Nonreciprocal Translocation

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Theodore Kushnick - One of the best experts on this subject based on the ideXlab platform.

K. H. Ramesh - One of the best experts on this subject based on the ideXlab platform.

  • Short communication Association of MLL amplification with poor outcome in acute myeloid leukemia
    2020
    Co-Authors: Robert W. Maitta, Linda A. Cannizzaro, K. H. Ramesh
    Abstract:

    Chromosomal rearrangements and amplification of the MLL gene at 11q23 are common abnormal- ities found in patients with severe myelodysplastic disorders and lymphoid and acute myeloid leukemias. MLL rearrangements are associated with aggressive disease in both children and adults, with current evidence suggesting that MLL alterations are associated with a poor prognosis. We report the clinical, cytogenetic and histologic findings of a patient who presented with a de novo diagnosis of AML-M4 and who fits the profile of patients presenting with MLL alterations, such as old age at presentation, rapid progression, therapeutic refractoriness, and poor outcome. Two bone marrow specimens taken 1 month apart show the rapid deterioration of the patient's cytoge- netic abnormalities at the 11q23 locus, with amplification of MLL that was originally seen as a homogeneously staining region (hsr) on chromosome 11. In the second biopsy the hsr and MLL amplification appears as Nonreciprocal Translocation of multiple copies in the form of marked amplification of MLL on chromosome 16 in a background of increasing chromosomal aberrations. This case suggests that either the MLL amplification and Translocation alone or in conjunction with other flanking oncogenes may have played an important role in poor patient outcome. 2009

  • Association of MLL amplification with poor outcome in acute myeloid leukemia
    Cancer Genetics and Cytogenetics, 2009
    Co-Authors: Robert W. Maitta, Linda A. Cannizzaro, K. H. Ramesh
    Abstract:

    Abstract Chromosomal rearrangements and amplification of the MLL gene at 11q23 are common abnormalities found in patients with severe myelodysplastic disorders and lymphoid and acute myeloid leukemias. MLL rearrangements are associated with aggressive disease in both children and adults, with current evidence suggesting that MLL alterations are associated with a poor prognosis. We report the clinical, cytogenetic and histologic findings of a patient who presented with a de novo diagnosis of AML-M4 and who fits the profile of patients presenting with MLL alterations, such as old age at presentation, rapid progression, therapeutic refractoriness, and poor outcome. Two bone marrow specimens taken 1 month apart show the rapid deterioration of the patient's cytogenetic abnormalities at the 11q23 locus, with amplification of MLL that was originally seen as a homogeneously staining region (hsr) on chromosome 11. In the second biopsy the hsr and MLL amplification appears as Nonreciprocal Translocation of multiple copies in the form of marked amplification of MLL on chromosome 16 in a background of increasing chromosomal aberrations. This case suggests that either the MLL amplification and Translocation alone or in conjunction with other flanking oncogenes may have played an important role in poor patient outcome.

Yasuji Koyama - One of the best experts on this subject based on the ideXlab platform.

  • Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae
    Fungal Biology and Biotechnology, 2018
    Co-Authors: Tadashi Takahashi, Masahiro Ogawa, Atsushi Sato, Yasuji Koyama
    Abstract:

    Background Translocated chromosomal duplications occur spontaneously in many organisms; segmental duplications of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae . However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication. Results Double-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by Nonreciprocal Translocation of a 1.4-Mb duplicated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT . This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions. Conclusion In this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae .

  • Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae
    Fungal biology and biotechnology, 2018
    Co-Authors: Tadashi Takahashi, Masahiro Ogawa, Atsushi Sato, Yasuji Koyama
    Abstract:

    Translocated chromosomal duplications occur spontaneously in many organisms; segmental duplications of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae. However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication. Double-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by Nonreciprocal Translocation of a 1.4-Mb duplicated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT. This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions. In this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae.

James E. Haber - One of the best experts on this subject based on the ideXlab platform.

  • Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends.
    PLOS Genetics, 2010
    Co-Authors: John R. Lydeard, Zachary Lipkin-moore, Suvi Jain, Vinay V. Eapen, James E. Haber
    Abstract:

    In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a Nonreciprocal Translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition.

  • Chromosome breakage and repair.
    Genetics, 2006
    Co-Authors: James E. Haber
    Abstract:

    FROM time to time one of my colleagues working at a medical school commiserates with me because I spend ∼40 hr a year lecturing to undergraduates. I always reply that teaching has compelled me to learn a lot of material that I would not have known about had I taught only my specialized subject. These forays into the “beyond” were instrumental in moving my research in new directions. Three articles that I published in Genetics in the early 1980s (McCusker and Haber 1981; Haber and Thorburn 1984; Haber et al. 1984) were the result of learning about, in order to teach, classic genetic experiments in Drosophila and maize. When I arrived at Brandeis I was assigned to teach genetics, a subject I had never studied as either an undergraduate or a graduate student2. I was fortunate to team up in teaching with Jeff Hall, a master geneticist, who taught me much of the lore of Drosophila and maize, to add to the yeast genetics that my lab and I were slowly learning3. I was particularly interested in Barbara McClintock's study of the “Activator (Ac)/Dissociator (Ds)” transposable elements whose excisions led to cycles of breakage–fusion–bridge (BFB) of broken chromosome ends that generated chromosomal truncations. Her studies resonated strongly with the behavior of apparently broken chromosomes that we were studying in budding yeast as a consequence of mating-type gene switching. The study of yeast mating-type (MAT) gene switching has provided gainful employment for many scientists interested in cell-type regulation, gene silencing, chromosome architecture, and DNA repair. Haploid cells express either the MATa or the MATα allele and mate with cells of the opposite type, but cells expressing both MATa and MATα are nonmating. But most unusual was that homothallic MATa cells could switch to MATα, or vice versa, as often as every cell division. Takano and Oshima (1970; Oshima and Takano 1971) showed that switching depended on two distant loci that they viewed as “controlling elements,” similar to those defined by McClintock in maize. Hicks et al. (1977) made the insightful suggestion that these two loci were in fact unexpressed copies of mating-type information (now called HMLα and HMRa) that could be transposed to replace the original MAT allele. Extending the mutational analysis of Mackay and Manney (1974) and the sometimes-published work of the inventive Don Hawthorne (Hawthorne 1963; see also Herskowitz 1988), Strathern et al. (1981) proposed that MATα encoded both a repressor of a-specific genes (MATα2) and a positive regulator of α-specific genes (MATα1). Thus a matα1 matα2 mutant proved to be a-like. The transposition/replacement of MAT alleles occurs very frequently in homothallic cells, expressing the HO gene encoding a site-specific endonuclease, but is very rare in heterothallic strains where HO is inactive. One way we tried to study these rare events was by mating two heterothallic MATα strains together, on the assumption that if one of them switched to MATa, it would readily conjugate with a MATα cell, as first shown by Hawthorne (1963). We took up this approach (McCusker and Haber 1981) and found that indeed ∼25% of MATα × MATα matings did appear