Molecular Cloning and Analysis of L(1)ogre, a Locus of Drosophila Melanogaster with Prominent Effects on the Postembryonic Development of the Central Nervous System

Previous genetic studies have shown that wild-type function of the l(1)ogre (lethal (1) optic ganglion reduced) locus is essential for the generation and/or maintenance of the postembryonic neuroblasts including those from which the optic lobe is descended. In the present study molecular isolation and characterization of the l(1)ogre locus was carried out to study the structure and expression of this gene in order to gain information about the nature of l(1)ogre function and its relevance to the development of the central nervous system. About 70 kilobases (kb) of genomic DNA were isolated that spanned the region where l(1)ogre was known to reside. Southern analysis of a l(1)ogre mutation and subsequent P element-mediated DNA transformation mapped the l(1)ogre+ function within a genomic fragment of 12.5 kb. Northern analyses showed that a 2.9-kb message transcribed from this 12.5-kb region represented l(1)ogre. A 2.15-kb portion of a corresponding cDNA clone was sequenced. An open reading frame (ORF) of 1,086 base paris was found, and a protein sequence of 362 amino acids with one highly hydrophobic segment was deduced from conceptual translation of this ORF.     

The action of theI(1)npr-1 + locus on theDrosophila glue geneSgs-3 is cell-autonomous

The 2B5 chromosomal locus inDrosophila contains a gene,I(1)npr-1 ⁺, whose product required intrans for the expression of the larval salivary gland-specific geneSgs-3. We have addressed the question as to whether this factor acts in a cell-autonomous manner or not. This was made possible by the use of a transformant strain that makes anSgs-3-E. coli β-galactosidase (lacZ) fusion protein, under the control of anSgs-3 promoter, allowing the cellular examination of gene expression by a histochemical assay for enzyme activity. Using genetic methods, larvae that were mosaic for the loss of function mutationl(l)npr-1 were generated. The expression of theSgs-3-lacZ fusion gene was assayed histochemically in such larvae. Our results strongly indicate a cell-autonomous requirement for the product ofl(1)npr-1+. This is in contrast to another factor, the hormone ecdysterone, which is also required forSgs-3 expression, but acts in a non-autonomous manner     

Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis

NEM-sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF-1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identified as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.     

The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus.

Two temperature-sensitive mutations, ams-1 and rne-3071, in the ams (altered mRNA stability) gene have been used extensively to investigate the processing and decay of RNA in Escherichia coli. We have sequenced these temperature-sensitive alleles and found that the mutations are separated by only 6 nucleotides and cause conservative amino acid substitutions next to a possible nucleotide-binding site within the N-terminal domain of the Ams protein. Computer analysis revealed that the region altered by the mutations has extensive sequence similarity to a predicted gene product from the mre (murein pathway cluster e) locus of E. coli, which has been implicated previously in determining bacterial cell shape.     

Analysis of a cDNA from the neurologically active locus shaking-B (Passover) of Drosophila melanogaster.

We have isolated and sequenced a cDNA from the shaking-B locus of Drosophila melanogaster. The cDNA contains an open reading frame with extensive homology to another D. melanogaster gene, l(1)ogre. This suggests the existence of a new family of proteins required for the development and maintenance of the D. melanogaster nervous system.     

Two Genetically and Molecularly Distinct Functions Involved in Early Neurogenesis Reside within the Enhancer of Split Locus of Drosophila Melanogaster

