The Effects of Mutagen-Sensitive Mutants of DROSOPHILA MELANOGASTER in Nonmutagenized Cells

The effects of 13 mutagen-sensitive (mus) mutants (representing seven loci) on mitotic chromosome stability in nonmutagenized cells have been examined genetically. To do this, mus-bearing flies heterozygous for the recessive somatic-cell marker, multiple wing hairs (mwh), were examined for increased frequencies of mwh clones in the wing blade. Mutants at the mus-103, mus-104 and mus-106 loci do not affect the frequency of mwh clones, while mus-101, mus-102, mus-105 and mus-109 alleles cause increases in the frequency of mwh clones. These data show that the wild-type alleles of latter four loci specify functions that are required for chromosome stability in nonmutagenized cells. Analysis of the size distribution of mwh clones produced by these mutants suggests that most chromosome instability caused by these mutants is the consequence of chromosome breakage; in the presence of mus-105 and mus-109 alleles a small fraction of the mwh clones are produced by an event (mitotic recombination, mutation, nondisjunction) that produces euploid clones. To inquire whether any of the extant alleles of the mus-101, mus-102, mus-105 and mus-109 loci might be leaky alleles of loci that carry out essential mitotic functions, chromosome stability in females homozygous for alleles of these loci has been compared to that of females carrying one dose of a mutant over a deficiency for that mus locus. These comparisons show that the extant alleles at the mus-101, mus-109 and mus-105 loci are all leaky mutants. It is suggested that all three of these loci may specify essential mitotic functions.     

Timing of mitotic chromosome loss caused by the ncd mutation of Drosophila melanogaster.

We studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin-like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX-XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin-like proteins in Drosophila.     

The Absence of Somatic Effects of P-M Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

The wings and abdomens of dysgenic and nondysgenic control flies were scored for the presence of clones of cells mutant for first and third chromosome markers. These exceptional clones can arise from mitotic recombination, de novo mutation or deletion, and P-M hybrid dysgenesis has been shown to increase the frequency of parallel processes occurring in germ-line cells. Particular attention was given to careful genetic and molecular characterization of all stocks and to providing adequate and appropriate controls so that even very small increases in somatic clone frequency due to P-M hybrid dysgenesis would be detected. No difference was found in the frequency, size distribution or anatomical distribution of mutant somatic clones correlated to hybrid dysgenesis, confirming previous indications. The potential adaptive significance of a germ-line restriction of P-M hybrid dysgenesis is discussed.     

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.     

Genetic interactions between mei-S332 and ord in the control of sister-chromatid cohesion.

The Drosophila mei-S332 and ord gene products are essential for proper sister-chromatid cohesion during meiosis in both males and females. We have constructed flies that contain null mutations for both genes. Double-mutant flies are viable and fertile. Therefore, the lack of an essential role for either gene in mitotic cohesion cannot be explained by compensatory activity of the two proteins during mitotic divisions. Analysis of sex chromosome segregation in the double mutant indicates that ord is epistatic to mei-S332. We demonstrate that ord is not required for MEI-S332 protein to localize to meiotic centromeres. Although overexpression of either protein in a wild-type background does not interfere with normal meiotic chromosome segregation, extra ORD+ protein in mei-S332 mutant males enhances nondisjunction at meiosis II. Our results suggest that a balance between the activity of mei-S332 and ord is required for proper regulation of meiotic cohesion and demonstrate that additional proteins must be functioning to ensure mitotic sister-chromatid cohesion.     

The odd-even effect in mitotically unstable B chromosomes in grasshoppers

The odd-even effect, by which B chromosomes are more detrimental in odd numbers, has been reported in plants and animals. In grasshoppers, there are only a few reports of this effect and all were referred to as traits related to the formation of aberrant meiotic products (AMPs). Here we review the existing information about B chromosome effects on AMPs, chiasma frequency and the number of active nucleolus organizer regions (NORs) per cell. Polysomy for A chromosomes and B chromosomes are two kinds of chromosome polymorphism frequently found in grasshoppers. In some aspects, e.g. meiotic behaviour and mitotic instability leading to individual mosaicism (in the case of mitotically unstable Bs), polysomic As show similar characteristics to B chromosomes. In fact, polysomy is regarded as one of the main mechanisms for B chromosome origin. Here we review some features of meiotic behaviour in known cases of polysomy and mitotically unstable Bs in grasshoppers, in looking for possible causes for the odd-even effect. In all these traits, the odd-even effect was apparent, although its appearance was not universal in any case, with variation among species or populations within the same species. The equational division and lagging of the extra chromosomes, when univalents, could favour the appearance of abnormal meiotic products, and the formation of bivalents, when there are two or more extra chromosomes, inhibits this process. Therefore, the odd-even effect might be a consequence of the concomitant operation of both aspects of extra chromosome meiotic behaviour. The possibility that the odd-even effect might result from an increase in cell stress generated by odd numbers is suggested.     

Spontaneous Mitotic Recombination and Evidence for an X-Ray-Inducible System for the Repair of DNA Damage in DROSOPHILA MELANOGASTER

Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.     

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6^cs, ca^nd, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6^cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca^nd, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.     

Wolbachia-mediated male killing is associated with defective chromatin remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.     

A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.

The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F(1) genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin.     

The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases

S. pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3. We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11. These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences. dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted. The gene products identified by anti-dis2 serum are enriched in nuclei. By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S. cerevisiae; the latter complements S. pombe dis2-11. These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1. The implications of these findings are discussed with regard to mitotic control.     

The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death

The segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.     

