Drosophila Germ Cells Research Articles

Tudor and its domains: germ cell formation from a Tudor perspective

In many metazoan species, germ cell formation requires the germ plasm, a specialized cytoplasm which often contains electron dense structures. Genes required for germ cell formation in Drosophila have been isolated predominantly in screens for maternal-effect mutations. One such gene is tudor (tud); without proper tud function germ cell formation does not occur. Unlike other genes involved in Drosophila germ cell specification tud is dispensable for other somatic functions such as abdominal patterning. It is not known how TUD contributes at a molecular level to germ cell formation but in tud mutants, polar granule formation is severely compromised, and mitochondrially encoded ribosomal RNAs do not localize to the polar granule. TUD is composed of 11 repeats of the protein motif called the Tudor domain. There are similar proteins to TUD in the germ line of other metazoan species including mice. Probable vertebrate orthologues of Drosophila genes involved in germ cell specification will be discussed.

Increased spontaneous DNA damage in Cu/Zn superoxide dismutase (SOD1) deficientDrosophila

The superoxide dismutases (SODs) protect oxygen-using cells against reactive oxygen species, the potentially toxic by-products of respiration, oxidative metabolism, and radiation. We have previously shown that genetic disruption of CuZn SOD (SOD1) in Drosophila imparts a recessive phenotype of reduced lifespan, infertility, and hypersensitivity to oxidative stress. We now show that the absence of SOD1 increases spontaneous genomic damage. The increase in spontaneous mutation rate occurs in SOD1-null mutants in somatic cells as well as in the germ line. Further, we show that specific DNA repair-defective mutations, which are easily tolerated in SOD1(+) flies, lead to high mortality when introduced into the SOD1-null homozygous mutant background.

Selective Activation of STAT5 Unveils Its Role in the Maintenance of Hematopoietic Stem Cells and the Development of Myeloproliferative Disorder.

Recent studies have implicated the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the maintenance of stem cells, such as mouse embryonic stem cells and Drosophila germ cells. We have previously reported that thrombopoietin (TPO) can support in vitro self-renewal division of murine hematopoietic stem cells (HSCs) (CD34−/lowc-Kit+Sca-1+lineage marker-negative; CD34−KSL cells). Signal transducers and activators of transcription 5 (STAT5) is one of the major signaling molecules that mediate TPO signals. All these findings suggest that STAT5 could be an attractive candidate for therapeutic manipulation of HSCs. Cytokines activate JAK/STAT5 pathway along with other signaling pathways, causing difficulty to dissect STAT5-specific functions in hematopoietic stem cells (HSCs). Here we took advantage of constitutively active STAT5 mutants to selectively activate STAT5 signaling pathway in HSCs. The mutants used are STAT5A 1*6 that harbors two amino acid mutations S710F and H298R in the effecter domain, and STAT5A #2 that harbors a point mutation N642H in the SH2 domain. Retroviral transduction of either STAT5 1*6 or STAT5#2 mutant into purified CD34−KSL HSCs caused a drastic expansion of multipotential progenitors in vitro and promoted multi-lineage differentiation in vitro. During 7 days of culture supplemented with SCF and TPO, the number of high proliferative potential colonies (HPPC) increased ten-fold compared with the GFP control and half of them were derived from multipotential progenitor cells. Notably, even in the culture supplemented with SCF only, expression of STAT5 mutants in HSCs supported a similar mode of expansion of progenitors cells and multi-lineage differentiation, indicating that activation of STAT5 can substitute major biological effects of TPO in HSCs. In all in vitro experiments, STAT5 1*6 showed stronger effects than STAT5#2. To evaluate the effect of STAT5A mutants in the maintenance of long-term bone marrow repopulating HSC ex vivo, cultured transduced cells corresponding to 30 initial CD34−KSL HSCs were transplanted into lethally irradiated mice 7 to 10 days after transduction. Although rapid hamatopoietic repopulation was observed with HSCs expressing STAT5A 1*6, mice developed myeloproliferative disease (MPD) and succumbed to death within two months. In contrast, HSCs expressing STAT5A #2 presented significantly higher long-term repopulating capacity than the GFP control. These data indicate that selective activation of STAT5 maintains long-term repopulating ability of HSCs ex vivo. Oncostatin M, a well known STAT5 target gene, has been postulated to be involved in the development of MPD and was actually induced STAT5A 1*6-expressing cells. However, transplantation of OSM−/− HSCs expressing STAT5A 1*6 similarly caused a lethal MPD in wild-type mice, indicating that Oncostatin is not the main target for STAT5 in MPD development. Taken together, our findings establish a role for STAT5 in the self-renewal of HSCs and provide STAT5 as novel target for therapeutic manipulation of HSCs ex vivo.

