Evidence from somatic cells for crossing-over in humans

Chromosomal and gene marker studies of somatic cells in a family with multiple chromosome translocations show apparent instability of the translocations in transmission from generation to generation. This instability is attributed to meiotic crossing-over between the involved chromosomes. Technical advances in chromosome analysis combined with human gene map information in the study of this unusual family demonstrate for the first time that crossing-over involves exchange of chromatin between nonsister chromatids of homologous chromosomes.     

Mechanisms for gene conversion and homologous recombination: The double-strand break repair model and the successive half crossing-over model

Two mechanisms for gene conversion and homologous recombination were discussed. (1) The double-strand break repair model. A double-strand break is expanded to a gap, which is then repaired by copying a homologous sequence. The gene conversion is often accompanied by crossing-over of the flanking sequences. We obtained evidence for this model in Red pathway of bacteriophage lambda and RecE pathway of E. coli. (2) The successive half crossing-over model. Half crossing-over leaves one recombinant duplex and one or two end(s) out of two parental duplexes. The resulting ends are, in turn, recombinogenic. Successive rounds of the half crossing-over mechanism explains why apparent plasmid gene conversion in RecF pathway of E. coli is not accompanied by crossing-over. This model can explain chromosomal gene conversion if we assume that the donor is first replicated. Gene conversion during mating-type switching in yeast, antigenic variation in unicellular microorganisms, and chromosomal gene conversion in mammalian somatic cells are explained by this model. Distinguishing between these two mechanisms is important in understanding recombination in yeast and mammalian cells and also in its application to gene targeting.     

Somatic crossing-over in the housefly

Summary X-rays were found to produce genetically marked cells and cell clones in heterozygous larvae of Musca domestica. The results favor the conclusion that these marked clones originated by somatic crossing-over. The technique should prove useful in developmental analyses of the housefly.     

Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans.

Thirteen pesticides, aminotriazole, benomyl, captafol, captan, dalapon-Na, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, neburon, picloram and tordon were tested for ability to induce (1) point mutations to 8-azaguanine resistance, (2) mitotic crossing-over, and (3) mitotic non-disjunction and haploidization in Aspergillus nidulans. Tests were performed at three different pHs, i.e. 4.5, 7, 8.2. Three of the pesticides, captan , captafol and dichlorvos induced point mutations; dichlorvos also induced a high frequency of mitotic crossing-over and non-disjunction; benomyl induced a very high frequency of non-disjunction whereas aminotriazole induced weakly both types of somatic segregation.     

Mitotic crossing-over by anticancer drugs in Saccharomyces cerevisiae strain D5

Treatment with an anticancer drug causing mitotic crossing-over could lead to expression of recessive genes, previously masked in a heterozygote. Used clinically, such drugs might cause an increased risk of cancer in cases of familial tumours, such as Wilm's tumour or retinoblastoma. Potentially, novel forms of drug resistance could also be unmasked by such a recombinogenic event. We have estimated the extent of this potential problem in current clinical drugs by comparing a range of antitumour agents for ability to cause mitotic crossing-over in Saccharomyces cerevisiae strain D5. We have compared these data with ability to cause an increase in total aberrant colonies in the same experiments. Although many of the agents known to cause point mutation also have some ability for mitotic crossing-over, there are also point mutagens which have little recombinogenic potential. Conversely, some effective recombinogens appear to be either very specific or rather ineffective point mutagens. Although the most generally effective agents in the present experiments were alkylating agents, several other types of drug including DNA-cutting agents, topoisomerase inhibitors, other DNA-binding drugs and antimetabolites may stimulate mitotic crossing-over. None of the mitotic inhibitors or the DNA minor groove binding drugs tested caused recombinogenic events. It would seem that the ability to induce mitotic crossing-over is an important endpoint in its own right. Assays for this event might provide an important complement to other assays commonly required for registration of new pharmaceuticals.     

