On the Interpretation of Mutagenically Induced Mosaicism in Drosophila

This paper draws attention to the formal parallelism that exists between chromosomal-loss mosaicism and mutagenically induced mosaicism in Drosophila and suggests that, although the underlying processes by which these two types of genetic mosaics are generated are very different, the more refined methodology employed in developmental analyses of genetically induced chromosomal-loss mosaics may be profitably extrapolated to mutagenesis studies. Results obtained from various studies of genetically induced mosaics and from a previous EMS mutation induction experiment at the yellow locus are utilized to illustrate this methodology and to estimate the total mutagenicity rates of EMS.-The following are some of the tentative conclusions that have been drawn in this report regarding an EMS concentration that produced 31% F(2) lethals in the standard X-linked recessive lethal test: (1) The frequency of cuticular mosaics is at least 5 times that of F(3) lethals. (2) At least 60% of all cuticular mosaics go undetected in the standard X -linked recessive lethal test since their mutant tissue does not extend into the germ line. (3) The frequency of EMS-induced cryptic mosaics is probably less than 10% the frequency of cuticular mosaics. (4) Some EMS-induced mutations are probably bona fide completes; if confirmed, this inference must be taken into consideration in estimating the total mutagenicity rates of this agent and in molecular interpretations of its mechanism of action. (5) The fact that the proportion of mutant tissue in EMS-induced mosaics is greater than 25% is consistent with the suggestion that the action of EMS is occasionally delayed until after the first cleavage division of the embryo. (6) Such an EMS concentration causes on the average more than 5 independent genetic alterations in the entire haploid genome of an X-bearing sperm.-This report clarifies the experimental evidence that must be generated, and the methodology that can be used to analyze this evidence, if it is of interest to render these and related conclusions regarding the effect of EMS on D. melanogaster more accurate, or if it is of interest to conduct a similar analysis for other mutagens that cause a significant degree of mosaicism.     

Ionizing radiation and genetic risks. II. Nature of radiation-induced mutations in experimental mammalian in vivo systems

This paper reviews data on the nature of spontaneous and radiation-induced mutations in the mouse. The data are from studies using a variety of endpoints scorable at the morphological or the biochemical level and include pre-selected as well as unselected loci at which mutations can lead to recessive or dominant phenotypes. The loci used in the morphological recessive specific-locus tests permit the recovery of a wide spectrum of induced changes. Important variables that affect the nature of radiation-induced mutations (assessed primarily using tests for viability of homozygotes) include: germ cell stage, type of irradiation and the locus. Most of the results pertain to irradiated stem cell spermatogonia. The data on morphological specific-locus mutations show that overall, more than two-thirds of the X- or gamma-ray-induced mutations are lethal when homozygous. This proportion may be lower for those that occur spontaneously, but the numbers of tested mutants are small. For spontaneous mutations, there is evidence for the occurrence of mosaics and for proviral insertions. Most or all tested induced enzyme activity variants, dominant visibles (recovered in specific-locus experiments) and dominant skeletal mutations are lethal when homozygous and this is true of 50% of dominant cataract mutations, but again, the numbers of tested mutants are small. Electrophoretic mobility variants, which are known to be due to base-pair changes, are seldom induced by irradiation. At the histocompatibility loci, no radiation-induced mutations have been recovered, presumably because deletions are incompatible with survival even in heterozygotes. All these findings are consistent with the view that in mouse germ cells, most radiation-induced mutations are DNA deletions. Some mutations (in the morphological specific-locus tests) which had previously been inferred to be deletions on the basis of genetic analyses have now been shown to be DNA deletions by molecular methods. However, the possibility cannot be excluded that at least a small proportion of induced mutations may be intragenic changes. The data on the rates of induction of recessive lethals and of dominant skeletal and dominant cataract mutations (and proportions of the latter two which are homozygous lethal) can be used to estimate the proportions of recessive lethals which are expressed as skeletal abnormalities or cataracts. These calculations show that about 10% of recessive lethals manifest themselves as skeletal and less than 0.2% as cataract mutations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental Genetics of the 2c-D Region of the Drosophila X Chromosome

