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.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

Germline hypermutability in Drosophila and its relation to hybrid dysgenesis and cytotype

In its hypermutable state, an unstable singed allele, sn^w, mutates in the germline to two other alleleic forms at a total frequency usually between 40 and 60%. In its stable state, the mutation rate of sn^w is essentially zero. Its state depends on an extrachromosomal condition indistinguishable from a property called cytotype previously studied as a component of hybrid dysgenesis. Of the two known systems of hybrid dysgenesis, denoted P-M and I-R, sn^w hypermutability is determined by the P-M system and appears to be independent of the I-R system. Cytotype, as defined by the control of sn^w mutability, is self-reproducing in the cytoplasm or nucleoplasm of the germline through at least two generations. However, it is not entirely autonomous, being ultimately determined by the chromosomes after sufficiently many generations of backcrossing. This combination of chromosomal and extrachromosomal transmission agrees well with previous studies on cytotype. Temperature differences have little effect on the mean mutation rates, but they have a pronounced effect on the intrinsic variance among individuals. The latter effect suggests that high temperatures reduce germ-cell survival during the development of dysgenic flies. Chromosomal rearrangements produce no apparent effects on the behavior of sn^w. Hypermutability is thought to be caused by the excision or other alteration of an inserted genetic element in the sn^w gene. This element might be a copy of the "P factor," which is though to be a mobile sequence capable of causing female sterility and other dysgenic traits in the P-M system.     

OM Mutations in DROSOPHILA ANANASSAE Are Linked to Insertions of a Transposable Element

It has been hypothesized that Om mutability in Drosophila ananassae (involving spontaneous mutation at 20 loci, resulting in semidominant, nonpleiotropic eye morphology defects) was due to insertion of a transposable element, tom. One particularly unstable X-linked Om allele produced several derivatives, one of which has a more extreme Om phenotype and was accompanied by a singed bristle mutant, sn ^9g. DNA probes from the sn locus of D. melanogaster were used to clone the homologous region of D. ananassae. Analysis of sn^9g DNA detected a 6.5-kb insert. Genomic Southern blotting and in situ hybridization techniques showed that this insert is repetitive and dispersed. The existence of the tom element is supported by genetic mapping that established homology between the 6.5-kb sn^9g insert and Om mutants at the four X-linked loci tested.     

Unstable alleles of the singed locus in Drosophila melanogaster with reference to a transposon marked with a visible mutation

Summary The gene clw can be integrated into another site in the X chromosome to form the mutable two gene transposon system sn::Tn-clw. When brought under the common control of the transposon, sn and clw can concomitantly mutate and manifest themselves. Tn-clw was found to move from the sn region to a position of±52 m.u. This transposition was associated with changes in sn::Tn-clw:(1) loss of the instability property by the sn ⁺ allele; (2) appearance of a new lethal allele, clw ⁹, designated as a transposon allele. Based on analysis of the direction and frequency of the sn-clw mutations, the unstable genes of the sn locus were grouped in three pleiades. Interallelic mutations occurred regularly at frequencies of 0.1%–5.6% with changes in the manifestation of the clw mutation specific to each pleiade. Transition from one pleiade to another was rare (5×10^–4), and it was associated with a new phenotypic expression of the sn and clw alleles. The mutational differences between the pleiades are presumably related to differences in the localization of Tn-clw within the sn locus.It was shown that the presence of Tn promotes recombination events in the rightmost portions of the sn-oc interval.     

