David T. Suzuki

Developmental properties of shibirets1: A pleiotropic mutation affecting larval and adult locomotion and development☆☆☆

The sex-linked recessive mutation, shibirets1, causes rapid paralysis in adults and larvae at 29°C whereas normal mobility occurs at 22°C. The influence of the mutation on development was analyzed by studying the effects of brief heat pulses administered at various developmental stages. Mutant animals had a polyphasic temperature-sensitive period (TSP) for lethality and a polyphasic effective lethal phase (LP). In addition, heat shocks caused a broad spectrum of phenotypically distinct defects, each characteristic having its own distinct TSP. Some of the processes affected were: early embryogenesis including gastrulation, and the development of eyes, bristles, legs, wings, and the neuromuscular system. The developmental properties of shibirets1 allowed us to make a number of observations concerning determination and pattern formation and to conclude that the lesion affects a fundamental cell process common to many cell types.

Activity of a transfer RNA modifying enzyme during the development of Drosophila and its relationship to the su(s) locus

Abstract The Chromatographie elution profiles of radioactively labeled asparaginyl, aspartyl, histidyl and tyrosyl-tRNAs from different developmental stages of Drosophila melanogaster have been examined. The relative proportions of certain chromatographically distinct forms of these four tRNAs are altered in a similar way during the life cycle of wild-type flies. These four tRNAs show a further quantitative change in flies of the genotype su(s) 2 v;bw . Two-dimensional thin-layer chromatograms of pancreatic RNase digests of the two major tRNA Asp Chromatographie forms revealed almost identical patterns. Two-dimensional thin-layer chromatograms of piperidine hydrolysates of these tRNAs revealed that one contained a nucleotide similar to Qp, an unidentified nucleotide occurring in Escherichia coli tRNA. It was concluded that these Chromatographie forms are homogeneic † , the difference between them being the extent of the modification of one nucleotide. The su(s) 2 v;bw mutant appears to be altered in some aspect of this modification process.

Analysis of tRNAs during the development of Drosophila

Abstract Conditions for the chromatography of radioactively labeled Drosophila aminoacyl-tRNAs on reversed-phase 5 columns have been established. All 20 aminoacyl-tRNAs from first instar, third instar, and adult flies were compared. While some of the aminoacyl-tRNAs remain essentially unchanged during the development of Drosophila, others show marked quantitative changes. These changes are discussed in relation to possible control mechanisms during development.

Behavioral mutants of Drosophila melanogaster

SummarySex-linked behavioral mutants were induced in Drosophila melanogaster with ethyl methanesulfonate (EMS) and isolated by direct visual observation of abnormal phenotypes. The four behavioral phenotypes used were flight-reduction, hyperactivity, hypoactivity and stress-sensitivity, and are easily discernable in either single or small populations of mutant flies. In one screen, forty-two behavioral mutants were recovered from strains derived from 800 mutagen-treated X chromosomes. In a second screen, 139 behavioral mutants were obtained from 2369 X chromosomes. The high rate at which behavioral mutants were recovered in the second screen, when compared to new visibles (28) and new temperature-sensitive lethals (124), suggests that the isolation of behavioral mutations on the autosomes of Drosophila and in the genomes of larger insects should be practical.

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC; tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5–7, 72F1–2, 83F-84A; tRNA3Met, 46A1–2, 61D1–2, 70F1–2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84D3–4, 92B1–9; tRNA4Val, 56D3–7, 70BC.

Temperature-sensitive mutations in Drosophila melanogaster. XI. Male sterile mutants of the Y chromosome.

Abstract Eight temperature-sensitive (ts) male sterile mutations have been induced by ethyl methanesulfonate treatment of Y chromosomes derived from a selected temperature-resistant Amherst wild-type stock of Drosophila melanogaster. Males carrying such mutated Y chromosomes (Yts) are sterile when raised at 29°C but fertile when reared at 22°C. Complementation tests of the mutants with Y chromosome fragments, deletions, and inter se localized all eight to the long arm of the chromosome in four different complementation groups. When Yts-bearing males, reared to adulthood at 22°C, were subjected to a 48-hr regimen at 29°C and mated to fresh virgin females daily, a significant reduction in fertility resulted 5 days after initiation of 29°C treatments. This period of sterility was transient (48–72-hr duration) and corresponded to a temperature-sensitive period (TSP) of spermatogenesis during the primary spermatocyte stage. A more precise definition of the TSP utilized exposure of subadult males to 29°C at selected developmental periods during which only certain germ cell stages are present. Upon eclosion adult males were subjected to a similar schedule of consecutive matings of 12-hr duration in order to detect any delay in the appearance of fertility. Different ts males could be distinguished by the resultant pattern of sterility, and the TSP of different mutations thus localized to either primary spermatocyte or immediately post-meiotic stage. Associated with Yts-mediated sterility, spermiogenesis is defective at restrictive temperature as evidenced by the production of nonmotile sperm and a failure to transfer such sperm to the female during copulation. In addition, electron microscopy detected a variety of ultrastructural abnormalities, including defects of axoneme formation, irregularities of Nebenkern derivative development, and failures of separation from the syncitial state or mature cyst with subsequent degeneration.