We have conducted a genetic and developmental analysis of genes within the 2C-D area of the X chromosome. Phenotypes of 33 mutations representing nine adjacent complementation groups including eight recessive lethals and one visible homeotic mutation (polyhomeotic) are described. Germline clonal analysis of the eight zygotic lethals has revealed three types of gene requirements: normal activity at two pupal lethal loci (corkscrew and C204) and one larval lethal locus (ultraspiracle) is required for normal embryogenesis; normal activity at three larval lethal loci (DF967, VE651 and Pgd) is required for normal oogenesis; and activity at only one locus (EA82), a larval lethal, appears to have no maternal requirement. Ambiguous results were obtained for the GF316 lethal complementation group. Analysis of mitotic figures of the pupal lethals indicates that C204 disrupts an essential mitotic function. This result correlates with the preblastoderm arrest observed among embryos derived from germline clones of C204. Embryos derived from germline clones of corkscrew (csw) exhibit a "twisted" phenotype. The recessive lethal ultraspiracle (usp) disrupts the organization of the posterior tip of the larval both zygotically and maternally: second instar usp/Y larvae derived from heterozygous usp/+ mothers possess an extra set of spiracles, whereas usp/Y embryos derived from females possessing a germline clone (usp/usp) exhibit a localized ventral defect in the ninth or posterior eighth abdominal segment. Analysis of the phenotypes of deficiency-hemizygous embryos indicates the presence of an embryonic zygotic lethal locus, as yet unidentified, which produces central nervous system and ventral hypoderm degeneration. Additional information on the genetic organization of loci within the adjacent 2E area are also described.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

I−R hybrid dysgenesis in Drosophila melanogaster: nature and site specificity of induced recessive lethals

This paper presents results of the genetic and cytological analysis of 144 sex-linked recessive lethals, plus 1 non-lethal. All of them were induced by I−R hybrid dysgenesis. This collection of mutants was pooled from experiments involving inducer chromosomes that differ in the chrosomal position of their I elements. Our results show that 30% of the recessive lethals are associated with chromosomal rearrangements which depend on the strength of the I−R interaction. These lethals are induced on both inducer- and reactive-origin chromosomes, and their frequency is dependent on the structure of the inducer chromosome used. The I−R-induced lethals occur along the entire length of the X chromosome. These sites probably correspond to specific loci which are more or less homologous with I. The complementation relationshups showed that some specific loci were more frequently involved in all the lethal mutations tested. The most sensitive loci are, in order of observation: l(1)J1, ct, f, ma¹ and m. Among induced recessive lethals considered to be point mutation, complementation tests showed that many of them are in fact multilocius deficiencies which can be detected only at the molecular level.

It seems that the production of I−R rearrangements (cytologically visible or not) may be the most important mechanism leading to lethal mutations. These mutations probably occur during the transposition of I elements, hence their importance from an evolutionary standpoint.     

Genetic Analysis of the Centromeric Heterochromatin of Chromosome 2 of DROSOPHILA MELANOGASTER: Deficiency Mapping of Ems-Induced Lethal Complementation Groups

Until recently, little was known of the genetic constitution of the heterochromatic segments of the major autosomes of Drosophila melanogaster . Our previous report described the genetic dissection of the proximal, heterochromatic region of chromosome 2 of Drosophila melanogaster by means of a series of overlapping deficiencies generated by the detachment of compound second autosomes (Hilliker and Holm 1975). Analysis of these deficiencies by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for the existence of at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. These data in conjunction with cytological observations demonstrated that light and rolled and three loci lying between them are located within the proximal heterochromatin of the second chromosome.——The present report describes the further analysis of this region through the induction with ethyl methanesulphonate (EMS) of recessive lethals allelic to the 2L and 2R proximal deficiencies associated with the detachment products. Analysis of the 118 EMS-induced recessive lethals and visible mutations recovered provided evidence for seven loci in the 2L heterochromatin and six loci in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these loci, one in 2L and two in 2R fall near the heterochromatic-euchromatic junctions of 2L and 2R respectively. None of the 113 EMS lethals behaved as a deficiency, implying that the heterochromatic loci uncovered in this study represent nonrepetitive cistrons. Thus functional genetic loci are found in heterochromatin, albeit at a very low density relative to euchromatin.     

Heterogeneity of Lethals in a "Simple" Lethal Complementation Group

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster, eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua 1 and pur 1, which yield auxotrophic mutations.--The eight new mutations have been characterized phenotypically in double heterozygotes with gua 1, pur 1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2. The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.--Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

Recessive lethals induced by ethyl methanesulfonate and diethyl sulfate in Drosophila melanogaster post-meiotic male cells pre-treated with butylated hydroxytoluene

The response of Drosophila melanogaster male germ cells to the induction of mutation by ethyl methanesulfonate (EMS) and diethyl sulfate (DES) and the influence of pre-treatments with butylated hydroxytoluene (BHT) were studied. Careful sampling of cell stages revealed that fully mature motile sperm were less sensitive to the induction of sex-linked recessive lethals by EMS than late spermatids, and that the remaining cell stages presented a fairly homogeneous response to the mutagen. The frequency of lethals induced by DES could be grouped into two plateaus: the first one, with a higher mutation rate, comprised motile and immotile sperm and late spermatids, the second one, medium and early spermatids. No sparing action of BHT was detected in any of the developing germ cells treated with EMS or DES, whereas an increase in sex-linked recessive lethal frequency was observed in some experiments in early spermatids. The enhancement of damage is attributed to impairment of repair achieved through the ability of BHT to modify enzymic activity.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. I. Recessive Lethal Mutations

The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.     