Male-Sterilizing Interactions between Duplications and Deficiencies for Proximal X-Chromosome Material in DROSOPHILA MELANOGASTER

The genetic limits of sixty-four deficiencies in the vicinity of the euchromatic-heterochromatic junction of the X chromosome were mapped with respect to a number of proximal recessive lethal mutations. They were also tested for male fertility in combination with three Y chromosomes carrying different amounts of proximal X-chromosome-derived material (B^SYy⁺, y⁺Ymal¹²⁶ and y⁺Ymal⁺). All deficiencies that did not include the locus of bb and a few that did were male-fertile in all male-viable Df(1)/Dp(1;Y) combinations. Nineteen bb deficiencies fell into six different classes by virtue of their male-fertility phenotypes when combined with the duplicated Y chromosomes. The six categories of deficiencies are consistent with a formalism that invokes three factors or regions at the base of the X, one distal and two proximal to bb, which bind a substance critical for precocious inactivation of the X chromosome in the primary spermatocyte. Free duplications carrying these regions or factors compete for the substance in such a way that, in the presence of such duplications, proximally deficient X chromosomes are unable to command sufficient substance for proper control of X-chromosome gene activity preparatory to spermatogenesis. We conclude that there is no single factor at the base of the X that is required for the fertility of males whose genotype is otherwise normal.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.     

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.     

Genetic Characterization of the Region between 86f1,2 and 87b15 on Chromosome 3 of DROSOPHILA MELANOGASTER

The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

Genetic Change in Mutations at the T/t-Locus in the Mouse

Recessive lethal or semilethal alleles at the T/t locus in the mouse generate new t-variants, with characteristics different from the parent allele at a rate of about 10^-3. Almost invariably the variant chromosome carries marker genes derived from the opposite parental chromosome. New t-mutations obtained in this way are sometimes recessive lethals that are indistinguishable from those in already known complementation groups. Most derived t-mutations are viable, however. This paper summarizes data on the rate and types of variants produced by members of each of the six lethal complementation groups, and by semilethal alleles. It appears that particular complementation groups preferentially generate certain types of variants, and that in general, the pattern of variant production runs "uphill," that is, to less abnormal states. The data are compatible with the hypothesis that t-mutations represent some extent of altered chromosome and that variants are produced by loss of abnormal material.     

The Action of the Notch Locus in DROSOPHILA MELANOGASTER. II. Biochemical Effects of Recessive Lethals on Mitochondrial Enzymes

We show that six mapped recessive lethal point mutations of the Notch locus affect mitochondrial enzyme activities: NADH oxidase, NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. The mutant N^264-40, which has the same morphological and embryological effects as the Notch⁸ deletion, demonstrates the same biochemical effects and dosage relations as Notch⁸. The other five mapped recessive lethals also affect four enzymic activities. They show specific patterns of activity that depend in several cases on the wild-type chromosome in the heterozygous females. That effect occurs with mutants located in the extreme right part of the Notch locus where some mutations, according to other authors, show temperature-sensitive expression.     

Allelic Negative Complementation at the Abruptex Locus of DROSOPHILA MELANOGASTER

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.     

A Cytogenetic Analysis of the Chromosomal Region Surrounding the α-Glycerophosphate Dehydrogenase Locus of DROSOPHILA MELANOGASTER

The chromosomal region surrounding the structural gene for α-glycerophosphate dehydrogenase (αGpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. αGpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the αGpdh locus.     

Gene mapping within the T/t complex of the mouse. I. t-lethal genes are nonallelic

The t haplotypes of mouse chromosome 17 are natural polymorphisms in wild populations that contain mutations that affect or control such diverse functions as tail length, embryonic lethality and maturation and function of male germ cells. The major impediment to dissecting the genetics of this complex region has been its unusual property of recombination suppression in heterozygotes with wild-type chromosomes. Recently it was shown that recombination suppression does not occur in heterozygotes containing two different t haplotypes, which suggested that t chromosomes may be mismatched with respect to wild-type but share sequences that permit crossing-over between them. Thus for the first time questions of allelism and map positions of the t-lethal mutations can be addressed. We report here the results of three experiments that analyzed the t^w12 haplotype trans to either t^w5, t^w32 or t^w18. In all cases these lethal mutations were nonallelic to t^w12. These results, together with evidence for functional relatedness, suggest the t-lethals may be a gene family spread out over more than 15 centiMorgans of chromosome 17.     

