Translocations induced by fast neutrons and X-rays in Delia antiqua

Summary A comparison was made using X-rays and fast neutrons for the induction of translocations in Delia antiqua. Using the same radiation dose, no difference in efficiency between the two radiation types could be observed. However, with fast neutrons many multiple translocations were induced, including a quadruple translocation involving 4 out of 5 autosomes. One male linked translocation was also induced.The reciprocal translocations were assigned into two classes: symmetrical and asymmetrical, and ten of the latter were chosen for inbreeding to produce homozygotes. Asymmetrical exchanges were chosen so that translocation homozygotes could be differentiated cytologically from the normal karyotype. In seven different translocations, homozygous larvae were observed, but often at a low frequency. In four of these lines, viable adult homozygotes were observed. Subsequent random sib-crossing failed to produce a homozygous line.     

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.     

Effect of induced chromosomal rearrangements on the fertility and viability of Anopheles atroparvus

Partial sterility in 3 strains of malarial mosquito Anopheles atroparvus with heterozygous chromosomal rearrangements In2(R;L) 1+In2L1; In3(R;L) 1+D3R1; T(2R;3R)1 was studied. Fertility of these lines was decreased 2.5 to 5 times, as compared with the control. No homozygotes for rearrangements were detected. Causes for partial sterility of insects with heterozygous insertions, translocations and the significance of constructing homozygous strains are discussed.     

Breakpoint mapping and haplotype analysis of three reciprocal translocations identify a novel recurrent translocation in two unrelated families: t(4;11)(p16.2;p15.4)

The majority of constitutional reciprocal translocations appear to be unique rearrangements arising from independent events. However, a small number of translocations are recurrent, most significantly the t(11;22)(q23;q11). Among large series of translocations there may be multiple independently ascertained cases with the same cytogenetic breakpoints. Some of these could represent additional recurrent rearrangements, alternatively they could be identical by descent (IBD) or have subtly different breakpoints when examined under higher resolution. We have used molecular breakpoint mapping and haplotyping to determine the origin of three pairs of reciprocal constitutional translocations, each with the same cytogenetic breakpoints. FISH mapping showed one pair to have different breakpoints and thus to be distinct rearrangements. Another pair of translocations were IBD with identical breakpoint intervals and highly conserved haplotypes on the derived chromosomes. The third pair, t(4;11)(p16.2;p15.4), had the same breakpoint intervals by aCGH and fosmid mapping but had very different haplotypes, therefore they represent a novel recurrent translocation. Unlike the t(11;22)(q23;q11), the formation of the t(4;11)(p16.2;p15.4) may have involved segmental duplications and sequence homology at the breakpoints. Additional examples of recurrent translocations could be identified if the resources were available to study more translocations using the approaches described here. However, like the t(4;11)(p16.2;p15.4), such translocations are likely to be rare with the t(11;22) remaining the only common recurrent constitutional reciprocal translocation.     

Frequent occurrence of large duplications at reciprocal genomic rearrangement breakpoints in multiple myeloma and other tumors

Using a combination of array comparative genomic hybridization, mate pair and cloned sequences, and FISH analyses, we have identified in multiple myeloma cell lines and tumors a novel and recurrent type of genomic rearrangement, i.e. interchromosomal rearrangements (translocations or insertions) and intrachromosomal inversions that contain long (1–4000 kb; median ∼100 kb) identical sequences adjacent to both reciprocal breakpoint junctions. These duplicated sequences were generated from sequences immediately adjacent to the breakpoint from at least one—but sometimes both—chromosomal donor site(s). Tandem duplications had a similar size distribution suggesting the possibility of a shared mechanism for generating duplicated sequences at breakpoints. Although about 25% of apparent secondary rearrangements contained these duplications, primary IGH translocations rarely, if ever, had large duplications at breakpoint junctions. Significantly, these duplications often contain super-enhancers and/or oncogenes (e.g. MYC) that are dysregulated by rearrangements during tumor progression. We also found that long identical sequences often were identified at both reciprocal breakpoint junctions in six of eight other tumor types. Finally, we have been unable to find reports of similar kinds of rearrangements in wild-type or mutant prokaryotes or lower eukaryotes such as yeast.     

