS-PCR) was developed in the present investigation. The DGAT1 gene was recently identified as underlying variation in milk production traits. To date, PCR techniques such as PCR-RFLP have been used for detecting the DGAT1 K232A polymorphism, despite being expensive and laborious. The method proposed here, a tetra-primer ARMS-PCR, showed 1009/o sensitivity and specificity when compared with PCR-RFLP results obtained in a sample of 80 animals tested in a double-blind system. Our results suggest that the use of tetra-primer ARMS-PCR for DGAT1 K232A polymorphism genotyping could greatly reduce costs providing information for both research purposes and for dairy cattle breeders who perform DGAT1 genotyping for gene-assisted selection. [ABSTRACT FROM AUTHOR]

Kappa-casein (κ-casein) is one of the most abundant milk proteins. Its main function is to avoid the aggregation of casein micelles, keeping them, and therefore calcium phosphate, in pockets in solution. In bovines, a κ-casein functional polymorphism has been associated with fat, calcium, and protein milk contents and faster curd contraction in cheese production. Quicker curd contraction reduces the loss of milk solids, enhancing cheese yield. This polymorphism induces a double amino acid substitution (Thr136Ile and Ala148Asp). The polymorphism is normally detected by PCR-RFLP, which is a laborious method. An interesting methodological alternative is the tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR). A tetra-primer ARMS-PCR for the detection of this κ-casein polymorphism has been described. However, specificity was not achieved, probably due to problems with primer design. We developed a new tetra-primer ARMS-PCR for the detection of the κ-casein polymorphism. This new method was validated in a double-blind test, by comparison with the results obtained for 50 Guzerá bulls formerly genotyped by PCR-RFLP. This new method achieved 100% sensitivity and specificity. We conclude that this method is a useful, cost-efficient alternative for the detection of functional κ-casein polymorphisms.

Polymorphisms in the TP53 gene codon 72 (Arg72Pro) influence apoptosis induction and DNA damage repair. We evaluated how variants of protein p53 (p53Arg and p53Pro) affect cell death and DNA damage repair by analyzing the frequencies of karyorrhexis and micronuclei. There were significant differences in the frequency of karyorrhexis between the three p53 genotypes (Arg/Arg, Arg/Pro, and Pro/Pro), between samples taken before and after radiotherapy, and between patients and controls. The frequency of micronucleated cells increased significantly after radiotherapy. There were no significant differences in the micronucleus frequency in healthy tissues of these patients compared to controls, or in the comparisons between the three genotypes. We conclude that Arg72Pro polymorphism influences cell apoptotic capacity. This is the first study investigating karyorrhexis and micronuclei, as indicators of apoptosis after radiotherapy, and how these indicators are influenced by the TP53 polymorphism Arg72Pro.

Phenylalanine hydroxylase deficiency is a trait inherited in an autosomal recessive pattern; the associated phenotype varies considerably. This variation is mainly due to the considerable allelic heterogeneity in the phenylalanine hydroxylase enzyme locus. We examined the genotype-phenotype correlation in 54 phenylketonuria (PKU) patients from Minas Gerais, Brazil. Two systems were used. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation and the second was a correlation analysis. An AV was assigned to each mutation: AV = 1 for classical PKU mutation; AV = 2 for moderate PKU mutation; AV = 4 for mild PKU mutation, and AV = 8 for non-PKU hyperphenylalaninemia mutation. The observed phenotype for AV analysis was the clinical diagnosis established by the overloading phenylalanine test. Among the 51 PKU patients that we analyzed based on this trait, in 51% the predicted phenotype did not match the observed phenotype; the highest degree of concordance was found in patients with null/null genotypes. The genotype was observed to be a good predictor of the clinical course of the patients and significant correlations were found between phenylalanine values at first interview and predicted residual activity, genotype and arbitrary value sum.

