Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR–RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.     

Molecular cloning and function analysis of insulin-like growth factorbinding protein 1a in blunt snout bream(Megalobrama amblycephala)

Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.     

Molecular characterization and association analysis of porcine CA3

Carbonic anhydrase 3 (CA3) is a member of the carbonic anhydrase family, which plays an important role in various cell processes. In this paper, molecular characterization revealed that CA3 genomic DNA consists of seven exons and six introns, spans about 10.5 kb and maps to porcine chromosome 4q11-->q14. Results of expression profiles showed that the expression levels of CA3 increased in skeletal muscles from prenatal 33- to 65-day-old Chinese Tongcheng pigs. These levels subsequently decreased to a steady state in prenatal 90-day-old, postnatal 2-day-old, postnatal 28-day-old, and pregnant 65-day-old pigs. The expression patterns of Chinese Tongcheng pig embryos were different from that of Landrace pig embryos. CA3 was expressed at higher levels in skeletal muscle and liver than in kidney, lung, stomach, intestine, and brain, but was not detected in heart and spleen. Statistical analysis showed the CA3 gene polymorphism was different between Chinese indigenous and introduced commercial western pig breeds, and was associated with intramuscular fat content and percentage of ham of pigs.     

Molecular cloning,sequence characterization,polymorphism and association analysis of porcine ROPN1 gene

The full-length cDNA sequence of one porcine gene, ROPN1, was isolated using the rapid amplification of cDNA ends (RACE) method based on one pig EST sequence which was highly homologous to the coding sequence of human ROPN1 gene. The porcine ROPN1 gene encodes a protein of 212 amino acids which shares high homology with the rhophilin associated protein 1 (ROPN1) of eight species: gray short-tailed opossum (96%), horse (95%), cattle (94%), mouse (93%), rat (92%), chimpanzee (85%), human (85%) and rhesus monkey (85%). Phylogenetic analysis revealed that the porcine ROPN1 gene has a closer genetic relationship with the ROPN1 gene of gray short-tailed opossum. Polymorphism analysis showed that there was a T/C mutation at the position of 536 bp of mRNA and this leaded to the amino acid alteration from the Arg residue to the Cys residue. PCR-Hae III-RFLP was established to detect this T/C mutation and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of first parity (P < 0.01) and all parities (P < 0.05) in Large White sows, and also significantly associated with the litter size of all parities in Landrace sows (P < 0.01). Therefore, ROPN1 gene could be a useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.     

Molecular cloning and SNP association analysis of chicken PMCH gene

The pre-melanin-concentrating hormone (PMCH) gene is an important gene functionally concerning the regulations of body fat content, feeding behavior and energy balance. In this study, the full-length cDNA of chicken PMCH gene was amplified by SMART RACE method. The single nucleotide polymorphisms (SNPs) in the PMCH gene were screened by comparative sequence analysis. The obtained non-synonymous coding SNPs (ncSNPs) were designed for genotyping firstly. Its effects on growth, carcass characteristics and meat quality traits were investigated employing the F₂ resource population of Gushi chicken crossed with Anak broiler by AluI CRS-PCR–RFLP. Our results indicated that the cDNA of chicken PMCH shared 67.25 and 66.47 % homology with that of human and bovine PMCH, respectively. The deduced amino acid sequence of chicken PMCH (163 amino acids) were 52.07 and 50.89 % identical to those of human and bovine PMCH, respectively. The PMCH protein sequence is predicted to have several functional domains, including pro-MCH, CSP, IL7, XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. gga-miR-18a, gga-miR-18b, gga-miR-499 microRNA targeting site was predicted in the 3′ untranslated region of chicken PMCH mRNA. In addition, a total of seven SNPs including an ncSNP and a synonymous coding SNP, were identified in the PMCH gene. The ncSNP c.81 A > T was found to be in moderate polymorphic state (polymorphic index = 0.365), and the frequencies for genotype AA, AB and BB were 0.3648, 0.4682 and 0.1670, respectively. Significant associations between the locus and shear force of breast and leg were observed. This polymorphic site may serve as a useful target for the marker assisted selection of the growth and meat quality traits in chicken.     