Lethals, Steriles and Deficiencies in a Region of the X Chromosome of CAENORHABDITIS ELEGANS

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.     

Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus.

In plant genomes as well as other eukaryotic genomes, meiotic recombination does not occur uniformly. At the level of the gene, high recombination frequencies are often observed within genetic loci in maize, but this feature of intragenic recombination is not seen at the csr1 locus in Arabidopsis. These observations suggest that meiotic recombination in plant genomes varies considerably among species. In the present study we investigated meiotic recombination at the wx locus in rice. The mutation sites of wx mutants induced by ethyl methanesulfonate (EMS) treatment or gamma-ray irradiation and a spontaneous wx mutant were physically characterized, and the genetic distances between those wx mutation sites were estimated by pollen analysis. Based on these results, the recombination frequency at the wx locus in rice was estimated as 27.3 kb/cM, which was about 10 times higher than the average for the genome, suggesting that there was a radically different rate of meiotic recombination for intra- and intergenic regions in the rice genome.     

The capacity of Drosophila for detecting relevant genetic damage

For the detection and study of mutagenic agents, Drosophila offers many advantages. It is a higher organism with a short generation time that is cheap and easy to breed in large numbers. The simple genetic testing methods provide unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations, but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test thus deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one-fifth of the genome is tested for newly induced forward mutations, including small deletions.Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver. An additional advantage, in this respect, is the capacity of Drosphila for detecting short-lived activation products, because intracellular activation occurs within the spermatids and spermatocytes. These properties make the test for recessive sex-linked lethals a useful tool for verifying results obtained in the pre-screening of potential mutagens with fast microbial assay systems.In studies on non-disjunction, detailed genetic analysis of the induced changes is possible, and these may shed light on the mechanisms involved.A new adaptation of the bithorax transvection method by Mendelson permits the recovery of high yields of chromosome aberrations in a fast one-generation test.     

Effect of storage and dose on MMS-induced deletions. Complementation analysis of X-chromosomal recessive lethals in the zeste-white and maroon-like regions of Drosophila melanogaster

The effect of storage on MMS-induced recessive lethals in the zeste-white (3A1-3C2) and the maroon-like (18F4-20F) regions was studied by complementation analysis. (1) Without any exception, all 52 mutants (from unstored spermatozoa) mapped in the zeste-white region were restricted to single complementation units. Furthermore, none of an additional 15 lethals, sampled from sperm that had been stored in females for 9-12 days, was associated with a deletion. (2) Of 34 mutations induced by 8.5 X 10(-2) mM MMS in the maroon-like (mal) region, 4 spanned 2 or more complementation units, and thus are considered to be deletions. A high dose of 2.5 mM MMS provided 55 lethals for analysis of which 4 were deletions. There was no evidence for any difference in the frequency of deletions as the MMS concentration was enhanced from 8.5 X 10(-2) mM to 2.5 mM. However, with storage, 47.1% lethals (16 of 34 mutants induced by 2.5 mM MMS) mapped in the mal region were found to involve large structural changes. (3) A high proportion of double mutants in both the zeste-white (z w) and the maroon-like regions was found among the chromosomes analyzed. These double mutants have one lethal positioned within the region studied and the other outside it. Clearly, the proportion of double mutants increased with dose, from 6.3 to 41.7% in z w and from 14.7 to 61.8% in the mal section. Apurinic sites in DNA reacted with MMS are considered as the likely primary lesions responsible for the storage effect on MMS-induced recessive lethals in the mal region. Thus, the ability of MMS to produce delayed deletion lethals seems to correlate with preference for alkylation of base nitrogens. An interesting aspect for further analysis is the apparent infrequency in the zeste-white region of alkylation-induced chromosomal breakage, as observed by various investigators for MMS, EMS and MNNG.     