Use of a Yeast Site-Specific Recombinase to Produce Female Germline Chimeras in Drosophila

We describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.     

Modification of chromosome instability in Aspergillus nidulans

Strains of Aspergillus nidulans with a chromosome segment in duplicate show instability at mitosis; their colonies produce faster-growing sectors which arise from nuclei with spontaneous deletions in either duplicate segment. In an attempt to probe the deletion process, the effects of mutations causing sensitivity to UV treatment, and those of manganous ions, have been studied in strains carrying either Dp(I,II) or Dp(III,VIII). For comparison, the effects of Mn2+ on balanced and unbalanced diploids have also been examined. The uvsE allele, which decreases intragenic mitotic crossing over in diploids, increased deletion frequency in strains with either duplication. The uvsB allele, which increases intragenic mitotic crossing over in diploids, increased deletion frequency only in Dp(I,II) strains; in addition, by causing early mitotic crossing over between the homologous segments, it produced some novel deletion products. Mn2+ substantially decreased the deletion frequency in Dp(I,II) strains and decreased mitotic crossing over in diploids; it had no effect on Dp(III,VIII) strains. The results suggest that in haploid duplication strains there are two classes of spontaneous DNA lesions, recombinogenic and non-recombinogenic, both of which, failing repair, lead to deletion.     

Chromosome studies in polycythemia vera.

Polycythemia vera (PV) represents an apparent monoclonal stem cell proliferation with a frequent transition to full neoplastic behavior. Up to 26% of untreated PV patients can be expected to have some chromosome abnormalities in the marrow at the time of diagnosis, and 10--15% have an abnormal cell line or clone. Both structural and numerical aberrations occur. Aneuploidy is the most common type of chromosome abnormality, however, with hyperdiploid clones occurring more frequently than hypodiploid clones. Chromosomes 1, 8, 9 and 20 are involved in a non-random pattern, and aberrations of all the F group, or at least the No. 20 chromosome seem to be associated to some extent with diseases involving erythroid hyperplasia. Leukemia develops in a certain percentage of patients regardless of the type of treatment they have received, but the relationship, if any, between the chromosome abnormalities and the development of leukemia is still uncertain. The abnormal clones that occur in PV appear to be quite stable and there is no indication at this time that they correlate with a prognosis of leukemic transformation.     

Studies of mitotic and centromeric abnormalities in Roberts syndrome: Implications for a defect in the mitotic mechanism

Roberts syndrome is an inherited human condition that is of particular interest because separation of centromeres and constitutive heterochromatin is observed in metaphase chromosomes. In this study we investigated the frequency of other cytological abnormalities in three Roberts syndrome patients. Our findings when taken with previous cytological reports emphasize that there are other features that are equally characteristic of Roberts syndrome: (1) aneuploidy with random chromosome loss and (2) micronuclei and/or nuclear lobulations of 8%–24% of interphase cells. We observed abnormal chromosome movement involving one or all the chromosomes during anaphase. Evidence is presented suggesting that aneuploidy, micronuclei and abnormal nuclear morphology are a direct result of lagging chromosomes. The cytological features documented for Roberts syndrome indicate that this is a human mitotic mutant.by T.C. Hsu     

Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster

Single cells were marked by X-ray-induced somatic recombination at the blastoderm stage (3 hr) and at 7 and 10 hr of development, when the embryos are relatively insensitive to radiation damage. The resulting clones of genetically marked cells were analyzed on the adult cuticle. For the three stages examined there is a decrease in clone size with developmental age indicating proliferation of the primordial disc cells which give rise to the adult cuticular structures. The extent of the clones has been used to study the specificity of determination in the primordial disc cells at the time of induction. A significant fraction of the clones induced at all three stages extend from the antenna into the head. In the thorax, some of the clones induced at 3 hr extend from the second leg into the wing, thus indicating that these cells are not yet disc-specifically determined. No clones, however, were found extending from the first leg into the second or from the second into the third, and by 7 hr all thoracic clones were restricted to a single disc. The results provide evidence that determination at the blastoderm stage is not yet disc-specific.     

The distribution of chromosome damage, non-reciprocal translocations and clonal aberrations in lymphocytes from Chernobyl clean-up workers

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.     

Establishing interspecific mosaic genome lines between Drosophila ananassae and Drosophila pallidosa by means of parthenogenesis

Strong sexual isolation exists between the closely related species Drosophila ananassae and D. pallidosa, but there is no obvious post-mating isolation; both sexes of the hybrids and their descendants appear to be completely viable and fertile. Strains exhibiting parthenogenesis have been derived from wild populations of both species. We intercrossed such strains and established iso-female lines after the second generation of parthenogenesis. These lines are clones, carrying homozygous chromosomes that are interspecific recombinants. We established 266 such isogenic lines and determined their genetic constitution by using chromosomal and molecular markers. Strong pseudo-linkage was seen between loci on the left arm of chromosome 2 and on the right arm of chromosome 3; the frequency of inheriting the two chromosome regions from the same species was significantly larger than expected. One possible cause of pseudo-linkage is female meiotic bias, so that chromosomes of the same species origin tend to be distributed to the same gamete. But this possibility is ruled out; backcross analysis indicated that the two chromosome regions segregated independently in female hybrids. The remaining possibility is elimination of low-fitness flies carrying the two chromosome regions from different species. Thus, genetic incompatibility was detected in the species pair for which no hybrid breakdown had previously been indicated. The 'interspecific mosaic genome' lines reported here will be useful for future research to identify genes involved in speciation and phenotypic evolution.