Molecular correlation of the genetic aspects of the function of the neurogenic gene Enhancer of split [E(spl)] has previously been hampered by the densely transcribed nature of the chromosomal region within which it resides. We present data indicating that two distinct molecular species contribute to E(spl) function. Analysis of new E(spl) alleles has allowed us to define two complementing functions within the locus. Subsequent phenotypic analysis of different E(spl) deficiencies combined with P element-transformed constructs has demonstrated that these two functions correspond to: (1) a family of helix-loop-helix (HLH) protein-encoding genes and (2) the single copy gene E(spl) m9/10, whose product shares homology with G-protein beta subunits. The zygotically active E(spl) HLH genes can, at least partially, substitute for one another's functions and their total copy number determines the activity of the locus. E(spl) m9/10 acts synergistically with the E(spl) HLH genes and other neurogenic genes in the process of neurogenesis. The maternal component of E(spl) m9/10 has the most pronounced effect in neurogenesis, while its zygotic component is predominantly required during postembryonic development. The lethality of trans-heterozygotes of null E(spl) deficiency alleles with a strong Delta point mutation is a result of the concomitant reduction in activity of both E(spl) HLH and m9/10 functions. Immunocytochemical localization of the E(spl) m9/10 protein has revealed that it is a ubiquitously distributed nuclear component in embryonic, larval and imaginal tissues.     

The Genetics of the Dras3-Roughened-Ecdysoneless Chromosomal Region (62b3-4 to 62d3-4) in Drosophila Melanogaster: Analysis of Recessive Lethal Mutations

The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.     

The Drosophila Tumorous lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus

The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant mutation in the hopscotch (hop) locus, which is required for cell division and for proper embryonic segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of larval and pupal development.     

The Drosophila Tumorous lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus

The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant mutation in the hopscotch (hop) locus, which is required for cell division and for proper embryonic segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of larval and pupal development.     

The Interaction of Two Complex Loci, Zeste and Bithorax in DROSOPHILA MELANOGASTER

It has been found that certain alleles of the zeste locus (z(a) 1-1.0) have no phenotype of their own, but interact with certain alleles at the bithorax locus (bx 3-58.8). This interaction takes the form of an enhancement of the homeotic bx phenotype to a more extreme form-i.e., the metathorax is transformed into mesothorax in varying degrees depending on the bx allele used. This enhancement is somewhat reminiscent of the transvection effect described by Lewis (1954). The characterization of the interaction thus far has shown that the enhancement only effects bx alleles which arise spontaneously, whereas the origin of the z(a) allele is unimportant. The gene claret nondisjunctional was used for the production of gynandromorphs which showed that the enhancing ability of z(a), like the eye pigment change caused by z, is autonomous. The enhancement of one specific allele (bx(34e)), which is temperature-sensitive, has allowed a delineation of the temperature-sensitive period of the bithorax locus to a period extending from the middle of the second larval instar to the middle of the third larval instar. These results, as well as those of other enhancer and suppressor systems in Drosophila, have revealed the possibility of the involvement of heterocyclic compounds in the control of cell determination and fate in Drosophila melanogaster.     

Inositol Mutants of SACCHAROMYCES CEREVISIAE: Mapping the ino1 Locus and Characterizing Alleles of the ino1, ino2 and ino4 Loci

An extensive genetic analysis of inositol auxotrophic mutants of yeast is reported. The analysis includes newly isolated mutants, as well as those previously reported (Culbertson and Henry 1975). Approximately 70% of all inositol auxotrophs isolated are shown to be alleles of the ino1 locus, the structural gene for inositol-1-phosphate synthase, the major enzyme involved in inositol biosynthesis. Alleles of two other loci, ino2 and ino4, comprise 9% of total mutants, with the remainder representing unique loci or complementation groups. The ino1 locus was mapped by trisomic analysis with an n + 1 disomic strain constructed with complementing alleles at this locus. The ino1 locus is shown to be located between ura2 (11.1 cm) and cdc6 (21.8 cm) on chromosome X. An extended map of chromosome X of yeast is presented. Unlike most yeast loci, but similar to the his1 locus, the ino1 locus lacks allelic representatives that are suppressible by known suppressors. This finding suggests that premature termination of translation of the ino1 gene product may be incompatible with cell viability.     

