A new strategy for the detection of subtelomeric rearrangements

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.     

Chromosome Rearrangement by Ectopic Recombination in Drosophila Melanogaster: Genome Structure and Evolution

Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure. A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments. Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences. The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.     

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.     

A 1.5-Mb-resolution radiation hybrid map of the cat genome and comparative analysis with the canine and human genomes

We report the construction of a 1.5-Mb-resolution radiation hybrid map of the domestic cat genome. This new map includes novel microsatellite loci and markers derived from the 2X genome sequence that target previous gaps in the feline-human comparative map. Ninety-six percent of the 1793 cat markers we mapped have identifiable orthologues in the canine and human genome sequences. The updated autosomal and X-chromosome comparative maps identify 152 cat-human and 134 cat-dog homologous synteny blocks. Comparative analysis shows the marked change in chromosomal evolution in the canid lineage relative to the felid lineage since divergence from their carnivoran ancestor. The canid lineage has a 30-fold difference in the number of interchromosomal rearrangements relative to felids, while the felid lineage has primarily undergone intrachromosomal rearrangements. We have also refined the pseudoautosomal region and boundary in the cat and show that it is markedly longer than those of human or mouse. This improved RH comparative map provides a useful tool to facilitate positional cloning studies in the feline model.     

Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells

DNA double-strand breaks (DSBs) may be caused by normal metabolic processes or exogenous DNA damaging agents and can promote chromosomal rearrangements, including translocations, deletions, or chromosome loss. In mammalian cells, both homologous recombination and nonhomologous end joining (NHEJ) are important DSB repair pathways for the maintenance of genomic stability. Using a mouse embryonic stem cell system, we previously demonstrated that a DSB in one chromosome can be repaired by recombination with a homologous sequence on a heterologous chromosome, without any evidence of genome rearrangements (C. Richardson, M. E. Moynahan, and M. Jasin, Genes Dev., 12:3831-3842, 1998). To determine if genomic integrity would be compromised if homology were constrained, we have now examined interchromosomal recombination between truncated but overlapping gene sequences. Despite these constraints, recombinants were readily recovered when a DSB was introduced into one of the sequences. The overwhelming majority of recombinants showed no evidence of chromosomal rearrangements. Instead, events were initiated by homologous invasion of one chromosome end and completed by NHEJ to the other chromosome end, which remained highly preserved throughout the process. Thus, genomic integrity was maintained by a coupling of homologous and nonhomologous repair pathways. Interestingly, the recombination frequency, although not the structure of the recombinant repair products, was sensitive to the relative orientation of the gene sequences on the interacting chromosomes.     

Sensitivity of multiple color spectral karyotyping in detecting small interchromosomal rearrangements

Multiple color spectral karyotyping (SKY) has been proven to be a very useful tool for characterization of the complex rearrangements in cancer cells and the de novo constitutional structural abnormalities. The sensitivity of SKY in detecting interchromosomal alterations was assessed with 10 constitutional translocations involving subtelomeric regions. Among the 13 small segments tested, 9 were clearly visualized and 8 were unambiguously identified by SKY. Fluorescence in situ hybridizations (FISH) with subtelomeric probes confirmed the reciprocity in three of the four translocations in which a small segment was not detectable by SKY. On the basis of resolution level of G-banding and the information obtained from the FISH analysis, the minimum alteration that SKY can detect is estimated to be 1,000-2,000 kbp in size with the currently available probes. This study has demonstrated the power, but also the limitations, of SKY in detecting small interchromosomal alterations, particularly those in subtelomeric regions.     

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats.

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10-20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5-6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.     

Putative interchromosomal rearrangements in the hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotype ‘Chinese Spring’ revealed by gene locations on homoeologous chromosomes

Background

Chromosomal rearrangements are a major driving force in shaping genome during evolution. Previous studies show that translocated genes could undergo elevated rates of evolution and recombination frequencies around these genes can be altered. Based on the recently released genome sequences of Triticum urartu, Aegilops tauschii, Brachypodium distachyon and bread wheat, an analysis of interchromosomal translocations in the hexaploid wheat genotype ‘Chinese Spring’ (‘CS’) was conducted based on chromosome shotgun sequences from individual chromosome arms of this genotype.

