Identification of polymorphisms associated with economic traits is important for successful marker-assisted selection in cattle breeding. The family of mammalian sirtuin regulates many biological functions, such as life span extension and energy metabolism. SIRT2, a most abundant sirtuin in adipocytes, acts as a crucial regulator of adipogenic differentiation and plays a key role in controlling adipose tissue function and mass. Here we investigated single nucleotide polymorphisms (SNPs) of bovine SIRT2 in 1226 cattle from five breeds and further evaluated the effects of identified SNPs on economically important traits of Nanyang cattle. Our results revealed four novel SNPs in bovine SIRT2, one was located in intronic region and the other three were synonymous mutations. Linkage disequilibrium and haplotype analyses based on the identified SNPs showed obvious difference between crossbred breed and the other four beef breeds. Association analyses demonstrated that SNPs g.17333C > T and g.17578A > G have a significantly effect on 18-months-old body weight of Nanyang population. Animals with combined genotype TTGG at the above two loci exhibited especially higher body weight. Our data for the first time demonstrated that polymorphisms in bovine SIRT2 are associated with economic traits of Nanyang cattle, which will be helpful for future cattle selection practices.
The big-headed turtle (Platysternon megacephalum) is critically endangered because of overharvesting, illegal trade, and habitat destruction. Assessment of genetic variability in existing populations becomes very important to the taxonomy and conservation of this species. Here we describe 14 microsatellite loci isolated from an enriched genomic library of the big-headed turtle, and the polymorphisms of these loci were assessed in 28 individuals from Huizhou, Heyuan, Zhaoqing, and Shaoguan of Guangdong, China. The range of polymorphism information content is 0.305-0.738, and no evidence of significant linkage disequilibrium was found among any pairs of loci. These 14 new polymorphic microsatellite loci can be used in population genetics, taxonomy, phylogeography, behavior ecology, and conservation efforts of Platysternon megacephalum.
Sparganosis is a zoonotic disease caused by the spargana of Spirometra, and snake is one of the important intermediate hosts of spargana. In some areas of China, snake is regarded as popular delicious food, and such a food habit potentially increases the prevalence of human sparganosis. To understand the prevalence of Spirometra in snakes in food markets, we conducted a study in two representative cities (Guangzhou and Shenzhen), during January-August 2013. A total of 456 snakes of 13 species were examined and 251 individuals of 10 species were infected by Spirometra, accounting for 55.0% of the total samples. The worm burden per infected snake ranged from 1 to 213, and the prevalence in the 13 species was 0?96.2%. More than half (58.1%) of the spargana were located in muscular tissue, 25.6% in subcutaneous tissue, and 16.3% in coelomic cavity. The results indicated that Spirometra severely infected snakes in food markets in Guangzhou and Shenzhen, implying that eating snakes has great health risk and improper cooking methods may increase the risk of Spirometra infection in humans in China. Additional steps should be considered by the governments and public health agencies to prevent the risk of snake-associated Spirometra infections in humans.
As the master regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARG) is required for the accumulation of adipose tissue and hence contributes to obesity. A previous study showed that the substitution of +20A>G in PPARG changed the 7(th) amino acid from Asp to Gly, creating a mutant referred to as PPARG Asp7Gly. In this study, association analysis indicated that PPARG Asp7Gly was associated with lower body height, body weight and heart girth in cattle (P<0.05). Overexpression of PPARG in NIH3T3-L1 cells showed that the Asp7Gly substitution may cause a decrease in its adipogenic ability and the mRNA levels of CIDEC (cell death-inducing DFFA-like effector c) and aP2, which are all transcriptionally activated by PPARG during adipocyte differentiation. A dual-luciferase reporter assay was used to analyze the promoter activity of CIDEC. The results confirmed that the mutant PPARG exhibited weaker transcriptional activation activity than the wild type (P<0.05). These findings likely explain the associations between the Asp7Gly substitution and the body measurements. Additionally, the Asp7Gly mutation may be used in molecular marker assisted selection (MAS) of cattle breeding in the future.