L(1)trol and L(1)devl, Loci Affecting the Development of the Adult Central Nervous System in Drosophila Melanogaster

Adult optic lobes of Drosophila melanogaster are composed of neurons specific to the adult which develop postembryonically. The structure of the optic lobes and aspects of its development have been described, and a number of mutants that affect its development have been identified. The focus of every screen to date has been on disruption of adult structure or function. Although these loci were originally identified on the basis of viable mutants, some have proven capable of giving rise to lethal alleles. It seems reasonable to assume that mutants which strongly affect development of the imaginal-specific central nervous system may evidence abnormalities during the late larval or pupal stages when the adult central nervous system is undergoing final assembly and might show a lethal phase prior to eclosion (as is true for mutations at the previously defined l(1)ogre locus). We have carried out the first screen of autosomal and sex-linked late larval and pupal lethals to identify mutations that affect the development of the optic lobes. Our screen yielded nine mutants that could tentatively be grouped into three classes, depending on the neuroblast population affected and imaginal disc phenotypes. Two of these, including one that is allelic to l(1)zw1, were chosen for further analysis.     

Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

Circadian rhythms in Drosophila melanogaster: analysis of period as a function of gene dosage at the per (period) locus

Mutations at the per (period) locus of Drosophila melanogaster affect the period of its circadian rhythms. An analysis of published data on strains having duplications and deletions of the per locus indicates that the period is a logarithmic function of the level of the per gene product. The analysis also indicates that period is relatively insensitive to the level of that gene product; a presumed 300% increase in the gene product level produces only a 4.6% decrease in the period. The period of a strain transformed with per+ DNA conforms to the same logarithmic relationship if the level of mRNA in the transformant, which is one-tenth that in the wild type, is considered equivalent to a gene dosage one-tenth the wild type dose of 2, or 0.2. The periods of strains having various doses of mutant per alleles which shorten (pers) or lengthen (per1) the period can be fitted to the same logarithmic function. The analysis may provide an explanation for the partial dominance of pers over per+ and the dominance of per+ over per1, since it suggests that the per1 gene product is nearly inactive while the pers gene product is more than 34 times as active as the wild type product. Analysis of periods of strains heterozygous at the per locus suggests that the per gene product may be a multimeric protein. Three possible roles for the per gene product in circadian rhythmicity are discussed, including a role in synchronizing rhythm-producing cells.     

Endogenous interferon specifically regulates Newcastle disease virus-induced cytokine gene expression in mouse macrophages.

In macrophages from inbred mice, the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the If-1 locus, which carries the allele for either high (h) or low (l) IFN production. Here, we report that the activity of genes within the If-1 locus is influenced by macrophage-derived endogenous IFN. In addition to various other biological effects, we observed that endogenous IFN specifically downregulated NDV-induced IFN and interleukin 6 production. Preculture of bone marrow-derived macrophages (BMM) from BALB/c (If-1l) mice in macrophage colony-stimulating factor plus anti-IFN-beta provoked a 30- to 50-fold increase in NDV-induced cytokine production compared with induced control cultures in macrophage colony-stimulating factor alone, whereas only a 4- to 6-fold increase was observed in anti-IFN-beta-treated BMM from C57BL/6 (If-1h) mice. This resulted in nearly complete abrogation of the genetically determined difference in the response to NDV. The increase was specific for NDV and was marked by strong additional activation of IFN-alpha genes. Studies using BMM from B6.C-H28c If-1l congenic mice gave results identical to those obtained with BALB/c BMM. Addition of 20 IU of recombinant IFN-alpha 4 to anti IFN-beta-treated macrophages from B6.C-H28c mice 20 h prior to NDV infection strongly downregulated the IFN-alpha, IFN-beta, and interleukin 6 responses. The genetic difference between macrophages from If-1h and If-1l mice was thus reestablished, since the same treatment caused only weak reduction of NDV-induced cytokine gene expression in BMM from C57BL/6 mice. These data suggest that the If-1h and If-1l alleles harbor IFN-inducible genes that, following activation, specifically suppress subsequent cytokine gene expression in response to NDV.     

