Mitotic Recombination Events Research Articles

Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

Autonomous and non-autonomous phenotypic expression of Notch mutant cells in Drosophila embryogenesis.

Transplantation of single homozygous Notch - cells into the ventral neuroectoderm of Drosophila wild-type embryos reveals a non-autonomous behaviour of these cells. However, mitotic recombination events induced in heterozygous cells in imaginal discs lead to generation of homozygous Notch - cells which differentiate cell autonomously. We have tested various possible explanations for the non-autonomous behaviour of Notch mutant cells following transplantation. We confirm the previous results using different Notch alleles. Moreover, we find that increasing the number of wild-type Notch copies in a cell increases probability that it will take on an epidermal fate. However, single Notch mutant cells behave differently depending on whether they are placed in the ventral neuroectoderm, the procephalic neuroectoderm or the proctodeal anlage. Following transplantation into host embryos devoid of mesoderm, almost all single Notch mutant cells behave autonomously. These results suggest an influence of the mesoderm on ectodermal development. Finally, we provide further evidence that DELTA acts as the signal source in lateral inhibition.

Isolation and molecular characterization of spontaneous mutants of lymphoblastoid cells with extended loss of heterozygosity

The human major histocompatibility complex comprising the HLA class I and II genes provides a versatile source of natural heterozygous loci. This polymorphic genetic system allows analysis of the mechanistic aspects of loss of heterozygosity (LOH), a major phenomenon observed at tumor suppressor genes in human cancer cells. Four lymphoblastoid cell lines, ORI, TK6, WI-L2-NS and VH, were used to adjust current HLA immunoselection protocols to quantify loss of HLA-A2 in human lymphoblastoid cell lines. The modified selection protocol was used to isolate independent spontaneous HLA-A2 mutants from the lymphoblastoid cell line ORI. The frequency of spontaneous loss of HLA-A2 in ORI was 1.7×10 −5. By HLA typing 35 spontaneous HLA-A2 mutants, we showed that 74% of the HLA-A2 mutants also lost expression of the HLA-B allele, which is located on the same haplotype as HLA-A2. Microsatellites on both arms of chromosome 6 were used for molecular characterization of the spontaneous HLA-A2 mutants. Loss of heterozygosity at various loci on the p-arm or loss of an entire chromosome 6 was found in 80% of the mutants. Surprisingly, it appeared that a presumed mitotic recombination event in the cell line ORI itself had resulted in homozygosity of all markers distal from the HLA locus up to the telomere. This greatly limited the detection of mitotic recombination, resulting in LOH up to the telomere, on the short arm of chromosome 6 in this cell line. However, gene dosage analysis detected two copies of the remaining D6S265 allele in mutants which showed LOH at various loci along the p-arm. This suggested that recombination resulted in LOH in these mutants. The lymphoblastoid cell line TK6 did contain informative microsatellites along the complete chromosome 6. Mutants of TK6 either retained heterozygosity of all p-arm markers, showed LOH of all p-arm markers or showed loss from a breakpoint up to the telomere. These data indicate that recombination and chromosome loss both are important mechanisms involved in loss of the HLA-A2 allele in vitro. Such mechanisms may be involved in LOH in vivo and contribute to loss of tumor suppressor alleles.

Interstitial Deletion Constitutes the Major Mechanism for Loss of Heterozygosity on Chromosome 20q in Polycythemia Vera

