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1.
Finette BA 《DNA Repair》2006,5(9-10):1049-1064
Pediatric acute lymphocytic leukemia (ALL) is a multifactorial malignancy with many distinctive developmentally specific features that include age specific acquisition of deletions, insertions and chromosomal translocations. The analysis of breakpoint regions involved in these leukemogenic genomic rearrangements has provided evidence that many are the consequence of V(D)J recombinase mediated events at both immune and non-immune loci. Hence, the direct investigation of in vivo genetic and epigenetic features in human peripheral lymphocytes is necessary to fully understand the mechanisms responsible for the specificity and frequency of these leukemogenic non-immune V(D)J recombinase events. In this review, I will present the utility of analyzing mutagenic V(D)J recombinase mediated genomic rearrangements at the HPRT locus in humans as an in vivo model system for understanding the mechanisms responsible for leukemogenic genetic alterations observed in children with leukemia. 相似文献
2.
Activation of V(D)J recombination induces the formation of interlocus joints and hybrid joints in scid pre-B-cell lines 总被引:2,自引:0,他引:2
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V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/+) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/+ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations. 相似文献
3.
Su W Boursier L Padala A Sanderson JD Spencer J 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(4):2360-2366
Human intestinal lamina propria plasma cells are considered to be the progeny of chronically stimulated germinal centers located in organized gut-associated lymphoid tissues such as Peyer's patches and isolated lymphoid follicles. We have sampled human colonic lamina propria plasma cells and naive and memory B cell subsets from human Peyer's patches by microdissection of immunohistochemically stained tissue sections and used PCR methods and sequence analysis to compare IgVlambdaJlambda rearrangements in the plasma cell and B cell populations. Rearrangements that were either in-frame or out-of-frame between V and J were compared. Usage of IgVlambda families in the in-frame rearrangements from the plasma cells resembled that observed in the mantle cells, suggesting that antigenic selection for cellular specificity does not dramatically favor any particular Vlambda segment. However, in marked contrast, out-of-frame rearrangements involving Vlambda1 and Vlambda2 families are rarely observed in intestinal plasma cells, whereas rearrangements involving Vlambda5 are increased. This resulted in significantly biased ratios of in-frame:out-of-frame rearrangements in these Vlambda families. Out-of-frame rearrangements of IgVlambdaJlambda from plasma cells, including those involving the Vlambda5 family, have a significant tendency not to involve Jlambda1, consistent with the hypothesis that this population includes rearrangements generated by secondary recombination events. We propose that modification of out-of-frame rearrangements of IgVlambdaJlambda exists, probably a consequence of secondary rearrangements. This may be a mechanism to avoid translocations to susceptible out-of-frame IgVlambdaJlambda rearrangements during somatic hypermutation. 相似文献
4.
Langerak AW Nadel B De Torbal A Wolvers-Tettero IL van Gastel-Mol EJ Verhaaf B Jäger U van Dongen JJ 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(6):3878-3888
In addition to the classical Vkappa-Jkappa, Vkappa-kappa deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jkappa recombination signal sequence (RSS) or intronRSS elements can occur in the Igkappa (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JkappaRSS-intronRSS, Vkappa-intronRSS, and JkappaRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JkappaRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2-1.0 vs < 0.03). The initial JkappaRSS-intronRSS signal joint formation (except for Jkappa1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Ckappa deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins. 相似文献
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6.
Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected. 相似文献
7.
V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements. 相似文献
8.
The epidermal growth factor receptor (EGFR) is a membrane-bound, 170 kDa, protein tyrosine kinase that plays an important role in tumorigenesis. The EGFR gene, which is composed of over 168 kb of sequence, including a 123-kb first intron, is frequently amplified and rearranged in malignant gliomas leading to the expression of oncogenic deletion (DM) and tandem duplication (TDM) mutants. The most common DM in gliomas is EGFRvIII, which arises from recombination between introns 1 and 7 with deletion of exons 2 through 7 and intervening introns. In addition, some human gliomas express 180- to 190-kDa TDM, which are constitutively active and highly oncogenic. Both DM and TDM arise by recombination of introns that contain sequences with homology to the recombination signal sequence (RSS) heptamers and nonamers present in the V(D)J region of the immunoglobin and T lymphocyte antigen receptor genes. V(D)J RSS have also been identified in certain proto-oncogenes like bcl-2 that are involved in translocations associated with the development of human lymphomas and in other genes such as hypoxanthine-guainine phosphoribosyl transferase (HPRT) in which deletion mutations and intron rearrangements are a common phenomenon. Together with the expression of recombination associated gene (RAG) and nonhomologous end-joining (NHEJ) proteins in gliomas, these observation suggest that aberrant activity of the V(D)J recombinase may be involved in the activation of proto-oncogenes in both liquid and solid tumors. 相似文献
9.
Inversions produced during V(D)J rearrangement at IgH, the immunoglobulin heavy-chain locus. 总被引:2,自引:0,他引:2
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Diversity in immunoglobulin antigen receptors is generated in part by V(D)J recombination. In this process, different combinations of gene elements are joined in various configurations. Products of V(D)J recombination are coding joints, signal joints, and hybrid junctions, which are generated by deletion or inversion. To determine their role in the generation of diversity, we have examined two sorts of recombination products, coding joints and hybrid junctions, that have formed by inversion at the mouse immunoglobulin heavy-chain locus. We developed a PCR assay for quantification and characterization of inverted rearrangements of DH and JH gene elements. In primary cells from adult mice, inverted DJH rearrangements are detectable but they are rare. There were approximately 1,100 to 2,200 inverted DJH coding joints and inverted DJH hybrid junctions in the marrow of one adult mouse femur. On day 16 of gestation, inverted DJH rearrangements are more abundant. There are approximately 20,000 inverted DJH coding joints and inverted DJH hybrid junctions per day 16 fetal liver. In fetal liver cells, the number of inverted DJH rearrangements remains relatively constant from day 14 to day 16 of gestation. Inverted DJH rearrangements to JH4, the most 3' JH element, are more frequently detected than inverted DJH rearrangements to other JH elements. We compare the frequencies of inverted DJH rearrangements to previously determined frequencies of uninverted DJH rearrangements (DJH rearrangements formed by deletion). We suggest that inverted DJH rearrangements are influenced by V(D)J recombination mechanistic constraints and cellular selection. 相似文献
10.
