High-mobility-group family genes from Lampetra japonica reveal their early origin and molecular evolution in the vertebrate lineage

High-mobility group family (HMG) genes are ubiquitous in vertebrates, including mammals, birds, amphibians and fishes. To elucidate the molecular phylogeny of the HMG genes in the primitive vertebrate, we have cloned three homologues of HMG-box genes, called Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX, from a cDNA library generated from lymphocyte-like cells of the Japanese lamprey (Lampetra japonica), an Agnathan that occupies a critical phylogenetic position between invertebrates and vertebrates. The open reading frames of Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX contained 627 bp, 585 bp and 678 bp, respectively. The analysis of the deduced amino acid sequences indicated that these three putative Lj-HMGB proteins contain four domains: HMG-box A, HMG-box B, an acidic carboxyl-terminal tail and a linker. A phylogenetic analysis revealed that the Lj-HMGB proteins fall outside the vertebrate clade; Lj-HMGBX is descended from a gene ancestral to the mammalian HMGB1/2/3. This discovery implies that there was a gene duplication event in the HMGB1/2/3 gene family that occurred after the divergence of the vertebrates (Cyclostomata) from the Cephalochordata and Urochordata at least 450 million years ago (MYA). The Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX genes were detected in most tissues of the lamprey by RT-PCR. Our findings provide insight into the phylogeny of the HMGB genes in vertebrates.     

Evidence for a quantitative trait locus affecting low levels of apolipoprotein B and low density lipoprotein on chromosome 10 in Caucasian families

High plasma apolipoprotein B (apoB) and LDL cholesterol levels increase cardiovascular disease risk. These highly correlated measures may be partially controlled by common genetic polymorphisms. To identify chromosomal regions that contain genes causing low plasma levels of one or both parameters in Caucasian families ascertained for familial hypobetalipoproteinemia (FHBL), we conducted a whole-genome scan using 443 microsatellite markers typed in nine multigenerational families with at least two members with FHBL. Both variance components and regression-based linkage methods were used to identify regions of interest. Common linkage regions were identified for both measures on chromosomes 10q25.1-10q26.11 [maximum log of the odds (LOD) = 4.2 for LDL and 3.5 for apoB] and 6q24.3 (maximum LOD = 1.46 for LDL and 1.84 for apoB). There was also evidence for linkage to apoB on chromosome 13q13.2 (LOD = 1.97) and to LDL on chromosome 3p14.1 at 94 centimorgan (LOD = 1.52). Bivariate linkage analysis provided further evidence for loci contributing to both traits (6q24.3, LOD = 1.43; 10q25.1, LOD = 1.74). We evaluated single nucleotide polymorphisms (SNPs) in genes within our linkage regions to identify variants associated with apoB or LDL levels. The most significant finding was for rs2277205 in the 5' untranslated region of acyl-coenzyme A dehydrogenase short/branched chain and LDL (P = 10(-7)). Three additional SNPs were associated with apoB and/or LDL (P < 0.01). Although only the linkage signal on chromosome 10 reached genome-wide statistical significance, there are likely multiple chromosomal regions with variants that contribute to low levels of apoB and LDL and that may protect against coronary heart disease.     

氨肽酶N的表达及其与结石形成的关系(英文)

 为研究大鼠高胆固醇饮食时 ,肝脏氨肽酶N(APN)在实验结石形成中可能的结石发生作用 ,采用 1.2 %胆固醇饮食 4周 ,诱发新西兰兔胆囊结石形成 .根据兔APN基因cDNA序列设计引物 ,提取肝脏总RNA .利用RT PCR检测肝脏APNmRNA水平的变化 ,用组织化学方法观察肝脏毛细胆管膜上APN的表达 .观察新西兰兔胆囊结石形成过程中肝脏APN的mRNA水平的变化、APN表达及胆汁中APN活性、胆脂、总蛋白含量的变化 ,探讨APN在胆石形成中可能的作用 .经成石饲料饲养后 ,随着胆汁饱和度增加和APN活性加强 ,胆囊结石组肝脏APNmRNA水平较对照组明显增高 ,胆囊结石组胆汁中总胆固醇、CSI、总蛋白浓度及APN活性均明显高于对照组 ,且胆汁中APN活性与肝脏APN的表达及胆汁CSI增高呈正相关 .结果提示 ,当存在胆汁过饱和的情况下 ,APN很可能作为促成核因子在胆结石形成早期发挥重要作用     

RNAi knockdown of Oryza sativa root meander curling gene led to altered root development and coiling which were mediated by jasmonic acid signalling in rice

Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice.     