The genetic effects of mitomycin C in Drosophila melanogaster ☆: I. Induced mutations and XY chromosomal interchanges

Abstract The genetic effects of the alkylating agent mitomycin C (MC) were investigated. MC was found to induce sex-linked recessive lethals in all stages of oogenesis with the highest frequency occurring in meiotic broods. MC-Induced Xue5f8Y and Yue5f8Y interchanges were recovered from both males and females, as well as females homozygous for c3G , thus indicating that the mechanism involved in the generation of these interchanges is independent of spontaneous meiotic crossing-over. These interchanges occur predominantly in gonial cells as indicated by brood analysis. The increased frequency of MC-induced interchanges in gonial broods has been shown to result from gonially induced events that are multiplied by subsequent mitotic divisions. Also, the increased number of females yielding interchanges in the gonial broods suggests that gonial cells are more sensitive to the effects of MC on inducing interchanges. Genetic evidence concerning the division behavior of gonial stem cells is presented.

The genetic effects of mitomycin C in Drosophila melanogaster ☆: II. Induced meiotic recombination

Abstract The antibiotic, mitomycin C (MC), induces non-homologous chromosomal interchanges in gonial cells of Drosophila males and females. In both males and females, the interchanges were recovered in a non-random distribution within the gonial broods of individual families. MC also increased the frequency of crossing-over in Drosophila females especially in the centromeric regions of gonial cells, yet did not induce crossing-over in males. The MC-induced increase in crossing-over in females was not a result of gonially induced events that were multiplied mitotically in stem cells as was the case with MC-induced interchanges. MC was also mutagenic in both sexes especially in those cells which had completed the first meiotic prophase. Since MC can either mono- or bifunctionally alkylate DNA, it was proposed that excision of monofunctionally alkylated bases would give rise to single-strand “nicks”, whereas excision of two bases involved in a bifunctionally alkylated crosslink would generate double-strand “cuts”. By this model, single-strand nicks could act as the precondition for crossing-over and would produce crossovers when the necessary factors for spontaneous meiotic crossing-over are present. Double-strand “cuts”, on the other hand, could generate translocation-type interchanges independently of the crossover apparatus in both males and females. A test of this prediction was made with the monofunctional alkylating agent ethyl methanesulfonate (EMS). Results were consistent with the model and revealed that gonial cells just preceding the stage at which crossing-over occurs, are most sensitive to EMS.

Temperature-sensitive mutations in Drosophila melanogaster: X. Developmental analysis of the paralytic mutation, parats☆☆☆★

Abstract The recessive, sex-linked mutation, paralytic temperature-sensitive (parats), causes a reversible temperature dependent paralysis in adult flies only. When adult parats flies are shifted from 22°C to 29°C, they are paralyzed in less than 5 sec; a shift back to 22°C restores mobility almost instantly. Somatic cuticular mosaics of mutant and wild-type tissue were created by loss of an unstable ring X chromosome. The behavior of 310 (out of 371) of these mosaics was analyzed at 29°C. The relationship between the location of mutant tissue and the pattern of aberrant behavior at 29°C was analyzed in detail with a view to an anatomical localization of the defect caused by parats. Many mosaics fell into one of the following ten categories whose behavior is discussed in detail: abdomen only, complete head, bilateral head, complete thorax, bilateral thorax, leg only, complete head-thorax, complete head-bilateral thorax, bilateral head-complete thorax, and bilateral head-thorax. The mosaic data suggest that the mutation is autonomous (i.e., mutant cells are not affected by surrounding wild-type tissue) and that it represents a lesion in nervous tissue. The results from electroretinograms and optomotor tests suggest that the mutation does not affect the ability of the eye to receive and transduce light into impulses in the retinula cells, but that the impulses either do not reach or cannot be interpreted by the brain. Thus, parats affects the central nervous system. Survival of completely parats flies with prolonged exposure to high temperature suggests that vital organs continue to function during paralysis. Indeed, the heart was observed to beat quite normally. This indicates that parats does not effect the stomodeal ganglion of the autonomic nervous system. It is concluded that parats affects at least two components of the central nervous system: (1) the head including the brain and subesophageal ganglion, and (2) the fused thoracic ganglia located in the ventral region of the thorax.

Temperature-sensitive mutations in Drosophila melanogaster,: VI. Temperature effects on development of sex-linked recessive lethals

Abstract The developmental effects of four temperature-sensitive lethal mutations on the X chromosome of Drosophila melanogaster were analyzed. By shifting cultures from 29°C (restrictive temperature) to 22°C (permissive) and vice versa at different stages, the temperature-sensitive period (TSP) of each mutation was defined: (1) l(1)E34ts was a pupal lethal with a monophasic TSP initiated at the time of pupation; (2) l(1)E7ts was a late pupal and early adult lethal with a TSP during the pupal period in males, whereas females had a prolonged TSP or succession of TSPs extending from the embryonic through the larval stages; (3) l(1)E25ts was polyphasic with a succession of TSPs in the embryo, in the period between first and second larval instars and in adult females; (4) l(1)E12ts was sensitive to 29°C at all stages of development. The potential limitations and utility of ts mutations in the analysis of development were discussed.