A model for somatic pairing derived from somatic crossing over with third chromosome rearrangements in Drosophila melanogaster

Summary X-ray induced crossing-over between the left arms of third chromosome pairs heterozygous for rearrangements was used to measure the somatic pairing of these arms. mwh spots were scored in the wing blades. A significantly reduced number of crossover spots is found in flies heterozygous for inversions or translocations located in the 3L arm. This decrease from control frequencies was experimentally demonstrated to be due to inviability of some crossover spots which are aneuploid as a result of crossing-over within the inverted sequence or between the translocation piece and its centromere. By the same token, some euploid crossover spots are produced by crossing-over outside of the inverted region or between the translocation breakpoint and the mwh locus. Thus, these results are evidence that the euchromatic non-centric portions of the 3L arms are somatically paired in the presence of rearrangements.A model of somatic pairing in which chromosomes are normally found in a hair pin configuration is proposed to account for these results and for some previous results with X-chromosomes which indicated that inversion heterozygotes did not pair somatically.Supported in part by USPHS grant GM 16096 to J.R.M.     

Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans

Thirteen pesticides, aminotriazole, benomyl, captafol, captan, dalapon-Na, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, neburon, picloram and tordon were tested for ability to induce (1) point mutations to 8-azaguanine resistance, (2) mitotic crossing-over, and (3) mitotic non-disjunction and haploidization in Aspergillus nidulans. Tests were performed at three different pHs, i.e. 4.5, 7, 8.2. Three of the pesticides, captan, captafol and dichlorvos induced point mutations; dichlorvos also induced a high frequency of mitotic crossing-over and non-disjunction; benomyl induced a very high frequency of non-disjunction whereas aminotriazole induced weakly both types of somatic segregation.     

Polymorphism of human chromosomes 1, 9, 16, Y: Variations,segregation and mosaicism

Summary A study was carried out on C-banded chromosomes 1, 9, 16, Y from an unselected population and from 30 normal families. We found: a) great variability in length and position of the C-bands; b) somatic mosaicism involving C-bands; c) variants in children that were not present in parental patterns. The possible role of crossing-over in generating the last two phenomena is discussed.     

Induction of somatic and male crossing-over by bleomycin in Drosophila melanogaster

Bleomycin (BLM) is well known as an antibiotic as well as for its antineoplastic activity. A clinical preparation of BLM was tested for its recombinogenicity in a higher eukaryotic organism, Drosophila melanogaster. Feeding of the F1 larvae on a medium with BLM increased somatic crossing-over spots on female tergites and induced recombination in male germ cells. However, nonlinear dose-response curves were obtained. Malformed tergites were also observed in females treated with BLM.     

Genotoxic effects of niclosamide in Aspergillus nidulans

A 2-5-month treatment with niclosamide, a widely used drug in developing countries, has been reported to induce lymphosarcomas in toad liver and kidney. The genotoxic effects of this drug have also been evaluated in Salmonella typhimurium, in somatic and germinal cells of mice and in human lymphocytes exposed in vitro and in vivo. The present study shows that niclosamide is also capable of inducing mitotic crossing-over and non-disjunction in Aspergillus nidulans, which points to the wide potential of this drug as a genotoxic agent.     

Cytological analysis of X-ray-induced aberrations in spermatocytes I and the somatic ganglia of male Drosophila melanogaster.

The correlation between damage induced in somatic cells and that induced in germinal cells of the same genetic pool of individuals is important in establishing the possibility of extrapolation of this damage to the offspring. We have used Drosophila melanogaster to compare cytologically the frequency of aberrations induced in the cells of somatic ganglia and in the spermatocytes I of males irradiated with 625 R X-rays. The spermatocytes were more sensitive than the gangliar cells to radiation: the aberrations induced in the spermatocytes I were three times more frequent than those induced in the ganglia. The distribution of the aberrations was, however, similar. The most important variable element lies in our observing no symmetrical exchanges in the spermatocytes. This result seems to us to be related to the fact that in the males of D. melanogaster there is no crossing-over.     

