Robertsonian translocation (rob(1;29)) is the most frequent structural chromosomal abnormality in cattle. Heterozygous carriers have a normal phenotype but show a 3-5% decrease in fertility. Chromatin decondensation was evaluated similar to the inactive X chromosome when submitted to demethylating agent. Based on this result, and the concept that imprinted genes are essential in embryonic development, we decided to query genes located on BTA1 and BTA29 that could undergo genome imprinting. The collagen typeVIII-α1 (Col8A1) acted on extracellular matrix structural proteins. DNA bisulfite conversion and sequentiation methods were used to compare its differential methylation patterns. It was performed on eight Creole cattle DNA blood samples from normal and rob(1;29) carriers. An in silico screening for CpG islands in its promoter uncovered a single region of 454 bp prone to methylation. BiQ-Analizer software was used to show the selective conversion of unmethylated cytosines to uracils obtaining the following results: unmethylated CpGs: 0.000 (0 cases), methylated CpGs: 0.802 (77 cases) and CpGs not present: 0.198 (19 cases). No differences between samples were observed in this highly methylated region. This technique was successfully applied so it is a straightforward methodology that can be utilized to evaluate different tissue associated to specific gene expression.

Bovine presents a complex genome due to high number of repetitive sequences involved in macro and micro-rearrangements that are essential in the bovids evolution. However, it has a morphologically simple karyotype, with acrocentric autosomes (2n=60,XX and 60,XY). The Robertsonian translocation rob(1;29), which produces embryo mortality, has been widely described in different breeds of Bos taurus but the complexity of its mechanism and its consequences in chromatin changes, are still not clear. Clastogenic agents are used as an approach to study this rearrangement in chromatin structure. Particularly, the aphidicolin (APC) inhibits the eukaryotic DNA polymerase alpha during replication. This fact permit to identify regions rich in dCTP due to a competition and spreading of the enzyme. Lymphocyte macro culture of 2 female Uruguayan Creole cattle carriers of rob(1;29) were APC-inducted (0,3uM) in one cell cycle. A new break point, rob(1;29)p13/21 (relative distance: p2c/p1=0.45), is located in a late replication region (RBG- band). Two fragile sites (c-fra) in the long arm of rob(1;29) (q13/ 21 and q43) and two other c-fra in BTAX (q12 and q31) were scored. C-fra: rob(1:29)q13/21 y Xq12 were located proximal to centromere in later replication region while rob(1;29)q43 and Xq31 were located proximal to telomere in early replication region. A high incidence in c-fra proximal to centromere in both biarmed chromosomes (BTAXc2=11.3 P<0.001; rob(1;29)c2=4.66P<0.05) was found. Comparing APC-effect: BTA1q13/21 and rob(1;29)q13/21, the high damage found in the second was equivalent to 75% of heterozygosis. This region (RBG- band) has a microsatellite associated to this rearrangement and tissue specific genes.

Los bovinos presentan un genoma complejo debido al número alto de secuencias repetidas involucradas en la formación de macro y micro-rearreglos, que fueron esenciales en la evolución de los bóvidos. Sin embargo presentan un cariotipo de morfología sencilla, con autosomas acrocéntricos (2n=60,XX y 60,XY). La translocación Robertsoniana rob(1;29) está ampliamente descripta en diferentes razas de Bos taurus, determinando mortalidad embrionaria temprana. La complejidad del reordenamiento y sus consecuencias en cambios de la cromatina, no son bien conocidas. Para estudiar este reordenamiento se utilizó afidicolina (APC), un agente clastogénico que inhibe a la ADN polimerasa alfa durante la replicación y permite identificar regiones ricas en dCTP debido a competencia y desplazamiento de la enzima. Se realizaron macro cultivos linfocitarios de 2 hembras de bovinos Criollos Uruguayos portadores de la rob(1;29). Se indujeron con APC (0,3uM) durante un ciclo celular. Se localizó un nuevo punto de ruptura, rob(1;29)p13/21 (distancia relativa: p2c/p1=0.45) en una región de replicación tardía (RBG-). Se analizaron dos sitios frágiles (c-fra) en el brazo largo de la rob(1;29) (q13/21 y q43) y otros dos c-fra en BTAX (q12 y q31). Los c-fra: rob(1:29)q13/21 y Xq12 se ubicaron próximos al centrómero en regiones de replicación tardía, mientras que rob(1;29)q43 y Xq31 se ubicaron próximos al telómero en regiones de replicación temprana. Se encontró una alta incidencia de c-fra proximal al centrómero en ambos cromosomas (BTAXc²=11.3 P<0.001; rob(1;29)c²=4.66P<0.05). Comparando los efectos de APC: BTA1q13/2 y rob1;29q13/21, se encontró mayor daño en el segundo, equivalente a un 75% de heterocigosis. Esta región (RBG-) contiene un microsatélite asociados al reordenamiento y genes tejido-específicos.

