Identification and characterization of proteins that interact with Drosophila melanogaster protein kinase CK2.

D. melanogaster CK2 (DmCK2) is a highly conserved protein kinase that is composed of catalytic, alpha, and regulatory, beta, subunits associated as an alpha2beta2 heterotetramer. In order to analyze the functions of CK2 in this metazoan model, we have used the two hybrid approach to identify interacting proteins. One of these cDNAs, DmA24, encodes a novel polypeptide with no homologs in GenBank, and is notable in that it contains a bipartite nuclear localization signal and two sites for phosphorylation by CK2. In situ hybridization to polytene chromosomes indicates that the DmA24 gene is located at the 61 D interval of chromosome II a region that also harbors 3 additional genes with similar structure. DmA24p interacts with DmCK2alpha, but not with DmCK2beta, demonstrating that this interaction is specific for the catalytic subunit of CK2. In addition, the protein is phosphorylated by the holoenzyme purified from Drosophila embryos. These studies identify DmA24p as a potentially new physiological partner of DmCK2. In addition, we also report the results of a large-scale screen that has identified a new set of DmCK2-interacting proteins. Most notable among these are Surf6, a nucleolar protein involved in RNA processing, and Spalt, a homeotic protein.

Cytological location and further characterization of the Third chromosome resistance gene in Drosophila melanogaster.

Mutations in the Third chromosome resistance (Tcr; 3-39.6) gene confer dominant resistance to alpha-methyl dopa and suggest the gene is involved in catecholamine metabolism. Evidence for involvement in catecholamine metabolism comes from the three phenotypes associated with the mutant Tcr chromosomes--dominant resistance, dominant rescue of partially complementing l(2)amd alleles, and recessive lethal phenotypes. Only dominant resistance to alpha s-methyl dopa, however, was mapped to the Tcr locus. Both recessive lethality and dominant rescue of l(2)amd alleles have now been mapped to the Tcr gene and, through the isolation of a new deletion in the region, we demonstrate these phenotypes are due to a loss of Tcr function. This deletion places the Tcr gene in the 69B4-5 to 69C8-11 region. Additionally, we have tested and verified three predictions of the biochemical model proposed by Bishop, Sherald, and Wright (1989) for the function of the Tcr protein.

The Drosophila gene asteroid encodes a novel protein and displays dosage-sensitive interactions with Star and Egfr.

The asteroid gene of Drosophila was found to lie within 189 bp of Star. Asteroid cDNA clones were isolated and sequenced and a single putative open reading frame was identified that encodes a novel protein of 815 amino acids with a calculated molecular mass of 93 kilodaltons. Using cDNA probes, asteroid transcripts were localized to the proliferative tissues of embryos and to the mitotically active tissue anterior to the morphogenetic furrow in eye imaginal discs. Ribonuclease protection assays identified a mutation of asteroid that acts as a dominant enhancer of Star mutations and also enhances the Ellipse mutation, EgfrE1. Based on these data, a model for asteroid gene function in EGF receptor signaling is presented.

Developmental timing and tissue specificity of heterochromatin-mediated silencing.

Heterochromatic position-effect variegation (PEV) describes the mosaic phenotype of a euchromatic gene placed next to heterochromatin. Heterochromatin-mediated silencing has been studied extensively in Drosophila, but the lack of a ubiquitous reporter gene detectable at any stage has prevented a direct developmental characterization of this phenomenon. Current models attribute variegation to the establishment of a heritable silent state in a subset of the cells and invoke differences in the timing of silencing to explain differences in the patch size of various mosaic patterns. In order to follow the course of heterochromatic silencing directly, we have generated Drosophila lines variegating for a lacZ reporter that can be induced in virtually all cells at any developmental stage. Our data indicate that silencing begins in embryogenesis and persists in both somatic and germline lineages. A heterogeneity in the extent of silencing is also revealed; silencing is suppressed in differentiated tissues but remains widespread in larval imaginal discs containing precursor cells for adult structures. Using eye development as an example, we propose that the mosaic phenotype is determined during differentiation by a variegated relaxation in heterochromatic silencing. Though unpredicted by prevailing models, this mechanism is evident in other analogous systems.

Genomic imprinting of chromatin in Drosophila melanogaster.