An acquired deletion of the long arm of chromosome 20 is a recurrent abnormality in myeloproliferative disorders, particularly polycythemia vera and myelodysplastic syndromes. The association of 20q deletions with myeloid "stem cell" disorders suggests that the deletions mark the site of one or more genes, loss or inactivation of which plays a role in the regulation of normal hematopoietic progenitors. We have recently performed a detailed molecular analysis of 20q deletions in peripheral blood (PB) granulocytes and defined a commonly deleted region of 16 to 21 centimorgan (cM). To further reduce the size of the common deleted region we have searched for small deletions or mitotic recombination events, neither of which would be detected by conventional cytogenetics. We have studied 48 patients with polycythemia vera and four patients with idiopathic myelofibrosis. In each case, cytogenetic analysis had either failed or had shown no abnormalities of chromosome 20. Seventeen microsatellite markers that span the common deleted region were used to search for loss of heterozygosity in granulocyte DNA. No instance of microsatellite instability was observed in a total of 880 comparisons of granulocyte and T-cell DNA. Granulocyte DNA from four patients exhibited allele loss on 20q. In each case the allele loss was caused by an interstitial deletion because heterozygosity at distal markers was retained and because quantitative Southern blotting demonstrated hemizygosity. Loss of heterozygosity in PB granulocytes would be masked by the presence of significant numbers of normal granulocytes not derived from the malignant clone. Therefore, the human androgen receptor assay (HUMARA) was used to determine granulocyte clonality. In 21 of 27 informative female patients the majority of the granulocytes were clonally derived. In 5 patients the granulocytes appeared polyclonal and in 1 patient unilateral X inactivation was observed in both granulocytes and T cells. These results show that, in the vast majority of patients presented here, the failure to detect loss of heterozygosity cannot be attributed to the presence of normal polyclonal granulocytes. Our results therefore show that allele loss on chromosome 20q in polycythemia vera does not commonly involve mitotic recombination or chromosome loss and that microsatellite instability is a rare event in this disorder.

Induction of somatic mutation and recombination by four inhibitors of eukaryotic topoisomerases assayed in the wing spot test of Drosophila melanogaster.

Four inhibitors of eukaryotic topoisomerases were investigated for genotoxic effects in the wing spot test of Drosophila melanogaster. As a somatic mutation and recombination test (SMART) this assay assesses mitotic recombination and mutational events of various kinds. We studied camptothecin as a topoisomerase I inhibitor, as well as ellipticine as an intercalating inhibitor and teniposide and etoposide as two non-intercalating inhibitors of topoisomerase II. Wing spots were induced in flies trans-heterozygous for the recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) as well as in flies heterozygous for mwh and the multiply inverted TM3 balancer chromosome. All four compounds proved significantly genotoxic in this test. The spot induction frequencies formally standardized to the millimolar unit of exposure dose decreased in the order camptothecin > teniposide > ellipticine greater, similar etoposide in the mwh/flr3 inversion-free genotype. In the mwh/TM3 genotype, in which mitotic crossing over is suppressed because of the inversion-heterozygosity, the observed spot frequencies were considerably reduced, but to different extents. In this genotype, spot induction by ellipticine was not statistically significant, and it was determined that >99% of the spots are due to mitotic recombination in mwh/flr3 flies. For the other compounds, spot induction in the inversion-heterozygous genotype was significant. The relative contribution of recombination to total spot induction in the inversion-free genotype was 88% for camptothecin. It was significantly lower for the two epipodophyllotoxins teniposide (71%) and etoposide (59%). Only suggestions can be proffered at present as to how these proportions could be related to the primary damage produced by the respective compounds on the chromosomes.

Chromosome polymorphisms close to the cm-ADE1 locus of candida maltosa.

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succino-carboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE1; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it residues.

Multiple pathways for homologous recombination in Saccharomyces cerevisiae.

The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.

DNA structure-dependent requirements for yeast RAD genes in gene conversion.

In Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR). The donors have a chromatin structure inaccessible for both transcription and HO cleavage. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.

Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for both nucleotide excision repair and certain mitotic recombination events. Here, model recombination and repair intermediates were used to show that Rad1-Rad10-mediated cleavage occurs at duplex-single-strand junctions. Moreover, cleavage occurs only on the strand containing the 3' single-stranded tail. Thus, both biochemical and genetic evidence indicate a role for the Rad1-Rad10 complex in the cleavage of specific recombination intermediates. Furthermore, these data suggest that Rad1-Rad10 endonuclease incises DNA 5' to damaged bases during nucleotide excision repair.

Organization of chromosomes in Plasmodium falciparum: a model for generating karyotypic diversity.