Results

A total of 720 genes representing putative interchromosomal rearrangements was identified. They were distributed across the 42 chromosome arms. About 59% of these translocated genes were those involved in the well-characterized translocations involving chromosomes 4A, 5A and 7B. The other 41% of the genes represent a large numbers of putative interchromosomal rearrangements which have not yet been described. The number of the putative translocation events in the D subgenome was about half of those presented in either the A or B subgenomes, which agreed well with that the times of interaction between the A and B subgenomes almost doubled that between either of them and the D subgenome.

Conclusions

The possible existence of a large number of interchromosomal rearrangements detected in this study provide further evidence that caution should be taken when using synteny in ordering sequence contigs or in cloning genes in hexaploid wheat. The identification of these putative translocations in ‘CS’ also provide a base for a systematic evaluation of their presence or absence in the full spectrum of bread wheat and its close relatives, which could have significant implications in a wide array of fields ranging from studies of systematics and evolution to practical breeding.

     

Multiplex-fluorescence in situ hybridization for chromosome karyotyping

Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes approximately 14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use.     

Increased risk of trisomy 21 in offspring of carriers of balanced non-contributing autosomal rearrangements is not explained by interchromosomal effect

Only relatively recently the suggestion that interchromosomal effect (ICE) may be present in man had stopped to be argued. At once it became evident that this phenomenon is inherent to a proportion of balanced chromosome rearrangement carriers, predominantly to patients with fertility problems. It is important to establish whether ICE operates in genome of fertile rearrangement carriers and to determine what kind of rearrangement and how far increases a risk of aneuploidy offspring. Using own and literature data 1) we have assessed rates of inherited non-contributing balanced rearrangements in patients with trisomy 21 (T21) and rates of both mutant and inherited non-contributing balanced rearrangements in parents of offspring with T21 and 2) we have analyzed a parental origin of T21 in affected offspring of carriers of balanced rearrangement. We have found that carriers of balanced reciprocal translocation or inversion, but not robertsonian translocation, are at increased risk of T21 offspring. However these data do not support the existence of ICE in its common sense, i.e. as an effect of rearrangement on other chromosome’s segregation at the carrier’s meiosis. Probably the data obtained suggest an effect of paternal rearrangements on maternal chromosomes segregation after fertilization.     

Physical and genetic characterization reveals a pseudogene, an evolutionary junction, and unstable loci in distal Xq28

A large portion of human Xq28 has been completely characterized but the interval between G6PD and Xqter has remained poorly understood. Because of a lack of stable, high-density clone coverage in this region, we constructed a 1.6-Mb bacterial and P1 artificial chromosome (BAC and PAC, respectively) contig to expedite mapping, structural and evolutionary analysis, and sequencing. The contig helped to reposition previously mismapped genes and to characterize the XAP135 pseudogene near the int22h-2 repeat. BAC clones containing the distal int22h repeats also demonstrated spontaneous rearrangements and sparse coverage, which suggested that they were unstable. Because the int22h repeats are involved in genetic diseases, we examined them in great apes to see if they have always been unstable. Differences in copy number among the apes, due to duplications and deletions, indicated that they have been unstable throughout their evolution. Taking another approach toward understanding the genomic nature of distal Xq28, we examined the homologous mouse region and found an evolutionary junction near the distal int22h loci that separated the human distal Xq28 region into two segments on the mouse X chromosome. Finally, haplotype analysis showed that a segment within Xq28 has resisted excessive interchromosomal exchange through great ape evolution, potentially accounting for the linkage disequilibrium recently reported in this region. Collectively, these data highlight some interesting features of the genomic sequence in Xq28 and will be useful for positional cloning efforts, mouse mutagenesis studies, and further evolutionary analyses.     

