Antibody class switching mediated by yeast endonuclease-generated DNA breaks

Antibody class switching in activated B cells uses class switch recombination (CSR), which joins activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) within two large immunoglobulin heavy chain (IgH) locus switch (S) regions that lie up to 200 kilobases apart. To test postulated roles of S regions and AID in CSR, we generated mutant B cells in which donor Smu and accepter Sgamma1 regions were replaced with yeast I-SceI endonuclease sites. We found that site-specific I-SceI DSBs mediate recombinational IgH locus class switching from IgM to IgG1 without S regions or AID. We propose that CSR evolved to exploit a general DNA repair process that promotes joining of widely separated DSBs within a chromosome.     

Common mechanism of chromosome inversion in B- and T-cell tumors: relevance to lymphoid development

An inversion of chromosome 14 present in the tumor cells of a patient with childhood acute lymphoblastic leukemia of B-cell lineage was shown to be the result of a site-specific recombination event between an immunoglobulin heavy-chain variable gene and the joining segment of a T-cell receptor alpha chain. This rearrangement resulted in the formation of a hybrid gene, part immunoglobulin and part T-cell receptor. Furthermore, this hybrid gene was transcribed into messenger RNA with a completely open reading frame. Thus, two loci felt to be normally activated at distinct and disparate points in lymphocyte development were unified and expressed in this tumor.     

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.     

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.     

Homologous recombination catalyzed by mammalian cell extracts in vitro

An assay was developed to detect recombination events taking place in an in vitro reaction. Extracts of cultured mouse preB lymphocytes were found to catalyze homologous recombination between substrate DNA molecules but not site-specific recombination between cloned mouse immunoglobulin D and J genes. Addition of deoxyribonucleoside triphosphates increased the frequency of homologous recombination. This recombination activity was not observed in two differentiated lymphocyte cell lines.     

Two pairs of recombination signals are sufficient to cause immunoglobulin V-(D)-J joining

The minimum sequence requirements for antigen receptor V-(D)-J joining were studied by constructing recombination-substrates containing synthetic recombination signals and introducing them into a recombination-competent pre-B cell line. Two sets of heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences were shown to be sufficient to cause the V-(D)-J joining, if the 12- and 23-base pair spacer rule is satisfied. A point mutation in the heptamer sequence, or a change in the combination of the two spacer lengths, drastically reduced the recombination.     

AID-driven deletion causes immunoglobulin heavy chain locus suicide recombination in B cells

Remodeling of immunoglobulin genes by activation-induced deaminase (AID) is required for affinity maturation and class-switch recombination in mature B lymphocytes. In the immunoglobulin heavy chain locus, these processes are predominantly controlled by the 3' cis-regulatory region. We now show that this region is transcribed and undergoes AID-mediated mutation and recombination around phylogenetically conserved switchlike DNA repeats. Such recombination, which we term locus suicide recombination, deletes the whole constant region gene cluster and thus stops expression of the immunoglobulin of the B cell surface, which is critical for B cell survival. The frequency of this event is approaching that of class switching and makes it a potential regulator of B cell homeostasis.     

A novel mechanism of somatic rearrangement predicted by a human T-cell antigen receptor beta-chain complementary DNA

The T-cell antigen receptor is a cell surface molecule vital in mediating the cellular immune response. The arrangement and rearrangement of the gene segments encoding the beta-chain polypeptide of the receptor are similar to those of immunoglobulin gene segments. The two constant region genes of the human T-cell antigen receptor are 8 kilobases apart with a cluster of joining segments located 5' of each constant region gene. Although most beta-chain gene rearrangements involve the variable, diversity, and joining segments, analysis of a beta-chain complementary DNA clone suggests the occasional occurrence of another type of rearrangement.     

Active T-cell receptor genes have intron deoxyribonuclease hypersensitive sites

The T-cell receptor beta-chain gene has a nuclease hypersensitive site in several kinds of T cells, which does not appear in B cells expressing immunoglobulins. Conversely, the kappa immunoglobulin gene shows a known hypersensitive site at its enhancer element in B cells, as expected, but this site is absent in T cells. As is the case with immunoglobulin genes, the T-cell receptor site lies within the gene, in the intron separating joining and constant region segments. These nuclease hypersensitive DNA configurations in the introns of active T-cell receptor and immunoglobulin genes may arise from control elements that share ancestry but have diverged to the extent that each normally acts only in lymphoid cells which use the proximal gene product.     