A reliable, non-invasive PCR method for giant panda (Ailuropoda melanoleuca) sex identification

We developed an inexpensive, fast and reliable PCR method for sex identification of giant panda (Ailuropoda melanoleuca) by using one pair of primers to co-amplify homologous fragments with size polymorphism that located at amelogenin (AMEL) exon 5. In giant panda, a 63 bp deletion in exon 5 of Y-linked allele provides a significant discrimination between AMELX and AMELY, thus the amplification products can be distinguished simply by agarose gel electrophoresis, exhibiting sex-specific banding patterns (male: 237 bp, 174 bp; female: 237 bp). Both blood and feces samples from known-sex giant pandas were successfully amplified. Cross species test also revealed that this method could be applied to other Ursidae species. These authors contributed equally to this work.     

Genetic differentiation of Pseudoregma bambucicola population based on mtDNA COII gene

mtDNA COII gene sequences were identified and analyzed using different types of software, namely, MEGA5.0, DNAMAN, and DnaSP5.0 in four Chinese provinces, namely, Sichuan, Zhejiang, Guizhou and Shanghai. Analysis of molecular genetic variation and its genetic structure and differentiation, combined with NJ tree, MP tree analysis and analysis of molecular variance (AMOVA), at Fst = 0.0582 conclude that the genetic differentiation is low, gene flow is Nm = 8.0911, and gene exchange is sufficient. However, for the geographic populations of Pseudoregma bambucicola in the four provinces, their gene exchange is relatively weak at Nm = 0.8284, whereas the genetic differentiation is high at Fst = 0.3764. Based on the data, total nucleotide diversity between the populations is 0.00158 ± 0.00021. The results showed that the total population of Tajima’s D and Fu’s Fs results are D = ?0.885 and Fs = 0.226, respectively. The experimental numerical results showed that this total population is not significant (P > 0.10), indicating that nine different geographic populations are short-term. No expansion occurred in the internal population. This study provided a theoretical and practical basis for the comprehensive prevention and control of P. bambucicola.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

细胞外组蛋白在脓毒症、类风湿性关节炎、急性肺损伤等多种疾病的发生发展中起关键作用,但由于缺乏合适的标准品,至今无法对患者体内的胞外组蛋白进行精确定量,导致在多种感染性疾病中无法根据血清组蛋白含量对疾病进行精确分级,也无法据此合理用药。同时,对患者体内胞外组蛋白精确定量也有助于确定细胞毒性机制研究的使用剂量。本研究用大肠杆菌表达单体变性组蛋白H3和H4,亲和纯化后用梯度稀释和透析方法,可以得到复性的组蛋白单体H3、H4以及H3/H4复合物。通过对蛋白质在纯化过程中稳定性的比较,发现H3/H4复合物较单体更为稳定。 以该复合物（50 μg/mL）处理HUVEC细胞,细胞存活率约为20%,与小牛胸腺组蛋白（200 μg/mL）的毒性类似。 该复合物引起的细胞毒性可被人血清白蛋白以浓度依赖的形式（0.625~10 mg/mL）缓解,提示其构象基本正确。 因此,重组组蛋白H3/H4复合物可以作为精确定量组蛋白的标准品,对基于组蛋白含量的疾病分级和组蛋白毒性机制的研究均有应用价值。     

鲈鱼IgM重链基因cDNA的克隆及其原核表达

采用同源克隆和RACE技术扩增到鲈鱼 (Lateolabrax japonicus) 免疫球蛋白M (Immunoglobulin M,IgM) 重链 (Heavy chain,H) 基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。