One hundred and fifteen unrelated Mangalarga Marchador horses were sampled from three geographically distinct regions of Minas Gerais State, Brazil (South, Southeast, and Northeast) and tested for 10 microsatellite loci. Genetic diversity and population structure parameters were estimated with ARLEQUIN 3.0, CERVUS 2.0, POPGENE 1.31, GENEPOP on the web, STRUCTURE 2.0, and SPAGEDI 1.2 software packages. Under Hardy-Weinberg assumptions, seven markers were at equilibrium (LEX014, LEX017, LEX019, SGCV23, TKY321, VHL20, and VIASH39), while two (ASB3 and LEX031) presented significant homozygote excess. Seventy-four alleles were identified in these nine markers, with a mean of 8.22 alleles. Mean heterozygosity was 0.637 and polymorphism information content was 0.662. Markers ASB3, LEX019, SGCV23, TKY321, and VHL20 were highly informative (PIC >0.7) and may be useful for eventual expansion of parentage test panels. The F(ST) value (0.0562) indicated relatively little geographical structure. However, based on a Bayesian-based cluster analysis under a three-cluster model, 94% of the 115 individuals were correctly assigned to the subpopulations from where they were sampled. Mean pairwise f was relatively high (0.11), and in spite of the efforts towards non-consanguineous sampling, 1% of the pairs of individuals shared over 50% of the alleles. These results strongly suggest that the population is genetically structured. Under a conservation genetics approach, two strategies are recommended: avoidance of crosses between highly endogamic individuals and stimulation of crosses between individuals from those regions for which low genetic flow was identified.

Frequencies of kappa-casein gene alleles were determined in 1316 animals from the Brazilian Bos indicus genetic groups (Sindhi cows, Gyr sires, Gyr cows, Guzerat sires, Guzerat cows, Nellore sires, and Gyr x Holstein crossbreds) by means of polymerase chain reaction-restriction fragment length polymorphism analysis using two independent restriction nucleases (Hinf I and HaeIII). The genotyping of kappa-casein alleles (A and B) is of practical importance, since the B allele is found to correlate with commercially valuable parameters of cheese yielding efficiency. The frequencies of the B allele of kappa-casein among breeds ranged from 0.01 to 0.30. The Sindhi breed had the highest frequency for the B allele (0.30), while the frequencies of this allele in other breeds ranged from 0.01 to 0.18. A wide variation in the B allele frequency among B. indicus breeds was found suggesting that molecular selection for animals carrying the B allele could impact breeding programs for dairy production.

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Factors affecting pregnancy rate of 5627 Zebu embryos in crossbred females with unknown proportions of Holstein and Zebu breeding were examined. After evaluation for developmental stage, quality, and viability, embryos were immediately transferred to recipients. Pregnancy diagnosis was conducted approximately 53 d after transfer; pregnancy rate was coded as a binomial event and analyzed using logistic regression models. Maximum likelihood methodology and the likelihood ratio statistic were used to estimate regression coefficients and test hypotheses. Explanatory variables were year of transfer (1992-1999), season of transfer (summer, autumn, winter and spring), breed of the embryo (Guzerat, Gyr or Nellore), stage of the embryo (morula, early blastocyst, blastocyst, expanded blastocyst, and hatching blastocyst), quality of the embryo (excellent, good or regular), and donor-recipient synchrony (estrus in the recipient occurred 2-3 d before, 1 d before, the day of, 1 d after, or 2-3 d after estrus in the donor). Average pregnancy rate was 63.7%. Pregnancy rates were not significantly affected by breed of embryo. The best multiple-logistic model to explain the pregnancy result included the effects of year and season of transfer, embryo stage and quality, and estrous synchrony between donor and recipient (P

Recent reports identified DGAT1 (EC 2.3.1.20) harboring a lysine to alanine substitution (K232A) as a candidate gene with a strong effect on milk production traits. Our objective was to estimate the frequency of the DGAT1 K232A polymorphism in the main Zebu and Taurine breeds in Brazil as well as in Zebu x Taurine crossbreds as a potential QTL for marker-assisted selection. Samples of 331 animals from the main Brazilian breeds, Nellore, Guzerat, Red Sindhi, Gyr, Holstein, and Gyr x Holstein F1 were genotyped for DGAT1 K232A polymorphism (A and K alleles) using the PCR-RFLP technique. The highest frequency of the A allele was found in the Holstein sample (73%) followed by Gyr x Holstein F1 (39%). Gyr and Red Sindhi showed low frequencies of A alleles (4 and 2.5%, respectively). The A allele was not found in the Nellore and Guzerat samples. Our results could be used to guide association studies between this locus and milk traits in these breeds.