Molecular cloning and sequencing of porcine<Emphasis Type="Italic"> C5</Emphasis> gene and its association with immunological traits

The complement system helps in the lysis of invading pathogens and modulates the inflammatory as well as the humoral and cellular immune responses. C5 mediates many potent inflammatory and cytolytic events after proteolytic activation by complement convertase enzymes. Hence, to investigate the role of pig C5 (pC5) as a candidate gene for disease resistance in pigs, the complete cDNA of pC5 was sequenced, screened for single nucleotide polymorphisms (SNPs), and an association analysis with various immunological parameters measured in F2 animals of a pig resource population based on a cross of Duroc and Berlin miniature pigs (DUMI) was carried out. In total, 5,422 bp of pC5 cDNA was sequenced, which codes for the 1,677-amino-acid precursor of C5. Four polymorphic sites were detected, one of which was segregating in the DUMI population in three genotypic patterns: AA, AC and CC. Classical (CH50) and alternative (AH50) complement activities, C3c levels, haptoglobin (HP) acute phase protein levels, and antibody titers against Mycoplasma (Mk) and Aujesky (ADV) vaccines were measured in the resource population. Association analysis between C5 and the immunological parameters was carried out using repeated measures mixed and general linear model analysis. The homozygote AA was found to be significantly different from the other two genotypes with respect to AH50 and CH50, whereas genotype CC was found to be significantly different from the other genotypes for C3c and HP levels. No significant difference could be seen between genotypes for antibody titers against vaccinations. Association of C5 with complement activity traits and acute phase proteins promotes pC5 as a candidate gene for innate disease resistance.Nucleotide sequence data reported are available in GenBank database under the accession numbers AY332748 and AY349423     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

The human hyaluronan synthase genes: genomic structures,proximal promoters and polymorphic microsatellite markers

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.     

Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases

We have investigated the molecular basis of selective and complete C1s deficiency in 2-year-old girl with complex autoimmune diseases including lupus-like syndrome, Hashimoto's thyroiditis, and autoimmune hepatitis. This patient's complement profile was characterized by the absence of CH50 activity, C1 functional activity <10%, and undetectable levels of C1s Ag associated with normal levels of C1r and C1q Ags. Exon-specific amplification of genomic DNA by PCR followed by direct sequence analysis revealed a homozygous nonsense mutation in the C1s gene exon XII at codon 534, caused by a nucleotide substitution from C (CGA for arginine) to T (TGA for stop codon). Both parents were heterozygous for this mutation. We used the new restriction site for endonuclease Fok-1 created by the mutation to detect this mutation in the genomic DNA of seven healthy family members. Four additional heterozygotes for the mutation were identified in two generations. Our data characterize for the first time the genetic defect of a selective and complete C1s deficiency in a Caucasian patient.     

绵羊钙调蛋白调节基因Calponin h1 的分子克隆

在一项研究中我们发现雌激素体在胚胎发育后期对绵羊子宫平滑肌Calponin (CaP) 基因的活动有明显上调作用,而CaP一直被作为观察其他基因表达水平变化的基准参照基因（Reference Gene）。迄今为止, 绵羊CaP尚未完整克隆,为进一步了解其结构和功能,根据人、小鼠和家猪的同源保守区序列设计锚定寡核苷酸引物,通过5′-RACE及3′-RACE方法克隆了绵羊子宫平滑肌组织全长CaP h1 cDNA (GenBank登录号： AY327118), 在cDNA序列的基础上, 又通过PCR-SSP方法获得了CaP h1基因除内含子1、2之外的其余4个内含子全部序列 （GenBank登录号分别为：AY771807,AY771808, AY771809, AY771810） 。DNA序列测定和分析表明,绵羊子宫平滑肌CaP h1 cDNA全长1499bp, 编码297个氨基酸,5′-UTR及3′-UTR分别为79bp和529bp。CaP h1基因组DNA的克隆和序列分析表明,绵羊CaP全长约8kb,由 7个外显子和6个内含子组成。 同源序列比较发现,该基因外显子在不同物种间相对保守;与人类、野猪、小鼠、大鼠和鸡Calponin mRNA同源性分别为88％、92％、81％、79％和81％,但不同物种间内含子存在较大差异(>50%)。本研究填补了绵羊CaP基因分子克隆的空白,为进一步研究该基因的功能及子宫平滑肌收缩的调节机理奠定了基础。     

Porcine growth differentiation factor 9 gene polymorphisms and their associations with litter size

