Expression profiling of candidate genes for abdominal fat mass in domestic chicken <Emphasis Type="Italic">Gallus gallus</Emphasis>

The quantitative traits of mass and percentage of abdominal fat in chicken and various types of obesity in mammals are homologous and functionally similar. Therefore, the genes involved in obesity development in humans and laboratory rodents as well as those responsible for pig lard thickness could be involved in abdominal fat deposition in broilers. Expression of candidate genes FABP1, FABP2, FABP3, HMGA1, MC4R, PPARG, PPARGC1A, POMC and PTPN1 was studied in fat, liver, colon, muscle, pituitary gland, and brain in chicken (broilers) using real-time PCR. Significant difference in the HMGA1 gene expression in the liver of broiler chicken with high (3.5 ± 0.18%) and low (1.9 ± 0.56%) abdominal fat concentration has been revealed. The expression of this gene was been shown to correlate with the amount (0.7, P ≤ 0.01) and mass (0.7, P ≤ 0.01) of abdominal fat. The PPARG gene expression in liver in the same chicken subsets was also significantly different. Correlation coefficients of the gene expression with the abdominal fat amount and mass were respectively 0.55 (P ≤ 0.05) and 0.57 (P ≤ 0.01). Based on these results, we suggest that the HMGA1 and PPARG genes are involved in abdominal fat deposition. The search for single nucleotide polymorphisms (SNPs) in the HMGA and PPARG regulatory regions could facilitate identifying genetic markers for broiler breeding according to the mass and percentage of abdominal fat.     

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110 000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.     

Characterization of the bovine ampkγ1 gene

AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits.     

Characterization and chromosomal localization of the chicken avidin gene family

Chicken avidin is a biotin-binding protein expressed under inflammation in several chicken tissues and in the oviduct after progesterone induction. The gene encoding avidin belongs to a family that has been shown to include multiple genes homologous to each other. The screening and chromosomal localization studies performed to reveal the structure and organization of the complete avidin gene family is described. The avidin gene family is arranged in a single cluster within a 27-kb genomic region. The cluster is located on the sex chromosome Z on band q21. The organization of the genes was determined and two novel avidin-related genes, AVR6 and AVR7, were cloned and sequenced.     

Libraries of Large-Insert Genomic Clones as a Tool for Molecular Cytogenetic Analysis of Avian Genome

Integration of molecular and cytegenetic levels of investigation results in complex understanding of structural and functional genome organization. Gridded libraries of large-insert genomic clones represent a powerful tool of the genome analysis. Their utilization provides coordination of data on molecular organization of nucleic acids with cytogenetic data on the chromosome structure. These libraries played an important role in sequencing of genomes of human, mouse, and other organisms as an instrument linking molecular biological and cytogenetic data via construction of contigs and their localization on the chromosomes. They also enabled analysis of orthology between the mammalian genomes. The existing avian libraries fit molecular cytogenetic analysis of the class Aves genome, and can be successfully used for the isolation and characterization of large genomic fragments. This provides utilization of these libraries not only for the chromosome mapping, but also for positional cloning and search for candidate genes for quantitative traits.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 581–589.Original Russian Text Copyright © 2005 by Sazanov, Romanov, Smirnov.     

Associations between two novel rSNPs in 5'-flanking region of the bovine casein gene cluster and milk performance traits

Ten evolutionary conservative sequences with high identity level to homological sequences in other mammal species were revealed in 5'-flanking region of casein's genes cluster. Five novel SNPs located inside of the evolutionary conservative regions were identified. The binding sites were revealed to be present in one allelic variant of four detected SNPs. So these SNPs were considered as rSNPs. Significant differences of allelic frequencies were revealed between beef cow's group and dairy cow's group in two rSNPs (NCE4, NCE7, p<0.001). Different alleles of those two rSNPs were shown to be associated with some milk performance traits in Black-and-White Holstein dairy cows. Significant difference of protein percentage has been found between cows with G/G and A/A genotypes (P<0.05) and A/G and A/A genotypes (P<0.05) for NCE4 polymorphism. The groups of animals with genotypes G/G and A/G for NCE7 polymorphism were significantly different in milk yield at the first lactation (kg) (P<0.01), milk fat yield (kg) (P<0.05) and milk protein yield (kg) (P<0.01). For the last trait the difference was significant also between cows with genotypes G/G and A/A for rSNP NCE7 (P<0.05).     

Three-dimensional structure of respiratory complex I from Escherichia coli in ice in the presence of nucleotides

Complex I (NADH:ubiquinone oxidoreductase) is the largest protein complex of bacterial and mitochondrial respiratory chains. The first three-dimensional structure of bacterial complex I in vitrified ice was determined by electron cryo-microscopy and single particle analysis. The structure of the Escherichia coli enzyme incubated with either NAD(+) (as a reference) or NADH was calculated to 35 and 39 A resolution, respectively. The X-ray structure of the peripheral arm of Thermus thermophilus complex I was docked into the reference EM structure. The model obtained indicates that Fe-S cluster N2 is close to the membrane domain interface, allowing for effective electron transfer to membrane-embedded quinone. At the current resolution, the structures in the presence of NAD(+) or NADH are similar. Additionally, side-view class averages were calculated for the negatively stained bovine enzyme. The structures of bovine complex I in the presence of either NAD(+) or NADH also appeared to be similar. These observations indicate that conformational changes upon reduction with NADH, suggested to occur by a range of studies, are smaller than had been thought previously. The model of the entire bacterial complex I could be built from the crystal structures of subcomplexes using the EM envelope described here.