To determine the distribution of Vlambda and Jlambda as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vlambda and Jlambda or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vlambda and VH was noted among the productive rearrangements. The distribution of productive Vlambda rearrangements was skewed, with significantly greater representation of the Jlambda distal cluster C Vlambda genes and the Vlambda distal Jlambda7 element, consistent with the possibility that there was receptor editing of the Vlambda locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vlambda1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity. 相似文献
11.
Murray JM O'Neill JP Messier T Rivers J Walker VE McGonagle B Trombley L Cowell LG Kelsoe G McBlane F Finette BA 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(8):5393-5404
V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans. 相似文献
12.
Homozygous mutation at the scid locus in the mouse results in the aberrant rearrangement of immunoglobulin and T-cell receptor gene segments. We introduced a retroviral vector containing an inversional immunoglobulin rearrangement cassette into scid pre-B cells. Most rearrangements were accompanied by large deletions, consistent with previously characterized effects of the scid mutation. However, two cell clones were identified which contained perfect reciprocal fragments and wild-type coding joints, documenting, on a molecular level, the ability of scid pre-B cells to generate functional protein-coding domains. Subsequent rearrangement of the DGR cassette in one of these clones was accompanied by a deletion, suggesting that this cell clone had not reverted the scid mutation. Indeed, induced rearrangement of the endogenous kappa loci in these two cell clones resulted in a mixture of scid and wild-type V-J kappa joints, as assayed by a polymerase chain reaction and DNA sequencing. In addition, three immunoglobulin mu- scid pre-B cell lines showed both scid and wild-type V-J kappa joins. These experiments strongly suggest that the V(D)J recombinase activity in scid lymphoid cells is diminished but not absent, consistent with the known leakiness of the scid mutation. 相似文献
13.
14.
V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called "trans-rearrangements." Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk. 相似文献
15.
16.
The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans. 相似文献
17.
Thymocytes in mutant mice with severe combined immunodeficiency (scid thymocytes) show ongoing recombination of some T-cell receptor delta gene elements, generating signal joints quantitatively and qualitatively indistinguishable from those in wild-type fetal thymocytes. Excised D delta 2-J delta 1 and D delta 1-D delta 2 rearrangements are detectable at levels equivalent to or greater than those in thymocytes from wild-type mice on fetal day 15. Signal junctional modification, shown here to occur frequently in wild-type adult but not newborn excised D delta 2-J delta 1 junctions, can occur normally in adult scid thymocytes. Excised D delta 1-D delta 2 scid junctions, similar to wild-type thymocytes, include pseudonormal coding junctions as well as signal junctions. Inversional D delta 1-D delta 2 rearrangements, generating conventional hybrid junctions, are also reproducibly detectable in scid thymus DNA. These hybrids, unlike those reported for artificial recombination constructs, do not show extensive nucleotide loss. In contrast to the normal or high incidences of D delta 1-, D delta 2-, and J delta 1-associated signal junctions in scid thymocytes, V delta 1, V gamma 3, and V gamma 1.2 signal products are undetectable in scid thymocytes or are detectable at levels at least 10-fold lower than the levels in wild-type fetal thymocytes. These findings confirm biased T-cell receptor element recombination by V(D)J recombinase activity of nontransformed scid thymocytes and indicate that analysis of in vivo-mediated gene rearrangements is important for full understanding of how the scid mutation arrests lymphocyte development. 相似文献
18.
The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism 总被引:57,自引:0,他引:57
B A Malynn T K Blackwell G M Fulop G A Rathbun A J Furley P Ferrier L B Heinke R A Phillips G D Yancopoulos F W Alt 《Cell》1988,54(4):453-460
Abelson murine leukemia virus-transformed precursor B lymphocytes from scid (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their Ig heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and JH locus to form aberrant "DJH" rearrangements and secondary rearrangement of sequences from within the VH locus to the aberrant "DJH" intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments. 相似文献
19.
Marculescu R Vanura K Montpellier B Roulland S Le T Navarro JM Jäger U McBlane F Nadel B 《DNA Repair》2006,5(9-10):1246-1258
A large number of lymphoid malignancies is characterized by specific chromosomal translocations, which are closely linked to the initial steps of pathogenesis. The hallmark of these translocations is the ectopic activation of a silent proto-oncogene through its relocation at the vicinity of an active regulatory element. Due to the unique feature of lymphoid cells to somatically rearrange and mutate receptor genes, and to the corresponding strong activity of the immune enhancers/promoters at that stage of cell development, B- and T-cell differentiation pathways represent propitious targets for chromosomal translocations and oncogene activation. Recent progress in the understanding of the V(D)J recombination process has allowed a more accurate definition of the translocation mechanisms involved, and has revealed that V(D)J-mediated translocations result both from targeting mistakes of the recombinase, and from illegitimate repair of the V(D)J recombination intermediates. Surprisingly, V(D)J-mediated translocations turn out to be restricted to two specific sub-types of lymphoid malignancies, T-cell acute lymphoblastic leukemias, and a restricted set of mature B-cell Non-Hodgkin's lymphomas. 相似文献
20.
V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites. 总被引:13,自引:11,他引:2
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V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations. 相似文献