Inactivation of rabbit immunoglobulin lambda chain variable region genes by the insertion of short interspersed elements of the C family

Two rabbit germ-line genes encoding immunoglobulin lambda light chain V regions were cloned from a rabbit genomic liver DNA library and characterized. One, V lambda 1, is separated by at least 8 kb from any other V lambda gene. The second, V lamdba 4, forms part of a three-gene cluster with two functional V lambda genes recently reported. Both V lambda 1 and V lambda 4 have structural features rendering them pseudogenes. The coding regions have frame-shift mutations which would yield defective protein products; both genes are also interrupted by the insertions of short, interspersed repetitive elements of the C family. In the V lambda 1 gene, the 369-bp insert is located upstream of the gene between the putative TATA box and the leader exon, whereas in gene V lambda 4, the 360-bp insert interrupts the FR2 at codon 48c. In addition, the sequence of the complement-determining region 3 of gene V lambda 1 is very similar to the mouse DSP2.6 sequence.     

Enrichment of short interspersed transposable elements to embryonic stem cell‐specific hypomethylated gene regions

Embryonic stem cells (ESCs) have a distinctive epigenome, which includes their genome‐wide DNA methylation modification status, as represented by the ESC‐specific hypomethylation of tissue‐dependent and differentially methylated regions (T‐DMRs) of Pou5f1 and Nanog. Here, we conducted a genome‐wide investigation of sequence characteristics associated with T‐DMRs that were differentially methylated between ESCs and somatic cells, by focusing on transposable elements including short interspersed elements (SINEs), long interspersed elements (LINEs) and long terminal repeats (LTRs). We found that hypomethylated T‐DMRs were predominantly present in SINE‐rich/LINE‐poor genomic loci. The enrichment for SINEs spread over 300 kb in cis and there existed SINE‐rich genomic domains spreading continuously over 1 Mb, which contained multiple hypomethylated T‐DMRs. The characterization of sequence information showed that the enriched SINEs were relatively CpG rich and belonged to specific subfamilies. A subset of the enriched SINEs were hypomethylated T‐DMRs in ESCs at Dppa3 gene locus, although SINEs are overall methylated in both ESCs and the liver. In conclusion, we propose that SINE enrichment is the genomic property of regions harboring hypomethylated T‐DMRs in ESCs, which is a novel aspect of the ESC‐specific epigenomic information.     

Prothrombin activation by thrombolytic agents

Previous studies showed an early increase of fibrinopeptide A and thrombin-anti-thrombin III complex (TAT) plasma levels during thrombolytic therapy. This might be caused not only by liberation of preformed thrombin from the thrombus or reperfused tissue, but also by activation of prothrombin.In 11 patients with acute myocardial infarction treated with 1.5 million U streptokinase (SK) over 30 min, and in 6 patients with deep vein thrombosis receiving standard dose urokinase (UK) infusion, TAT and the prothrombin fragment F1+2 increased significantly 3h after commencing thrombolytic therapy, and decreased again thereafter.In vitro, TAT and F1+2 levels increased in anticoagulated normal plasma after incubation with SK, UK or tissue-type plasminogen activator (t-PA). By incubation of purified prothrombin with SK+plasminogen (PLG), UK, UK+PLG, t-PA, t-PA+PLG, or plasmin, F1+2 immunoreactivity was generated. Also the generation of thrombin-like activity from purified prothrombin could be demonstrated by means of a chromogenic substrate assay with inhibition of non-specific cleavage by purified plasminogen activator inhibitor type 2 (PAI-2) or α₂-antiplasmin (APL).These results demonstrate an activation of prothrombin associated with stimulation of fibrinolysis. Further studies should clarify the clinical significance of this phenomenon, and whether it could have implications for optimizing the concomitant anticoagulation in order to prevent reocclusion in patients undergoing thrombolysis.     

