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.     

Facilitated isolation of rare recombinants by ligase chain reaction: selection for intragenic crossover events in the Drosophila optomotor-blind gene

Ligase chain reaction (LCR) was evaluated as a tool for the detection of point mutations. For the mutation studied, the specificity of the method is sufficient to detect the mutant allele in the presence of a 200-fold molar excess of the wild-type sequence. LCR was therefore employed in a genetic recombination experiment as a probe for a recessive lethal point mutation. LCR greatly facilitated the isolation of a rare recombinant originating from a crossover event in the 40 kb interval separating the lethal mutation and an enhancer trap insertion in the optomotor-blind locus. The recombinant will allow the study of gene control in situ, in a largely unperturbed regulatory environment.     

Facilitated isolation of rare recombinants by ligase chain reaction: selection for intragenic crossover events in the Drosophila optomotor-blind gene

Ligase chain reaction (LCR) was evaluated as a tool for the detection of point mutations. For the mutation studied, the specificity of the method is sufficient to detect the mutant allele in the presence of a 200-fold molar excess of the wild-type sequence. LCR was therefore employed in a genetic recombination experiment as a probe for a recessive lethal point mutation. LCR greatly facilitated the isolation of a rare recombinant originating from a crossover event in the 40 kb interval separating the lethal mutation and an enhancer trap insertion in the optomotor-blind locus. The recombinant will allow the study of gene control in situ, in a largely unperturbed regulatory environment.     

Frequency of gamma-Ray Induced Specific Locus and Recessive Lethal Mutations in Mature Germ Cells of the Zebrafish, BRACHYDANIO RERIO

Specific locus and recessive lethal mutations are induced by γ-rays with approximately first order kinetics in the zebrafish (Brachydanio rerio) with frequencies of 4 x 10^-5 r^-1 and 4 x 10^-3 r^-1, respectively. The surprisingly low ratio (100:1) of recessive lethals to specific locus mutations may be due to the induction of large deficiencies by γ-rays.     

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.     

Genetic Analysis of Delta, a Neurogenic Gene of Drosophila melanogaster

We report here the results of a genetic analysis of the gene Delta (Dl) of Drosophila melanogaster. Dl has been mapped to the band 92A2, on the basis of two pieces of evidence: (1) this band is the common breakpoint of several chromosomal aberrations associated with Dl mutations and (2) recombination mapping of alleles of five different lethal complementation groups that are uncovered by Df( 3R)Dl(FX3) (breakpoints at 91F11; 92A3). Dl was found to map most distally of all five complementation groups. The analysis of a large number of Dl alleles demonstrates the considerable genetic and functional complexity of Dl. Three types of Dl alleles are distinguishable. Most alleles behave as amorphic or hypomorphic recessive embryonic lethal alleles, which in addition cause various defects in heterozygosity over the wild-type allele. The defects are due to haplo-insufficient expression of the locus and can be suppressed by a duplication of the wild-type allele. The second class is comprised of three alleles with antimorphic expression. The phenotype of these alleles can only be reduced, rather than suppressed, by a duplication of the wild-type allele. The third group is comprised of three visible, predominantly hypomorphic alleles with an antimorphic component of phenotypic expression. The pattern of interallelic complementation is complex. On the one hand, there is a group of hypomorphic, fully penetrant embryonic lethal alleles which complement each other. On the other hand, most alleles, including all amorphic alleles, are viable over the visible ones; alleles of antimorphic expression, however, are lethal over visible alleles. These results are compatible with a rather complex genetic organization of the Dl locus.     

