In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wild-type p53 could be transiently phosphorylated at multiple sites

<p>Abstract</p> <p>Background</p> <p>Infected cells recognize viral replication as a DNA damage stress and elicit the host surveillance mechanism to anti-virus infection. Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through phosphorylation modification for their own purpose. But there is rarely research about p53 phosphorylation status in the context of HPV-E6. Therefore, we investigated whether p53 could be phosphorylated by HPV-E6.</p> <p>Methods</p> <p>We used a mammalian green fluorescence protein (GFP) expression system to express HPV-18E6 with GFP fusion proteins (GFP-18E6) in wild-type (wt) p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and subcellular location of E6 protein. By immunofluorescence technique and immunoblotting, we determined the positive phosphorylated sites of p53 and observed the distribution of phosphorylated p53 in the context of GFP-18E6.</p> <p>Results</p> <p>GFP-18E6 was predominantly located in nuclei of wt p53 cell lines, and it could induce transient phosphorylation of p53 at multiple sites, such as Ser¹⁵, Ser²⁰, and Ser³⁹². All the three sites of phosphorylated p53s were localized in nuclei together with GFP-18E6.</p> <p>Conclusion</p> <p>In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wt p53 could be transiently phosphorylated at multiple sites.</p

Recombinational landscape of porcine X chromosome and individual variation in female meiotic recombination associated with haplotypes of Chinese pigs

<p>Abstract</p> <p>Background</p> <p>Variations in recombination fraction (θ) among chromosomal regions, individuals and families have been observed and have an important impact on quantitative trait loci (QTL) mapping studies. Such variations on porcine chromosome X (SSC-X) and on other mammalian chromosome X are rarely explored. The emerging assembly of pig sequence provides exact physical location of many markers, facilitating the study of a fine-scale recombination landscape of the pig genome by comparing a clone-based physical map to a genetic map. Using large offspring of F₁females from two large-scale resource populations (Large White ♂ × Chinese Meishan ♀, and White Duroc ♂ × Chinese Erhualian ♀), we were able to evaluate the heterogeneity in θ for a specific interval among individual F₁females.</p> <p>Results</p> <p>Alignments between the cytogenetic map, radiation hybrid (RH) map, genetic maps and clone map of SSC-X with the physical map of human chromosome X (HSA-X) are presented. The most likely order of 60 markers on SSC-X is inferred. The average recombination rate across SSC-X is of ~1.27 cM/Mb. However, almost no recombination occurred in a large region of ~31 Mb extending from the centromere to Xq21, whereas in the surrounding regions and in the Xq telomeric region a recombination rate of 2.8-3.3 cM/Mb was observed, more than twice the chromosome-wide average rate. Significant differences in θ among F₁females within each population were observed for several chromosomal intervals. The largest variation was observed in both populations in the interval <it>UMNP71-SW1943</it>, or more precisely in the subinterval <it>UMNP891-UMNP93</it>. The individual variation in θ over this subinterval was found associated with F₁females' maternal haplotypes (Chinese pig haplotypes) and independent of paternal haplotype (European pig haplotypes). The θ between <it>UMNP891 </it>and <it>UMNP93 </it>for haplotype 1122 and 4311 differed by more than fourteen-fold (10.3% vs. 0.7%).</p> <p>Conclusions</p> <p>This study reveals marked regional, individual and haplotype-specific differences in recombination rate on SSC-X. Lack of recombination in such a large region makes it impossible to narrow QTL interval using traditional fine-mapping approaches. The relationship between recombination variation and haplotype polymorphism is shown for the first time in pigs.</p

Cloning, mapping and molecular characterization of porcine progesterone receptor membrane component 2 (PGRMC2) gene.

Progesterone plays an important role in sow reproduction by stimulating classic genomic pathways via nuclear receptors and non-genomic pathways via membrane receptors such a progesterone receptor membrane component 2 (PGRMC2). In this work, we used radiation hybrid mapping to assign PGRMC2 to pig chromosome 8 and observed that this receptor has two transcripts in pigs. The full-length cDNA of the large transcript is 1858 bp long and contains a 669-bp open reading frame (ORF) encoding a protein of 223 amino acids. The shorter transcript encodes a protein of 170 amino acids. The porcine PGRMC2 gene consists of three exons 446 bp, 156 bp and 1259 bp in length. The promoter sequence is GC-rich and lacks a typical TATA box. Several putative cis-regulatory DNA motifs were identified in the 208-bp upstream genomic region. Five single nucleotide polymorphisms (SNPs) were detected in introns* and the 3' UTR. RT-PCR indicated that the PGRMC2 gene is expressed ubiquitously in all pig tissues examined

Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones

BACKGROUND: In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system [1]. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. RESULTS: Seventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (H(T)), and effective allele number (N(E)) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions. CONCLUSION: The sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 bases

Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones

Background: In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system [1]. Traditional isolation methods using partial genomic libraries are time-consuming and costintensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. Results: Seventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (HT), and effective allele number (NE) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions. Conclusion: The sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 base

Fine mapping of a QTL for ear size on porcine chromosome 5 and identification of high mobility group AT-hook 2 (HMGA2) as a positional candidate gene

