The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining

In this study, the joining sequences between chromosomes 14 and 18 on the 14q+ chromosomes of a patient with pre-B-cell leukemia and four patients with follicular lymphoma carrying a t(14;18) chromosome translocation were analyzed. In each case, the involved segment of chromosome 18 has recombined with the immunoglobulin heavy-chain joining segment (JH) on chromosome 14. The sites of the recombination on chromosome 14 are located close to the 5' end of the involved JH segment, where the diversity (D) regions are rearranged with the JH segments in the production of active heavy-chain genes. As extraneous nucleotides (N regions) were observed at joining sites and specific signal-like sequences were detected on chromosome 18 in close proximity to the breakpoints, it is concluded that the t(14;18) chromosome translocation is the result of a mistake during the process of VDJ joining at the pre-B-cell stage of differentiation. The putative recombinase joins separated DNA segments on two different chromosomes instead of joining separated segments on the same chromosome, causing a t(14;18) chromosome translocation in the involved B cells.     

RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.     

DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes

Immunoglobulin K genes are constructed during lymphocyte differentiation by the joining of two DNA elements, VK and JK, to form both a VKJK coding unit and a reciprocal recombination product. The two products formed in single VK-to-JK joining events can be directly isolated through the use of a retrovirally introduced recombination substrate. The structural analysis of a number of recombinants and the derivation of secondary recombination products define some of the basic features of the mechanism of immunoglobulin gene assembly.     

Virus population dynamics and acquired virus resistance in natural microbial communities

Viruses shape microbial community structure and function by altering the fitness of their hosts and by promoting genetic exchange. The complexity of most natural ecosystems has precluded detailed studies of virus-host interactions. We reconstructed virus and host bacterial and archaeal genome sequences from community genomic data from two natural acidophilic biofilms. Viruses were matched to their hosts by analyzing spacer sequences that occur among clustered regularly interspaced short palindromic repeats (CRISPRs) that are a hallmark of virus resistance. Virus population genomic analyses provided evidence that extensive recombination shuffles sequence motifs sufficiently to evade CRISPR spacers. Only the most recently acquired spacers match coexisting viruses, which suggests that community stability is achieved by rapid but compensatory shifts in host resistance levels and virus population structure.     

球孢白僵菌硝酸还原酶基因同源重组载体的构建

根据已报道球孢白僵菌(Beauveria bassiana)硝酸还原酶基因(nitrate reductase,NR)序列设计引物,从球孢白僵菌D1-5菌株基因组DNA中扩增并克隆该基因上下游片段NR1和NR2,成功构建了球孢白僵菌NR基因同源重组敲除载体,以期敲除球孢白僵菌的NR基因,建立白僵菌同源重组基因敲除体系。     

Endonucleolytic activity that cleaves immunoglobulin recombination sequences

An endonucleolytic activity has been identified in nuclear extracts of chick embryo bursa and mouse fetal liver cells. The activity introduces a double-strand cut in the vicinity of the recombination site of immunoglobulin joining gene segments. The cleavage occurs at the dinucleotide pair TG-AC. This activity is a good candidate for the putative endonuclease involved in recombination of the immunoglobulin variable, diversity, and joining regions. It is distinct from the endonuclease activities previously reported by others.     

Structure of a NHEJ polymerase-mediated DNA synaptic complex

Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway required to maintain genome stability. Many prokaryotes possess a minimalist NHEJ apparatus required to repair DSBs during stationary phase, composed of two conserved core proteins, Ku and ligase D (LigD). The crystal structure of Mycobacterium tuberculosis polymerase domain of LigD mediating the synapsis of two noncomplementary DNA ends revealed a variety of interactions, including microhomology base pairing, mismatched and flipped-out bases, and 3' termini forming hairpin-like ends. Biochemical and biophysical studies confirmed that polymerase-induced end synapsis also occurs in solution. We propose that this DNA synaptic structure reflects an intermediate bridging stage of the NHEJ process, before end processing and ligation, with both the polymerase and the DNA sequence playing pivotal roles in determining the sequential order of synapsis and remodeling before end joining.     