MMS induction of different types of genetic damage in Aspergillus nidulans: a comparative analysis in mutagenesis.

Methyl methanesulphonate (MMS) was used to test the induction of gene mutation, somatic crossing-over and mitotic non-disjunction in A. nidulans. Gene mutation was tested by inducing mutants resistant to 8-azaguanine and revertants of methG1 in a haploid strain. Somatic crossing-over was tested in heterozygous diploids, both with a selective method, i.e. inducing homozygosis to FPA resistance in a heterozygous fpa A1/+ strain, and with a non-selective method, i.e. identifying the frequencies of colour sectors. This latter method was also used to estimate the induction of non-disjunction because additional markers were present which permitted us to distinguish the two types of colour segregant. Generally, 3 different experimental procedures were used, namely the "plate test", i.e. plating of conidia in agar media containing MMS, and two types of "liquid test", i.e. brief treatment of quiescent or pre-germinated conidia in MMS solution before they were plated on agar media. Point mutations were induced with about equal efficiency with each method, whereas crossing-over was induced preferentially when germinating conidia were exposed to MMS. On the other hand, non-disjunction was induced in germinating and quiescent spores with equal efficiency, but such segregants were not recovered with the selective (fpa) method. The results are discussed for both their practical use in the mutagenic testing procedure and their theoretical implication.     

The inability of aminoazotoluene to induce somatic crossing-over or mutation in Glycine max (L.) merrill

Seeds from varieties T219 and L65–1237 of Glycine max heterozygous for the gene combination Y₁₁y₁₁ controlling chlorophyll development were soaked in aqueous solutions of aminoazotoluene (AAT) for varying time periods. Analysis of the simple leaves and the first compound leaf for spots indicated that this drug did not increase the frequency of mutation or somatic crossing-over in this system.     

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.     

multiSNV: a probabilistic approach for improving detection of somatic point mutations from multiple related tumour samples

Somatic variant analysis of a tumour sample and its matched normal has been widely used in cancer research to distinguish germline polymorphisms from somatic mutations. However, due to the extensive intratumour heterogeneity of cancer, sequencing data from a single tumour sample may greatly underestimate the overall mutational landscape. In recent studies, multiple spatially or temporally separated tumour samples from the same patient were sequenced to identify the regional distribution of somatic mutations and study intratumour heterogeneity. There are a number of tools to perform somatic variant calling from matched tumour-normal next-generation sequencing (NGS) data; however none of these allow joint analysis of multiple same-patient samples. We discuss the benefits and challenges of multisample somatic variant calling and present multiSNV, a software package for calling single nucleotide variants (SNVs) using NGS data from multiple same-patient samples. Instead of performing multiple pairwise analyses of a single tumour sample and a matched normal, multiSNV jointly considers all available samples under a Bayesian framework to increase sensitivity of calling shared SNVs. By leveraging information from all available samples, multiSNV is able to detect rare mutations with variant allele frequencies down to 3% from whole-exome sequencing experiments.     

Use of alpha- and beta-tubulin mutants for the study of spontaneous and induced chromosomal mis-distribution in Aspergillus nidulans

The effect of two different mutations, one involving an alpha-tubulin (tubA) and the other a beta-tubulin (benA33) gene, on somatic segregation has been investigated in diploid strains of A. nidulans. Both mutations, particularly benA33, increase the level of spontaneous chromosomal mis-distribution (CMD) phenomena, without affecting the frequency of crossing-over. The employment of homozygous strains for each of the two mutations in sensitivity tests toward various chemicals, allowed the clear identification of those interfering with microtubule assembly-disassembly processes (i.e. chloral hydrate, diamide, aminocarb, N-ethyl-maleimide, p-chlormercuribenzoate). Such compounds turned out to be very efficient and specific inducers of CMD in a somatic segregation assay performed using the wild-type strain P1. The same assay, when carried out with some of these compounds but employing a tubA/tubA strain, revealed a marked proneness toward CMD to be associated with such mutation, which is known to confer microtubule hypostability.     

