Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis

Immunologically defined alleles of the pig apolipoprotein B (ApoB) locus (apoB) are correlated with different blood cholesterol levels and predisposition towards premature coronary heart disease. We show here that these alleles are associated with differences in the apoB gene by identifying six restriction fragment length polymorphisms at the pig apoB locus. We have sequenced a 2.4-kb fragment encompassing exons 11 through 14 of one allele, and 7.1 kb from the 3' one-third of exon 26 to about 1 kb past the last exon from another allele. The decoded amino acids of the pig and human ApoB proteins are identical at 70% of these positions. One region close to the C-terminus of the protein is surprisingly different in pigs and humans (57% identity) but the C-terminal region is relatively well conserved (74% identity). Neither of the two putative low-density lipoprotein (LDL) receptor-binding sites is completely conserved in pigs and humans, but identical stretches of amino acids occur near these sites more frequently than in the other sequenced regions. We compare the nucleotide sequences of the region encompassing the putative LDL receptor-binding sites from four pig alleles, including one implicated directly in atherosclerosis. None of the differences appears to account for the hypercholesterolemic phenotype. We conclude that significant differences in the physiology of LDL particles result from changes outside the putative receptor-binding region.     

The robust phylogeny of Korean wild boar (<Emphasis Type="Italic">Sus scrofa coreanus</Emphasis>) using partial D-loop sequence of mtDNA

In order to elucidate the precise phylogenetic relationships of Korean wild boar (Sus scrofa coreanus), a partial mtDNA D-loop region (1,274 bp, NC_000845 nucleotide positions 16576-1236) was sequenced among 56 Korean wild boars. In total, 25 haplotypes were identified and classified into four distinct subgroups (K1 to K4) based on Bayesian phylogenetic analysis using Markov chain Monte Carlo methods. An extended analysis, adding 139 wild boars sampled worldwide, confirmed that Korean wild boars clearly belong to the Asian wild boar cluster. Unexpectedly, the Myanmarese/Thai wild boar population was detected on the same branch as Korean wild boar subgroups K3 and K4. A parsimonious median-joining network analysis including all Asian wild boar haplotypes again revealed four maternal lineages of Korean wild boars, which corresponded to the four Korean wild boar subgroups identified previously. In an additional analysis, we supplemented the Asian wild boar network with 34 Korean and Chinese domestic pig haplotypes. We found only one haplotype, C31, that was shared by Chinese wild, Chinese domestic and Korean domestic pigs. In contrast to our expectation that Korean wild boars contributed to the gene pool of Korean native pigs, these data clearly suggest that Korean native pigs would be introduced from China after domestication from Chinese wild boars.     

牛ACAD8基因结构及其多态性与生产性状的关联分析

乙酰辅酶A脱氢酶(ACAD)是由核基因编码存在于线粒体中黄色酶的一个家族,它催化脂肪酸的α和β-氧化,该家族第八成员负责催化缬氨酸分解代谢。本研究中获得了牛的ACAD8基因的mRNA和基因组DNA序列并通过基因组DNA与mRNA的比对确定了其基因结构。其mRNA序列包含一个1,251 bp的开放阅读框,一个37 bp 的5′非翻译区和一个 444 bp 的 3′非翻译区;其基因组DNA全长13,814 bp,包含11个外显子和10个内含子。我们从样本中随机选取6个个体进行扩增测序,结果表明牛的ACAD8基因13,408位(GenBank登录号:DQ435445)一个A-G单核苷酸多态。运用PCR-RFLP对不同的基因型进行了区分。对该SNP与来自5个品种共178头无亲缘关系的牛的生产性状进行了关联分析,结果表明:该A-G单核苷酸多态对日增重和嫩度有显著影响(P < 0.05)。具有G等位基因的牛比具有A等位基因的牛生长较快,其肉质也较嫩。因此,牛ACAD8基因在该位点的单核苷酸多态可以作为提高生长速度和牛肉嫩度的标记。     

