A Cytogenetic Analysis of Chromosomal Region 31 of Drosophila Melanogaster

Cytogenetic region 31 of the second chromosome of Drosophila melanogaster was screened for recessive lethal mutations. One hundred and thirty nine new recessive lethal alleles were isolated that fail to complement Df(2L)J2 (31A-32A). These new alleles, combined with preexisting mutations in the region, define 52 complementation groups, 35 of which have not previously been described. Among the new mutations were alleles of the cdc2 and mfs(2)31 genes. Six new deficiencies were also isolated and characterized identifying 16 deficiency subintervals within region 31. The new deficiencies were used to further localize three loci believed to encode non-histone chromosomal proteins. Suvar(2)1/Su(var)214, a dominant suppressor of position-effect variegation (PEV), maps to 31A-B, while the recessive suppressors of PEV mfs(2)31 and wdl were localized to regions 31E and 31F-32A, respectively. In addition, the cytological position of several mutations that interact with heterochromatin were more precisely defined.     

Cytogenetic Analysis of Segregation Distortion in Drosophila Melanogaster: The Cytological Organization of the Responder (Rsp) Locus

The segregation distortion phenomenon occurs in Drosophila melanogaster males carrying an SD second chromosome and an SD+ homolog. In such males the SD chromosome is transmitted to the progeny more frequently than the expected 50% because of an abnormal differentiation of the SD+-bearing sperms. Three major loci are involved in this phenomenon: SD and Rsp, associated with the SD and SD+ chromosome, respectively, and E(SD). In the present work we performed a cytogenetic analysis of the Rsp locus which was known to map to the centromeric heterochromatin of the second chromosome. Hoechst- and N-banding techniques were used to characterize chromosomes carrying Responder insensitive (Rspi), Responder sensitive (Rsps) and Responder supersensitive (Rspss) alleles. Our results locate the Rsp locus to the h39 region of 2R heterochromatin. This region is a Hoechst-bright, N-banding negative heterochromatic block adjacent to the centromere. Quantitative variations of the h39 region were observed. The degree of sensitivity to Sd was found to be directly correlated with the physical size of that region, demonstrating that the Rsp locus is composed of repeated DNA.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

Genetic Analysis of the Claret Locus of Drosophila Melanogaster

The claret (ca) locus of Drosophila melanogaster comprises two separately mutable domains, one responsible for eye color and one responsible for proper disjunction of chromosomes in meiosis and early cleavage divisions. Previously isolated alleles are of three types: (1) alleles of the claret (ca) type that affect eye color only, (2) alleles of the claret-nondisjunctional (cand) type that affect eye color and chromosome behavior, and (3) a meiotic mutation, non-claret disjunctional (ncd), that affects chromosome behavior only. In order to investigate the genetic structure of the claret locus, we have isolated 19 radiation-induced alleles of claret on the basis of the eye color phenotype. Two of these 19 new alleles are of the cand type, while 17 are of the ca type, demonstrating that the two domains do not often act as a single target for mutagenesis. This suggests that the two separately mutable functions are likely to be encoded by separate or overlapping genes rather than by a single gene. One of the new alleles of the cand type is a chromosome rearrangement with a breakpoint at the position of the claret locus. If this breakpoint is the cause of the mutant phenotype and there are no other mutations associated with the rearrangement, the two functions must be encoded by overlapping genes.     

Cytogenetic Analysis of the Second Chromosome Heterochromatin of Drosophila Melanogaster

This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures.     

The Rosy Locus in Drosophila Melanogaster: Xanthine Dehydrogenase and Eye Pigments

The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.     

A Functional and Structural Analysis of the Sex Combs Reduced Locus of Drosophila Melanogaster

We have undertaken a developmental genetic analysis of the homeotic gene Sex combs reduced (Scr) of Drosophila melanogaster by examining embryonic and adult phenotypes of mutations affecting Scr gene function. Molecular mapping of Scr breakpoint lesions has defined a segment of greater than 70 kb of DNA necessary for proper Scr gene function. This region is split by the fushi tarazu (ftz) gene, with lesions affecting embryonic Scr function molecularly mapping to the region proximal (5') to ftz and those exhibiting polyphasic semilethality predominantly mapping distal (3') to ftz. Gain-of-function mutations are associated with genomic rearrangements and map throughout the Scr locus. Our analysis has revealed that the Scr locus encompasses genetic elements that are responsible for functions in both the embryonic and larval to adult periods of development. From these studies, we conclude that Scr is a complex genetic locus with an extensive regulatory region that directs functions required for normal head and thoracic development in both the embryo and the adult and that the regulation of Scr during these two periods is distinct.     

Genetic Analysis of the Additional Sex Combs Locus of Drosophila Melanogaster

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.     