La translocación Robertsoniana (rob1; 29) está ampliamente distribuida en razas comerciales y en reservas genéticas de bovinos Criollos americanos. Se ha descrito un enlentecimiento en el desarrollo de embriones portadores de esta aneuploidía, frente a embriones normales. La acción de la 5-aza-C, como agente desmetilante, permitiría descondensar la heterocromatina constitutiva o facultativa. En este trabajo se realiza inducción con el análogo de base 5-aza-C(10mM, 2 hrs) en cultivos linfocitarios de una hembra y un macho portadores de la rob1; 29, frente a bovinos Criollos normales. Se controla la acción desmetilante del inductor al identificar la despiralización del cromosoma X de replicación tardía en hembras y permitir el análisis de la despiralización de la cromatina en múltiples regiones de los autosomas (grandes, medianos, pequeños); de la rob1; 29 y del BTA1. Se discute la correlación existente entre regiones desmetiladas con la descondensación de la heterocromatina facultativa (condicional), relacionándola con la inestabilidad genómica, y la reprogramación epigenética.

The Robertsonian translocation (rob1; 29) is widely spread in commercial breeds and specially in genetic reserve of American Creole cattle. It is also described a delay on embryo development in front of normal ones. The action of 5-aza-C, as an hypomethylated agent, could permitted to decondensate the constitutive or facultative heterochromatin. In this work we made induction with the 5-aza-C(10mM, 2 hrs) analogs, in lymphocyte cultures of female and male carriers and normal Creole cattle. The DNA hypometilation is found in the inactive X chromosome of late replication as it is incorporated during the last hours of cell culture. The decondensing effects of 5-aza-C analogs is observed in multiple regions of the autosomes chromatin, the rob1; 29 and the BTA1. A correlation between hypometilated regions and decondensed of facultative (conditional) heterochromatin is related with genomic instability, and epigenetic reprogramming.

The Uruguayan Creole cattle genetic reserve consists of a herd of about 600 animals (bulls, cows and calves) located in an indigenous habitat of 650 hectares. In a previous study, a random sample from this herd showed high heterozygosity and a Hardy-Weinberg equilibrium for markers of major genes related to milk production. To study its genetic diversity we genotyped a sample of bulls (N = 19 out of 23 for the whole herd) using the PCR reaction with a set of 17 microsatellite markers. Between two and seven different alleles were identified per microsatellite in a total of 73 alleles. The expected mean heterozygosity (He) per locus was between 0.465 and 0.801, except for microsatellite HEL13 which gave a He value of 0.288. The expected mean heterozygosity was 0.623 and the polymorphic information content (PIC) was between 0.266 for HEL13 and 0.794 for CSSM66. The genetic diversity found in polymorphic markers in the breeding bulls of this Creole cattle population supports previous genetic analyses using major production genes and indicate that further studies should be carried out on this population to provide data of interest to cattle production.

The ancestors of Uruguayan Creole cattle were introduced by the Spanish conquerors in the XVII century, following which the population grew extensively and became semi-feral before the introduction of selected breeds. Today the Uruguayan Creole cattle genetic reserve consists of 575 animals. We used the tetra primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) to analyze the kappa-casein, beta-casein, alphaS1-casein and alpha-lactoalbumin gene polymorphisms and restriction fragment length polymorphism PCR (RFLP-PCR) for the beta-lactoglobulin and the acylCoA:diacyl glycerol acyltransferase 1 (DGAT1) genes. The kappa-casein and beta-lactoglobulin genes presented very similar A and B allele frequencies, while the alphas1-casein and alpha-lactoalbumin gene B alleles showed much higher frequencies than the corresponding A alleles. The beta-casein B allele was not found in the population sampled. There was a very high frequency of the DGAT1 gene A allele which is associated with low milk fat content and high milk yield. All loci were in Hardy-Weinberg equilibrium and the level of heterozygosity agreed with the high genetic diversity observed in a previous analysis of this population. Preservation of the allelic richness observed in the Uruguayan Creole cattle should be considered for future dairy management and livestock genetic improvement. The results also emphasize the value of the tetra primers ARMS-PCR technique as a rapid, easy and economical way of genotyping cattle breeds for milk gene single nucleotide polymorphisms.