Detection of quantitative trait loci for androstenone, skatole and boar taint in a cross between Large White and Meishan pigs

'Boar taint' is a strong perspiration-like, urine-like unpleasant odour given off upon heating or cooking of meat from some intact (uncastrated) male pigs. Data from the F(2) generation of a Large White (LW) x Meishan (MS) crossbred population were analysed to detect quantitative trait loci (QTL) for traits associated with boar taint. Fat samples from 178 intact male pigs slaughtered at 85 +/- 5 kg were analysed for the major contributors to boar taint (androstenone, indole and skatole). Fat and lean samples from cooked meat were scored for boar, abnormal and pork flavour and odour by a trained sensory panel (SP). A scan with 117 markers covering the whole genome was performed in the F(2) individuals, together with their F(1) parents and purebred grandparents. At the 5% chromosomal significance threshold (approximately equal to the genome-wide suggestive significance threshold), QTL were detected for the laboratory estimate of androstenone on chromosomes 2, 4, 6, 7 and 9. However, only on chromosome 6 were there QTL for boar flavour (BF) traits in the same or adjacent marker intervals as a QTL for the laboratory estimate of androstenone. On chromosome 14, QTL were detected for the laboratory estimates of indole and skatole, the SP score for skatole and the scores for BF in lean and BF in fat. In all five cases, the MS allele generally increased the estimate or score, compared with the LW allele, but it appeared that desirable and undesirable alleles were present in both breeds. This locus on chromosome 14 has considerable potential for use to reduce the incidence of boar taint, especially if further research can identify the causative polymorphism or strongly associated markers.     

6个中国猪地方品种和3个瑞典猪DNA分子系统发育相关关系

线粒体DNA遗传多样性用于评价6个中国地方猪种和3个瑞典家猪系统发育关系。采用PCR和序列分析方法得到了来自9个品种140头猪的线粒体中控制区440bp和细胞色素b基因798bp核苷酸序列。系统发育分析结果表明：6个中国地方猪种起源于亚洲野猪。中国地方猪种和欧洲野猪的线粒体DNA核苷酸序列变异发生在413000－875000年以前,而亚洲紧猪的变异仅发生在7000－156000上以前,由于2000年以前或18世纪初中国猪种导入欧洲家猪,因此瑞典家猪既属于欧洲类也属于亚洲类。     

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus)

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (k1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109–111);2) the four positions N-terminal to the interdomain disulfide bond (167–170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z48308     

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.     

Effects of hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 polymorphisms on fat androstenone level and gene expression in Duroc pigs

A high level of androstenone in porcine adipose tissue is a major factor contributing to boar taint. Porcine hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 (3β‐HSD, also known as HSD3B1) plays a key role in the hepatic metabolism that catalyzes androstenone to β‐androstenol. Therefore, 3β‐HSD is a candidate gene for boar taint. This study aimed to investigate functional 3β‐HSD polymorphisms in Duroc pigs. We found eight single nucleotide polymorphisms (SNPs) in the full‐length porcine 3β‐HSD. Four of the SNPs had restriction enzyme sites, and we genotyped them in 147 uncastrated male Duroc pigs using a polymerase chain reaction–restriction fragment length polymorphism method. Pigs with the GG genotype at the g.165262G>A locus (SNP5) had significantly lower androstenone levels than did those with other genotypes (P = 0.030). SNP5 also was associated with differences in 3β‐HSD mRNA levels: pigs with the GG genotype had higher levels than those with other genotypes (P = 0.019). The SNP5 polymorphism could affect the hepatic catabolism of androstenone and consequently impact androstenone accumulation in the adipose tissue. Therefore, SNP5 in the 3β‐HSD of Duroc pigs could be a useful selective marker for decreasing boar taint.     

野猪及其与家猪杂种猪Myostatin基因3'编码区的克隆和序列分析

Myostatin, which is a highly conservative gene among breeds, is a negative regulator of muscle. The 3' coding region of wild boar and crossbred pig myostatin was cloned by RT - PCR and sequenced. Compared with that of GenBank, the homology of the nucleotide sequence between wild bear and crossbred pig is identical in this region indicating that domestic pigs were evolved from wild boar and there was not changed in this region during the evolution processes.     