<p>Abstract</p> <p>Background</p> <p>Ear size and shape are distinct conformation characteristics of pig breeds. Previously, we identified a significant quantitative trait locus (QTL) influencing ear surface on pig chromosome 5 in a White Duroc × Erhualian F₂resource population. This QTL explained more than 17% of the phenotypic variance.</p> <p>Methods</p> <p>Four new markers on pig chromosome 5 were genotyped across this F₂population. RT-PCR was performed to obtain expression profiles of different candidate genes in ear tissue. Standard association test, marker-assisted association test and F-drop test were applied to determine the effects of single nucleotide polymorphisms (SNP) on ear size. Three synthetic commercial lines were also used for the association test.</p> <p>Results</p> <p>We refined the QTL to an 8.7-cM interval and identified three positional candidate genes i.e. <it>HMGA2</it>, <it>SOX5 </it>and <it>PTHLH </it>that are expressed in ear tissue. Seven SNP within these three candidate genes were selected and genotyped in the F₂population. Of the seven SNP, <it>HMGA2 </it>SNP (JF748727: g.2836 A > G) showed the strongest association with ear size in the standard association test and marker-assisted association test. With the F-drop test, F value decreased by more than 97% only when the genotypes of <it>HMGA2 </it>g.2836 A > G were included as a fixed effect. Furthermore, the significant association between g.2836 A > G and ear size was also demonstrated in the synthetic commercial Sutai pig line. The haplotype-based association test showed that the phenotypic variance explained by <it>HMGA2 </it>was similar to that explained by the QTL and at a much higher level than by <it>SOX5</it>. More interestingly, <it>HMGA2 </it>is also located within the dog orthologous chromosome region, which has been shown to be associated with ear type and size.</p> <p>Conclusions</p> <p><it>HMGA2 </it>was the closest gene with a potential functional effect to the QTL or marker for ear size on chromosome 5. This study will contribute to identify the causative gene and mutation underlying this QTL.</p

Genome-wide identification of QTL for age at puberty in gilts using a large intercross F2 population between White Duroc and Erhualian

Puberty is a fundamental development process experienced by all reproductively competent adults, yet the specific factors regulating age at puberty remain elusive in pigs. In this study, we performed a genome scan to identify quantitative trait loci (QTL) affecting age at puberty in gilts using a White Duroc × Erhualian intercross. A total of 183 microsatellites covering 19 porcine chromosomes were genotyped in 454 F2 gilts and their parents and grandparents in the White Duroc × Erhualian intercross. A linear regression method was used to map QTL for age at puberty via QTLexpress. One 1% genome-wise significant QTL and one 0.1% genome-wise significant QTL were detected at 114 cM (centimorgan) on SSC1 and at 54 cM on SSC7, respectively. Moreover, two suggestive QTL were found on SSC8 and SSC17, respectively. This study confirmed the QTL for age at puberty previously identified on SSC1, 7 and 8, and reports for the first time a QTL for age at puberty in gilts on SSC17. Interestingly, the Chinese Erhualian alleles were not systematically favourable for younger age at puberty

Problematika Penyelesaian Sengketa Kewenangan Lembaga Negara Oleh Mahkamah Konstitusi

According to Article 24C verse (1) of the 1945 Constitution, Constitutional court has an authority to examine the dispute among the state institution in which its authority is given by the constitution directly. But there is a certain problem in practice which is related to definition of “state institution” and “authorities are granted the Constitution” in the 1945 Constitution. This condition opens a debate the interpretation in executing the settlement on authority dispute among the institutions. In addition, should be considered the settlement of disputes the authority of institutions, whose authority derived from regulation other than the Constitution Menurut ketentuan Pasal 24C ayat (1) UUD NRI Tahun 1945, penyelesaian sengketa kewenangan lembaga negara yang kewenangannya diberikan oleh UUD merupakan kewenangan Mahkamah Konstitusi. Namun dalam praktiknya, proses penyelesaian sengketa kewenangan lembaga negara menghadapi problem tersendiri seiring tidak adanya batasan ruang lingkup dan definisi “lembaga negara” dan frasa “kewenangannya diberikan UUD” secara pasti dalam UUD NRI Tahun 1945. Situasi ini pada akhirnya menimbulkan multitafsir yang berpotensi mengakibatkan tidak efektifnya penyelesaian sengketa kewenangan lembaga negara di Indonesia. Selain itu, perlu dipikirkan mekanisme penyelesaian sengketa kewenangan lembaga yang kewenangannya bersumber dari peraturan selain UUD

Sexually dimorphic genetic architecture of complex traits in a large-scale F2 cross in pigs

BACKGROUND: It is common for humans and model organisms to exhibit sexual dimorphism in a variety of complex traits. However, this phenomenon has rarely been explored in pigs. RESULTS: To investigate the genetic contribution to sexual dimorphism in complex traits in pigs, we conducted a sex-stratified analysis on 213 traits measured in 921 individuals produced by a White Duroc × Erhualian F(2) cross. Of the 213 traits examined, 102 differed significantly between the two sexes (q value <0.05), which indicates that sex is an important factor that influences a broad range of traits in pigs. We compared the estimated heritability of these 213 traits between males and females. In particular, we found that traits related to meat quality and fatty acid composition were significantly different between the two sexes, which shows that genetic factors contribute to variation in sexual dimorphic traits. Next, we performed a genome-wide association study (GWAS) in males and females separately; this approach allowed us to identify 13.6% more significant trait-SNP (single nucleotide polymorphism) associations compared to the number of associations identified in a GWAS that included both males and females. By comparing the allelic effects of SNPs in the two sexes, we identified 43 significant sexually dimorphic SNPs that were associated with 22 traits; 41 of these 43 loci were autosomal. The most significant sexually dimorphic loci were found to be associated with muscle hue angle and Minolta a* values (which are parameters that reflect the redness of meat) and were located between 9.3 and 10.7 Mb on chromosome 6. A nearby gene i.e. NUDT7 that plays an important role in heme synthesis is a strong candidate gene. CONCLUSIONS: This study illustrates that sex is an important factor that influences phenotypic values and modifies the effects of the genetic variants that underlie complex traits in pigs; it also emphasizes the importance of stratifying by sex when performing GWAS. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12711-014-0076-2) contains supplementary material, which is available to authorized users