基于叶绿体基因trnL-trnF、trnH-psbA和trnT-trnL序列的甘薯种质遗传多样性分析

【目的】利用叶绿体基因组（cpDNA）的间隔区序列（trnL-trnF、trnH-psbA和trnT-trnL）对甘薯种质进行遗传多样性分析,为其种质保护及开发利用提供理论依据。【方法】以从我国12个省份收集的52份甘薯种质为材料,从10个cpDNA间隔区序列的引物中筛选出能扩增单一、清晰明亮且稳定的序列引物,利用其PCR扩增筛选出间隔区序列,并进行测序及序列拼接。利用DnaSP 5.0进行序列特征分析,采用MEGA X计算52份甘薯种质材料的遗传距离,并构建系统发育进化树。【结果】筛选获得7对扩增结果较理想的引物,其PCR扩增产物经测序分析,共获得3个有效标记（trnL-trnF、trnH-psbA和trnT-trnL）。三者的拼接序列长度为2239 bp,共有7个变异位点,2个单一突变位点,5个简约信息位点,11个插入/缺失位点。在52份甘薯种质材料中,trnL-trnF、trnH-psbA和trnT-trnL序列的变异位点数量（Vs）分别为1、1和5个,单倍型数目（H）分别为2、4和5个,拼接序列的单倍型数目为10个;核苷酸多样性（π）和单倍型多样性（H_dπ）最高的序列分别为trnT-trnL（π=0.00052）和trnH-psbA（H_d=0.535）。trnL-trnF、trnH-psbA和trnT-trnL序列的Tajima’s D、Fu and Li’s D*和Fu and Li’s F*均无显著差异（P>0.05）,符合中性进化模式。基于拼接序列构建的系统发育进化树显示,52份甘薯种质材料的遗传距离为0~1.1848,平均遗传距离0.1018,其分为五大类,其中第Ⅰ类~Ⅳ类仅含有少量种质,其余41份种质归为第Ⅴ类。【结论】52份甘薯种质材料的遗传变异较为丰富,但种质材料间的遗传多样性低,与cpDNA特性和甘薯遗传背景狭窄有关。基于trnL-trnF、trnH-psbA和trnT-trnL的拼接序列更能准确分析甘薯种质的遗传多样性,且有效划分不同类群,为甘薯集团育种提供候选材料。     

CRISPR/Cas9系统在植物基因组定点编辑中的研究进展

当外源DNA通过转基因技术导入植物细胞后,会以同源重组或非同源重组两种不同的方式整合到基因组中,进而获得相应的目标性状。外源DNA与受体细胞序列相同或相近的位点发生重新组合,从而整合到受体细胞的染色体上称之为同源重组;当发生了DNA双链断裂的细胞为了避免DNA或染色体断裂而造成DNA降解或对生命力的影响,而强行将2个DNA断端彼此连接在一起时则为非同源重组。发生非同源重组的细胞其基因组常出现核苷酸片段的插入和/或缺失以及其他突变等多种情况,使得研究者无法得到精确控制的突变结果;而发生同源重组的细胞基因组序列通常不变,通过加入同源重组的供体DNA,可以实现对基因组的精确修饰和改造。由于在植物中产生自发同源重组的概率很低,对植物基因组进行精确修饰和改造非常困难,位点特异性核酸酶的出现和应用,大大提升了同源重组的效率,使基因组编辑变得更加高效和精确,从而使得对包括植物在内的任何物种进行基因组编辑都将成为可能。锌指核酸酶（ZFN）和TALE核酸酶（TALENs）是能够使DNA的靶位点产生DNA双链断裂进而实现基因组定点编辑的常用系统,但在具体应用中发现这两种系统存在着许多缺陷和不足,如脱靶效应、与基因组进行特异结合与染色体位置及邻近序列有关等,另外技术难度大、构建组装时间长也限制了其应用。CRISPR/Cas系统广泛存在于细菌及古生菌中, 是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。Ⅱ型CRISPR/Cas系统经过密码子优化等改造后已成为继锌指核酸酶ZFNs和TALENs后的新型高效定点编辑的新技术,具有突变效率高、制作简单、易操作及成本低的特点。目前,该技术成功应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确编辑,编辑的类型包括基因的定点插入、小片段的缺失、多个位点同时突变、基因定点的indel突变等。目前,CRISPR/Cas系统在植物中的应用还比较有限,但该技术为植物基因工程的发展呈现了美好的前景。文中首先简要介绍了CRISPR/Cas系统的组成和基本原理,进而详细综述了该技术在植物内源基因和外源基因定点编辑中的应用,主要列举了自CRISPR/Cas系统改造成功以来利用该系统对单子叶和双子叶植物进行基因组定点编辑的案例,最后对基因组编辑技术在农业和植物基因工程上的应用进行了展望,希望能够为开展该领域研究的科研工作者提供参考。     

Comparison of fine-scale recombination rates in humans and chimpanzees

We compared fine-scale recombination rates at orthologous loci in humans and chimpanzees by analyzing polymorphism data in both species. Strong statistical evidence for hotspots of recombination was obtained in both species. Despite approximately 99% identity at the level of DNA sequence, however, recombination hotspots were found rarely (if at all) at the same positions in the two species, and no correlation was observed in estimates of fine-scale recombination rates. Thus, local patterns of recombination rate have evolved rapidly, in a manner disproportionate to the change in DNA sequence.     