Mutations induced by a hormonal imbalance in Drosophila melanogaster

Pterin treatment of chrysalids in diapause modified the juvenile hormone-ecdysone ratio. The treatment of Drosophila donor mutant with a mixture of reduced folic acid, pterins and extract of Pierides chrysalids in diapause induced the formation of short sequences, by the intermediary of variations in hormonal balance. The effect of this variation was seen in the germinal lines especially at the gonial stage, where recessive, visible or lethal mutations were induced in the form of clusters. Genetically active fractions were found in the 4S–8S and 18S–28S sedimentation zones after saccharose gradient ultracentrifugation. The short DNA sequences coded for tRNA and rRNA. Consequently, it is these DNA sequences from the Drosophila donor mutant that altered the genetic information of the host.2 tpes of recessive visible mutation appeared: those affecting the differentiation of the imaginal discs and those affecting the pigment biosynthetic chain. Recessive lethals were induced by treatment.3 hypotheses are proposed: the first suggests the formation of a short DNA sequence complexing at a specific locus in the acceptor. The second involves transporable factors belonging to the acceptor itself, behaving as a particular transporable factor. The third supposes the induction of alterations at loci of rRNA and tRNA synthesis at the origin of perturbations in protein synthesis. The present data do not allow us to choose between 3 hypotheses.In conclusion, it seems that a ‘hormonal imbalance’ can have grave consequences not only for the individual itself but also for its descendants.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Characterization of third chromosome dominant alpha-methyl dopa resistant mutants (Tcr) and their interactions with l(2)amd alpha-methyl dopa hypersensitive alleles in Drosophila melanogaster

In Drosophila melanogaster two alleles at the Third chromosome resistance locus (Tcr; 3-39-6) were isolated in a screen of EMS mutagenized third chromosomes for dominant resistance to dietary alpha-methyl dopa, alpha-MD, a structural analogue of DOPA. Both alleles of Tcr are recessive lethals exhibiting partial complementation. Almost half (48.3%) of the Tcr40/Tcr45 heterozygotes die as embryos but some survive past adult eclosion. Both the embryonic lethal phenotype and the adult phenotype suggest that Tcr is involved in cuticle synthesis. Tcr mutants suppress the lethality of partially complementing alleles at the alpha-MD hypersensitive locus, l(2)amd. The viability of Tcr40/Tcr45, however, is not increased by the presence of a l(2)amd allele. The possibility that the Tcr and l(2)amd mutations reveal a catecholamine metabolic pathway involved in cuticle structure is discussed.     

HIGH INBREEDING DEPRESSION,SELECTIVE INTERFERENCE AMONG LOCI,AND THE THRESHOLD SELFING RATE FOR PURGING RECESSIVE LETHAL MUTATIONS

The evolutionary dynamics of recessive or slightly dominant lethal mutations in partially self-fertilizing plants are analyzed using two models. In the identity-equilibrium model, lethals occur at a finite number of unlinked loci among which genotype frequencies are independent in mature plants. In the Kondrashov model, lethals occur at an infinite number of unlinked loci with identity disequilibrium produced by partial selfing. If the genomic mutation rate to (nearly) recessive lethal alleles is sufficiently high, such that the mean number of lethals (or lethal equivalents) per mature plant maintained at equilibrium under complete outcrossing exceeds 10, selective interference among loci creates a sharp discontinuity in the mean number of lethals maintained as a function of the selfing rate. Virtually no purging of the lethals occurs unless the selfing rate closely approaches or exceeds a threshold selfing rate, at which there is a precipitous drop in the mean number of lethals maintained. Identity disequilibrium lowers the threshold selfing rate by increasing the ratio of variance to mean number of lethals per plant, increasing the opportunity for selection. This theory helps to explain observations on plant species that display very high inbreeding depression despite intermediate selfing rates.     

Tissue-Specific and Complex Complementation Patterns in the Punch Locus of DROSOPHILA MELANOGASTER

Mutations in the Punch locus result in loss of GTP cyclohydrolase activity, but all mutations do not affect the enzyme in the same way. There are at least three classes of Punch mutations. One class results in a dominant eye color, recessive lethal phenotype. A second class of mutations also causes a recessive lethal phenotype, but heterozygous mutants have normal eye color. They show loss of GTP cyclohydrolase function in all tissues where activity can be measured. Alleles comprising a third class are recessive eye color mutations that are homozygous viable. Individuals with this third type of mutation show loss of enzyme activity in the eye, but show normal or near-normal activity elsewhere. In order to examine the organization and function of this locus further, we have performed interallelic complementation tests on 25 Punch mutations, monitoring viability and enzyme activity in prepupae and adults. Most allele combinations are lethal. Those that complement do so in ways that are tissue-or stage-specific and unpredictable. Tests of mutants with tissue-specific phenotypes and of individuals mutant for complementing Punch lethal alleles lead us to conclude that Punch is a complex locus, both with respect to its organization and to its products.     

Metronidazole (Flagyl): lack of induction of sister-chromatid exchanges in CHO-K1 cells

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.