A mouse translocation associated with Caspr5-2 disruption and perinatal lethality

We have previously described the paralogous mouse genes Caspr5-1, -2, and -3 of the neurexin gene family. Here we present the cytogenetic and molecular mapping of a null mutation of Caspr5-2 which was caused by reciprocal translocation between chromosomes 1 and 8 with breakpoints at bands 1E2.1 and 8B2.1, respectively. The translocation disrupts Caspr5-2 between exons 1 and 2 and causes stillbirth or early postnatal lethality of homozygous carriers. Because no other candidate genes were found, the disruption of Caspr5-2 is most likely the cause of lethality. Only rarely do homozygotes survive the critical stage, reach fertility, and are then apparently normal. They may be rescued by one of the two other Caspr5 paralogs. Caspr5-2 is expressed in spinal cord and brain tissues. Despite giving special attention to regions where in wild-type fetuses maximum expression was found, no malformation that might have caused death could be detected in fetal homozygous carriers of the translocation. We, therefore, suspect that Caspr5-2 disruption leads to dysfunction at the cellular level rather than at the level of organ development.     

A mouse translocation associated with Caspr5-2 disruption and perinatal lethality

We have previously described the paralogous mouse genes Caspr5-1, -2, and -3 of the neurexin gene family. Here we present the cytogenetic and molecular mapping of a null mutation of Caspr5-2 which was caused by reciprocal translocation between chromosomes 1 and 8 with breakpoints at bands 1E2.1 and 8B2.1, respectively. The translocation disrupts Caspr5-2 between exons 1 and 2 and causes stillbirth or early postnatal lethality of homozygous carriers. Because no other candidate genes were found, the disruption of Caspr5-2 is most likely the cause of lethality. Only rarely do homozygotes survive the critical stage, reach fertility, and are then apparently normal. They may be rescued by one of the two other Caspr5 paralogs. Caspr5-2 is expressed in spinal cord and brain tissues. Despite giving special attention to regions where in wild-type fetuses maximum expression was found, no malformation that might have caused death could be detected in fetal homozygous carriers of the translocation. We, therefore, suspect that Caspr5-2 disruption leads to dysfunction at the cellular level rather than at the level of organ development.

     

High frequency of spontaneous translocations revealed by FISH in cells from patients with the cancer-prone syndromes ataxia telangiectasia and Nijmegen breakage syndrome.

The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 +/- 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 +/- 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 +/- 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 +/- 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 +/- 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.     

Molecular cytogenetic investigations define a subgroup of thyroid adenomas with 2p21 breakpoints clustered to a region of less than 450 kb

Structural rearrangements involving chromosome band 2p21 characterize a cytogenetic subgroup of benign thyroid tumors. To narrow down the breakpoints of these aberrations, we established two cell lines from benign thyroid tumors showing translocations involving 2p21. These two cell lines and one additional primary tumor were used for FISH-studies with 18 BAC clones. All breakpoints were mapped to a cluster of about 450 kb.     

Chromosome Rearrangements That Involve the Nucleolus Organizer Region in Neurospora

In ~3% of Neurospora crassa rearrangements, part of a chromosome arm becomes attached to the nucleolus organizer region (NOR) at one end of chromosome 2 (linkage group V). Investigations with one inversion and nine translocations of this type are reported here. They appear genetically to be nonreciprocal and terminal. When a rearrangement is heterozygous, about one-third of viable progeny are segmental aneuploids with the translocated segment present in two copies, one in normal position and one associated with the NOR. Duplications from many of the rearrangements are highly unstable, breaking down by loss of the NOR-attached segment to restore normal chromosome sequence. When most of the rearrangements are homozygous, attenuated strands can be seen extending through the unstained nucleolus at pachytene, joining the translocated distal segment to the remainder of chromosome 2. Although the rearrangements appear genetically to be nonreciprocal, molecular evidence shows that at least several of them are physically reciprocal, with a block of rDNA repeats translocated away from the NOR. Evidence that NOR-associated breakpoints are nonterminal is also provided by intercrosses between pairs of translocations that transfer different-length segments of the same donor-chromosome arm to the NOR.     

Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.

Insertional mutagenesis was investigated in a transgenic mouse strain (HUGH/4) derived from a fertilized egg injected with plasmid DNA containing the human growth hormone gene. Lethality occurred in homozygous embryos and was traced to the egg cylinder stage on days 4 to 5 of gestation, shortly after implantation. The mutation is on chromosome 12 and is distinct in location and integration pattern from another mutation also leading to lethality of homozygotes in the egg cylinder stage. Based on this and other evidence, relatively many genes may be recruited to activity near the time of implantation and may therefore present a large target of vulnerability to mutagenesis. The single insert in HUGH/4, consisting of approximately three tandem copies of plasmid sequences, is flanked by mouse cellular sequences that have undergone rearrangements, including a probable deletion. The data suggest the hypothesis that DNA rearrangements, which appear to be commonplace in transgenic mice, may arise because the initial insertional complex is unstable; stepwise changes may then be generated until a more stable conformation is achieved.     

Chromosome structure of Triticum timopheevii relative to T. turgidum.