Cell death-inducing DFFA-like effector c (CIDEC) protein, also known as fat specific protein 27 (Fsp27), is localized to lipid droplets. CIDEC protein is required for unilocular lipid droplet formation and optimal energy storage in addition to controlling lipid metabolism in adipocytes and hepatocytes. Research found that Ad-36 could induce lipid droplets in the cultured skeletal muscle cells and this process may be mediated by promoting CIDEC expression. The content of intermuscular fat is an important index for evaluation of beef quality, so the CIDEC gene appeared to be a candidate gene for regulation of intermuscular fat, however similar research for the bovine CIDEC gene is lacking. This paper examined the tissue expression profile of CIDEC gene in cattle using real-time RT-PCR to suggest that bovine CIDEC is highly expressed in adipose tissue. In addition, the Bovine CIDEC gene was cloned and inserted into the eukaryotic expression vector pET-28a(+), whereupon recombinant bovine CIDEC protein was induced and identified by Western-blot. A phylogenetic analysis showed that the animo acid sequence of bovine CIDEC was closer to mammalian CIDEC than rasorial CIDEC. We found ten single nucleotide polymorphisms sites (SNPs) in bovine CIDEC gene, of which SNP 2, 3, 4, 6 and 9, and SNP 8 and 10 were in complete linkage disequilibrium, respectively. SNP 1, 2 and 10 were used in further haplotype studies. Eight different haplotypes were identified in 973 cattle, of which haplotype 8 predominated with frequencies ranging from 42.90 to 54.30 %. This research provides a basis for future functional studies of CIDEC in cattle.
Adiponectin modulates lipid and glucose metabolism in adipose tissues and is also related to bone metabolism. Polymorphisms in the ADIPOQ gene likely have an impact on growth traits in cattle. In this study, we examined the relationship between ADIPOQ polymorphisms and body measurement parameters in Chinese beef cattle. First, we sequenced ADIPOQ and 1.2 kb of DNA upstream of its promoter, and we found 14 polymorphisms. With the luciferase reporter assay, we showed that the two polymorphisms SNP PR_-135 A>G and PR_-68 G>C, which are located in the core region of promoter, influence promoter activity of ADIPOQ. Second, we identified three haplotypes involved in these two polymorphic sites: A (A-135/C-68), B (A-135/G-68), and C (G-135/G-68). Haplotypes B and C are major haplotypes in five Chinese populations of cattle (Qinchuan, Nanyang, Jiaxian, Hazakh, and Chinese Holstein). We studied the effects of these three haplotypes on body measurements, gene expression, and promoter activity, and we found that the genotypes are associated with body measurement parameters in Qinchuan cattle. Individuals with genotype BC (AG/GG) had significantly higher body height and heart girth than others, and this result may be interpreted by the following two observations. The promoter activity with haplotype B (A/G) is significantly higher than those with A (A/C) and C (G/G) in driving reporter gene transcription; the ADIPOQ mRNA level in cattle with genotype BC (AG/GG) is relatively lower than that in cattle with genotype BB (AA/GG).
Growth is under complex genetic control and uncovering the molecular mechanisms how the genes and polymorphisms affect economic growth traits, are important for successful marker-assisted selection and more efficient management strategies in commercial cattle populations. SIRT1 is a NAD(+)-dependent deacetylase that belongs to the class III histone deacetylases. It plays an important role in numerous fundamental cellular processes including gene silencing, DNA repair, and metabolic regulation. In addition, SIRT1 acts as an inhibitor of adipogenesis and has been associated with body weight regulation. The objective of the present study was to identify single nucleotide polymorphisms (SNPs) of bovine SIRT1 using 1255 animals representing the five main Chinese breeds and to determine if these SNPs are associated with economically important traits in Nanyang cattle. The approach consisted of resequencing SIRT1 using a panel of DNA from unrelated animals of five different breeds and the process revealed five novel SNPs. SNPs g.17324T>C and g.17491G>A exhibited a high degree of linkage disequilibrium in all tested breeds. Seven major haplotypes accounting for 91.2% of the alleles were observed and the haplotype GCCGA was the most common haplotype in NY, QC, LX and JX breeds. An association analysis was performed between the five SNPs and six performance traits. SNP g.-274C>G was demonstrated to have a strong effect on 24-months-old body weight and g.17379A>G polymorphism was related to 6 and 12-months-old body weight in NY population, although these effects did not remained significant after the Bonferroni correction. Our results provide evidence that polymorphisms in SIRT1 are associated with growth efficiency traits, and may be used for marker-assisted selection and management in feedlot cattle.