Genomic organization of the mouse Msh4 gene producing bicistronic, chimeric and antisense mRNA

By degenerate PCR and screening of mouse testis cDNA library, we have identified seven cDNAs from the meiotic recombination gene Msh4. Variant alpha and probably beta are likely involved in meiotic DNA recombination. Other variants have distinctive structures; variant epsilon, theta; and iota form a bicistronic operon, while variant delta contains antisense RNA for the endoplasmic reticulum (ER) chaperon Hspa5 gene and small open reading frame (ORF) identical to epsilon ORF2. Analysis of the exon-intron structures revealed unusual genomic organizations: the first three exons of delta and the first exon of epsilon are respectively mapped to the Hspa5 locus on chromosome 2 and the Pcbp3 locus on chromosome 10; the remaining exons of both variants are mapped to the Msh4 locus on chromosome 3. The first exon of variant beta is located on chromosome 16, while the others are located on chromosome 3. Synthesis of these mRNAs is assumed to require interchromosomal trans-splicing alone (beta and epsilon) or in combination with converse-splicing (delta). Most Msh4 variant mRNAs are mainly expressed in testis, but a small amount of each variant except for epsilon is also expressed in brain, heart, thymus, ovary and embryonic head. Enhanced green fluorescent protein (EGFP) fusion experiments showed that all the ORFs are translated, and most of those proteins are localized to a particular subcellular compartment. It also appeared that expression of variant delta induces cell death. This study suggests that the dynamic interchromosomal (intergenic) trans-splicing generates functional diversity of the mouse Msh4 gene.     

Characterization of normal and rearranged human chromosomes by simultaneous Q- and R-banding with chromomycin A3

Simultaneous Q- and R-type banding patterns in human chromosomes have been achieved by staining with chromomycin A3. Some peculiarities of these patterns as compared to the patterns induced by other fluorochromes are described. The resolution power of this technique in analyzing structural rearrangements of human chromosomes is discussed.     

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.     

Expansion and contraction of the DUP240 multigene family in Saccharomyces cerevisiae populations

The influence of duplicated sequences on chromosomal stability is poorly understood. To characterize chromosomal rearrangements involving duplicated sequences, we compared the organization of tandem repeats of the DUP240 gene family in 15 Saccharomyces cerevisiae strains of various origins. The DUP240 gene family consists of 10 members of unknown function in the reference strain S288C. Five DUP240 paralogs on chromosome I and two on chromosome VII are arranged as tandem repeats that are highly polymorphic in copy number and sequence. We characterized DNA sequences that are likely involved in homologous or nonhomologous recombination events and are responsible for intra- and interchromosomal rearrangements that cause the creation and disappearance of DUP240 paralogs. The tandemly repeated DUP240 genes seem to be privileged sites of gene birth and death.     

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G²). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.     

Pathway utilization in response to a site-specific DNA double-strand break in fission yeast

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+). Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.     

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution

In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation. Received: 20 April 1998 / Accepted: 26 May 1998     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

Genome organization influences partner selection for chromosomal rearrangements

Chromosomal rearrangements occur as a consequence of the erroneous repair of DNA double-stranded breaks, and often underlie disease. The recurrent detection of specific tumorigenic rearrangements suggests that there is a mechanism behind chromosomal partner selection involving the shape of the genome. With the advent of novel high-throughput approaches, detailed genome integrity and folding maps are becoming available. Integrating these data with knowledge of experimentally induced DNA recombination strongly suggests that partner choice in chromosomal rearrangement primarily follows the three-dimensional conformation of the genome. Local rearrangements are favored over distal and interchromosomal rearrangements. This is seen for neutral rearrangements, but not necessarily for rearrangements that drive oncogenesis. The recurrent detection of tumorigenic rearrangements probably reflects their exceptional capacity to confer growth advantage to the rare cells that contain them. The abundant presence of neutral rearrangements suggests that somatic genome variation is also common in healthy tissue.