T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.     

Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.     

Beswitched. The looping out model for immunoglobulin class switching

During the switch in expression of an immunoglobulin class, the gene segment encoding the constant region of the heavy chain is replaced in a way that leads to a deletion. Three different models of how this deletion is generated have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, the products of the looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch.     

细胞因子对脐血B细胞免疫球蛋白类别转换的调节

为了解细胞因子对新生儿Ｂ细胞免疫球蛋白类别转换的调节作用,在体外细胞培养的基础上,采用反向酶联免疫斑点法观察了脐血单个核细胞及添加重组细胞因子后脐血Ｂ细胞免疫球蛋白释放细胞数量（ＩｇＳＣｓ）。结果：正常脐血单个核细胞仅产生少量的ＩｇＳＣｓ。用抗ＣＤ３单抗刺激丝裂霉素Ｃ处理后的脐血Ｔ细胞,并补充ｒＩＬ－２、ｒＩＬ－４、ｒＩＬ－１０及其组合,可诱导脐血Ｂ细胞释放ＩｇＡ、ＩｇＧ和ＩｇＭ。这些结果提示：细胞因子的补充可促进体外新生儿Ｂ细胞免疫球蛋白的类别转换     

Development of assay system for immunoglobulin production regulatory factors using whole cell cultures of mouse splenocytes

We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, beta-lactoglobulin, alpha-casein, and beta-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.     

A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation

Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%–43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.     

The repair and recombination enzyme ERCC1 is not required for immunoglobulin class switching

Class switch recombination (CSR) is a programmed gene rearrangement in which a B cell which is producing IgM and IgD antibody develops into an IgG-, IgA- or IgE-expressing cell. This is achieved by recombination between switch regions located 5' of each of the immunoglobulin heavy chain constant regions, except Cdelta. The mechanism of CSR has not been resolved but it is thought to involve a double-strand break followed by end joining. It has previously been suggested that the nucleotide excision repair protein ERCC1 may be involved in CSR due to its known roles in removal of 3' single-stranded tails in various types of recombination. In this study, we examined class switching in cultured splenocytes from ERCC1-deficient mice and found no evidence of any deficiency.     

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.     

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 × 10? M?1 · s?1 and 1.3 × 10? M?1 · s?1 for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 × 10? M?1 · s?1 for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.     

Detection of circular excised DNA deletion elements in Tetrahymena thermophila during development.

Extensive programmed DNA deletion occurs in ciliates during development. In this study we examine the excised forms of two previously characterized deletion elements, the R- and M-element, in Tetrahymena. Using divergently oriented primers in polymerase chain reactions we have detected the junctions formed by joining the two ends of these elements, providing evidence for the presence of circular excised forms. These circular forms were detected in developing macronuclear DNA from 12-24 h after mating began, but not in micronuclear or whole cell DNA of vegetative cells. They are present at very low abundance, detectable after PCR only through hybridization with specific probes. Sequence analysis shows that the circle junctions occur at or very near the known ends of the elements. There is sequence microheterogeneity in these junctions, which does not support a simple reciprocal exchange model for DNA deletion. A model involving staggered cuts and variable mismatch repair is proposed to explain these results. This model also explains the sequence microheterogeneity previously detected among the junction sequences retained in the macronuclear chromosome.     

DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy

DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 × 10⁶ daltons), duplex DNA circles of a smaller size (140 × 10⁶ daltons) with protruding tails were formed.     

Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.     

Characterization of a new class of circular DNA molecules in yeast

This paper describes the isolation of a pure population of covalently closed circular twisted DNA molecules from yeast. These molecules are homogeneous in size, that is consist of monomers of 2.2μ and of multiple length oligomers of n x 2.2μ. While no data rule out the mitochondrial origin of this DNA, its actual intracellular localization remains unknown; it displays the same buoyant density as the main nuclear DNA and therefore is not the heavy nuclear satellite DNA (γ-DNA described by Moustacchi and Williamson (1966)); although circular molecules represent only 1 to 5 % of the total DNA, they can be prepared in sizable and reproducible amounts by a method based on the use of mechanical disruption of yeast cells rather than lysis by snail gut juice.     

Superhelical turns of closed circular DNA in solution and in a gel: evidence for a conformational difference

We have compared the number of superhelical turns, tau, in circular covalently closed plasmid pBR beta G DNA obtained by four different methods, each based on one particular distinguishing principle. Three of the methods allow an unequivocal determination of tau under gel electrophoresis conditions, whilst the fourth enables us to determine its value in solution. We were able to detect a significant difference between the two environments, corresponding to an unwinding of the DNA duplex angle by 0.3 degrees when a sample is transferred from solution to gel. The possible existence of such an effect has been generally overlooked by previous investigators. Our result suggests that the previously reported value for the number of base-pairs per helical turn should be adjusted downwards by about 0.10, so that it applies to conditions in solution.     

The 9-bp deletion in region V of mitochondrial DNA: evidence of mutation recurrence

A deletion of one of the two copies of a 9-bp direct repeat sequence (CCCCCTCTA) in region V of mitochondrial DNA has previously been used as a polymorphic anthropological marker for people of east Asian origin, and to a lesser extent, in Oceanian and African populations. We report the presence of the 9-bp deletion in homoplasmy in skeletal muscle fibers and lymphocytes of a Spanish Caucasian individual. Other mitochondrial DNA polymorphisms associated with the 9-bp deletion characteristic of other populations were not present. Our results suggest that the 9-bp deletion probably originated independently in the maternal lineage of the propositus, and that it can thus be described as a recurrent mutation.     

The DNA glycosylases Ogg1 and Nth1 do not contribute to Ig class switching in activated mouse splenic B cells

During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes.