An unusual translocation of immunoglobulin gene segments in variants of the mouse myeloma MPC11

Immunoglobulin light chain genes of the mouse are composed in germ-line DNA of four separate segments, the leader, V (variable), J (joining) and C (constant) segments. In immunocompetent cells a V and J gene segment are joined by a site-specific recombination event. In variants of the mouse myeloma MPC11 a so-called kappa (k) light chain fragment is expressed which consists of the MOPC321 leader peptide, joined to the kappa constant region peptide. Using the Southern blotting technique we found that the gene coding for the light chain fragment has apkparently been generated by an aberrant translocation of a V gene segment identical or very similar to the MOPC321 V gene segment into the large intervening sequence between the J and the C gene segments. The resulting deletion of the splice signals of the J segments could be the reason for the observed splicing between leader and C region sequences, a phenomenon which may be of general interest for the understanding of the splicing mechanism.     

Sequences at the somatic recombination sites of immunoglobulin light-chain genes.

The entire nucleotide sequence of a 1.7-kilobase embryonic DNA fragment containing five joining (J) DNA segments for mouse immunoglobulin kappa chain gene has been determined. Each J DNA segment can encode amino acid residues 96--108. Comparison of one of the five J DNA sequences with those of an embryonic variable (V) gene and a complete kappa chain gene permitted localisation of a precise recombination site. The 5'-flanking regions of J DNA segments could form an inverted stem structure with the 3'-non-coding region of embryonic V genes. This hypothetical structure and gel-blotting analysis of total embryo and myeloma DNA suggest that the somatic recombination may be accompanied by excision of an entire DNA segment between a V gene and a J DNA segment. Antibody diversity may in part be generated by modulation of the precise recombination sites.     

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.     

A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation.

The active gene for a kappa light chain is formed by a somatic recombination event that joins one of several hundred variable region genes to one of a series of recombination sites (J-segments) encoded close to the kappa constant region gene. The nucleotide sequences of cloned germ line and somatically recombined genes define the precise organisation of these genetic segments and the site and nature of the recombination event that joined them. Apart from somatic recombination, no further alteration of ther germ line sequence has occurred. The J-segment is of special interest as it encodes signals for both DNA and RNA splicing and provides a means of generating further immunoglobulin gene diversity.     

高致病性猪链球菌双组分系统Ihk/Irr双基因缺失突变株的构建

目的构建Ⅱ型猪链球菌05ZYH33菌株双组分系统Ihk/Irr的双基因缺失突变株.方法首先对双组分系统Ihk/Irr基因进行序列分析;选取Ihk/Irr基因的相关片段克隆至p ET30a表达载体,进行可溶表达并注射家兔制备抗体;分别将ihk基因上游irr基因下游各1 kb的片段扩增,将两个片段连接起来;克隆至温敏型p JRS233的穿梭质粒中;利用两步同源重组法获得突变体;分别提取突变株的基因组DNA,RNA及菌体总蛋白,利用PCR,RT-PCR,Western-blot方法验证突变体.结果成功制备了双组分系统Ihk/Irr可溶性表达的蛋白,成功构建了重组的温敏型ikr-p JRS233重组质粒,成功将重组质粒转入猪链球菌05ZYH33中并获得突变株,基因组PCR,RTPCR及Western-blot分别证实了双组分系统Ihk/Irr双基因被成功敲除.结论成功构建重组温敏型的穿梭质粒,导入到猪链球菌05ZYH33菌株中经两步同源重组获得突变株,在基因组DNA,RNA及蛋白水平上验证了双组分系统Ihk/Irr双基因缺失株的成功构建.     

RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene

Both C kappa regions in MPC 11 cells are rearranged into active transcripion units, one producing a normal kappa chain and the other an internally deleted kappa fragment lacking a V region. The gene coding for the kappa fragment mRNA is aberrantly rearranged and lacks a site for V leads to C kappa splicing. An alternative splicing event which deletes the V region from the nuclear RNA precursor generates the kappa fragment mRNA.     

A novel type of aberrant recombination in immunoglobulin genes and its implications for V-J joining mechanism

Functional kappa light chain genes are formed during B-lymphocyte differentiation by the joining of initially separate V and J gene segments. It has been suggested that the intervening DNA is deleted, however the recent reports of what appear to be the reciprocal products of V and J recombination (back-to-back conserved V and J flanking sequences, called f-fragments) in DNA from mature lymphocytes make a simple deletion model unlikely. An alternative scheme involving unequal sister chromatid exchange has been proposed, supported by the evidence that the f-fragments seem to have segregated from the chromosome carrying the reciprocal complete kappa light chain gene (this and other schemes are briefly reviewed in ref. 8). We report here the analysis of a mouse myeloma (MOPC 41), in which a productive (kappa+) and a non-productive (kappa-) rearrangement has occured, which may help to clarify the mechanism of V-J joining. The aberrant rearrangement has led to the joining of a J1 gene segment to a sequence unrelated to any V gene (L10), and which in the germ line is flanked by a sequence resembling a V region recombination signal sequence. In this case no segregation of the reciprocal recombination products (kappa-41 and f41), which is a required step in sister chromatid exchange models, has taken place. An inversion model provides the simplest explanation of this J rearrangement.     

Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction

The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.     

ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks

Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.     

Deletion of the human T-cell receptor delta-gene by a site-specific recombination

The newly described T-cell receptor (TCR) delta locus is located inside the TCR alpha locus, between variable region (V)alpha and joining region (J)alpha. Although the delta and alpha TCR genes are physically linked on the same chromosome, they are sequentially expressed during T-cell development. This implies the existence of a highly efficient regulatory mechanism by which these two genes are independently rearranged. We have recently described a genetic element 'T early alpha' (TEA) in humans transcribed in foetal thymocytes, spliced alternatively to constant region (C)alpha, and located between the TCR-delta locus (5') and the group of J alpha segments (3'). Importantly, TEA flanks a common site of rearrangement in the thymus, and distinguishes cells using TCR-gamma/delta (TEA in germline configuration) from cells using TCR-alpha/beta (TEA deleted on both chromosomes). In order to understand this TEA-associated recombination we analysed genomic clones representing these thymic rearrangements. We show that the TEA-associated recombination deletes the delta locus before productive (V delta D delta J delta) rearrangement. The diversity (D)delta and J delta regions, which provide the major source of delta gene diversity, are eliminated as a consequence of delta gene deletion and cannot then be used in conjunction with an alpha-TCR. We propose that the TEA-associated deletion of TCR-delta precedes the formation of an alpha-TCR and could down-regulate TCR-delta formation in maturing thymus.     

Organization and sequences of the variable, joining and constant region genes of the human T-cell receptor alpha-chain

An essential property of the immune system is its ability to generate great diversity in antibody and T-cell immune responses. The genetic and molecular mechanisms responsible for the generation of antibody diversity have been investigated during the past several years. The gene for the variable (V) region, which determines antigen specificity, is assembled when one member of each of the dispersed clusters of V gene segments, diversity (D) elements (for heavy chains only) and joining (J) segments are fused by DNA rearrangement. The cloning of the beta-chain of the T-cell antigen receptor revealed that the organization of the beta-chain locus, which is similar to that of immunoglobulin genes, is also composed of noncontiguous segments of V, D, J and constant (C) region genes. The structure of the alpha-chain seems to consist of a V and a C domain connected by a J segment. We report here that the human T-cell receptor alpha-chain gene consists of a number of noncontiguous V and J gene segments and a C region gene. The V region gene segment is interrupted by a single intron, whereas the C region contains four exons. The J segments, situated 5' of the C region gene, are dispersed over a distance of at least 35 kilobases (kb). Signal sequences, which are presumably involved in DNA recombination, are found next to the V and J gene segments.     

Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements

The genes for the T-cell receptor, like the immunoglobulin genes, are rearranged as DNA. The mechanism of this rearrangement is not clear; unequal crossover between chromosomes and the looping-out and excision of the excess DNA have both been suggested. We isolated small polydisperse circular (spc) DNAs from mouse thymocytes and cloned them into a phage vector. Of the 56 clones we analysed, nine contained sequences homologous to T-cell receptor alpha-chain joining (J alpha) segments. We have characterized one of these clones; it contains one J alpha segment, and the product out of the recombination of a variable region of the alpha-chain gene (V alpha) with a J alpha gene segment. This is the first demonstration of the presence in extrachromosomal DNA of a reciprocal recombination product of any rearranging immunoglobulin or T-cell receptor gene. The finding verifies that V alpha-J alpha joining can occur by the looping-out and excision of chromosomal DNA.     

人类抗体重排机制的起源

 重组激活基因（RAG）蛋白介导的抗体重排是脊椎动物适应性免疫系统的核心,抗体重排机制的起源一直是免疫学研究的热点。本研究以有活化石之称的文昌鱼为对象,对多个文昌鱼基因组草图进行深度信息学分析,发现了一个全新DNA转座子ProtoRAG。进一步功能研究表明,ProtoRAG编码的蛋白能够介导自身的转座和宿主DNA的重排,其作用机制与人类抗体蛋白介导的抗体重排机制基本一致。因此,ProtoRAG就是研究人员长期搜寻的祖先RAG转座子的“分子活化石”,该发现为诺贝尔生理学或医学奖获得者利根川进提出的“抗体重排机制转座子起源”假说提供了最有力和最直接的证据。     

Double-stranded cleavage by cell extracts near recombinational signal sequences of immunoglobulin genes

The genes encoding the variable regions of murine immunoglobulin light chains are present in the germ line in two separate segments, V and J. During B lymphocyte differentiation these segments are brought together to form a single unit (for review see ref. 1). Although much is known about the structures of V and J segments, both in germ-line configuration and after rearrangement, essentially nothing is known about the biochemical mechanism of V-J recombination. One possible step in proposed mechanisms of immunoglobulin gene rearrangement is endonucleolytic cleavage of the participating DNA segments before joining. In an attempt to detect such an activity, we have developed an assay for the detection of site-specific double- or single-strand endonucleolytic activity in crude soluble extracts. Using this assay we have detected an activity in extracts of nuclei from mouse B-lymphoid lines and from mouse L cells that is capable of introducing duplex breaks near the recombinational signal sequences of immunoglobulin JK segments. We report the activity here because of its intrinsic interest although we lack any direct evidence that it has a role in V-J recombination.