Hub genes identification and association of key pathways with hypoxia in cancer cells: A bioinformatics analysis

Three human cancer cell lines (A549, HCT116, and HeLa) were used to investigate the molecular mechanisms and potential prognostic biomarkers associated with hypoxia. We obtained gene expression data from Gene Expression Omnibus (GEO) datasets GSE11704, GSE147384, and GSE38061, which included 5 hypoxic and 8 control samples. Using the GEO2R tool and Venn diagram software, we identified common differentially expressed genes (cDEGs). The cDEGs were then subjected to Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis by employing DAVID. The hub genes were identified from critical PPI subnetworks through CytoHuba plugin and these genes' prognostic significance and expression were verified using Kaplan-Meier analysis and Gene Expression Profiling Interactive Analysis (GEPIA), respectively. The research showed 676 common DEGs (cDEGs), with 207 upregulated and 469 downregulated genes. The STRING analysis showed 673 nodes and 1446 edges in the PPI network. We identified 4 significant modules and 19 downregulated hub genes. GO analysis revealed all of them were majorly involved in ribosomal large subunit assembly and biogenesis, rRNA processing, ribosome biogenesis, translation, RNA & protein binding frequently at the sites of nucleolus and nucleoplasm while 11 were significantly associated with a better prognosis of hypoxic tumors. Our research sheds light on the molecular mechanisms that underpin hypoxia in human cancer cell lines and identifies potential prognostic biomarkers for hypoxic tumors.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

Molecular analysis of chicken immune response genes

We have recently isolated immune response genes of the major histocompatibility B complex of the chicken (the B-L beta genes) by cross-hybridization in low stringency with an HLA class II beta chain probe. After reviewing the main results obtained, we present a detailed analysis of the region flanking the first gene characterized, B-L beta III. By Southern blot analysis with exon-specific probes, we demonstrate the presence of another related B-L beta gene 10 kb on the 3' side of B-L beta III, the B-L beta V gene. Moreover, retrospective analysis of the phage clones initially isolated with the HLA-DQ beta probe, using a chicken class I probe that we isolated by chromosome walking from the B-L beta genes, indicates that the B-L beta III gene is closely linked on its 5' side to a class I gene, B-FVI.     

Interaction of viruses with cellular response to hypoxia

Recent studies show that low oxygen tension levels in cell culture up-regulate the replication of human B19 parvovirus, Kaposi's sarcoma, and human immunodeficiency viruses as well as the expression of viral oncogenic proteins. The mechanisms of this regulation proceed with the major hypoxia-related factor, HIF-1 (hypoxia inducible factor-1). HIF-1 misregulation is implicated in the oncogenesis potential of some of these viruses.     

Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia

Mammalian cells have the ability to alter their gene expression in order to survive or adapt to a variety of environment stresses including hypoxic stress. Maintaining oxygen supply has been accepted as essential for cell survival and growth. To determine the cellular and molecular changes which take place under oxygen deprivation, an NS0 cell line producing a human-mouse chimeric antibody was cultured under hypoxic conditions (<1%). Various cellular parameters such as viability, productivity, metabolism, apoptosis and cell cycle were studied and notable changes were shown to be accompanied by changes in metabolic rates. When the cells where exposed to hypoxia for 48 h, cell growth was suppressed and cell death was detected. To better understand and explore the mechanisms underpinning these biological alterations and to identify the genes involved in the genetic reprogramming, genome-wide analyses were performed using GeneChip Mouse Genome arrays. The gene expression profiling generated by the microarray technique revealed that hypoxia, even in the early stages (12h), induces significant changes in gene expression in NS0 cells. The primary responses to hypoxia within the cells were: (1) the up-regulation of pathways such as glycolysis that ultimately lead to alternative routes of ATP generation and increased oxygen availability; and (2) the down-regulation of genes involved in purine/pyrimidine and one carbon pool metabolisms required for DNA and RNA synthesis. By combining gene expression and physiological changes under hypoxia, it was possible to explore the mechanisms of hypoxia-induced alterations in more depth.     

Molecular analysis of common polymorphisms within the human Tyrosinase locus and genetic association with pigmentation traits

We have compared the melanogenic activities of cultured melanocytes carrying two common TYR alleles as homozygous 192S‐402R wild‐type, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of protein and DOPAoxidase levels. Homozygous wild‐type strains on average had higher levels of TYR protein and enzyme activity than other genotypes. Homozygous 402Q/Q melanocytes produced significantly less TYR protein, displayed altered trafficking and glycosylation, with reduced DOPAoxidase. However, near wild‐type TYR activity levels could be recovered at lower growth temperature. In a sample population from Southeast Queensland, these two polymorphisms were present on four TYR haplotypes, designated as WT 192S‐402R, 192Y‐402R and 192S‐402Q with a double‐variant 192Y‐402Q of low frequency at 1.9%. Based on cell culture findings and haplotype associations, we have used an additive model to assess the penetrance of the ten possible TYR genotypes derived from the combination of these haplotypes.     

Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker-assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan-European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.