Characterization of the PGK2 Associated Microsatellite S0719 on SSC7 Suitable for Parentage and QTL Diagnosis

We isolated and characterized the highly polymorphic tetra-nucleotide microsatellite S0719 on SSC7q14-q15 adjacent to the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and assigned it to the USDA-MARC linkage map on SSC7 position 77.5 cM closely linked to markers SW859 (76.3 cM) and SWR2036 (79.0 cM). In a panel of 344 individuals representing 11 pig breeds (European, Chinese, and North American), a total of 32 alleles were observed, and the overall breeds' calculated PIC (polymorphism information content), H_E (heterozygosity), and N_E (effective allele number) were 0.94, 0.94, and 16.41. Breed-specific PIC and H_E ranged from 0.66 to 0.87, whereas N_E was as low as 2.95 and as high as 7.96. Considering the high allelic variation of S0719 within and among pig breeds (79% of the genotyped animals were heterozygous), the marker is useful for individual animal identification and parentage determination. Finally, S0719 is also a valuable STS marker for fine-mapping QTL on SSC7 as position 77.5 cM is located in 25 QTL intervals (http://www.animalgenome.org/QTLdb/).     

Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation

The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy.     

Screening of Pig (Sus scrofa) Bactericidal Permeability-Increasing Protein (BPI) Gene as Marker for Disease Resistance

Salmonella infection can cause septicemia, acute or chronic enteritis and wasting in weaned pigs, but may occur in other age groups. The bactericidal/permeability-increasing protein (BPI) gene plays an important role in the natural defense of the host and is found to be associated with resistance/susceptibility to Salmonella infection and identified as a candidate gene for disease resistance breeding in pig. This study was conducted to screen the resistance and/or susceptibility of pigs to Salmonella infection, to determine the genotype and evaluate presence of resistant allele of the BPI gene in population of pigs, and to establish genetic data for pig breeders for the improvement of Philippine pig industry. In this study, 389 blood samples from different pig breeds were collected from pig breeder farms in the Philippines. Genomic DNA was extracted from these samples and genotyping was done by PCR-RFLP analysis using AvaII restriction enzyme. Out of 389 pigs, the genotypic frequency showed that 98.4, 1.3, and 0.3% pigs are resistant (GG), heterozygous type (AG), and susceptible (AA), respectively. The application of BPI gene as marker for disease resistance will provide information to the pig industry to implement strategies for the identification of Salmonella infection-resistant pigs.     

The investigation of optimal bombardment parameters for transient and stable transgene expression in Sorghum

Summary This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r _s=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1⁻¹ bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.     

PIG7慢病毒表达载体的构建

目的:构建一个含有PIG7基因ORF区的表达载体pPRRL-PIG7-IRES,为研究PIG7基因的功能打下基础。方法:采用RT-PCR方法从经苯丁酸钠处理过的Kasumi-1细胞获得PIG7基因SIMPLE转录本的ORF片段,再用酶切-连接的方法将目的片段亚克隆入慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结果:PIG7基因ORF区成功亚克隆入了慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结论:成功完成了PIG7慢病毒表达载体的构建,为研究PIG7基因的功能打下了基础。     

Phylogenetic analysis and positive-selection site detecting of vascular endothelial growth factor family in vertebrates

Vascular endothelial growth factor (VEGF), known to play an important role in vascular homeostasis, vascular integrity and angiogenesis, is little known about the evolutionary relationship of its five members especially the role of gene duplication and natural selection in the evolution of the VEGF family. In this study, seventy-five full-length cDNA sequences from 33 vertebrate species were extracted from the NCBI's GenBank, UniProt protein database and the Ensembl database. By phylogenetic analyses, we investigated the origin, conservation, and evolution of the VEGFs. Five VEGF family members in vertebrates might be formed by gene duplication. The inferred evolutionary transitions that separate members which belong to different gene clusters correlated with changes in functional properties. Selection analysis and protein structure analysis were combined to explain the relationship of the site-specific evolution in the vertebrate VEGF family. Eleven positive selection sites, one transmembrane region and the active sites were detected in this process.     

PIG11与热休克蛋白60的相互作用介导肝癌HepG2细胞凋亡

为了探讨候选肝癌抑癌蛋白PIG11(p53-induced gene 11,PIG11)诱导细胞凋亡的机制,首次在HepG2细胞株中鉴定了11个PIG11结合蛋白,热休克蛋白60(heat shock protein 60,Hsp60)为其中之一．采用免疫共沉淀联合Western blot 技术对Hsp60进行了验证．用Western blot检测其蛋白质表达,结果显示：pLXSN-PIG11-HepG2细胞中Hsp60蛋白表达较pLXSN-HepG2、HepG2细胞组下调(n=3,P < 0.01)．选取与Hsp60关系密切的Bax蛋白进行研究,Western blot结果显示PIG11高表达可引起胞浆Bax向线粒体转位．以上结果表明,PIG11蛋白能与HepG2细胞中的Hsp60结合,促进Hsp60-Bax的分离,引起Bax从胞液到线粒体转位,激活线粒体凋亡途径,这可能是其诱导HepG2细胞凋亡的主要机制之一．     

Genetic Diversity of Chinese Native Pigs Inferred from Protein Electrophoresis

We examined protein polymorphism of 20 nativepig breeds in China and 3 introduced pig breeds. Thirtyloci have been investigated, among which six loci werefound to be polymorphic. Especially, the polymorphismof malate dehydrogenase (MDH), adenylate kinase(AK), and two new alleles of adenosine deaminase (ADA)had not been reported in domestic pigs and wild pigs.The percentage of polymorphic loci (P), the meanheterozygosity (H), and the mean number ofalleles (A) are 0.200, 0.065, and 1.300, respectively.The degree of genetic variability of Chinese pigs as awhole was higher than that of goats, lower than thatof cattle and horses, and similar to that ofsheep. Using the gene frequencies of the 30 loci, Nei'sgenetic distance among the 20 native breeds in China and3 introduced pig breeds was calculated by theformula of Nei. The program NEIGHBOR in PHYLIP3.5c was chosen to construct an UPGMA tree and a NJtree. Our results show that, of the total geneticvariation found in the native pig breeds in China, 31%(0.31) is ascribable to genetic differencesamong breeds. About 69% of the total genetic variationis found within breeds. Most breeds are in linkagedisequilibrium. The patterns of genetic similaritiesbetween the Chinese native pig breeds were notin agreement with the proposed pig type classification.     

Genetic Diversity of Yunnan Local Pig Breeds Inferred from Blood Protein Electrophoresis

Protein electrophoresis was used to examine theblood protein polymorphism in Yunnan local pig breeds,i.e., the Saba pig, Dahe pig, and Diannan small-ear pigbreeds. Of 38 genetic loci surveyed, 9 were found to be polymorphic. The percentage ofpolymorphic loci (P) varies from 0.1875 to 0.2121, andthe mean individual heterozygosity (H) varies from0.0712 to 0.1027 in three pig breeds. The resultsindicate that blood protein polymorphism in Yunnan pigbreeds is high. Yunnan local pig breeds have a wealth ofgenetic diversity at the level of blood proteins.