Mitotic crossing-over as an important mechanism of floral sectoring in tradescantia

A purplle flowered Tradescantia hirsuticaulis is described exhibiting three classes of somatic sectors: red-only, red/blue twin spots and blue-only. The twin spots are shown to be the results of a single event since the association of red and blue cells in the same stamenis non-random and intervening purple cells occur far too infrequently to permit hairs containing both red and blue sectors to arise by sequential, facilitated, red-only and blue-only events.As in T. clone 02, deletion, as evidenced by the presence of micronuclei (chromosome fragments), is indicated to be one mechanism producing some kinds of sectors (red-only) in response to ionizing radiation.The predominant, if not exclusive, mechanism of spontaneous sectoring and important mechanism of even 60 R-induced sectoring in this T. hirsuticaulis (and by inference, in other Tradescantia) is argued to be mitotic crossing-over, since other mechanisms operating on its genotype do not allow for twin spots arising as the result of one event. This is supported by the prediction of all three types of sectors in the proper order of frequency as well as by the calculation of a “coefficientof coincidence” and a radiation response that are similar to those found for mitotic crossing-over in Drosophila.     

The kinetics of X-ray-induced somatic crossing-over in Drosophila melanogaster and the effects of dose fractionation

Nonlinear dose-response curves have been obtained with the X-ray induction of somatic crossover spots on Drosophila tergites, possibly reflecting the formation of independent breaks in two homologous chromatids in G₂ with restitution by crossing-over. Interrupting the irradiation dose for some time reduces the number of crossover spots observed compared with that found on flies irradiated continuously with the same total energy. The effect of dose fractionation is to separate the two breaks in time so that the pair of ends at the first break in the chromatid are rejoined to their original configuration before a new pair of ends is available for interaction. The amount of repair observed with 10 min separation is comparable to that observed from stage-7 oocytes.     

The pattern of polytene chromosome conjugation and crossing-over in interspecific hybrids of Drosophila

Summary The pairing of polytene chromosomes was investigated in the hybrids between three closely related species of Drosophila belonging to the virilis species group. It was found that within the same hybrid different chromosome bands lost the ability to pair by differing degrees. Furthermore, the same chromosome sections paired with different frequencies depending on the hybrid involved. This study revealed that poor polytene chromosome pairing in the hybrids is not due to specific genetic interaction in the hybrids, but depends solely on the properties of the homologous loci themselves. It was also of interest to find whether the pattern of polytene chromosome somatic pairing resembled in some way the picture of chromosome synapsis during meiosis. To obtain evidence for this, crossing-over in the hybrid 5th chromosome was analyzed both genetically and cytologically (from salivary gland chromosome observations). It was found that the sections of the fifth chromosome which were characterized by a high frequency of conjugation in the salivary glands of hybrids also exhibited a high frequency of crossing-over in hybrid females. It may be concluded that sections of the polytene chromosome characterized by a low frequency of conjugation behave in the same manner in meiosis, and thus rarely take part in genetic recombination.     

Segregation after mitotic crossing-over in isodicentric X chromosomes

Segregation after mitotic crossing-over in an isodicentric (idic) X chromosome with one active and one inactive centromere has given rise to two new cell lines, one in which the idic(Xpter) chromosome has two active centromeres (most of these chromosomes also have an inversion) and another in which neither centromere is active. The two X chromosomes are attached at the telomeres of their short arms. Similar segregation has given rise to two other cell lines with idic(Xq-) chromosomes. Other observations on segregation after mitotic crossing-over are reviewed. Unequal crossing-over has apparently played a major role in the evolution of various genes and heterochromatin. Retinoblastoma and Wilms tumor are in some cases associated with homozygosity of a chromosome segment resulting from mitotic crossing-over. Similarly, the high incidence of cancer in Bloom syndrome may be caused by mitotic crossing-over leading to homozygosity or amplification of oncogenes.