Estimation of the levels of radiation-induced <Emphasis Type="Italic">P</Emphasis>-element transposition in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> experimental populations and laboratory strains

When experimental P + M populations were exposed to chronic γ-irradiation (0.31 mGy/h), the highest instability level of the singed-weak (sn _w ) locus was observed in F₃–F₁₀ with a subsequent decrease and stabilization of the mutation rate. The sn ^w mutation rate was within the range of spontaneous variation in conditions of P-M hybrid dysgenesis and irradiation of males of the Harwich laboratory strain with active P elements at 1.61 mGy/h. The instability of the sn ^w locus was significantly higher at lower dose rates (0.23 and 0.31 mGy/h), suggesting a nonlinear dose-effect relationship.     

Position Effects and Variegation Enhancers in an Autosomal Region of DROSOPHILA MELANOGASTER

A dominant eye color mutation was found associated with a third chromosome inversion broken distally at or near the karmoisin (kar) locus in 87C and proximally within centric heterochromatin. Suppressibility of the mutant phenotype by an extra Y chromosome indicated that this was an example of dominant position-effect variegation. When heterozygous with deficiencies uncovering the kar locus, this inversion chromosome was found to be lethal unless a region in 87EF was also deleted. Extra Y chromosomes rescued inversion/deletion heterozygotes, while removal of the Y chromosome from heterozygous males deficient for the region in 87EF was lethal. Thus, a variegating lethal lies near the breakpoint in 87C, and a wild-type gene that enhances its variegation lies in 87EF. Furthermore, deletion of the region in 87EF was found to strongly suppress white-mottled-4 (w^m4) variegation, while deletion of another region in 87BC suppressed less strongly. These results indicate that essential genes on autosomes are sensitive to position effects, and loci that enhance variegation, as defined by deficiency mapping, are very common.     

Revertants of Dominant Mutations Associated with the Antennapedia Gene Complex of DROSOPHILA MELANOGASTER: Cytology and Genetics

Using X-ray mutagenesis we have induced and recovered phenotypic revertants of four dominant mutations thought to be associated with the Antennapedia complex of Drosophila melanogaster. These include seven revertants of Antennapedia-73b (Antp^73b), six of Extra Sex Combs of Wakimoto (Scx^w), three of Deformed (Dfd) and one of Humeral (Hu). Fifteen of the 17 revertants are associated with chromosomal aberrations and localize Antp^73b, Scx ^w and Hu to polytene chromosome bands 84B1,2. The Dfd lesion is apparently located in or adjacent to bands 84A4,5. Since all of the dominants are reverted by events that delete their respective chromosomal loci, we conclude that all four are the result of a gain-of-function lesions. Complementation analysis of the various revertant chromosomes has shown that Scx^w and Hu are dominant allelic variants of the Antp locus. The Dfd lesion represents a dominant mutation at a locus just proximal to Antp and previously only occupied by recessive lethal mutations. Characterization of the revertants of Scx^w and a comparison with the properties of the original mutation has revealed that the original lesion has effects on both the Antp and Sex Combs Reduced (Scr) loci and that these defects are in some cases separable by the reverting event.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Cytogenetic analysis of the cSOD microregion in Drosophila melanogaster

This report describes the genetic organization of a euchromatic region on the third chromosome of Drosophila melanogaster extending cytologically from 68A2 to C1, an interval comprising 10 or 11 polytene chromosome bands. The gene for cytoplasmic superoxide dismutase (cSOD) maps within this interval, as does low xanthine dehydrogenase (lxd).--Recessive lethal mutations were generated within the region by ethyl methanesulfonate mutagenesis and by hybrid dysgenesis. These lethals fall into 11 functional groups, which were partially ordered by complementation with deletions having breakpoints within the region. The distribution of dysgenesis-induced mutations in the region is highly nonrandom, the majority being within a single group. The mutability of this gene is comparable to that of singed (sn), a documented "hot-spot" for P-element insertion.--One of the EMS-induced lethals, l-108, fulfills biochemical criteria expected of a hypomorphic allele of cSOD. To our knowledge this is the first such allele recovered of this gene, and it should prove very useful in an analysis of the in vivo function of cytoplasmic SOD. Indeed, it has been demonstrated that cSOD is almost certainly a vital gene.