to result from such switches, leading to stable MATa/MATα diploids. However, most of the events were different and were often genetically unstable, giving rise to colonies with multiple phenotypes. Many matings arose from the creation of an at least transient a-like cell (presumably lacking expression of both MATα1 and MATα2), allowing it to mate with another α-mater. In some cases the resulting diploids were 2n-1 aneuploids that had lost the entire, presumably broken, chromosome. In many instances the diploids that were still heterozygous for markers distal to the MAT locus, but now α-mating. The most interesting group resulted in the loss of the all the markers distal to and including MATα. Many of these colonies gave evidence of continuing genomic instability, with the loss of additional markers on chromosome III. Among nine stable derivatives we analyzed in detail, eight were homozygous for MATα and more distal markers, but one contained a truncation of the right arm from at least MAT to the end of the chromosome. This last type was reminiscent of outcomes described in maize after the transposition of the Ds element. The truncation in yeast was one of the first examples of the apparent acquisition of a new telomere, either by de novo addition or by a Nonreciprocal Translocation. Before the days of genome sequencing, it was not possible to be more precise. Viewed from today's perspective, we imagine that the diploids still heterozygous for the right arm repaired the chromosome break by gene conversion, becoming MATα/MATα. Those that became homozygous for the distal region likely resulted from break-induced replication (BIR, also known as recombination-dependent DNA replication) in which one end of the double-strand break (DSB) established a replication fork that could copy >100 kb to the end of the homologous, template chromosome (Morrow et al. 1997; Kraus et al. 2001; Davis and Symington 2004; Malkova et al. 2005). It is also possible that some of the diploids showing loss of heterozygosity for markers distal to MAT arose from reciprocal exchanges accompanying gene conversion because we did not recover instances in which the distal regions became homozygous wild type. In the same year that John McCusker examined MATα × MATα matings in heterothallic strains, Barbara Weiffenbach was studying similar genomic instability in homothallic strains (Weiffenbach and Haber 1981). Malone and Esposito (1980) had shown that MAT switching was lethal in the absence of the RAD52 recombination gene, suggesting that the process had similarities to the repair of X-ray-induced damage. It was not until 1982 that Strathern et al. (1982) showed that MAT switching did indeed involve a DSB. I had isolated a recessive mutation, swi1-1, that reduced, but did not eliminate, MAT switching (Garvik and Haber 1977); hence we could study a population of MATα rad52 cells in which several percent of the cells in each generation attempted to switch; such cells became a-like. This made it possible for us to examine newly generated broken chromosomes, importantly with all the breaks initiating at the same place. We mated these cells with a heterothallic MATα strain and recovered diploids in which the broken chromosome was repaired by gene conversion, by BIR, or by new telomere addition. We again found that many of these diploids exhibited genomic instability, so that the colonies that arose were sectored for markers on chromosome III. Again, there were cases where the broken chromosome was completely lost and others in which there were stable outcomes in which markers distal to MAT were hemizyous or homozygous4.

