Characterization of Lethal Drosophila melanogaster alpha-Actinin Mutants

We have partially characterized four Drosophilamelanogaster alpha-actinin gene mutants,I(1)2Cb¹, I(1)2Cb²,I(1)2Cb⁴, and I(1)2Cb⁵. Wedemonstrate that in each case the mutation is caused bya chromosomal rearrangement that precludes normal proteinsynthesis. In the absence of alpha-actinin, fliescomplete embryogenesis and develop into flaccid larvaethat die within approximately 24 hr. These larvae have noticeable muscle dysfunction at hatching,although they, nevertheless, are capable of escapingfrom the egg membranes and of subsequent crawlingmovements. During larval development muscles degenerate, progressively limiting mobility and ultimatelycausing death. Electron microscopy of mutant musclefibers reveals that myofibrils are grossly disrupted inone day old larvae and that electron-dense structures reminiscent of those seen in human nemalinemyopathies are present throughout larval life. Our workrigorously demonstrates that alpha-actinin deficienciesare the cause of I(1)2Cb muscle defects. We anticipate that the alpha-actinin mutants described hereinwill facilitate in vivo tests of spectrin superfamilyprotein domain functions using a combination of directedmutagenesis and germline transformation.     

Drosophila basement membrane collagen col4a1 mutations cause severe myopathy

Recent data from clinical and mammalian genetic studies indicate that COL4A1 mutations manifest with basement membrane defects that result in muscle weakness, cramps, contractures, dystrophy and atrophy. In-depth studies of mutant COL4A1-associated muscle phenotype, however, are lacking and significant details of the muscle-specific pathomechanisms remain unknown. In this study, we have used a comprehensive set of Drosophila col4a1 and col4a2 mutants and a series of genetic and mutational analyses, gene, protein expression, and immunohistochemistry experiments in order to establish a Drosophila model and address some of these questions. The Drosophila genome contains two type IV collagen genes, col4a1 and col4a2. Mutant heterozygotes of either gene are viable and fertile, whereas homozygotes are lethal. In complementation analysis of all known mutants of the locus and a complementation matrix derived from these data we have identified the dominant lesions within the col4a1, but not within the col4a2 gene. Expression of a col4a1 transgene partially rescued the dominant and recessive mutant col4a1 alleles but not the col4a2 mutations that were all recessive. Partial complementation suggested that col4a1 gene mutations have strong antimorph effect likely due to the incorporation of the mutant protein into the triple helix. In col4a1 mutants, morphological changes of the oviduct muscle included severe myopathy with centronuclear myofibers leading to gradual development of female sterility. In larval body wall muscles ultrastructural changes included disturbance of A and I bands between persisting Z bands. In the most severely affected DTS-L3 mutant, we have identified four missense mutations within the coding region of the col4a1 gene two of which affected the Y within the Gly-X-Y unit and a 3' UTR point mutation. In conclusion, our Drosophila mutant series may serve as an effective model to uncover the mechanisms by which COL4A1 mutations result in compromised myofiber-basement membrane interactions and aberrant muscle function.     

The "image" of the regulatory gene in experiments with Drosophila

The mutants referred to as facultative dominant lethals were selected in the progeny of gamma-irradiated Drosophila males. The mutant males were viable and fertile, though their crosses with females of the yellow line yielded no daughters. The mutations obtained differed from the common mutations by (1) extremely varying penetrance of F1 hybrids from crosses with various lines; (2) the uncertain relationships between the mutant and normal alleles; (3) the different expression in somatic and germ cells; (4) the dependence of the expression on the sex of the parent carrying the donor mutations; (5) the mass morphosis formation and (6) the frequent reversal to the norm. These mutations are assigned to the regulatory group and their specific expression (see above) can be helpful in identifying regulatory gene mutations. We assume that the specific expression of the mutations studied is related to specific properties of the regulatory genes. These properties are as follows: (1) only one out of two homologous regulatory genes located on one homolog is in an active state, (2) in the haploid chromosome set the regulatory gene is represented by several alleles (cys-alleles); (3) only one allele ensures the regulatory gene activity.     

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.     

Genetic Analysis of Rough Sheath1 Developmental Mutants of Maize

Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1(+) allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data).     

Dominant Suppressors of Yeast Actin Mutations That Are Reciprocally Suppressed

A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.     

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.     

Drosophila non-muscle alpha-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

The α-Glycerophosphate in DROSOPHILA MELANOGASTER II. Genetic Aspects

Seven alleles of the alpha-Glycerophosphate dehydrogenase-1 (alphaGpdh-1) locus of Drosophila melanogaster have been described. These include two naturally occurring electrophoretic variants, one EMS-induced electrophoretic variant, and four EMS-induced "null" or "zero" mutants. With the electrophoretic variants, the locus was mapped to II-20.5 +/- 2.5. A complementation matrix was prepared utilizing the null mutants. Three of the four mutants and a deletion of the locus (Grell 1967) exhibit dosage dependency. The dosage independent mutant exhibits complementation with two of the other null alleles. Flies genetically deficient in alpha-glycerophosphate dehydrogenase are fertile, but their relative viability is severely diminished. Such flies also lose the ability to sustain flight, an observation consistent with the enzyme's function in energy production. The levels of mitochondrial alpha-glycerophosphate oxidase, measured in flies genetically deficient in the cytoplasmic enzyme, were normal.     

Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.     

Dopa decarboxylase from Drosophila melanogaster

Summary Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to -methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.     

A large-scale screen for mutagen-sensitive loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.     

A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea.

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc(29DE) gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc(95E) mutants, which are extremely sensitive to caffeine. Although huc(95E) is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc(95E) gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc(29D) and huc(95E) as a first step toward molecularly characterizing the relevant genes.