Salmonella Pomona, a highly pathogenic serotype, can cause severe human salmonellosis, especially in children. Turtles and other reptiles are reservoirs for S. Pomona, and these cold-blooded animals remain a source of human Salmonella infections. Since the 1980s, this serotype has become a significant public health concern because of the increasing number of cases of S. Pomona infection in humans. To date, outbreaks of Salmonella Pomona infection in humans have mainly occurred in the United States, with some in other countries (e.g. Belgium, Germany, Canada), and most of the infections in humans were associated with turtles and other reptiles. In China, S. Pomona was first isolated from the feces of an infant in Shanghai in 2000, and two further cases of S. Pomona infection in humans were later found in Guangzhou. No one knew the source of S. Pomona in China. In this study, for the first time we isolated S. Pomona from free-living exotic red-eared sliders in the wild in China. Salmonella serotype (S. Pomona) was isolated from 16 turtle samples. The total carrying rate of S. Pomona in the collected red-eared sliders was 39% (n=41) overall: 40% (n=25) in juveniles and 38% (n=16) in adult turtles. This study suggests that the widespread exotic red-eared sliders may impact on public health and ecosystems of China by transmitting S. Pomona. Additional steps should be considered by the governments and public health agencies to prevent the risk of turtle-associated Salmonella infections in humans in China.
Deleted in breast cancer 1 (DBC1, KIAA1967, p30 DBC) is a novel protein that has been recently shown to bind and regulate SIRT1. Loss of function of DBC1 increased SIRT1 deacetylase activity, which promotes "browning" of WAT by deacetylating peroxisome proliferator-activated receptor (PPAR?) on Lys268 and Lys293. In the present study, we have cloned and characterized the bovine DBC1 gene. Two transcript variants of bovine DBC1 were identified, designated DBC1-A and DBC1-B, respectively, which were both located in nucleus. Protein sequence analysis showed that DBC1-A was well conserved across species. Expression analysis of DBC1 in seven different tissues of calves and bulls by RT-PCR indicated that the two transcripts were ubiquitously expressed. However, the relatively level of DBC1-A was higher when compared to DBC1-B in all examined tissues. Surprisingly, the expression of DBC1-A was extraordinary high in calves adipose tissue, which implicated its potential key role in regulating calve adipocyte development. These findings provide new insight into our understanding of the biochemical characteristics and physiological role of bovine DBC1.
Understanding the function of noncoding regions in the genome, such as introns, is of central importance to evolutionary biology. One approach is to assay for the targets of natural selection. On one hand, the sequence of introns, especially short introns, appears to evolve in an almost neutral manner. Whereas on the other hand, a large proportion of intronic sequence is under selective constraint. This discrepancy is largely dependent on intron length and differences in the methods used to infer selection. We have used a method based on DNA strand asymmetery that does not require comparison with any putatively neutrally evolving sequence, nor sequence conservation between species, to detect selection within introns. The strongest signal we identify is associated with short introns. This signal comes from a family of motifs that could act as cryptic 5 splice sites during mRNA processing, suggesting a mechanistic justification underlying this signal of selection. Together with an analysis of intron length and splice site strength, we observe that the genomic signature of splicing-coupled selection differs between long and short introns.
Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.
Understanding the mechanisms controlling transcription of a gene requires the identification and characterization of its cis-acting regulatory elements. A highly useful approach to the identification and characterization of cis-acting elements has been the systematic coupling of genomic fragments to reporter constructs, so called "promoter bashing". The expression from such reporters must be normalized for differences in transient transfection efficiency between cells and replicates. A novel dual color fluorescent reporter system to assay the promoter activity of a genomic DNA fragment of interest was established by cloning a Discosoma red fluorescent protein gene and a green fluorescent protein gene into a single vector, giving a system in which the ratio between red and green fluorescence is proportional to promoter activity. This system allows real time quantitative monitoring of promoter activity. We validated this approach by assaying the cis-acting regulatory potential of the peroxisome proliferators-activated receptor gamma2 gene.
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