  • RAD51-independent break-induced replication to repair a broken chromosome depends on a distant enhancer site
    Genes & Development, 2001
    Co-Authors: Anna Malkova, Laurence Signon, Christopher B. Schaefer, Maria L. Naylor, James F. Theis, Carol S. Newlon, James E. Haber
    Abstract:

    Break-induced DNA replication (BIR) plays a key role in the repair of double-strand breaks (DSBs) in eukaryotic chromosomes. BIR is likely to be important in restarting DNA replication after the collapse of a replication fork (for reviews, see Haber 1999; Michel 2000), but it also functions to repair DSBs created in other ways. In Saccharomyces cerevisiae, when there is homology with another chromosomal template only centromere-proximal to the DSB, repair may occur by recombination-dependent DNA replication, forming a Nonreciprocal Translocation (Bosco and Haber 1998). Similar types of events have been documented when linearized plasmids are introduced into yeast cells and initiate DNA replication from a template chromosome, travelling as much as several hundred kilobases to the end of the chromosome (Dunn et al. 1984; Morrow et al. 1997). BIR also may account for the analogous ALT mechanism of telomere maintenance of human tumor cells in the absence of telomerase, as well as similar types of telomere maintenance in budding yeast (Lundblad and Blackburn 1993; Le et al. 1999; Teng and Zakian 1999; Dunham et al. 2000; Teng et al. 2000). BIR is a recombination-dependent mechanism to initiate DNA synthesis. By creating a DSB in G1 cells, we showed that this replicative process can occur before the initiation of normal DNA replication, at least in wild-type cells (Bosco and Haber 1998). BIR may be closely related to the mechanism of gene conversion induced by a DSB, based on gene conversion that appears to involve both leading- and lagging-strand DNA synthesis (Holmes and Haber 1999). Both processes may initiate in the same way, by the invasion of a single-stranded DNA end that is produced by 5′ to 3′ resection of the DSB, to establish a modified replication fork. In gene conversion, this process would terminate when the second end of the DSB engaged the replication structure, whereas in BIR, the replication process would continue to the end of the chromosome. In some circumstances, BIR and gene conversion appear to be alternative, competing outcomes of DSB-initiated recombination (Esposito 1978; Voelkel-Meiman and Roeder 1990; Malkova et al. 2000). Nevertheless, there are significant differences between BIR and gene conversion (Malkova et al. 1996). Surprisingly, BIR can occur in the absence of the Rad51p strand exchange protein, whereas gene conversion is completely abolished (Fig. ​(Fig.1).1). However, BIR still requires the RAD52 protein (Malkova et al. 1996) and thus is apparently a recombination-dependent process. Similarly, in strains in which Rad54p, Rad55p, or Rad57p is deleted, BIR still occurs, but gene conversion is eliminated (Signon et al. 2001). In the absence of these recombination proteins, BIR is a relatively inefficient process, occurring in ∼10–15% of cell divisions. In colonies derived from single cells experiencing a DSB, ∼80% of them are sectored; some cells have lost the broken chromosome entirely whereas others retained it, by BIR ( see Fig. ​Fig.1).1). The RAD51- and RAD54-independent pathway of BIR depends on RAD59, TID1(RDH54), MRE11, RAD50, and XRS2 (Signon et al. 2001). Double mutant combinations rad51Δ rad50Δ, rad51Δ rad59Δ, and rad54Δ tid1Δ all severely impaired in BIR, although not as strongly as in rad52Δ strains (Signon et al. 2001). Figure 1 Repair of a DSB in a MATa/MATα-inc diploid. In the absence of Rad51p, gene conversion (A) is virtually eliminated. Repair occurs by break-induced replication, producing Ade+Thr− cells that may either retain a URA3 marker (B) or ... Essentially all DSB-induced mitotic recombination requires the Rad52 protein (reviewed in Pâques and Haber 1999), which has strand-annealing activity in vitro (Mortensen et al. 1996). Rad52p physically and genetically interacts with Rad51p and with the single-strand DNA-binding protein complex, RPA. However, several types of DSB-induced recombination can occur in the absence of Rad51p, Rad54p, Rad55p, and Rad57p. These events include single-strand annealing (SSA) of homologous sequences flanking a DSB (Ivanov et al. 1996) and maintenance of chromosome ends in the absence of telomerase (Le et al. 1999). On centromeric plasmids, but not on chromosomes, even gene conversion between inverted repeats can occur without Rad51p (Ivanov et al. 1996). It has been suggested that such recombination could occur by two Rad51p-independent processes, BIR coupled to SSA (Kang and Symington 2000). In recombining plasmids, the requirement for Rad51p depends on a favorable chromatin structure of the recombining sequences (Sugawara et al. 1995). When donor sequences are heterochromatic, Rad51p is required, whereas when the sequences are in a less constrained chromatin state, Rad51p is not needed. Rad52p is needed in every situation. These experiments suggest that successful BIR might only be able to be initiated at special locations along the chromosome, at sites that are sufficiently accessible to allow strand invasion in the absence of Rad51p. We asked if BIR initiated only at a limited number of locations. Surprisingly, repair did not initiate anywhere in the first 13 kb proximal to the DSB. In the absence of Rad51p, most of the BIR events on chromosome III depend on sequences adjacent to an origin of DNA replication, but the sequences needed for origin function are not required. Moreover, we show that this sequence does not provide an especially open site for strand invasion of the template, because it is only needed on the broken chromosome. This cis-acting site may enable the BIR process to perform the extensive replication that occurs from this point to the end of the chromosome, >130 kb away.

John E Wiley - One of the best experts on this subject based on the ideXlab platform.

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