Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein

The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd suggesting that it is structurally similar to mammalian complement factors C2 and B has been previously proposed. To test this model we performed SP6 RNA microinjection to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.     

Maintenance mechanism of polymorphism at the α-Gpdh locus in Drosophila melanogaster

Polymorphism at the alpha-Gpdh locus was studied in Drosophila melanogaster. Using two different lines, one marked by the F allele (FF line) another by the S allele (SS line), four populations were initiated, two in which the initial frequency of F was 0.1 and two in which it was 0.9. They have been observed for 34 generations. From the fifth generation on, the equilibrium frequency in the four cages was about 0.60. Viability has been measured during the evolution of te populations while F frequencies changed and recombinations between the FF and SS lines occurred. It has been evaluated in synthetic populations built with different frequencies: (1) from the original FF and SS lines and (2) from FF and SS lines extracted after 34 generations of joint evolution. In all three cases, the FF viability depended on the frequency of the F allele. The similarity of the three linear regressions implies that alpha-Gpdh locus or other closely linked loci is the target of the selection in the populations analyzed here.     

Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.     

Transvection at the vestigial locus of Drosophila melanogaster

Transvection is a phenomenon wherein gene expression is effected by the interaction of alleles in trans and often results in partial complementation between mutant alleles. Transvection is dependent upon somatic pairing between homologous chromosome regions and is a form of interallelic complementation that does not occur at the polypeptide level. In this study we demonstrated that transvection could occur at the vestigial (vg) locus by revealing that partial complementation between two vg mutant alleles could be disrupted by changing the genomic location of the alleles through chromosome rearrangement. If chromosome rearrangements affect transvection by disrupting somatic pairing, then combining chromosome rearrangements that restore somatic pairing should restore transvection. We were able to restore partial complementation in numerous rearrangement trans-heterozygotes, thus providing substantial evidence that the observed complementation at vg results from a transvection effect. Cytological analyses revealed this transvection effect to have a large proximal critical region, a feature common to other transvection effects. In the Drosophila interphase nucleus, paired chromosome arms are separated into distinct, nonoverlapping domains. We propose that if the relative position of each arm in the nucleus is determined by the centromere as a relic of chromosome positions after the last mitotic division, then a locus will be displaced to a different territory of the interphase nucleus relative to its nonrearranged homolog by any rearrangement that links that locus to a different centromere. This physical displacement in the nucleus hinders transvection by disrupting the somatic pairing of homologous chromosomes and gives rise to proximal critical regions.     

Mutagenesis at the cinnabar locus in Drosophila melanogaster

A study was undertaken to isolate mutations affecting the temporal appearance of kynurenine hydroxylase in Drosophila melanogaster. Such mutations, lacking or having reduced enzyme activity at the larval or pupal stage only, could represent changes in regulatory functions. Mutagenesis was carried out using EMS. Potential mutations were isolated from mass F₁ cultures. The screening of large numbers of individuals was made possible by the use of the mutant red, which allowed visual classification for the presence or absence of the enzyme at both stages. From a series of six mutagenesis experiments 111,561 chromosomes were tested, and 122 phenotypically mutant F₁ individuals were found. From these, 38 inheritable mutations were isolated which, by phenotypic observation, lacked or had reduced enzyme activity at the larval and pupal stages. Assay of enzyme activity levels in several of the mutants confirmed the phenotypic data. All of the 27 mutations that could be tested further are recessive and behave as cinnabar alleles. Complementation tests were performed between these 27 mutant stocks, and no complementation in the production of eye color has been seen between the mutants examined. When extended collection periods were used, a significantly higher percentage of inheritable mutations was isolated from the first 3 days of the screen. Over 80% of the F₁ phenotypic mutants could be classified as mosaics, which indicates that cinnabar can be autonomous under certain conditions. The failure to isolate mutations in possible regulatory function is discussed.     

Developmental analysis of the facets, a group of intronic mutations at the Notch locus of Drosophila melanogaster that affect postembryonic development

The activity of the Notch locus of Drosophila melanogaster during embryogenesis is necessary for the correct segregation of neural from epidermal lineages. The action of Notch is not confined to embryogenesis but is also essential for normal development during the postembryonic stages. Its action is pleiotropic, as revealed by the existence of several classes of mutations which affect various imaginal structures. Here, we examine a group of six recessive mutations, the facets (fa, fa3, fag, fag-2, fafx and fasw), which affect eye and optic lobe morphology and have been previously shown to be associated with the insertion of transposable elements into an intronic region of Notch. Using both somatic recombination and gynandromorph analysis, we find that their behavior in a mosaic analysis is not identical. While in the majority of alleles abnormal Notch function in the retina is sufficient to induce optic lobe abnormalities, in the case of fag-2, a considerable number of individuals having mosaic retinas exhibit normal optic lobe structure. All the facet alleles appear to behave in a cell-autonomous manner. A developmental analysis of the eye and optic lobe defects associated with the facet mutations support the contention that Notch may be involved not only in the formation of certain structures but also in their maintenance.     

Partial reversion at the bobbed locus of Drosophila melanogaster

In Drosophila melanogaster the tandemly arranged repetitive sequences coding for 18S and 28S rRNA are heterogenous at the level of the spacers between units and insertions that interrupt many 28S rRNA genes. This heterogeneity contrasts with the homogeneity of the regions transcribed into 18S and 28S rRNA. Homogenization and evolution of repetitive genes are usually explained by conversion, amplification events or unequal crossovers. In this paper we studied the change in rDNA patterns associated with partial reversion of bobbed mutations. In most cases, no increase in rDNA gene number, but a new repartition of gene types were found.     

Partial reversion at the bobbed locus of Drosophila melanogaster

In Drosophila melanogaster the tandemly arranged repetitive sequences coding for 18S and 28S rRNA are heterogenous at the level of the spacers between units and insertions that interrupt many 28S rRNA genes. This heterogeneity contrasts with the homogeneity of the regions transcribed into 18S and 28S rRNA. Homogenization and evolution of repetitive genes are usually explained by conversion, amplification events or unequal crossovers. In this paper we studied the change in rDNA patterns associated with partial reversion of bobbed mutations. In most cases, no increase in rDNA gene number, but a new repartition of gene types were found.     

Superunstable alleles at the cut locus in Drosophila melanogaster

Summary This is a detailed study of the reversions of the ct ^MR2 allele putatively carrying á mobile element (MR-transposon) in the cut locus. Stable, unstable and superunstable revertants have been identified. Besides, a series of multiple unstable visible and lethal ct mutations derived from the ct ^MR2 allele have been obtained. They are shown to include supermutable alleles. The results suggest that the MR-transposon is connected with at least three functions: excision; change of orientation; and change of position within the cut locus, these functions being disturbed in different ways in different unstable ct ⁺ and ct alleles. In some cases the mutant transitions are somehow strongly stimulated leading to superinstability, reaching the rate of 0.5.