Structure and function of the PHO82-pho4 locus controlling the synthesis of repressible acid phosphatase of Saccharomyces cerevisiae.

pho4 mutants of Saccharomyces cerevisiae, although rare among phosphatase-negative mutants isolated from wild-type strains, were isolated efficiently from pho80, pho85, or pho80 pho85 strains. The distribution of these pho4 mutants over the pho4 locus was determined by analyzing random spores of two- and three-factor crosses. The pho4-4 mutation confers temperature-sensitive synthesis of repressible acid phosphatase. An intragenic suppressor for the pho4-12 allele results in the temperature-sensitive synthesis of repressible acid phosphatase. Recombination between these sites occurs at 1.0 to 3.0%, the highest for any pair of sites within the pho4 locus. All these results strongly indicate that the information of the pho4 locus is translated into a protein. The PHO82 site was mapped inside the pho4 locus by random spore analysis. The order met10-pho4-1PHO82-1-pho4-9 on the right arm of chromosome VI was confirmed by tetrad analysis. Doubly heterozygous diploids, pho3 PHO82c PHO4+/pho3 pho82+ pho4, produce variable amounts of repressible acid phosphatase under repressive conditions depending on the combination of PHO82c and pho4 alleles. This phenomenon may reflect the constitutive production of the pho82+-pho4 product in the repressed condition, which interferes with the function of the PHO82c-PHO4+ product. The earlier model for the function of the PHO82-pho4 cluster, in which the PHO82 site acts as an operator of the pho4 gene, has been revised to a model in which the PHO82 site codes for the part of the pho4 protein that has affinity for the regulatory protein encoded by the pho80 and pho85 genes.     

Properties of mutationally altered RNA polymerases II of Drosophila

We tested and compared several in vitro properties of wild type and mutant RNA polymerases II from Drosophila melanogaster, using several different mutants of a single X-linked genetic locus, RpIIC4 (Greenleaf, A. L., Weeks, J. R., Voelker, R. A., Ohnishi, S., and Dickson, B. (1980) Cell 21, 785-792); the mutants tested included the original amanitin-resistant mutant, C4, which is nonconditional, plus the temperature-sensitive mutants A9, C20, E28, and 1Fb40. Using a tritium-labeled amanitin derivative, we demonstrated that C4 polymerase has a reduced binding affinity for amanitin. The C4 polymerase was as stable to thermal denaturation as the wild type enzyme, and the two enzymes had similar specific activities, ionic strength and Mn2+ requirements, and apparent Km values for UTP and GTP when assayed in the presence of Mn2+. However, with Mg2+ as the divalent cation, C4 polymerase was less active than wild type and had 2-fold higher apparent Km values for UTP and GTP. Three of the temperature-sensitive mutants, A9, C20, and E28, were derived from the amanitin-resistant mutant C4; the polymerase II activities from these mutants displayed resistance to alpha-amanitin in vitro identical with that of the C4 enzyme. C20, E28, and 1Fb40 polymerases were markedly less stable to thermal denaturation in vitro than wild type polymerase. The results presented indicate that the mutations at the RNA polymerase locus (RpIIC4-) directly alter the structure of the enzyme, providing conclusive evidence that the locus is a structural gene for a polymerase II subunit.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

Temperature-Dependent Expression of the APTEROUS Phenotype in DROSOPHILA MELANOGASTER

Mutations at the apterous (ap) locus in Drosophila melanogaster produce a variety of developmental defects, including several classes of wing abnormalities. We describe the wing phenotype produced by homozygotes and hemizygotes of three different temperature-sensitive apterous alleles grown at 16, 18, 20, 22, 25, and 29 degrees. We also describe the phenotype produced by each of these three alleles when heteroallelic with the non-temperature-sensitive apc allele. Constant-temperature and temperature-shift experiments show that each of the heteroallelic genotypes can produce several of the previously described apterous phenotypes and that the length of the temperature-sensitive period for a given phenotype depends on the allelic combinations used to measure it. We suggest that the stage-specific requirements of the tissue for gene product, rather than the time of gene expression per se, determine the temperature-sensitive periods for apterous and other loci. The results support the hypothesis that the various wing phenotypes produced by apterous mutations are due to quantitative reductions in the activity of gene product and that failure to meet specific threshold requirements for gene product can lead to qualitatively different phenotypes.