CHROMOSOMAL DNA SYNTHESIS IN DROSOPHILA MELANOGASTER

Analysis of labeling patterns in three chromosome segments of Drosophila melanogaster has shown that the replicative activity within chromosomes is temporally ordered. Moreover, specific labeling patterns on one chromosome occur with specific patterns on another chromosome with a very high degree of correlation. This circumstance leads to the conclusion that DNA synthesis among all the regions in the three chromosome segments studied is coordinated. The various labeling patterns observed in any one chromosome and the combinations of labeling patterns observed in all three chromosome segments can be arranged in ordered arrays, if one assumes that the DNA synthesis in each chromosome region will go to completion without stopping once it has started. Such arrays can serve as models for the temporal order of DNA synthesis among chromosome regions. They predict that in any one chromosome DNA replication begins and ends at very few loci and that synthesis at a larger number of points occurs at an intermediate time.     

Recombination and DNA Replication in the DROSOPHILA MELANOGASTER Oocyte

A method is described that permits the recovery of a well-synchronized population of oocytes. Utilizing this pupal system, the heat-responsive period for increasing crossing-over in the Drosophila genome has been defined for the X chromosome and a portion of chromosome 2. The response is initiated close to the time of oocyte formation (premeiotic interphase) and is terminated after ~36 hr. During the 36-hr period different regions show characteristic responses, which vary in degree, in duration, and in initiation and termination points, so as to generate the beginning of a thermal recombination map for the Drosophila genome. Centromere regions exhibit the greatest increases in crossing-over for their respective chromosomes but are distinctly asynchronous in time; interstitial regions respond the least. Correlated autoradiographic studies have localized DNA replication in the oocyte to a ~24-hr period, which also begins close to oocyte formation (premeiotic interphase); late labeling in restricted regions, undetectable with the present method, could extend the period, as could prolonged synthesis in the oocyte. The results demonstrate that DNA replication and the heat-sensitive period for enhancement of crossing-over are coincident processes over most and possibly all of their length.     

Location and Underreplication of Satellite DNA in DROSOPHILA MELANOGASTER

The two light nuclear satellites (PCsC1 = 1.672 and PCsC1 = 1.687) have been quantified in DNA isolated from the larvel imaginal discs and brains of Drosophila melanogaster with the genotypes X/O, X/X and X/Y. By comparing the results from these different genotypes, the amounts of the two satellites in the X and Y chromosomes and in the autosomes have been determined. The lightest satellite is not located to any appreciable extent in the X chromosome. The heterochromatic regions are not completely filled by these satellites. --Satellite DNA has also been quantified in DNA isolated from adults containing different genotypes. The two satellites are underreplicated to different extents. The apparent amount of underreplication for one of the satellites is different in different parts of the genome.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

The Characterization of α-Glycerophosphate Dehydrogenase Mutants in DROSOPHILA MELANOGASTER

Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM^- by solution immunoprecipitation experiments, but of these, only three are CRM^- by a more sensitive ¹²⁵ I-protein A-based radioimmune gel assay. The viability of the three CRM^- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdh^nGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed.     

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

Repair Replication and Photorepair of DNA in Larvae of DROSOPHILA MELANOGASTER

Repair replication of DNA has been studied in first instar larvae of Drosophila melanogaster with isopycnic centrifugation techniques. Larvae were fed BUdR, FUdR, streptomycin, penicillin, and Fungazone for two to four hours prior to exposure to UV, X-rays, MMS, or EMS. Feeding was continued for four hours in the presence of (3)HBUdR and DNA was isolated from whole larvae. Repair replication is stimulated by each of these agents. MMS is about 10 times as potent as EMS in stimulating repair synthesis. A dose of 200 ergs/mm(2) largely saturates the level of repair replication observed after UV irradiation. Repair replication rises between 0 and 80,000 R of X-rays before falling off. Semiconservative synthesis is seriously inhibited above a dose of 40,000 R of X-rays. Photorepair has been detected as a reduction in repair synthesis resulting from post-irradiation exposure to photoreactivating light. The same treatment has no detectable effect on X-ray-stimulated repair replication. Repair replication is insensitive to the presence of caffeine or hydroxyurea during the final incubation, although semiconservative synthesis is strongly inhibited by these agents. A mixture of BUdR and (3)HTdR can be used to replace (3)HBUdR in detecting repair replication.     

Molecular Characterization of Ribosomal Genes on the Ybb- Chromosome of DROSOPHILA MELANOGASTER

The question of whether the Ybb- chromosome contains ribosomal genes has been examined by using Southern blot analysis and comparing rDNA hybridization patterns for X/X and X/Ybb- DNA. The results demonstrate that the Ybb- chromosome contains sequences that hybridize to an rDNA probe under stringent conditions. Differential hybridization of some of these sequences with DNAs corresponding to different regions of a complete ribosomal gene repeat provides evidence that some of the genes on the Ybb- chromosome are type 2 repeats. Because data obtained by other workers suggest that type 2 repeats are transcribed only to low levels, these repeats may be classed as "nonfunctional". A further finding is that the ribosomal genes on the Ybb- chromosome do not undergo multiple rounds of DNA replication during polytenization of X/Ybb- cells.     

Recovery and Characterization of Temperature-Sensitive Mutations Affecting Adult Viability in DROSOPHILA MELANOGASTER

Temperature-sensitive (ts) autonomous cell-lethal mutations have been used extensively to study important developmental phenomena, such as pattern formation, in Drosophila. Their utility would be enhanced considerably if it were possible to establish which cell type is primarily affected by each lesion. To facilitate such an approach we have isolated and characterized 21 EMS- induced X-linked adult-lethal (adl) mutants, 16 of which are ts. Most of these lesions also elicit ts lethal effects during preimaginal development. They represent 19 different loci distributed randomly along the X chromosome. The general properties of these mutations are described. In addition, results of an in-depth analysis (focus mapping and, in some cases, temperature shift and heat-pulse studies) of four strains, adl-1^ts1, sesE, adl-2^{ts 1} and rex are reported. Two major temperature-sensitive periods (TSPs) of adl-1 lethality were resolved: one during the second half of embryogenesis and the other coinciding with pupariation. Mosaic analysis revealed separate mesodermal foci for leg paralysis. Developmental analysis of adl-1 embryos suggest that the adl-1 product may be required for maintenance of muscle tissue. Two discrete TSPs of sesE lethality exist: one during the second instar and the other extending from late third instar to early pupation. Mosaic analysis of sesE lethality resolved a pair of neural foci, each of which appears to incorporate three separate foci for leg paralysis. Mosaic analysis of adl-2^{ts 1} revealed the existence of paired lethal foci that appear to map to the vicinity of the subesophageal ganglion. Analysis of rex mosaics resolved separate mesodermal foci for leg paralysis.     

Genetics of Acetylcholinesterase in DROSOPHILA MELANOGASTER

Genes in Drosophila melanogaster that control acetylcholinesterase (AChE) were searched for by segmental aneuploidy techniques. Homogenates of flies containing duplications or deletions for different segments were assayed for enzyme activity. A region on the third chromosome was found for which flies having one does consistently gave lower AChE activity than euploid flies, which were in turn had lower activity than flies with three doses. The activity differences were in the approximate ratio 1:2:3. Fine structure deletion mapping within this region revealed a very small segment for which one-dose flies have approximately half-normal activity. To obtain putative AchE-null mutations, lethal mutations within this region were assayed. Four allelic lethals have approximately half-normal activity in heterozygous condition. These lethals probably define the structural locus (symbol: Ace) for AChE.