Single-strand conformation polymorphism analysis of candidate genes for reliable identification of alleles by capillary array electrophoresis

We investigated the reliability of capillary array electrophoresis-single strand conformation polymorphism (CAE-SSCP) to determine if it can be used to identify novel alleles of candidate genes in a germplasm collection. Both strands of three different size fragments (160, 245 and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temperatures (18 degrees C, 25 degrees C, 30 degrees C, and 35 degrees C). Mixtures of amplified fragments of either the intron interrupting the C-terminal WRKY domain of the Tc10 locus or the NBS domain of the TcRGH1 locus of Theobroma cacao were electroinjected into all 16 capillaries of an ABI 3100 Genetic Analyzer and analyzed three times at each temperature. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments. A statistical analysis of the means of the fragment mobilities demonstrated that single-stranded conformers of the fragments could be reliably identified by their mobility at all temperatures and size classes. The order of elution of fragments was not consistent over strands or temperatures for the intermediate and large fragments. If samples are only run once at a single temperature, small fragments could be identified from a single strand at a single temperature. A combination of data from both strands of a single run was needed to identify correctly all four of the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large fragments that differed by only a single single-nucleotide polymorphism (SNP) from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be < or =250 bp, samples should be analyzed at two different temperatures between 18 degrees C and 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures, and undenatured double-stranded (ds)DNA molecular weight standards, such as ROX 2500, should be included as internal standards.     

Expression of human β-defensin-2 with multiple joined genes in Escherichia coli

Human β-defensin (HBD)-2 is a small cationic peptide with a broad range of antimicrobial activity. In this study, multiple copies of the hBD2 gene were linked in tandem, and a number of different Escherichia coli expression vectors were evaluated, including pQE-30, pBV220, pET-28a(+), and pGEX-4T-2. No expression of multiple joined genes was detectable in the pQE-30 expression system, whereas in pBV220 with one or two joined hBD2 genes and in pET-28a(+) with one, two, or four copies, target proteins were expressed at a low level. Only when pGEX-4T-2 was applied as expression plasmid with one or two joined hBD2 genes were target proteins expressed in high level, and the expressed fusion proteins account for 26 and 16% of the total insoluble proteins, respectively. In the pGEX-4T-2 and pET-28a(+) expression systems, the effects of multiple joined genes on the growth of host strains and plasmid stability were examined. Host cells containing plasmid carrying fewer copies of hBD2 genes were faster in cell growth. Plasmid stability decreased with an increase in multiple joined genes, which was especially noticeable in the pET-28a(+) system. Furthermore, the presence of glucose in culture medium brought about a positive effect on plasmid stability when using pET28-nhBD2 as expression vectors.     

基因分类的统计方法

基因组学进行肿瘤分型中的基因筛选方法进行探讨,根据相关分析和95%参考值范围的概念确定筛选基因的方法,该方法利用了所有基因的信息,计算简便,在对白血病患者分型时取得了很好的效果.     

高尿酸血症和痛风易感基因单核苷酸多态性研究新进展及应用

尿酸盐转运子基因是近年全基因组扫描锁定的高尿酸血症和痛风易感基因, 如SLC22A12、SLC2A9、ABCG2基因等, 其编码肾尿酸盐转运系统相关蛋白质, 参与肾脏对尿酸的重吸收和分泌. 目前锁定的尿酸盐转运子基因可解释约7%的血尿酸水平变异, 有影响力的位点期待被发现. 已有研究将这些易感基因应用到药物基因组学及孟德尔随机化研究领域, 是未来新的发展方向.     

用原位杂交技术检测二价染色体上黄鳝rRNA基因多态性的研究

用地戈辛标记的原位杂交技术研究黄鳝二价体上ｒＲＮＡ基因的多态性．共检测到：数目和位置多态；杂交信号形态多态；ｒＲＮＡ基因的联合现象；７号二价体上可移动的ｒＲＮＡ基因位点；ｒＲＮＡ基因的串联重复；ｒＲＮＡ基因杂交信号周围的微信号密集等６种多态现象．还对本研究结果进行了详细地讨论．     

High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes inEscherichia coli

A novel strategy for constructing multiple joined genes of acidic partner-mediated antimicrobial peptide is described. This strategy allows the expression of antimicrobial peptide byEscherichia coli in a stable form and with high yield. Cecropin A (1–8)-melittin (1–10) (CAME) hybrid peptide was selected as a model of antimicrobial peptide. An acidic fragment from magainin intervening sequence was fused to the antimicrobial peptide as a partner to neutralize the lethal effects on the host cells. Multiple copies of the fusion peptide gene were tandemly linked and cloned into the expression vector pET21a. Multimers were expressed at high levels, reaching up to 36% of total cell proteins, and expression levels were proportional to the degree of multimerization. The fusion proteins were mainly expressed as inclusion bodies, probably owing to cysteine residues in the multimers. The target CAME peptide was obtained by cleaving the multimers with cyanogen bromide and purified by cation-exchange chromatography. Recombinant CAME peptide showed strong antimicrobial activities against both Gram-negative and -positive bacteria. These results might provide an efficient solution for high-level expression of various kinds of antimicrobial peptides that are toxic to the host.     

On application of directons to functional classification of genes in prokaryotes

Functional classification of genes represents one of the most basic problems in genome analysis and annotation. Our analysis of some of the popular methods for functional classification of genes shows that these methods are not always consistent with each other and may not be specific enough for high-resolution gene functional annotations. We have developed a method to integrate genomic neighborhood information of genes with their sequence similarity information for the functional classification of prokaryotic genes. The application of our method to 93 proteobacterial genomes has shown that (i) the genomic neighborhoods are much more conserved across prokaryotic genomes than expected by chance, and such conservation can be utilized to improve functional classification of genes; (ii) while our method is consistent with the existing popular schemes as much as they are among themselves, it does provide functional classification at higher resolution and hence allows functional assignments of (new) genes at a more specific level; and (iii) our method is fairly stable when being applied to different genomes.     

A Glossary for Chemical Approaches towards Unlocking the Trove of Metabolic Treasures in Actinomycetes

Actinobacterial natural products showed a critical basis for the discovery of new antibiotics as well as other lead secondary metabolites. Varied environmental and physiological signals touch the antibiotic machinery that faced a serious decline in the last decades. The reason was exposed by genomic sequencing data, which revealed that Actinomycetes harbor a large portion of silent biosynthetic gene clusters in their genomes that encrypt for secondary metabolites. These gene clusters are linked with a great reservoir of yet unknown molecules, and arranging them is considered a major challenge for biotechnology approaches. In the present paper, we discuss the recent strategies that have been taken to augment the yield of secondary metabolites via awakening these cryptic genes in Actinomycetes with emphasis on chemical signaling molecules used to induce the antibiotics biosynthesis. The rationale, types, applications and mechanisms are discussed in detail, to reveal the productive path for the unearthing of new metabolites, covering the literature until the end of 2020.