SIRT6 promotes DNA repair under stress by activating PARP1

Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.     

A common mechanism of chromosomal translocation in T- and B-cell neoplasia

The chromosomal breakpoint involved in the t(8;14)(q24;q11) chromosome translocation in the SKW-3 cell line, which directly involves the 3' flanking region of the c-myc gene, was cloned and sequenced. The breakpoint on chromosome 8 mapped to a position 3 kb 3' of c-myc while the chromosome 14 breakpoint occurred 36 kb 5' of the gene for the constant region of the alpha chain of the T-cell receptor (TCR). The translocation resulted in a precise rearrangement of sequences on chromosome 8 and what appears to be a functional J alpha segment on chromosome 14. Signal sequences for V-J joining occurred at the breakpoint positions on both chromosomes 14 and 8, suggesting that the translocation occurs during TCR gene rearrangement and that it is catalyzed by the enzymatic systems involved in V-J joining reactions. The involvement of c-myc in the translocation and the association of joining signals at the breakpoints provides a parallel to the situation observed in the translocations involving c-myc and the immunoglobulin loci in B-cell neoplasms and suggests that common mechanisms of translocation and oncogene deregulation are involved in B- and T-cell malignancies.     

Involvement of mammalian Mus81 in genome integrity and tumor suppression

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.     

CRISPR/Cas9介导基因组编辑技术在植物基因中的研究进展

CRISPR/Cas9介导的基因组编辑技术是在DNA双链的特定位置形成双链断裂,然后通过同源重组或非同源末端连接方式进行修复,造成基因组碱基局部缺失或插入而引起基因突变,它具有操作简单、突变效率高等优势。笔者归纳了CRISPR/Cas9系统的基本结构、分类及其在植物基因中的研究进展和未来的发展方向。     

大鼠热休克因子结合蛋白1原核表达载体的构建及表达

以重组克隆载体pGEM—T—hsbp1为模板,PCR扩增hsbp1,用限制性内切酶双酶切、T4连接酶连接、转化BL -21的方法构建pGEX-2TK-hsbp1原核表达载体,实现了hsbp1在BL21中的表达,SDS—PAGE检测表达的融合蛋白主要以可溶形式存在,并利用亲和层析得到融合蛋白。     

鱼类免疫球蛋白分子生物学研究进展

综述了近年来鱼类免疫球蛋白分子生物学的最新研究进展,主要涉及鱼类Ig基因序列分析、组成形式、重排机制以及转录调控4个方面.鱼类Ig重链和轻链基因克隆分析证实有多种不同的重链、轻链类型存在;重链和轻链基因由不同染色体上的多基因座编码,在不同的鱼类中具有不同的基因组织形式;重组信号序列在重链、轻链基因组中均有发现,其重排过程主要由重组活化酶进行调控;增强子和启动子在鱼类Ig转录调控中发挥重要作用,其中增强子序列在系统发育中具有保守性.Ig作为鱼体特异性体液免疫应答的主要因子,其分子生物学的深入研究对于阐明脊椎动物免疫系统进化具有重要意义.     

冬小麦对除草剂阔世玛的耐药性研究

为了解冬小麦对除草剂阔世玛的耐药性,以晋农190为材料,在冬小麦拨节前、后喷施不同浓度阔世玛,研究阔世玛浓度与倒2叶中丙二醛含量(MDA)、超氧化物歧化酶活性(SOD)和过氧化物酶活性(POD)的相关性。结果表明:拔节前、后处理均增加了倒2叶中MDA含量,并且二者在孕穗期、抽穗期和开花期呈显著正相关,随着阔世玛浓度的增大,冬小麦耐药性降低;拔节前喷施阔世玛未能直接引起SOD活性的升高;拔节前、后阔世玛浓度与POD活性在抽穗期和开花期呈极显著正相关,说明抽穗期和开花期冬小麦耐药性增强。从保护酶角度考虑,建议拔节前喷施0.15～0.30 kg/hm2或拔节后喷施0.30～0.45 kg/hm2阔世玛。     

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.