Chromosomes of the human malaria parasite, Plasmodium falciparum, are compartmentalized into conserved and polymorphic domains. The conserved domains are transcribed and localized within the central region o f a chromosome, whereas the polymorphic domains are transcriptionally silent and situated at chromosome ends. Unlike the central chromosomal domains, chromosome ends contain repetitive sequence elements. In this review, Michael Lonzer, Derik de Bruin, Samuel Wertheimer and Jeffrey Ravetch propose that the chromosome ends play a functional role in generating genetic diversity by promoting meiotic and mitotic recombination and chromosomal rearrangement events.

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase.

Yeast Saccharomyces cerevisiae strains have been constructed that co-express cDNAs coding for the human cytochrome P-450 enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochrome P-450 reductase (oxidoreductase). Microsomal fractions prepared from the strains were able to efficiently activate various drugs to Salmonella mutagens. These experiments demonstrated that a functional interaction occurred between the respective human enzymes in the yeast microsomes. For every drug tested, the microsomes containing CYP enzymes and oxidoreductase were 2- to 4-fold better in activation than the corresponding microsomes that contained CYP alone. Interestingly, co-expression of CYP1A2 with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-O-deethylase activity, a problem which is related to this specific substrate. Using the microsomes, it was demonstrated that aflatoxin B1 was activated to a mutagen not only by CYP1A2 but also by CYP1A1. In contrast, benzo[a]pyrene was exclusively activated by CYP1A1 whereas CYP1A2 was inactive. The drug 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was activated by CYP1A2 and to a lesser extent by CYP1A1. A strong substrate specificity was observed with the two structurally related heterocyclic arylamines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). MeIQx was activated efficiently by both CYP enzymes, whereas MeIQ was only activated by CYP1A2 and not by CYP1A1. The fact that microsomes from vector transformed control strains were unable to activate any of the drugs studied underlines the suitability of these microsomes for metabolic studies. Moreover, the presence of suitable marker genes in the yeast strains will enable us to study mitotic recombination and gene conversion events induced by drugs that require metabolic activation.

Using Mitotic Recombinant Mutant Clonal Lymphocytes for Physical Mapping of Polymorphic Loci on the Short Arm of Human Chromosome 6

Immunoselection has been used to identify human lymphocytes that have undergone spontaneous mutation resulting in the loss of expression of one of the codominant HLA-A alleles. Approximately 35% of such mutations are the consequence of mitotic recombination events. Mitotic recombination is the result of non-sister chromatid exchange that leaves the mutated cell homozygous for all loci distal to the crossover point. The location of the crossover has been regionalized for 99 independently derived mutant lymphocyte clones by identification of their loss or retention of heterozygosity at seven reference polymorphic loci on chromosome 6. If a polymorphic locus of unknown map position is studied in clones from this ordered set of mitotic recombinants, and clones that display loss of heterozygosity and retention of heterozygosity of the locus are observed, then the map position of the locus is between the appropriate reference loci of the ordered set. The newly mapped locus becomes a reference locus in turn. In this way the mitotic recombinant mutant clones can be used to generate an ordered set of crossover points with a theoretical resolution limited only by the number of mutants generated. In this paper such a set of mutants is used to refine or confirm the map position of eight polymorphic loci on chromosome 6.

Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease.

Damage-specific recognition and incision of DNA during nucleotide excision repair in yeast and mammalian cells requires multiple gene products. Amino-acid sequence homology between several yeast and mammalian genes suggests that the mechanism of nucleotide excision repair is conserved in eukaryotes, but very little is known about its biochemistry. In the yeast Saccharomyces cerevisiae at least 6 genes are needed for this process, including RAD1 and RAD10 (ref. 1). Mutations in the two genes inactivate nucleotide excision repair and result in a reduced efficiency of mitotic recombinational events between repeated sequences. The Rad10 protein has a stable and specific interaction with Rad1 protein and also binds to single-stranded DNA and promotes annealing of homologous single-stranded DNA. The amino-acid sequence of the yeast Rad10 protein is homologous with that of the human excision repair gene ERCC1 (ref. 3). Here we demonstrate that a complex of purified Rad1 and Rad10 proteins specifically degrades single-stranded DNA by an endonucleolytic mechanism. This endonuclease activity is presumably required to remove non-homologous regions of single-stranded DNA during mitotic recombination between repeated sequences as previously suggested, and may also be responsible for the specific incision of damaged DNA during nucleotide excision repair.

XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination.

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.

Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms

The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in micro-organisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic ‘petite’ mutation in haploid yeast cells.

Effects of Post-treatment Incubation on Recombinogenesis in Incision-proficient and Incision-deficient Strains of Saccharomyces cerevisiae: I. Recombinogenesis after UV- and .GAMMA.-Ray-irradiation.

After UV-irradiation of G1 phase cells, most gene conversion and crossing-over took place in cells of incision-proficient strains without post-irradiation incubation. In contrast, incision-deficient cells markedly induced both of these recombinational events only after irradiated cells were incubated for several hours in liquid growth medium before selection. These results indicate that when G1 cells are irradiated with UV recombinational events are initiated during that G1 phase in incision-proficient strains; whereas, they are not initiated in irradiated incision-deficient strains before the cells reach the S/G2 phase. Experimental gamma-ray results also suggest that post-irradiation incubation is not required for incision-proficient and -deficient strains to initiate mitotic recombinational events. Taken together, these results show that the formation of incision nicks and of post-replication gaps in DNA appears to be necessary for the initiation of recombinational events after UV treatment; whereas, it is not required after gamma-irradiation.

Genetic changes in skin tumor progression: Correlation between presence of a mutant ras gene and loss of heterozygosity on mouse chromosome 7

Initiation of tumorigenesis in mouse skin can be accomplished by mutagenesis of the H- ras gene by treatment with chemical carcinogens. A mouse model system has been developed to study the additional genetic events that take place during tumor progression. Skin carcinomas were induced in F1 hybrid mice exhibiting restriction fragment length polymorphisms at multiple chromosomal loci. Analysis of loss of heterozygosity in such tumors showed that imbalance of alleles on mouse chromosome 7, on which the H- ras gene is located, occurs very frequently in skin carcinomas. The chromosomal alterations detected, which included both nondisjunction and mitotic recombination events, were only seen in tumors that have activated ras genes. We conclude that gross chromosomal alterations that elevate the copy number of mutant H- ras and/or lead to loss of normal H- ras are a consistent feature of mouse skin tumor development.

Genetic and molecular analysis of recombination events in Saccharomyces cerevisiae occurring in the presence of the hyper-recombination mutation hpr1.

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.

Cytogenetics of Mendelian mutations associated with cancer proneness.

About 5% of Mendelian mutations displaying neoplastic tendencies are associated with chromosomal aberrations. The best established examples are retinoblastoma and del(13)(q14) and aniridia-Wilms' tumor and del(11)(p13). Evidence suggests that both mutations behave as dominant traits in the individual and as recessive traits in the cells. DNA analysis indicates that tumorigenesis arises from homozygosisty for the mutant allele at these loci, as a consequence of mitotic nondisjunction or from a mitotic recombination event. An additional argument for this conclusion is provided by the demonstration of duplication of 11p15 in some patients with the Beckwith-Wiedemann syndrome, which is complicated often by Wilms' tumor and other embryonal tumors. Data obtained with molecular probes have shown that also rhabdomyosarcoma and hepatoblastoma arise by homozygosity for a mutant allele at a locus on 11p, suggesting ontogenic relatedness of these tumor types. Additional examples of Mendelian mutations associated with chromosome deletions and neoplasia include Langer-Giedion syndrome with multiple exostoses and del(8)(q24.1), multiple endocrine neoplasia and del(20)(p12.2). While the presence of specific chromosome changes in subjects with high susceptibility to neoplasia does pinpoint the location of DNA sequences involved in the predisposition to certain types of cancers, selected Mendelian mutations associated with chromosome instability and cancer proneness may elucidate biological principles of cell proliferation and transformation. However, our current knowledge of mechanisms resulting in increased frequency of chromosome breakage and cancer susceptibility in ataxia-teleangiectasia, Fanconi's anemia, Bloom's syndrome, and similar conditions are still very incomplete.