Events In Drosophila Research Articles

Analyzing double-strand repair events in Drosophila.

AbstractTargeted manipulation of the genome is used to analyze gene expression, genome structure and protein structure. In yeast it has long been possible to introduce sequences into predetermined sites in the genome by double-strand break (DSB) repair (1). Until recently, such specificity has eluded investigators that use Drosophila as their model organism. Such a gene-targeting system is now available in Drosophila. The system is based on the repair of a specific DSB made by P element excision (2–12).KeywordsGene ConversionBreak SiteWhite GeneDonor ElementDonor SequenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Genetic analysis of punt, a type II Dpp receptor that functions throughout the Drosophila melanogaster life cycle.

TGF-beta (transforming growth factor-beta-) mediated signal transduction affects growth and patterning in a variety of organisms. Here we report a genetic characterization of the Drosophila punt gene that encodes a type II serine/threonine kinase TGF-beta/Dpp (Decapentaplegic) receptor. Although the punt gene was originally identified based on its requirement for embryonic dorsal closure, we have documented multiple periods of punt activity throughout the Drosophila life cycle. We demonstrate that potentially related embryonic punt phenotypes, defects in dorsoventral patterning and dorsal closure, correspond to distinct maternal and zygotic requirements for punt. In addition, we document postembryonic requirements for punt activity. The tight correspondence between both embryonic and postembryonic loss-of-function punt and dpp phenotypes implicates a role for Punt in mediating virtually all Dpp signaling events in Drosophila. Finally, our comparison of punt homoallelic and heteroallelic phenotypes provides direct evidence for interallelic complementation. Taken together, these results suggest that the Punt protein functions as a dimer or higher order multimer throughout the Drosophila life cycle.

Drosophila melanogaster as a Model System for Assessing Development under Conditions of Microgravity

More is known about the regulation of early developmental events in Drosophila than any other animal. In addition, its size and short life cycle make it a facile experimental system. Since developmental perturbations have been demonstrated when both oogenesis and embryogenesis occur in the space environment, there is a strong rationale for using this organism for the elucidation of specific gravity-sensitive developmental events.

Drosophila embryonic neuroblasts in culture: Autonomous differentiation of specific neurotransmitters

Isolated neuroblasts from gastrula-stage Drosophila embryos divide and differentiate in vitro to produce clonally derived clusters of neurons. Both serotonin and dopamine are expressed within these cultures in patterns that are similar to their distributions in vivo. Clusters containing serotonergic neurons are generally distinct from those with dopaminergic neurons, suggesting that different neuroblasts produce neurons with these phenotypes. The appearance of each transmitter correlates with transcription of dopa decarboxylase in the transmitter-positive cells. The developmental program leading to the appearance of either serotonergic or dopaminergic neurons is different for each transmitter type. Thus, serotonergic cells are progeny of early neuroblast divisions, whereas dopaminergics arise throughout the lineage. Inhibition of cell division, but not nuclear division, with cytochalasin B demonstrates that the expression of the serotonin phenotype requires a determined number of DNA replications. These experiments establish that neuroblasts, as soon as they are formed during early gastrulation events in Drosophila, are already determined for the subsequent expression of transmitter phenotype.

Molecular analysis of recombination events in Drosophila.

The locations of crossover junctions and gene conversion tracts, isolated in the rosy gene of Drosophila melanogaster, were determined using DNA sequencing and denaturing gradient gel electrophoresis. Frequent DNA sequence polymorphisms between the parental genes served as unselected genetic markers. All conversion tracts were continuous, and half of the reciprocal crossover events had conversion tracts at the crossover junction. These experiments have also identified the sequence polymorphisms responsible for altered gene expression in two naturally occurring rosy variants.

Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts.

Synthetic mRNA precursors from the Drosophila fushi tarazu (ftz) gene were shown to be accurately and efficiently spliced in Drosophila nuclear extracts derived from Kc tissue culture cells or 0- to 12-hr embryos. Splicing the ftz pre-mRNA requires ATP and low levels of Mg2+. The reaction proceeds with a lag of 20-30 min prior to appearance of spliced mRNA and appears to proceed in two steps. The first step is cleavage at the 5' splice site to generate a 5' exon (E1) fragment and an intron-3' exon (IVS-E2) species. The second step involves cleavage at the 3' splice site, ligation of the two exons (E1-E2), and intron (IVS) release. The excised intron (IVS) and intron-3' exon (IVS-E2) exhibit anomalous electrophoretic mobility, suggesting that they contain branched structures. Nuclease analysis using two-dimensional thin-layer chromatography indicates that both the IVS and IVS-E2 species possess branched trinucleotides in which a guanosine residue at the 5' end of the intron is linked in a 2'-5' phosphodiester bond to the 2' hydroxyl group of an adenosine residue in the intron. The site of branchpoint formation was localized by debranching the Drosophila lariat with mammalian (HeLa) cell debranching enzyme and by P1 and T2 nuclease analysis. These findings indicate that nuclear extracts derived from Drosophila cultured cells or embryos can accurately splice mRNA precursors and that the reaction mechanism is the same as has been observed in yeast and mammalian cells. This system provides an initial step toward the biochemical analysis of developmentally regulated pre-mRNA splicing events in Drosophila.