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5–13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from W e / W e embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.

A Noncoding RNA Is Required for the Repression of RNApolII-Dependent Transcription in Primordial Germ Cells

RNApolII-dependent transcription is repressed in primordial germ cells of many animals during early development and is thought to be important for maintenance of germline fate by preventing somatic differentiation [1]. Germ cell transcriptional repression occurs concurrently with inhibition of phosphorylation in the carboxy-terminal domain (CTD) of RNApolII, as well as with chromatin remodeling [2–4]. The precise mechanisms involved are unknown. Here, we present evidence that a noncoding RNA transcribed by the gene polar granule component (pgc) [5] regulates transcriptional repression in Drosophila germ cells. Germ cells lacking pgc RNA express genes important for differentiation of nearby somatic cells and show premature phosphorylation of RNApolII. We further show that germ cells lacking pgc show increased levels of K4, but not K9 histone H3 methylation, and that the chromatin remodeling Swi/Snf complex is required for a second stage in germ cell transcriptional repression. We propose that a noncoding RNA controls transcription in early germ cells by blocking the transition from preinitiation to transcriptional elongation. We further show that repression of somatic differentiation signals mediated by the Torso receptor-tyrosine kinase [6] is important for germline development.

Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

Influence of the yellow Locus on Sensitivity of Drosophila Germ Cells to Chemical Mutagens

The effect of the yellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the recessive sex-linked lethal mutations (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin wild; y; y ct v; y mei-9a) were treated by mutagenic agents and then crossed to Basc females. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that the yellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Basc males were crossed to females of various genotypes including y and y mei-9a ones. The crosses involving y females yielded F1 progeny with high spontaneous lethality, whereas in F2, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny. The results obtained using the above two approaches suggest that the yellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.

Germ cell-less expression in medaka (Oryzias latipes).

The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH revealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15-18, 2004.

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 ( mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 ( tsg101). Effects of mGCL-1 on the expression of MDM2–p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2–p53 axis by enhanced degradation of MDM2.

Germline mutations at microsatellite loci in homozygous and heterozygous mutants for mismatch repair and PCNA genes in Drosophila

Microsatellite instability (MSI) is a phenotype associated with the deficient repair of replication errors. Replication errors persist in defective mismatch repair (MMR) conditions, although alterations in components of the replication machinery, such as the proliferating cell nuclear antigen (PCNA) factor, could also increase the replication errors; therefore, MSI is expected in both situations. It also seems that heterozygous individuals for MMR genes have a high risk of cancer, as in the case of human non-polyposis colon carcinoma (HNPCC), characterised by MSI. Thus, here we investigate the effect of heterozygosity for a Msh2-null allele or for altered PCNA alleles, on the stability of microsatellite sequences. The study was carried out in Drosophila germ cells analysing the progeny of individual crosses. We found that one Msh2 disrupted allele is sufficient to produce MSI in germ cells. Although the MSI in Msh2 −/+ individuals was in the same order of magnitude as in Msh2 −/− individuals, the former manifested a MSI that was four-fold lower. To a lesser extent, PCNA homozygous and heterozygous mutants also show MSI in the germline, which reveals the importance of DNA replication factors to maintain genomic stability in vivo. Furthermore, the high MSI found both in heterozygous Msh2 and PCNA mutants suggests a high degree of genomic instability in individuals bearing a mutant allele of these genes, which could have important implications in cancer susceptibility.

Cell migration and programmed cell death of Drosophila germ cells.

Cell migration and programmed cell death are essential components of animal development and homeostasis, and the germ cells of Drosophila provide a simple genetic system to study the molecular mechanisms that govern these important cellular processes. Detailed descriptions of germ cell migration in Drosophila were accomplished long ago, but most genetic and molecular analyses of the process have occurred within the past 10 years. A few of the genes required for germ cell migration have been identified, and a very interesting picture is emerging. However, a process as complex as cell migration must involve the functions of many more molecules. In addition, cell migration and cell death mechanisms are often linked, as it is important to eliminate cells that are misplaced and could present a danger to the organism. In Drosophila, genes involved in germ cell migration can also affect programmed cell death. Currently, very little is known about how germ cells ectopic to the gonads are eliminated. To date, only four genes have been reported with roles in germ cell death, and three of these have additional functions in germ cell pathfinding. The nature of the cell death program has not been elucidated. Here, I provide a brief review of Drosophila germ cell migration and programmed cell death at both the descriptive and molecular levels. Many questions remain to be answered, but advances made in recent years are providing useful insights into these critical biological phenomena.