CO Ⅰ基因不同片段在苹果小卷叶蛾遗传分化中的应用研究

作者于2009年在青岛农业大学昆虫实验室内,利用引物C1-J-1718和2195扩增线粒体DNA的COⅠ基因并对所得到的片段进行序列分析,研究了苹果小卷叶蛾Adoxophyes orana Fischer von Roslerstamm胶东、北京、陕西3个地理种群的遗传分化,并比较了上述两个片段在种群水平系统发育研究中的应用价值。研究结果表明,C1-J-1718扩增序列更适合苹果小卷叶蛾的系统发育研究。胶东种群与北京种群的遗传距离为0.059,与陕西种群为0.049,北京种群与陕西种群为0.047,不同的地理种群遗传分化明显;胶东种群内青岛、威海、烟台地理种群间不存在遗传分化。研究结果还表明:北京种群与A.orana的遗传距离为0;陕西种群的与A.orana like的遗传距离仅为0.007。     

rRNA基因间隔区序列用于亲缘关系密切的根瘤菌种群鉴定及系统发育分析

通过rRNA基因内转录间隔区的碱基序列分析,对中华根瘤菌属Sinorhizobium、慢生根瘤菌属Bradyrhizobium和中慢生根瘤菌属Mesorhizobium种间亲缘关系十分密切的菌株进行区分．结果表明,rRNA基因间隔区序列能很好地区分种间亲缘关系十分密切的菌株．用rRNA基因间隔区序列构建的系统发育树与rRNA基因序列构建的系统发育树结果十分相似．     

由8个DNA片段推断云南金钱槭的系统位置

云南金钱槭是金钱槭属中的濒危物种,其系统地位迄今存在一定争议。本研究利用6个叶绿体基因片段(psbM-trnD、rbcL、trnD-trnT、rpl16、trnL-trnF与psbA-trnH)和2个核基因片段(ITS与CHS)数据,采用最大简约法(MP)及贝叶斯法(BI)对该种及若干近缘类群进行了系统发育分析,从而探讨云南金钱槭的系统位置。结果显示:1)基于6个叶绿体基因联合数据分析,云南金钱槭所在的金钱槭属形成单系群并与槭属彼此独立,互为姐妹属。2)基于CHS数据分析,金钱槭属物种自身形成单系群,但与槭属物种混在一起。3)基于ITS数据分析,金钱槭属为并系群,其中云南金钱槭内嵌于系统发育树的末端与梣叶槭聚在同一进化分枝。本研究中,不同数据集的结果出现冲突,可能是由于不同基因片段进化速率存在差异的影响。此外,核CHS基因在槭树科植物中的有效变异信息位点较少以及核ITS基因存在多拷贝的现象,均可能造成与6个叶绿体基因组合的分析结果出现差异。综合研究结果并参考形态学证据,倾向于云南金钱槭的系统位置继续归属于金钱槭属。进一步增加基因片段可能有助于云南金钱槭系统位置的明确。      

珙桐产黄酮内生真菌的分离和鉴定

采用珙桐(Davidia involucrata Baill.)组织分离产活性物质的内生真菌,以金黄色葡萄球菌、枯草杆菌、大肠杆菌为测试细菌进行内生真菌代谢物对细菌的抑制实验,用高效液相色谱(High Performance Liquid Chromatography,HPLC)分析内生真菌的活性物质成分。结果表明,从珙桐枝、叶中分离到126株内生真菌,其产物对3种细菌的抑菌率分别为65%、71%、25%,其中D37菌株对金黄色葡萄球菌的抑制作用最强。经HPLC分析证明,菌株D37的次生代谢产物中含有槲皮素、山奈酚2种黄酮类物质,每100 g干菌丝可产黄酮31.9 mg。根据菌株D37的形态学特征,结合核糖体基因居间序列(Internaltranscribed spacer,ITS)的分析结果,确定菌株D37为曲霉属,烟曲霉种(Aspergillus fumigatus)。从珙桐分离的产黄酮内生真菌D37可作为生产黄酮类药物的候选菌株,也为抗生素药物的筛选奠定了基础。     

Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX

Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. We report that the Nijmegen breakage syndrome protein (NBS1) and histone gamma-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of VDJ (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and gamma-H2AX form nuclear foci that colocalize with the T cell receptor alpha locus in response to recombination activating gene (RAG) protein-mediated VDJ cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and gamma-H2AX may be important for preventing oncogenic translocations.     

Target-selected inactivation of the zebrafish rag1 gene

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 F1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.     

大鲵2个驯养群体ITS2遗传多样性分析

对大鲵(Andrias davidianus)2个驯化种群的核DNA进行PCR扩增,获得大鲵核DNA上编码核糖体5.8S rRNA和28S rRNA基因的部分序列和完整的ITS2序列(606 bp)。运用DNA分析软件对大鲵2个驯养种群(重庆水产研究所长寿湖珍稀鱼类繁育中心及广汉珍稀鱼类养殖公司)进行了遗传多样性分析。结果显示,该序列平均T、C、A、G含量分别为15.6%、36.5%、12.5%和35.4%,其中G、C的含量C+G(平均为71.9%)显著高于A、T含量A+T(平均为28.1%)。所测序列中有271个碱基发生颠换,112个碱基发生转换,转换与颠换比值(Si/Sv)为0.41,颠换大于转换。10个体均为单倍型,单倍型多样度(H)均为1.000 0±0.126 0,平均核苷酸差异系数(K)为5.932 1±0.861 4,核苷酸多样性(Pi)为0.631 9±0.013 9。种群遗传分化度(Fst)为0.030 5,中性检验及聚类分析表明2个群体没有分化成单一的群体,2个驯养种群遗传多样性好。