The chromosome structure of four different wild populations and a cultivated line of Triticum timopheevii (2n = 28, AtAtGG) relative to Triticum turgidum (2n = 28, AABB) was studied, using genomic in situ hybridisation (GISH) and C-banding analysis of meiotic configurations in interspecific hybrids. Two wild accessions and the cultivated line showed the standard C-banding karyotype. The other two accessions are homozygous for translocation 5At/3G and translocations 1G/2G and 5G/6G. GISH analysis revealed that all the T. timopheevii accessions carry intergenome translocations 6At/1G and 1G/4G and identified the position of the breakpoint in translocation 5At/3G. C-banding analysis of pairing at metaphase I in the hybrids with T. turgidum provides evidence that four species-specific translocations (6AtS/1GS, 1GS/4GS, 4GS/4AtL, and 4AtL/3AtL) exist in T. timopheevii, and that T. timopheevii and T. turgidum differ in the pericentric inversion of chromosome 4A. Bridge plus acentric fragment configurations involving 4AL and 4AtL were identified in cells at anaphase I. This result suggests that the paracentric inversion of 4AL from T. turgidum does not exist in T. timopheevii. Both tetraploid species have undergone independent and distinct evolutionary chromosomal rearrangements. The position, intercalary or subdistal, of the breakpoints in species-specific translocations and inversions contrasts with the position, at or close to the centromere, of intraspecific translocations. Different mechanisms for intraspecific and species-specific chromosome rearrangements are suggested.     

The albino deletion complex and early postimplantation survival in the mouse

The albino deletion complex in the mouse represents 37 overlapping chromosomal deficiencies that have been arranged into at least twelve complementation groups. Many of the deletions cover regions of chromosome 7 that contain genes necessary for early embryonic development. The work reported here concentrates on two of these deletions (c6H, c11DSD), both of which were known to be lethal around the time of gastrulation when homozygous. A detailed embryological analysis has revealed distinct differences in the lethal phenotype associated with the c6H and c11DSD deletions. c6H homozygous embryos are grossly abnormal at day 7.5 of gestation, whereas c11DSD homozygous embryos appear abnormal at day 8.5 of gestation. There is no development of the extraembryonic ectoderm in c6H homozygotes, whereas extensive development of this tissue type occurs in c11DSD homozygotes. The visceral endoderm is abnormally shaped and the parietal endoderm appears to be overproduced in c6H homozygotes; these structures are not affected in c11DSD homozygotes. The embryonic ectoderm is runted in both types of embryo and it is not possible to obtain homozygous embryo-derived stem-cell lines for either deletion. Mesoderm formation occurs in the c11DSD but not in the c6H homozygotes. The c11DSD deletion chromosome complements the c6H chromosome in that the lethal phenotype of the compound heterozygote is similar to that of the c11DSD homozygote. These results suggest that a gene(s) necessary for normal development of the extraembryonic ectoderm is present in the c11DSD but deficient in the c6H deletion chromosome.     

Analysis of the functional significance of linkage group conservation in Drosophila

Linkage groups, as defined by chromosome arms in Drosophila melanogaster, appear to have remained largely intact within the genus Drosophila and, possibly, within the higher Diptera per se. We hypothesized that linkage group conservation might have a functional basis (possibly related to interphase chromosome arrangement). To test this hypothesis, a series of autosomal 2–3 translocations were synthesized, creating many new linkage groups. A total of 167 2–3 translocations were recovered, cytologically analyzed to determine their polytene chromosome breakpoints, and tested for homozygous viability and fertility. The breakpoints associated with homozygous viable translocations were randomly distributed throughout the genome, indicating that the linear continuity of the linkage groups could be disrupted quite extensively. Inter se complementation crosses between homozygous lethal translocations having similar breakpoints further confirmed this result, documenting that, at least with respect to homozygous viability, the linear integrity of the autosomal linkage groups was not of major functional significance. Fertility analysis of the homozygous translocations also indicated that sterility could not be a single major factor. Having concluded that linkage group conservation is not based on important functional interactions between specific linked chromosomal segments, or due principally to the sterility of new linkages, the problem of linkage group conservation remains unsolved. Several possible selective factors are discussed, principally segregational load and inbreeding depression, which may contribute to the elimination of new linkage rearrangements.     

Chromosomal distributions of breakpoints in cancer,infertility, and evolution

We extract 11 genome-wide sets of breakpoint positions from databases on reciprocal translocations, inversions and deletions in neoplasms, reciprocal translocations and inversions in families carrying rearrangements and the human-mouse comparative map, and for each set of positions construct breakpoint distributions for the 44 autosomal arms. We identify and interpret four main types of distribution: (i) a uniform distribution associated both with families carrying translocations or inversions, and with the comparative map, (ii) telomerically skewed distributions of translocations or inversions detected consequent to births with malformations, (iii) medially clustered distributions of translocation and deletion breakpoints in tumor karyotypes, and (iv) bimodal translocation breakpoint distributions for chromosome arms containing telomeric proto-oncogenes.     