Genetic Analysis of the Enhancer of Zeste Locus and Its Role in Gene Regulation in Drosophila Melanogaster

The Enhancer of zeste [E(z)] locus of Drosophila melanogaster is implicated in multiple examples of gene regulation during development. First identified as dominant gain-of-function modifiers of the zeste1-white (z-w) interaction, mutant E(z) alleles also produce homeotic transformations. Reduction of E(z)+ activity leads to both suppression of the z-w interaction and ectopic expression of segment identity genes of the Antennapedia and bithorax gene complexes. This latter effect defines E(z) as a member of the Polycomb-group of genes. Analysis of E(z)S2, a temperature-sensitive E(z) allele, reveals that both maternally and zygotically produced E(z)+ activity is required to correctly regulate the segment identity genes during embryonic and imaginal development. As has been shown for other Polycomb-group genes, E(z)+ is required not to initiate the pattern of these genes, but rather to maintain their repressed state. We propose that the E(z) loss-of-function eye color and homeotic phenotypes may both be due to gene derepression, and that the E(z)+ product may be a general repressing factor required for both examples of negative gene regulation.     

A Cytogenetic Analysis of the Punch-Tudor Region of Chromosome 2r in Drosophila Melanogaster

Eleven chromosomal deficiencies and several rearrangements in the Pu-tud region of chromosome 2R have been generated and examined cytologically. The Pu locus has been localized to chromosome bands 57C5-6 and tud to 57C7-8. Mutagenesis within the region defined by the deletion intervals has resulted in the isolation of 92 new lethal mutations. Seventy-six of these mutations have been separated into 16 complementation groups that have been ordered and placed cytologically by deletion mapping. All new alleles fully complement tud for both lethal and grandchildless phenotypes. The largest number of new mutations, a total of 25, are Pu alleles.     

A Genetic and Mosaic Analysis of a Locus Involved in the Anesthesia Response of Drosophila Melanogaster

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.     

Targeted Transposition at the Vestigial Locus of Drosophila Melanogaster

Targeted transposition is the replacement of one P element with another. We are exploiting this unique property of P elements to study the complex regulatory domain of the Dopa decarboxylase (Ddc) gene in Drosophila melanogaster. P element constructs targeted to the same site in the genome will be subjected to the same position effect. This allows the subtle effects typical of most mutations in the Ddc regulatory region to be measured in the absence of the variable influences of position effects which are associated with the current method of germline transformation. We have investigated some of the parameters affecting targeted transposition of a Ddc transposon, P[Ddc], into a P element allele at the vestigial locus. These events were detected by an increased mutant vg phenotype. The location of the donor transposon in cis or in trans to the target had little effect on the frequency of targeting. Likewise, the mobility of different donor elements, as measured by their rate of transposition to a different chromosome, varied nearly 20-fold, while the rate of targeted transposition was very similar between them. All targeted alleles were precise replacements of the target P element by P[Ddc], but in several cases the donor was inserted in the opposite orientation. The targeted alleles could be described as the result of a replicative, conversion-like event.     

A Genetic Analysis of the Stoned Locus and Its Interaction with Dunce, Shibire and Suppressor of Stoned Variants of Drosophila Melanogaster

The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mosaic analysis of a stoned lethal allele suggests that stoned functions either in the nervous system or in both the nervous system and musculature, but is not required for gross neural development. The behavioral alleles stn(ts) and stn(C), appear to be defective in a diametrically opposite sense, show interallelic complementation, and indicate distinct roles for the stoned gene product in the visual system and in motor coordination. A number of other neurological mutations have been investigated for their possible interaction with the viable stoned alleles. Mutations at two loci, dunce and shibire, act synergistically with the stn(ts) mutations to cause lethality, but fail to interact with stn(C). A third variant (Suppressor of stoned) has been identified which can suppress the debilitation associated with the stn(ts) mutations. These data, together with a previously identified interaction between the stn(ts) and tan mutants, indicate a central role for the stoned gene product in neuronal function, and suggests that the stoned gene product interacts, either directly or indirectly, with the neural cAMP second messenger system, with the synaptic membrane recycling pathway via dynamin, and with biogenic amine metabolism.     

Interaction of the Stubble-Stubbloid Locus and 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 instar. The region is coextensive with a complex genetic locus, the Broad-Complex (BR-C). The BR-C is a regulatory gene that contains two major functional domains, the br domain and the l(1)2Bc domain. BR-C mutants prevent metamorphosis, including morphogenesis of imaginal discs; br mutants prevent elongation and eversion of appendages and l(1)2Bc mutants prevent fusion of the discs. The Stubble-stubbloid (Sb-sbd) locus at 89B9-10 is best known for the effects of its mutants on bristle structure. Mutants of the BR-C and the Sb-sbd locus interact to produce severe malformation of appendages. Viable heteroallelic and homoallelic combinations of Sb-sbd mutants, including loss-of-function mutants, affect the elongation of imaginal disc appendages. Thus, the Sb-sbd(+) product is essential for normal appendage elongation. Sb-sbd mutants, however, do not affect eversion or fusion of discs. Correspondingly, only BR-C mutants deficient in br function interact with Sb-sbd mutants. The interaction occurs in deficiency heterozygotes using single, wild-type doses of the BR-C, of the Sb-sbd locus, or of both loci. These last results are formally consistent with the possibility that the BR-C acts as a positive regulator of the Sb-sbd locus. The data do not exclude other possible nonregulatory interactions between the two loci, e.g., interactions between the products of both genes.