Signals of Historical Interlocus Gene Conversion in Human Segmental Duplications

Standard methods of DNA sequence analysis assume that sequences evolve independently, yet this assumption may not be appropriate for segmental duplications that exchange variants via interlocus gene conversion (IGC). Here, we use high quality multiple sequence alignments from well-annotated segmental duplications to systematically identify IGC signals in the human reference genome. Our analysis combines two complementary methods: (i) a paralog quartet method that uses DNA sequence simulations to identify a statistical excess of sites consistent with inter-paralog exchange, and (ii) the alignment-based method implemented in the GENECONV program. One-quarter (25.4%) of the paralog families in our analysis harbor clear IGC signals by the quartet approach. Using GENECONV, we identify 1477 gene conversion tracks that cumulatively span 1.54 Mb of the genome. Our analyses confirm the previously reported high rates of IGC in subtelomeric regions and Y-chromosome palindromes, and identify multiple novel IGC hotspots, including the pregnancy specific glycoproteins and the neuroblastoma breakpoint gene families. Although the duplication history of a paralog family is described by a single tree, we show that IGC has introduced incredible site-to-site variation in the evolutionary relationships among paralogs in the human genome. Our findings indicate that IGC has left significant footprints in patterns of sequence diversity across segmental duplications in the human genome, out-pacing the contributions of single base mutation by orders of magnitude. Collectively, the IGC signals we report comprise a catalog that will provide a critical reference for interpreting observed patterns of DNA sequence variation across duplicated genomic regions, including targets of recent adaptive evolution in humans.     

Methods for Detecting Food-borne Enteroviruses

A method previously reported for detecting virus in a model system composed of cottage cheese contaminated with coxsackievirus type A9 has been adapted to detecting selected strains of enteroviruses in a variety of foods. Bentonite is omitted and serum is added for extracting virus from low-protein foods. Samples of foods, usually 25 g, must contain at least 3 to 4 plaque-forming units for a 50% probability of detecting virus. Sensitivity in detecting echovirus type 6 was lower than that for the other viruses used. After extraction from potato salad, poliovirus type 2 was completely reactivated if it had been neutralized with coproantibody, but it was only partially reactivated if neutralized with hyperimmune rabbit serum.     

微卫星DNA检测方法的研究

在检测牙鲆的微卫星变异时,对聚丙烯酰胺凝胶的种类、浓度及其银染方法进行了优化.实验总结了一套适用于微卫星检测的方法.该方法具有灵敏度高、凝胶透明度高、对环境污染小、条带清晰和染色时间短等特点,能显著提高检测分辨率,具有广泛的推广价值.     

内分泌干扰物的生物学检测和评价方法

内分泌干扰物(endocrine disrupters,EDs)是近年来环境科学研究的热点之一。EDs不仅对陆生动物和人类具有潜在的危害,还可以通过不同途径到达水环境中,影响水生动物的生长和繁殖等。因此,建立EDs快速、灵敏的检测和评价方法非常必要。此外,生物检测方法还能在一定程度上反映化学污染物对生物体的毒害效应。本文就研究EDs的离体和在体实验方法进行了介绍,并从个体、组织细胞、生化与分子(蛋白质、酶、RNA、激素)等不同水平上,重点综述了鱼类生物标志物在检测和评价EDs方面的研究进展。     