O-Alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stage of repair-proficient ring-X (RX) males were treated with methyl methane-sulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective ( mei-9 L1) or to repair-cor-competent females ( mei-9 +). Absence of the mei-9 + function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induced chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-enthyl- N′-nitro- N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9 L relative to mei-9 + was MMS > MNU > DMN = EMS > iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9 L1 females were plotted against those determined for mei-9 + females, straight lines of following slopes were obtained: MMS = 7.6, NU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O 4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.

Some aspects of the detection of potential mutagenic agents in drosophila

The Drosophila system is a valuable test for detecting and characterizing mutagenic agents. Tester strains are available or can be synthesized for determing almost all types of genetical change ranging from gene mutations to chromosome rearrangements in a great variety of cell types of both sexes. Metabolic activation of all groups of indirect mutagens tested so far (aryldialkyltriazenes, cyclophosphamides, nitrosamines, azo-, hydrazo- and azoxyalkanes, aflatoxins, and polycyclic hydrocarbons; about 35 representatives in all), gives strong although indirect support for the considerable metabolizing ability of Drosophila. This capability would be expected from comprehensive biochemical data on bioactivation of foreign compounds in other insects. From a comparison of which types of genetical change are induced at high, low and threshold concentrations, it appears that lethal tests remain the most reliable method for any screening program. Mutagenic agents such as diethylnitrosamine, hycanthone and certain triazene, which are highly efficient in the induction of recessive lethals (gene mutations and/or deficiencies), would not have been detected in Drosophila if chromosome brekage were the only indicator for mutagenic activity. Moreover, for several mono- and polyfunctional agents, the lowest dose which is still genetically active was definitely lowest for recessive lethals when compared with dominant lethals, chromosome rearrangements or loss. If a new mutagen is discovered by a screening procedure using Drosophila, an accurate picture of its ability to cause either or both gene mutations and chromosome aberrations can be drawn. Such work will be valuable in helping to clarify similar problems in mammalian systems. For instance, it was important to learn that mutagens of the nitrosamine type apparently fail to produce breakage events in Drosophila. Similarly, three cyclophosphamides appeared not to have chromosome breaking ability. However, from a more detailed study, in which a series of concentrations was used, it became obvious that a penetration effect or, more likely, a rate-limiting factor in bioactivation, was the cause of the negative results obtained with these agents.

Some aspects of the detection of potential mutagenic agents in drosophila

The Drosophila system is a valuable test for detecting and characterizing mutagenic agents. Tester strains are available or can be synthesized for determing almost all types of genetical change ranging from gene mutations to chromosome rearrangements in a great variety of cell types of both sexes. Metabolic activation of all groups of indirect mutagens tested so far (aryldialkyltriazenes, cyclophosphamides, nitrosamines, azo-, hydrazo- and azoxyalkanes, aflatoxins, and polycyclic hydrocarbons; about 35 representatives in all), gives strong although indirect support for the considerable metabolizing ability of Drosophila. This capability would be expected from comprehensive biochemical data on bioactivation of foreign compounds in other insects. From a comparison of which types of genetical change are induced at high, low and threshold concentrations, it appears that lethal tests remain the most reliable method for any screening program. Mutagenic agents such as diethylnitrosamine, hycanthone and certain triazene, which are highly efficient in the induction of recessive lethals (gene mutations and/or deficiencies), would not have been detected in Drosophila if chromosome brekage were the only indicator for mutagenic activity. Moreover, for several mono- and polyfunctional agents, the lowest dose which is still genetically active was definitely lowest for recessive lethals when compared with dominant lethals, chromosome rearrangements or loss. If a new mutagen is discovered by a screening procedure using Drosophila, an accurate picture of its ability to cause either or both gene mutations and chromosome aberrations can be drawn. Such work will be valuable in helping to clarify similar problems in mammalian systems. For instance, it was important to learn that mutagens of the nitrosamine type apparently fail to produce breakage events in Drosophila. Similarly, three cyclophosphamides appeared not to have chromosome breaking ability. However, from a more detailed study, in which a series of concentrations was used, it became obvious that a penetration effect or, more likely, a rate-limiting factor in bioactivation, was the cause of the negative results obtained with these agents.