Induction of mitotic crossing-over in diploid strains of Aspergillus nidulans using low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.     

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent.

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.     

On the mechanisms of induced somatic recombination by certain fungicides in Aspergillus nidulans.

Four fungicides interfered with the segregation of chromosomes at mitosis of Aspergillus nidulans by increasing the somatic recombination, shown as colour sectors in green colonies, in a strain heterozygous for spore colour mutations. In an attempt to discover the mechanisms by which these fungicides increased the somatic recombination, a prototrophic diploid strain, heterozygous for colour and several other appropriate markers in all chromosomes, was used which enabled the detection and classification of all colour recombinants to be made by genetic analysis. The fungicides investigated were: benomyl (methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate) a benzimidazole derivative, botran (2,6-dichloro-4-nitroaniline) and chloroneb (1,4-dichloro-2,5-dimethoxybenzene) of the aromatic hydrocarbon group of fungicides, and the antibiotic actinomycin D. At least three different mechanisms, non-disjunction, mitotic crossing-over and breakage-deletion, were found to be responsible for the recombinogenic activity of the compounds studied.     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

Unequal crossing over is the principal pathway of homologous recombination in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.     

CHPA, a cysteine- and histidine-rich-domain-containing protein, contributes to maintenance of the diploid state in Aspergillus nidulans

The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the chpA (cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid Aspergillus nidulans strains gives rise to chpA knockout haploids and heterozygous diploids but no chpA knockout diploids. A. nidulans chpA heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of chpA from diploid A. nidulans resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for chpA in maintenance of the diploid phase in A. nidulans. Furthermore, the human chpA homolog, Chp-1, was able to complement haploinsufficiency in A. nidulans chpA heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes.     

A Non segregation F2 Population Derived from the Cross of Triploid×Diploid in Rice

A non-segregating F2 population was obtained from the cross of tfiploid × diploid in rice ( Oryza sativa L. ). The maternal parent used in cross was SAR-3 which is an autotriploid occurring in the field, and the paternal was a restored line Sheng 47. The F2 population from the same F1 plant appears uniform, and so does F3. Results from coefficients of variation (CV) of F2, Ducan’s test of F3 and SSRs marker analysis showed that the F2 of SAR-3 × Sheng 47 was non-segregating. Markers for either parent could be present in F2 progenies. In some cases, both markers for each parent could be found on one chromosome. The authors presume that after crossing there might be recombination of chromosome followed by homozygotization. Stability of early generation could be utilized in breeding, as it is a new approach to shorten the breeding procedures.     

Unequal crossing over as the pathway of adaptive homologous recombination between the direct DNA repeats in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats within the extended tandem duplications in E. coli results from unequal sister-chromosome exchanges. This conclusion follows from the observations on the segregation of completely or partly homozygous diploid segregants by heterozygous duplications. The formation of diploid segregants with preserved heterozygosity for the unselected markers could also result from "symmetrical" intrachromosomal recombination. Analysis of the segregant genotypes, however, confirmed their formation via unequal crossing over. The data obtained indicated that in tandem duplications segregation of diploid recombinants of different types was preceded by the formation of triplications as the products of unequal sister-chromosome exchanges. In heterozygous duplications, unequal crossing over is manifested as a highly frequent adaptive exchange, providing the survival of the most part of the duplication-carrying cells on selective medium. It is suggested that adaptive mutagenesis can be the consequence of unequal sister crossing over.     

Alternative reproduction pathway inAspergillus nidulans

Recombinant haploid segregants were recovered in filamentous fungus Aspergillus nidulans (Eidam) G. Winter directly from the heterokaryons instead of diploid segregants (process described earlier as parameiosis). In spite of the reproductive complexity of A. nidulans, parameiosis has only now been observed in this fungus. Since parameiosis was characterized by the occurrence of genetic recombination inside heterokaryotic hyphae, master strains (uvs+) and uvs mutants with high rate of both mitotic exchanges or chromosome nondisjunction were used to form heterokaryons. Two groups of mitotic segregants were recovered directly from heterokaryons--aneuploids and stable haploids. Heterokaryons formed with uvs mutants produced a higher number of parameiotic segregants compared to the heterokaryons formed with uvs+ strains. Segregants were analyzed by nutritional markers, acriflavine resistance and conidial color. Normal meiotic behavior of haploid recombinants was observed.     

Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure.

Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.     

Unequal Crossing Over Is the Principal Pathway of Homologous Recombination in Tandem Duplications of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.__________Translated from Genetika, Vol. 41, No. 8, 2005, pp. 1038–1044.Original Russian Text Copyright © 2005 by Prokop’ev, Sukhodolets.     

Recombination between Homologous Chromosomes Induced by Unrepaired UV-Generated DNA Damage Requires Mus81p and Is Suppressed by Mms2p

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.     

A versatile and efficient gene-targeting system for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.     

A Test for Rare Male Mating Advantage with DROSOPHILA PSEUDOOBSCURA Karyotypes

Inbred diploid yeast strains heterozygous or homozygous for the rad18-2 allele and carrying markers for detection of mitotic recombination were constructed. The homozygous rad18-2/rad18-2 strain was highly sensitive to killing by UV light, showed greatly elevated frequencies of spontaneous and induced mitotic recombination and was more sensitive to trimethoprim than the wild-type diploid. The heterozygous strain RAD18/rad18-2 was intermediate in its response for these same phenotypic characters. These findings are discussed in the light of other studies in which incomplete dominance of genes involved in some aspect of DNA repair has been reported.     

Sister Chromatids Are Preferred over Homologs as Substrates for Recombinational Repair in Saccharomyces Cerevisiae

A diploid Saccharomyces cerevisiae strain was constructed in which the products of both homolog recombination and unequal sister chromatid recombination events could be selected. This strain was synchronized in G1 or in G2, irradiated with X-rays to induce DNA damage, and monitored for levels of recombination. Cells irradiated in G1 were found to repair recombinogenic damage primarily by homolog recombination, whereas those irradiated in G2 repaired such damage preferentially by sister chromatid recombination. We found, as have others, that G1 diploids were much more sensitive to the lethal effects of X-ray damage than were G2 diploids, especially at higher doses of irradiation. The following possible explanations for this observation were tested: G2 cells have more potential templates for repair than G1 cells; G2 cells are protected by the RAD9-mediated delay in G2 following DNA damage; sister chromatids may share more homology than homologous chromosomes. All these possibilities were ruled out by appropriate tests. We propose that, due to a special relationship they share, sister chromatids are not only preferred over homologous chromatids as substrates for recombinational repair, but have the capacity to repair more DNA damage than do homologs.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Meiotic parthenogenesis in a root-knot nematode results in rapid genomic homozygosity

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F(2) lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (<3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.     

Deletion of <Emphasis Type="Italic"> Aspergillus nidulans </Emphasis><Emphasis Type="Italic">aroC</Emphasis> using a novel blaster module that combines ET cloning and marker rescue

Blaster cassettes are of significant value in functional genomics, as they represent tools with which to inactivate duplicated or homologous genes in an individual organism. We have constructed a novel blaster module which allows repeated gene deletion in the filamentous fungus Aspergillus nidulans. Because bacterial resistance marker cassettes are employed as flanking repeats in direct orientation, the blaster cassette is suited for recombinogenic engineering by ET cloning in Escherichia coli. The functionality of the blaster module was demonstrated by deleting the chorismate mutase-encoding gene aroC of A. nidulans, followed by marker rescue based on mitotic recombination. The resulting aroCDelta strains are auxotrophic for phenylalanine but not tyrosine, and display a limited capacity for fruit body formation and ascosporogenesis, which depends on the phenylalanine/tyrosine supply. The data support the notion that amino acid status has a strong impact on cleistothecium development in A. nidulans.     

Bleomycin-induced mutation and recombination in Saccharomyces cerevisiae.

Clinical preparations of bleomycins (BM) were tested for their recombinogenicity and mutagenicity at relatively high survival levels in the simple eucaryote, Saccharomyces cerevisiae. More than a dozen test loci or genetic intervals were assayed for bleomycin-induced mutation or recombination. Treatments of stationary phase diploid yeast routinely results in 25--75% inactivation. The antibiotic was mildly to very highly recombinogenic and mutagenic, with one exception. The amount of bleomycin-induced mutation, gene conversion or crossing-over depended upon the particular genetic markers assayed. The drug was also potently recombinogenic in yeast cells growing in the presence of BM. These results contrast with the finding that this antitumor agent was not mutagenic in the Salmonella/mammalian microsome mutagenicity test; possible explanation of this difference are given.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.