Growth differentiation factor 9 (GDF9) is expressed in oocytes and is thought to be required for ovarian folliculogenesis.Given this function,GDF9 may be considered as a candidate gene controlling pig ovulate rate.In this study,the complete coding sequence was cloned (encoding a 444 amino acid),intron sequence and partial 5'-UTR of pig GDF9.RT-PCR results showed that GDF9 mRNA is expressed in a wide range of tissues of the ruttish Erhualian pig.The expression levels of GDF9 mRNA in pituitary,ovary,uterus and oviduct are higher in the Erhualian pigs than those in Duroc pigs,especially in pituitary with a significant difference (P＜0.05).Comparative sequencing revealed 12 polymorphisms,including 8 single nucleotide polymorphisms (SNPS) and one 314 bp indel in noncoding regions,and the other 3 SNPS in coding regions.Four polymorphisms,G359C,C1801T,T1806C and 314 bp indel,were developed as markers for further use in population variation and association studies.The G359C polymorphism segregates only in Chinese native pigs,Erhualian and Dahuabai,on the contrary,314 bp indel segregates only in Duroc and Landrace.C1801T and T1806C sites seem to be completely linked and segregate in Erhualian,Dahuabai and Landrace.In a word,GDF9 may be not associated with pig litter size in extensive populations as per the studies of allele distributions of the four polymorphisms and pilot association in four breeds.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

Alternative Complement Pathway in Schizophrenia

In the present study, we evaluated functional activity of the alternative pathway of complement in schizophrenia by measuring the alternative pathway hemolytic activity (AH50) of complement as well as hemolytic activity of the complement C3 component (C3H50) in the blood of patients with schizophrenia and healthy subjects. To assess the influence of neuroleptic treatment on measured parameters, both drug-free and medicated patients were examined. In addition, correlation analysis between AH50 and C3H50 has been performed. The results of the present study clearly demonstrate upregulation of the alternative complement cascade in schizophrenia and activator effect of neuroleptics on complement alternative pathway. Based upon the results obtained we hypothesize that hyperactivation of the alternative complement pathway in schizophrenia is stimulated by apoptotic cells.     

Single nucleotide polymorphism analysis of exons 3 and 4 of IGF-1 gene in pigs

The IGF-1 gene has been implicated as a candidate gene for the regulation of pig growth traits. We analyzed exons 3 and 4 of IGF-1 gene polymorphisms of the Banna mini-pig (28), the Tibetan mini-pig (30), the Junmu pig (55), and L. Yorkshire species (50) using PCR-SSCP. Three genotypes in exon 3 and 6 genotypes in exon 4 were observed, among which, one single nucleotide polymorphism, G201A, on exon 3 and two single nucleotide polymorphisms, A440G and T455C, on exon 4 were found. Statistical analysis of genotype frequencies revealed that the A allele was dominant in the large pig at the G201A locus (PIC = 0.20-0.34), and the AT alleles were dominant in the large pig at the A440G and T455C loci (PIC = 0.30-0.60). The genotype distribution between the various groups was significantly different (P< 0.01), with the highest heterozygosity seen in Junmu pigs at 0.223 and the lowest seen in L. Yorkshire at 0.098. The genetic distance of the Junmu pig from the L. Yorkshire is the smallest, the distance from the Tibetan miniature pigs is larger, and the distance from the Banna mini-pig is the largest. The IGF-1 gene polymorphism and heterozygosity results from various pig breeds indicate that IGF-1 is substantially polymorphic with significant difference of the polymorphic distribution and expression levels among various pig breeds. This information provides a theoretical basis for the genetic background of miniature pigs but also provides means to breed improved pig varieties.     

The murine Slp gene. Additional evidence that sex-limited protein has no biologic function

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

猪PRDX6基因编码区的多态性及遗传效应分析

PRDX6基因属于抗氧化蛋白超家族,主要在应答氧化压力、脂分解代谢、磷脂分解代谢中发挥作用。根据PRDX6基因的生理生化功能,文章选择其为影响猪肉质性状的候选基因,探索PRDX6基因的多态性与猪肉质性状的关系。首先分离了猪PRDX6的部分编码区序列,并在不同猪种间进行序列比对,发现编码区第4外显子有2个潜在的SNPs分别位于第417 bp处(C/T突变)和第423 bp处(A/G突变),但均没有引起氨基酸的改变。采用Pyrosequencing焦磷酸测序方法对这两个位点在6个猪种和247头F2"大白×梅山"资源家系中进行了遗传变异分析和性状关联分析。结果表明:417C/T位点国外品种均以C等位基因为主,而国内品种均以T等位基因为主,该位点与肌内脂肪、肌肉水分存在显著关联(P<0.05);423A/G位点国外品种均以A等位基因为主,国内品种以G等位基因为主,该位点与失水率、系水力、肌内脂肪及肌肉水分存在显著关联(P<0.05)。由此推断:这两个位点很可能是影响猪肉质性状(尤其是肌肉嫩度)的分子标记。