细胞程序性死亡的判定方法

胞程序性死亡有别于一般意义上的死亡──坏死,所以对其判定显得格外重要．国内外目前常从细胞形态、细胞膜完整性、DNA及某些重要生化指标等方面进行判定．     

两种萤火虫荧光素酶活性检测方法的一致性比较

目的：比较2种萤火虫荧光素酶活性检测方法的一致性。方法：分别采用化学发光技术（Che）及活体光学成像技术（Bio）,从细胞和动物水平检测在转染以萤火虫荧光素酶为报告基因的载体pCI-AAA-Fluc-neo后不同时间点,萤火虫荧光素酶的表达强度。结果：在细胞和动物水平,萤火虫荧光素酶的表达强度均随时间推移逐步递减。在HepG2细胞,萤火虫荧光素酶表达持续96h,活性从24h的2781±220mV（1．6×10^6±2．3×10^5光子）降至96h的49±3．5mV（6．4×10^4±2．5×10^4光子）。在动物水平得到相似的结果,BALB／c小鼠萤火虫荧光素酶表达持续20d,其活性从1d的16592±409mV（1．9×10^8±3．6×10^6光子）降至20d的798±139mV（3．37×10^5±3．8×10^4光子）。通过一致性检验,2种检测方法在细胞和动物水平的直线回归方程分别为lgChe=1．186·lgBio-3．764（r=-0．937,P〈0．001）和lgChe=0．451·lgBio＋0．64（r=0．915,P〈0．001）;进一步将理论数据与实验数据进行配对t检验,二者无统计学差异（P〉0．05）。结论：2种检测方法是一致的;从整个实验过程来看,活体光学成像技术较化学发光法更为简便、直观,可量化地对同一个体连续检测,减少了个体间差异和实验动物用量。     

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1^−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.     

基因芯片技术在病原细菌检测中的应用

基因芯片技术具有快速、高通量、平行化等优点,在病原细菌检测中有广泛的应用前景,选择细菌适宜的靶基因是芯片制备的关键之一。用细菌核糖体基因做靶基因的芯片技术,虽然应用广泛,但仍存在一些不足,随着基因组信息及基因功能的深入研究,包括毒力基因、耐药基因等具有较好种属特异性的细菌基因不断被发现,为芯片技术检测病原细菌提供了更多特异的靶基因,使检测结果更加灵敏、准确,在病原细菌研究中将发挥更大的作用。     

The Evolution of Multigene Families under Intrachromosomal Gene Conversion

A model for the evolution of the probabilities of genetic identity within and between loci of a multigene family in a finite population is formulated and investigated. Unbiased intrachromosomal gene conversion, equal crossing over between tandemly repeated genes, random genetic drift and mutation to new alleles are incorporated. Generations are discrete and nonoverlapping; the diploid, monoecious population mates at random. Formulas for the equilibrium values of the probabilities of identity and a cubic equation for the rate of convergence are deduced. Numerical examples indicate the following. The amount of homology at equilibrium generally decreases as the mutation rate, the population size and the number of repeats increase; it may increase or decrease with increasing crossover rate. The intralocus homology has an intermediate minimum, whereas the interlocus homology increases, as the rate of gene conversion increases. The intralocus homology decreases, whereas the interlocus homology increases, as the proportion of symmetric heteroduplexes increases. The characteristic convergence time can be sufficiently short to imply that intrachromosomal gene conversion may be an important mechanism for maintaining sequence homogeneity among repeated genes. The convergence time decreases as the conversion rate and the proportion of symmetric heteroduplexes increase; although exceptions occur, it generally increases as the population size and the number of repeats increase; it may increase or decrease with increasing crossover rate.     

Gene Duplication and Gene Conversion in the Caenorhabditis elegans Genome

A comprehensive analysis of duplication and gene conversion for 7394 Caenorhabditis elegans genes (about half the expected total for the genome) is presented. Of the genes examined, 40% are involved in duplicated gene pairs. Intrachromosomal or cis gene duplications occur approximately two times more often than expected. In general the closer the members of duplicated gene pairs are, the more likely it is that gene orientation is conserved. Gene conversion events are detectable between only 2% of the duplicated pairs. Even given the excesses of cis duplications, there is an excess of gene conversion events between cis duplicated pairs on every chromosome except the X chromosome. The relative rates of cis and trans gene conversion and the negative correlation between conversion frequency and DNA sequence divergence for unconverted regions of converted pairs are consistent with previous experimental studies in yeast. Three recent, regional duplications, each spanning three genes are described. All three have already undergone substantial deletions spanning hundreds of base pairs. The relative rates of duplication and deletion may contribute to the compactness of the C. elegans genome. Received: 30 July 1998 / Accepted: 12 October 1998     

植物基因分离方法

植物基因分离一直是当今生物技术研究的热点。分离作物重要农艺性状的功能基因利于对基因的结构和表达进行研究,并可以经转基因技术进行分子育种。根据中心法则,介绍了从DNA、RNA到蛋白质的三个层次进行植物基因分离的方法。     