During gametogenesis, chromosomes may become imprinted with information which facilitates proper expression of the DNA in offspring. We have used a position effect variegation mutant as a reporter system to investigate the possibility of imprinting in Drosophila melanogaster. Genetic crosses were performed in which the variegating gene and a strong modifier of variegation were present either within the same parental genome or in opposite parental genomes in all possible combinations. Our results indicate that the presence of the variegating chromosome and a modifier chromosome in the same parental genome can alter the amount of variegation formed in progeny. The genomic imprinting we observed is not determined by the parental origin of the variegating chromosome but is instead determined by the genetic background the variegating chromosome is subjected to during gametogenesis.

Evidence for intrinsic differences in the formation of chromatin domains in Drosophila melanogaster.

The results of an investigation into intrinsic differences in the formation of two different heterochromatic domains are presented. The study utilized two different position effect variegation mutants in Drosophila melanogaster for investigating the process of compacting different stretches of DNA into heterochromatin. Each stretch of DNA encodes for a gene that affects different aspects of bristle morphology. The expression of each gene is prevented when it is compacted into heterochromatin thus the genes serve as effective reporter systems to monitor the spread of heterochromatin. Both variegating mutants are scored in the same cell such that environmental and genetic background differences are unambiguously eliminated. Any differences observed in the repression of the two genes must therefore be the result of intrinsic differences in the heterochromatic compaction process for the two stretches of DNA. Studies of the effects different enhancers of variegation have upon the compaction of the two genes indicate each compaction event occurs independently of the other, and that different components are involved in the two processes. These results are discussed with regard to spreading heterochromatin and the role this process may play in regulating gene expression.

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

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

DdcDE1, a mutant differentially affecting both stage and tissue specific expression of dopa decarboxylase in Drosophila.

The isolation and characterization of a unique Dopa decarboxylase (Ddc) mutant in Drosophila melanogaster is reported. This mutant, DdcDE1, exhibits stage- and tissue-specific altered Ddc expression. Homozygous DdcDE1 embryos, central nervous systems (CNSs) at pupariation and newly eclosed adult epidermis all have approximately 5% as much specific dopa decarboxylase (DDC) activity as the pr control stock in which DdcDE1 was induced. In contrast, the DdcDE1 epidermis at pupariation has roughly 50% as much DDC activity as controls, a 10-fold increase over the relative activity detected in other tissues and stages. Although the adult cuticle lacks proper pigmentation as expected in flies with low DDC activity (less than or equal to 5%), the bristles unexpectedly have wild-type black pigmentation. This implies that the bristle forming cells have more DDC activity than the rest of the adult epidermis. This variegated phenotype, black bristles and pale cuticle, plus the fact that DdcDE1 was originally isolated in a reciprocal translocation between proximal X heterochromatin and the euchromatic left arm of the second chromosome, 42 bands from the Ddc locus, suggested that the mutant might be an example of position-effect variegation. All tests for position-effect variegation, including persistence of the mutant phenotype when DdcDE1 was removed from the translocation, were negative. At pupariation DDC cross-reacting material (CRM) levels are similar in DdcDE1 and wild-type controls, but in newly eclosed adults CRM levels are approximately 35% of wild-type controls. This suggests that DDC produced by DdcDE1 adults has less activity per DDC molecule than the DDC produced at pupariation by DdcDE1. If the DDC enzyme produced by DdcDE1 at adult eclosion had full DDC activity (35% DDC CRM = 35% DDC activity) then no mutant phenotype would be exhibited by DdcDE1 since flies with as little as 10% activity have a wild-type phenotype. DDC thermolability assays clearly demonstrate that DDC from DdcDE1 is more thermolabile than control DDC at both pupariation and adult eclosion. Furthermore, DDC from adults in both DdcDE1 and the pr control is more thermolabile than DDC from white prepupae. Mixing experiments indicate the difference in DDC thermolability between pr white prepupae and pr adults is not due to a difference in the white prepupal and adult supernatants. This suggests that in wild-type different isoforms of DDC are produced either by differences in post-translational modification or as a result of a different primary amino acid sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

The genetics of dopa decarboxylase in Drosophila melanogaster. IV. The genetics and cytology of the 37B10-37D1 region.

Of 204 mutations located in the 8-12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The chi methyl dopa hypersensitive gene, 1(2) and, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2) and, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. --Band 37C5 puffs at the time of pupariation; Puff Stages 8-10. Twelve of eighteen alleles of 1(2)37Cf have been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. --Of the 204 mutations considered here only one Ddcpl, affects the function of more than one gene. It eliminates Ddc+ and 1(2)37Ca+ function and at 30 degrees C reduces 1(2)37Ce+ function. It is not a deficiency but could be a polar mutant.