Base substitutions,targeted single-base deletions,and chromosomal translocations induced by bleomycin in plateau-phase mammary epithelial cells

Previous work showed that treatment of plateau-phase Chinese hamster ovary cells with the radiomimetic double-strand cleaving agent bleomycin induced very small deletions as well as interchromosomal reciprocal translocations, both of which could be ascribed to errors in end joining of DNA double-strand breaks. In an attempt to assess the possible role of TP53 in suppressing such repair errors, bleomycin-induced mutagenesis at the HPRT locus was examined in immortalized 184B5 human mammary epithelial cells (TP53(+)), and in a TP53-defective derivative, 184B5-E6tfxc6. For both cell lines, the most frequent bleomycin-induced mutations were base substitutions, with no apparent targeting to major bleomycin lesions. However, both lines also sustained single-base deletions that were targeted to expected sites of double-strand breaks, suggesting that they arose by end-joining repair of the breaks. Surprisingly, only a few large deletions or rearrangements, and no interchromosomal events involving the HPRT locus were detected among the mutants. The results suggest that in both cell lines, errors in double-strand break repair resulting in heritable large deletions and rearrangements are rare. Spectral karyotyping of bleomycin-treated 184B5 cells showed that a significant number of translocations were present shortly after bleomycin exposure, but their frequency decreased upon continued culture of the cells. Thus, for these cells, the lack of induced interchromosomal rearrangements can be explained in part by selection against such events as the cells proliferate.     

Distinguishing primary and secondary translocations in multiple myeloma

Multiple myeloma (MM) is a malignant post-germinal center tumor of somatically-mutated, isotype-switched plasma cells that accumulate in the bone marrow. It often is preceded by a stable pre-malignant tumor called monoclonal gammopathy of undetermined significance (MGUS), which can sporadically progress to MM. Five recurrent primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 have been identified in MGUS and MM tumors. The five partner loci include 11q13, 6p21, 4p16, 16q23, and 20q12, with corresponding dysregulation of CYCLIN D1, CYCLIN D3, FGFR3/MMSET, c-MAF, and MAFB, respectively, by strong enhancers in the IgH locus. The five recurrent translocations, which are present in 40% of MM tumors, typically are simple reciprocal translocations, mostly having breakpoints within or near IgH switch regions but sometimes within or near VDJ or JH sequences. It is thought that these translocations are caused by aberrant IgH switch recombination, and possibly by aberrant somatic hypermutation in germinal center B cells, thus providing an early and perhaps initiating event in transformation. A MYC gene is dysregulated by complex translocations and insertions as a very late event during the progression of MM tumors. Since the IgH switch recombination and somatic hypermutation mechanism are turned off in plasma cells and plasma cell tumors, the MYC rearrangements are thought to be mediated by unknown mechanisms that contribute to structural genomic instability in all kinds of tumors. These rearrangements, which often but not always juxtapose MYC near one of the strong immunoglobulin enhancers, provide a paradigm for secondary translocations. It is hypothesized that secondary translocations not involving a MYC gene can occur at any stage of tumorigenesis, including in pre-malignant MGUS tumor cells.     

Breakpoint mapping and array CGH in translocations: comparison of a phenotypically normal and an abnormal cohort

We report the analyses of breakpoints in 31 phenotypically normal and 14 abnormal carriers of balanced translocations. Our study assesses the differences between balanced translocations in normal carriers and those in abnormal carriers, focusing on the presence of genomic imbalances at the breakpoints or elsewhere in the genome, presence of cryptic chromosome rearrangements, and gene disruption. Our hypothesis is that all four features will be associated with phenotypic abnormalities and absent or much less frequent in a normal population. In the normal cohort, we identified neither genomic imbalances at the breakpoints or elsewhere in the genome nor cryptic chromosome rearrangements. In contrast, we identified candidate disease-causing imbalances in 4/14 abnormal patients. These were three breakpoint associated deletions and three deletions unrelated to the breakpoints. All six de novo deletions originated on the paternally inherited chromosome. Additional complexity was also present in one of these cases. Gene disruption by the breakpoints was present in 16/31 phenotypically normal individuals and in 5/14 phenotypically abnormal patients. Our results show that translocations in phenotypically abnormal patients are molecularly distinct from those in normal individuals: the former are more likely to be associated with genomic imbalances at the breakpoints or elsewhere and with chromosomal complexity, whereas the frequency of gene disruption is similar in both normal and abnormal translocation carriers.