DNA-damaging agents stimulate the formation of directed reciprocal translocations in Saccharomyces cerevisiae

DNA-damaging agents can stimulate the formation of directed reciprocal translocations of Saccharomyces cerevisiae containing his3 recombinational substrates to generate chromosomal rearrangements. Such agents were compared with those that can stimulate sister- chromatid recombination. We show that chemicals and environmental agents that produce a variety of DNA lesions, including bulky adduct, thymidine dimers, interstrand cross-links, double-strand breaks and alkylated bases, can stimulate recombination to yield reciprocal translocations. Of the agents teted, only the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguinidine (MNNG), and a bifunctional agent that causes bulky DNA adducts, 4-nitroquinoline-N-oxide (4-NQO), significantly stimulate sister-chromatid recombination in our assay. Factors that contribute to the stimulation of interchromosomal recombination include strain genetic background and ploidy.     

Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia

Reciprocal translocation t(9;22) is central to the pathogenesis of chronic myeloid leukemia. Some authors have suggested that Alu repeats facilitate this process, but supporting analyses have been sparse and often anecdotal. The purpose of this study was to analyze the local structure of t(9;22) translocations and assess the relevance of interspersed repeat elements at breakpoints. Collected data have been further compared with the current models of DNA recombination, in particular the single-strand annealing (SSA) and the nonhomologous end joining (NHEJ) processes. We developed a protocol for the rapid characterization of patient-specific genomic junctions and analyzed 27 patients diagnosed with chronic myeloid leukemia. Sequence analysis revealed microhomologies at the junctions of 21 patients of 27, while interspersed repeats were of relevance (P < 0.05) in at least 16 patients. These findings are more frequent than expected and give an indication that the main mechanisms involved in the t(9;22) translocation are the SSA and NHEJ pathways, both playing a role. Furthermore, our report is consistent with microhomologies facilitating the joining of DNA ends in the translocation process, and with both Alu and a variety of other repeat sequences pairing nonhomologous chromosomes during the SSA pathway.     

Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.     

Transcriptional activation of the IL31 gene by NFAT and STAT6

IL-31, a newly identified member of the IL-6 cytokine family, is involved in many pathological conditions, including atopic dermatitis and pruritis. In this study, we investigated how expression of IL-31 is regulated in T cells and mast cells. We observed that expression of IL-31 required a calcium signal and was dependent on the calcineurin-NFAT signaling pathway. Moreover, we found that IL-31 promoter contains a positive regulatory region that mediates calcium- and IL-4-dependent induction of the IL-31 gene and demonstrated that a change into an open chromatin conformation occurs in this region after stimulation with calcium and IL-4. Whereas IL-4 responsiveness required STAT6 binding sites, calcium responsiveness of IL-31 promoter required NFAT binding sites that bind NFATc1 and NFATc2 in vitro and in vivo. The induction of IL-31 promoter activity was impaired when these sites were mutated but was enhanced by CA-NFATc1 or STAT6 proteins and further increased synergistically by combinations of both proteins. Furthermore, the importance of STAT6 proteins was indicated by impaired, IL-4-mediated induction of IL-31 in STAT6-diminished Jurkat cells. Thus, our data demonstrate that calcium and IL-4 signals are required to mediate induction of IL-31 in Th2 cells and mast cells and that this induction appears to result from specific binding of NFAT and STAT6 proteins.     

A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements

Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions.     

Transcriptional activation by the E1A regions of adenovirus types 40 and 41

In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region 1A (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol acetyltransferase (CAT) gene controlled by the Ad2 E4 promoter. Cotransfection of HeLa cells with plasmids containing the E1A regions of Ad5, Ad40, and Ad41, respectively, and the CAT gene controlled by the Ad2 E4 promoter showed that activation of the E4 promoter by the E1A regions of Ad40 and Ad41 depends on the same sequence elements of the E4 promoter as activation by the Ad5 E1A gene products. The level of activation, however, is significantly lower. This might partly explain the reduced growth in HeLa cells of the two viruses.