Genetic relationship amongst the major non-coding regions of mitochondrial DNAs in wild boars and several breeds of domesticated pigs

We completed phylogenetic analysis of the major non-coding region of the mitochondrial DNA (mtDNA) from 159 animals of eight Euro-American and six East Asian domesticated pig breeds and 164 Japanese and five European wild boars. A total of 62 mtDNA haplotypes were detected. Alignment of these regions revealed nucleotide variations (including gaps) at 73 positions, including 58 sites with transition nucleotide substitutions, and two transversion substitutions. Phylogenetic analysis of the sequences could not organize domestic pig breeds into discrete clusters. In addition, many of the haplotypes found in members of diverged clustering groups were found primarily in Euro-American pig breeds, indicating extensive introgression of Asian domestic pigs into European breeds. Furthermore, phylogenetic analysis allocated the DNA sequences of non-coding regions into two different groups, and the deepest branchpoint of this porcine phylogeny corresponded to 86 000-136 000 years before present. This time of divergence would predate the historical period when the pig is thought to have been domesticated from the wild boar.     

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.     

A single nucleotide polymorphism in 3'-untranslated region contributes to the regulation of Toll-like receptor 4 translation

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.     

A genetic method to distinguish crossbred Inobuta from Japanese wild boars

To distinguish pig-wild boar crossbred Inobuta from Japanese wild boar populations, a genetic method by using mitochondrial DNA (mtDNA) haplotypes and the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) was developed. Sixteen mtDNA haplotypes from 152 wild boars from Kyushu, Shikoku and Honshu islands of Japan were distinct from those from Asian and European domestic pigs. Five alleles of GPIP were classified into two groups: 1). alleles GPIP*1, GPIP*3 and GPIP*3a from Japanese wild boars, Asian wild boars and domestic pigs; 2). alleles GPIP*4 and GPIP*4a from European wild boars and domestic pigs. An extensive genetic survey was done to distinguish the crossbred Inobuta from 60 wild boars hunted on Tsushima Island, Goto Islands, and Nagasaki and Ooita Prefectures. The mtDNA haplotypes from the 60 samples showed Japanese wild boars, but four wild boar samples from Nagasaki Prefecture had the European GPIP allele, GPIP*4. These results showed that nuclear DNA polymorphism analysis is useful, in addition to mtDNA haplotype assay, to detect "Inobuta" having the European genotype from Japanese wild boar populations.     

应用变性高效液相色谱技术建立胸苷酸合成酶基因多态检测平台

TS基因5′非翻译区(5′ untranslation region, 5′UTR)增强子区域(TS enhancer region, TSER)存在28 bp的2次(2R)、3次(3R)的串联重复多态, 在3R等位基因第二次重复中还存在一个G→C的单核苷酸多态性(single nucleotide polymorphisms, SNPs), 同时在3′非翻译区(3′ untranslation region, 3′ UTR)存在6个碱基片段缺失/插入多态。这些多态形式的存在影响了TS基因mRNA的稳定和翻译效率, 并可导致不同TS基因型肿瘤患者对以5-fuorouracil (5-FU)为基础的化疗疗效产生差异。为提高TS基因型临床检测的效率和准确性, 方便、快捷、准确和自动化区分各种纯合及杂合基因型, 设计多重PCR反应, 同时扩增TS基因5′ 和3′ 非翻译区多态所处片段。利用DHPLC技术建立TS基因多态性检测平台, 在非变性条件下, 通过优化DHPLC 洗脱梯度, 同时检测5′ TSER区的串联重复多态和3′ UTR片段长度多态; 在变性条件下, 检测5′ TSER区单核苷酸多态。同时采用PCR-RFLP和DNA 测序方法, 验证DHPLC分析结果。