Protein encoded by the Axin(Fu) allele effectively down-regulates Wnt signaling but exerts a dominant negative effect on c-Jun N-terminal kinase signaling

Axin plays an architectural role in many important signaling pathways that control various aspects of development and tumorigenesis, including the Wnt, transforming growth factor-beta, MAP kinase pathways, as well as p53 activation cascades. It is encoded by the mouse Fused (Fu) locus; the Axin(Fu) allele is caused by insertion of an IAP transposon. Axin(Fu/Fu) mice display varying phenotypes ranging from embryonic lethality to relatively normal adulthood with kinky tails. However, the protein product(s) has not been identified or characterized. In the present study, we conducted immunoprecipitation using brain extracts from the Axin(Fu) mice with specific antibodies against different regions of Axin and found that a truncated Axin containing amino acids 1-596 (designated as Axin(Fu-NT)) and the full-length complement of Axin (Axin(WT)) can both be generated from the Axin(Fu) allele. When tested for functionality changes, Axin(Fu-NT) was found to abolish Axin-mediated activation of JNK, which plays a critical role in dorsoventral patterning. Together with a proteomics approach, we found that Axin(Fu-NT) contains a previously uncharacterized dimerization domain and can form a heterodimeric interaction with Axin(WT). The Axin(Fu-NT)/Axin(WT) is not conducive to JNK activation, providing a molecular explanation for the dominant negative effect of Axin(Fu-NT) on JNK activation by wild-type Axin. Importantly, Axin(Fu-NT) exhibits no difference in the inhibition of Wnt signaling compared with Axin(WT) as determined by reporter gene assays, interaction with key Wnt regulators, and expression of Wnt marker genes in zebrafish embryos, suggesting that altered JNK signaling contributes, at least in part, to the developmental defects seen in Axin(Fu) mice.     

The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis.     

Dominant Maternal-Effect Mutations Causing Embryonic Lethality in Caenorhabditis Elegans

We undertook screens for dominant, temperature-sensitive, maternal-effect embryonic-lethal mutations of Caenorhabditis elegans as a way to identify certain classes of genes with early embryonic functions, in particular those that are members of multigene families and those that are required in two copies for normal development. The screens have identified eight mutations, representing six loci. Mutations at three of the loci result in only maternal effects on embryonic viability. Mutations at the remaining three loci cause additional nonmaternal (zygotic) effects, including recessive lethality or sterility and dominant male mating defects. Mutations at five of the loci cause visible pregastrulation defects. Three mutations appear to be allelic with a recessive mutation of let-354. Gene dosage experiments indicate that one mutation may be a loss-of-function allele at a haploin sufficient locus. The other mutations appear to result in gain-of-function "poison" gene products. Most of these become less deleterious as the relative dosage of the corresponding wild-type allele is increased; we show that relative self-progeny viabilities for the relevant hermaphrodite genotypes are generally M/+/+ greater than M/+ greater than M/M/+ greater than M/Df greater than M/M, where M represents the dominant mutant allele.     

Mutations Affecting Donor Preference during Mating Type Interconversion in Saccharomyces Cerevisiae

Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to α or α to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MATα cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MATα background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.     

The enhancer of split locus and neurogenesis in Drosophila melanogaster

Enhancer of split (E(spl)) is one of a group of so-called neurogenic genes of Drosophila. We describe two different types of E(spl) alleles, dominant and recessive, which exert opposite effects on both central and peripheral nervous system development. The only extant dominant allele determines a reduction in the number of central neurons and peripheral sensilla; this phenotype is not reduced by a normal complement of wild-type alleles. Since animals carrying a triploidy for the wild-type locus develop similar defects, the dominant allele is probably the result of a gain-of-function mutation. Several recessive alleles, obtained as revertants of the dominant allele, are loss-of-function mutations and determine considerable neural hyperplasia. The present evidence suggests that neural defects of E(spl) mutants are due to defective segregation of neural and epidermal lineages, leading to neural commitment of less or of more cells than in the wild type, depending upon whether the animals carry the dominant or any of the recessive alleles, respectively. Therefore, E(spl) formally behaves as a gene switching between neural and epidermal pathways.     

Genetic Analysis of B2t, the Structural Gene for a Testis-Specific β-Tubulin Subunit in DROSOPHILA MELANOGASTER

Genetic analysis of the B2t locus has resulted in the recovery of four recessive mutations in the B2t structural gene and a deficiency that deletes the locus. Two of the mutations were recovered as suppressors of B2t^D, a dominant male sterile mutation at the locus, and two were induced on wild-type chromosomes. All four mutant genes encode β₂-tubulin subunits that are synthesized at normal rates but do not accumulate. All mutants are completely male sterile as homozygotes.     