Germ cell-less Acts to Repress Transcription during the Establishment of the Drosophila Germ Cell Lineage

Previously, it has been shown that, during early Drosophila and C. elegans development, the germ cell precursors undergo a period of transcriptional quiescence [1–4]. Here, we report that Germ cell-less (GCL), a germ plasm component necessary for the proper formation of “pole cells,” the germ cell precursors in Drosophila[5, 6], is required for the establishment of this transcriptional quiescence. While control embryos silence transcription prior to pole cell formation in the pole cell-destined nuclei, this silencing does not occur in embryos that lack GCL activity. The failure to establish quiescence is tightly correlated with failure to form the pole cells. Furthermore, we show that GCL can repress transcription of at least a subset of genes in an ectopic context, independent of other germ plasm components. Our results place GCL as the earliest gene known to act in the transcriptional repression of the germline. GCL's subcellular distribution on the nucleoplasmic surface of the nuclear envelope [7] and its effect on transcription suggest that it may act to repress transcription in a manner similar to that proposed for transcriptional silencing of telomeric regions.

An Evolutionary Conserved Region in the vasa 3′UTR Targets RNA Translation to the Germ Cells in the Zebrafish

In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization. To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, we tested different vasa homologs for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish. Our results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline.

Phenotypes of Drosophila homologs of human XPF and XPG to chemically-induced DNA modifications

DmXPF ( mei9) and DmXPG ( mus201) mutants are Drosophila homologs of the mammalian XPF and XPG genes, respectively. For Drosophila germ cells, causal correlations exist between the magnitude of a potentiating effect of a deficiency in these functions, measured as the M NER−/ M NER+ mutability ratio, and the type of DNA modification. M NER−/ M NER+ mutability ratios may vary with time interval between DNA adduct formation and repair, mutagen dose and depend also on the genetic endpoint measured. For forward mutations, there is no indication of any differential response of DmXPF compared to DmXPG. Subtle features appeared from a class-by-class comparison: (i) Methylating agents always produce higher M NER−/ M NER+ ratios than their ethylating analogs; (ii) M NER−/ M NER+ mutability ratios are significantly enhanced for cross-linking N-mustards, aziridine and di-epoxide compounds, but not for cross-linking nitrosoureas. The low hypermutability effects with bifunctional nitrogen mustards, aziridine and epoxide compounds are attributed to unrepaired mono-alkyl adducts; (iii) The efficient repair of mono-alkyl-adducts at ring nitrogens in wild-type germ cells is evident from the absence of a dose–response relationship for ethylene oxide, propylene imine and methyl methanesulfonate (MMS). These chemicals become powerful germline mutagens when the NER system is disrupted. Systematic studies of the type performed on germ cells are not available for somatic cells of Drosophila. The sparse data available show large differences in the response of germ cells and somatic cells. The bifunctional agent mechlorethamine (MEC) but not the monofunctional MMS or 2-chloroethylamine cause in NER − XX♀ the highest potentiating effect on mitotic recombination. The causes of the discrepancy between the extraordinarily high activity of MEC in mus201 somatic cells and its low potentiating effect in germ cells is unknown at present.

In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanogaster.

DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the influence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-deficient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-proficient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC --> AT and AT --> GC transitions and AT --> TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-proficient conditions, were isolated in the NER-deficient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-deficient conditions, confirming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.

Cell-autonomous and somatic signals control sex-specific gene expression in XY germ cells of Drosophila.

When XX germ cells develop in a testis they become spermatogenic. Thus, somatic signals determine the sex of genetically female germ cells. In contrast, XY germ cells experimentally transferred to an ovary do not differentiate oogenic cells. Because such cells show some male characteristics when analyzed in adults, it was assumed that XY germ cells autonomously become spermatogenic. Recently, however, evidence showing that a female soma feminizes XY germ cells was reported. The conclusion was drawn that the sex determination of XY germ cells is dictated by the sex of the soma. We monitored the fate of XY germ cells placed in a female environment throughout development. Here we report that such germ cells respond to both cell-autonomous and somatic sex-determining signals, depending on the developmental stage. Analyzing the expression of sex-specific molecular markers, we first detected autonomous male-specific gene expression in XY germ cells embedded in female embryos and larvae. At later stages, however, we found that sex-specific regulation of gene expression within XY germ cells is influenced by somatic gonadal cells. After metamorphosis, XY germ cells developing in a female soma start expressing female-specific and male-specific markers. Transcription of female-specific genes is maintained, while that of male-specific genes is later repressed. We show that in such XY germ cells, the female-specific gene Sex-lethal (Sxl) is activated. Within the germline, Sxl expression is required for the activation of a further female-specific gene and the repression of male-specific genes. We thus report for the first time the existence of downstream targets of the gene Sxl in the germline.