基因功能注释的计算方法

对未知功能的基因进行注释的通常的方法是依据序列同源性分析。近年来,出现了多种不基于序列同源性的基因注释的计算方法,这些方法不依赖于核酸或蛋白质序列的相似性,所能预测的基因的功能属性也有所扩展,如,能够预测基因间相互作用关系。这些方法有效地减少实验材料、时间消耗。该文综述了几种这样的计算方法,包括原理、方法评估及存在的问题。     

Gene Conversion Among Paralogs Results in Moderate False Detection of Positive Selection Using Likelihood Methods

Previous studies have shown that recombination between allelic sequences can cause likelihood-based methods for detecting positive selection to produce many false-positive results. In this article, we use simulations to study the impact of nonallelic gene conversion on the specificity of PAML to detect positive selection among gene duplicates. Our results show that, as expected, gene conversion leads to higher rates of false-positive results, although only moderately. These rates increase with the genetic distance between sequences, the length of converted tracts, and when no outgroup sequences are included in the analysis. We also find that branch-site models will incorrectly identify unconverted sequences as the targets of positive selection when their close paralogs are converted. Bayesian prediction of sites undergoing adaptive evolution implemented in PAML is affected by conversion, albeit in a less straightforward way. Our work suggests that particular attention should be devoted to the evolutionary analysis of recent duplicates that may have experienced gene conversion because they may provide false signals of positive selection. Fortunately, these results also imply that those cases most susceptible to false-positive results—i.e., high divergence between paralogs, long conversion tracts—are also the cases where detecting gene conversion is the easiest. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metachromatic Agar-Diffusion Methods for Detecting Staphylococcal Nuclease Activity

Based on the metachromatic property of Toluidine Blue O, three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 mug/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue O are discussed.     

特定序列DNA检测的几种新方法

特定序列DNA的检测方法通常使用PCR、DNA杂交、连接酶反应等专法。近年来根据不同的啄理又发展出一些新方法,其中比较重要的是基于非酶连接反应、分子信号灯、纳米微粒、以及酶抑制剂-DNA-酶(IDE)的方法。     

Development of Diagnostic Methods for Detecting Ganoderma-infected Oil Palms

Two diagnostic methods have been applied for detecting the plant pathogenic fungus Ganoderma , a basidiomycete, causing the basal stem rot (BSR) diseaseof oil palms. One approach was the use of polyclonal antibodies (PAbs) raised against mycelial proteins of a single Ganoderma isolate and a mixture of nine different Ganoderma isolates. Both PAbs could detect Ganoderma in diseased oil palm root tissue by applying an indirect enzyme-linked immunosorbent assay. Low cross-reactions were observed with the five mainly occurring saprophytic fungi which could be isolated from diseased oil palms and were used as negative controls. The other approach was based on the polymerase chain reaction (PCR) in order to increase the sensitivity of the Ganoderma detection. The primers were generated from the internal transcribed spacer region 1 of rDNA of Ganoderma boninense and produced an PCR product of 167 bp in size. Fungal isolates and oil palm root samples were processed for PCR by three different DNA extraction methods. The most suitable extraction procedure was a simple alkaline extraction method. For a practical approach, a semiquantification was performed by assessing the PCR sensitivity limits of pure culture Ganoderma and naturally infected root samples.     

蛋白质O-GlcNAc修饰的检测技术

O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰是位于细胞浆和细胞核蛋白质的丝氨酸或苏氨酸上的一种翻译后修饰,在高等真核生物细胞中广泛存在.越来越多的研究表明,O-GlcNAc修饰在代谢调控、压力应激、细胞周期、凋亡、糖尿病、心血管疾病和癌症等多种生理和病理过程中发挥重要作用,因此, O-GlcNAc修饰已受到众多生命科学领域研究人员的关注.然而,由于O-GlcNAc修饰与传统的N聚糖和O聚糖修饰有所不同,常规糖基化修饰的检测方法并不适用于O-GlcNAc.本文对O-GlcNAc修饰的检测及其修饰位点的确定方法进行了综述,并分析了各种方法的优缺点.     

基因表达分析方法及其研究进展

近几年来,随着功能基因组学研究的兴起,基因表达研究的分析方法也在不断发展,主要有：差减杂交、差异显示、表达序列标签、基因表达的序列分析、微阵列杂交等。简要评述这五种方法的原理、优缺点等。