Genetic Analysis of Cold-Sensitive Ribosome Maturation Mutants of Escherichia coli

Four cold-sensitive mutants of Escherichia coli, which have defects in the maturation of the 50S ribosomal subunit, were isolated. Each of the mutations was shown to map at a different locus. The loci were assigned the name rim (ribosome maturation) and were shown to map as follows: rimA is co-transduced with ilvD and with pyrE; rimB is co-transduced with aroD; conjugation experiments limited rimD to a region between ilv and malB, and conjugation experiments limited rimC to the 22 to 30 min region of the chromosome. In merodiploids heterozygous for rimA, rimB, or rimD, the wild-type allele was shown to be dominant to the mutant allele. The observation that the rim loci lie outside the strA region and separate from each other, as well as the recessive character of the rim loci, suggests that the mutants may be defective in ribosome maturation factors rather than being defective in ribosomal structural proteins.     

H beta 58, an insertional mutation affecting early postimplantation development of the mouse embryo

The generation and analysis of insertional mutations affecting mouse embryogenesis provides a powerful method to identify new genes that function in early development. In this paper, we describe an insertional mutation that interferes with postimplantation mouse development beginning at the time of gastrulation. Embryos homozygous for the H beta 58 transgenic insertion developed normally through the early postimplantation, egg cylinder stage (day 6.5 of development). At the primitive streak stage (day 7.5), however, they began to display characteristic abnormalities, including a retardation in the growth of the embryonic ectoderm (the earliest identifiable defect), and in some cases abnormalities of the amnion and chorion. Homozygotes continued to develop for 2-3 more days, reaching the size of a normal 8.5 day embryo, and formed tissues representative of all three germ layers, including several differentiated cell types. The site of insertion was mapped, by a combination of cytogenetic and genetic methods, to chromosome 10, and it appeared to define a new genetic locus. The inserted transgene provided a probe to clone and characterize the mutant locus, as well as the corresponding wild-type locus. In addition to an insertion of 10-20 copies of the transgene, the mutant locus contained a deletion of 2-3 kb of DNA found at the wild-type locus, and possibly an insertion of mouse repetitive DNA. However, genomic sequences on both sides of the insertion site remained co-linear in the wild-type and mutant genomes, and no chromosomal abnormalities could be detected. Five single copy DNA probes spanning the insertion site were tested for their ability to hybridize to RNA from 8.5 day embryos; one of the probes (located within the region deleted from the mutant chromosome) hybridized to a 2.7 kb mRNA encoded at the H beta 58 locus, thus identifying a gene whose disruption appears to be responsible for the mutant phenotype.     

The unstable wDZL mutation of Drosophila is caused by a 13 kilobase insertion that is imprecisely excised in phenotypic revertants

We have analyzed the lesion in wDZL, a genetically unstable mutant allele of the eye color locus, white, of Drosophila melanogaster. We have cloned the DNA of the white locus region of flies carrying the wDZL allele and find a 13 kilobase insertion not present in the wild-type at the corresponding location. In 12 independent cases examined, reversion to a wild-type eye color phenotype correlates with the excision of a portion of this 13 kilobase insertion, indicating that the insertion is the cause of the mutation. The portion of the insertion that is excised in these eye color revertants is heterogeneous in size but appears to include the central 6 kilobases of the insertion in all cases. Many of these eye color revertants continue to undergo mutation at the white locus, indicating that the residual portion of the insertion in these revertants is sufficient to promote mutations.     

gammaTub23C interacts genetically with brahma chromatin-remodeling complexes in Drosophila melanogaster

The brahma gene encodes the catalytic subunit of the Drosophila melanogaster BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, we isolated the dominant-negative Pearl-2 allele of γTub23C. γTub23C encodes one of the two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. γ-Tubulin is a subunit of microtubule organizer complexes. We show that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by γTub23C mutations. The genetic interactions between γTub23C and chromatin-remodeling mutations suggest that γ-tubulin might have a role in regulating gene expression.     

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.