Targeted recovery of mutations in Drosophila.

Reverse genetic techniques will be necessary to take full advantage of the genomic sequence data for Drosophila and other experimental organisms. To develop a method for the targeted recovery of mutations, we combined an EMS chemical mutagenesis regimen with mutation detection by denaturing high performance liquid chromatography (DHPLC). We recovered mutant strains at the high rate of approximately 4.8 mutations/kb for every 1000 mutagenized chromosomes from a screen for new mutations in the Drosophila awd gene. Furthermore, we observed that the EMS mutational spectrum in Drosophila germ cells shows a strong preference for 5'-PuG-3' sites, and for G/C within a stretch of three or more G/C base pairs. Our method should prove useful for targeted mutagenesis screens in Drosophila and other genetically tractable organisms and for more precise studies of mutagenesis and DNA repair mechanisms.

Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells.

In many organisms, the primordial germ cells have to migrate from the position where they are specified towards the developing gonad where they generate gametes. Extensive studies of the migration of primordial germ cells in Drosophila, mouse, chick and Xenopus have identified somatic tissues important for this process and demonstrated a role for specific molecules in directing the cells towards their target. In zebrafish, a unique situation is found in that the primordial germ cells, as marked by expression of vasa mRNA, are specified in random positions relative to the future embryonic axis. Hence, the migrating cells have to navigate towards their destination from various starting positions that differ among individual embryos. Here, we present a detailed description of the migration of the primordial germ cells during the first 24 hours of wild-type zebrafish embryonic development. We define six distinct steps of migration bringing the primordial germ cells from their random positions before gastrulation to form two cell clusters on either side of the midline by the end of the first day of development. To obtain information on the origin of the positional cues provided to the germ cells by somatic tissues during their migration, we analyzed the migration pattern in mutants, including spadetail, swirl, chordino, floating head, cloche, knypek and no isthmus. In mutants with defects in axial structures, paraxial mesoderm or dorsoventral patterning, we find that certain steps of the migration process are specifically affected. We show that the paraxial mesoderm is important for providing proper anteroposterior information to the migrating primordial germ cells and that these cells can respond to changes in the global dorsoventral coordinates. In certain mutants, we observe accumulation of ectopic cells in different regions of the embryo. These ectopic cells can retain both morphological and molecular characteristics of primordial germ cells, suggesting that, in zebrafish at the early stages tested, the vasa-expressing cells are committed to the germ cell lineage.

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5′ or 3′ regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.

A novel method for the parallel monitoring of mitotic recombination and clastogenicity in somatic cells in vivo

Both homologous mitotic recombination (HMR), causing loss of heterozygosity (LOH) of the wild-type allele, and structural chromosome aberrations (CA) involve the formation of double-strand breaks in DNA. Whether the induction of CAs is always accompanied by HMR, or whether there exist DNA lesions specifically forming only one of the two end-points is unknown. Answering this fundamental question requires a system for the parallel detection of CAs and HMR, because only then is their analysis under strictly identical condition (dose, repair, genetic background) possible. We describe here a novel system for the parallel detection of HMR and loss of a whole chromosome as a measure of CA, utilizing somatic cells of Drosophila. In haploid germ cells of Drosophila, loss of a ring-shaped X-chromosome (rX) constitutes a frequent event providing an efficient method for measuring clastogenicity. For somatic cells, however, it was unclear whether the development of such a system would be feasible. The generally accepted notion has been that in XX female genotypes, loss of an entire X-chromosome acts as a cell lethal when generated at or shortly after blastoderm stage. However, here we show that rX-loss, if induced in pre-ommatidia cells of 3rd instar larvae, generates viable clones visible as small white patches in the red compound eye. To set up optimal conditions for the detection and quantification of rX-loss compared to HMR, several protocols were developed and tested against model carcinogens (methyl methanesulfonate, cisplatin and 7,12-dimethylbenz[ a]anthracene). Generally, we find striking differences in the efficiency of these carcinogens for recombination when compared with clastogenicity. The cross-linking agent cisplatin is 4- to 6-fold more clastogenic than recombinagenic. 7,12-Dimethylbenz[ a]-anthracene, on the contrary, produced less than a doubling effect for rX-loss but was highly active (20-times the background) for HMR. It appears therefore that both processes can be separated from each other. To the best of our knowledge, this is the first report suggesting, in terms of DNA adducts involved, qualitative differences between homologous recombination and clastogenic effects. Application of our system for studies on DNA repair may therefore provide new insight into the linkage of repair pathways in either of the two mechanisms.