Neutrons and X-rays,comparative studies with Drosophila melanogaster: 1. the viability and fertility of induced autosomal translocations

Studies on the genetic effects of neutrons and X-rays have produced evidence that may be interpreted as indicating that neutrons induce clusters of closely linked genetic changes. According to this interpretation, it is to be expected that neutron-induced translocations will have a higher rate of associated recessive lethality, compared with translocations induced by low-LET radiation such as X-rays. The experiment reported here was designed to test whether this expectation is fulfilled. The dose-frequency response with neutrons for the induction of autosomal translocation was established by exposing males from the Oregon-K stock and then sampling treated mature sperm. From the data obtained, it was estimated that 10 Gy neutrons should induce about the same frequency of autosomal translocations as 27 Gy X-rays. These 2 doses were used to induce translocations in the spermatozoa of males carrying lethal-free autosomes, derived from the Oregon-K stock. Induced translocations were tested for homozygous viability and fertility. When these criteria were used, no qualitative difference was detected between the translocations induced by neutrons and X-rays.     

Cell-stage-specific enhancement by caffeine of the frequency of chromatid aberrations induced by X-rays in neural ganglia of Drosophila melanogaster

Caffeine (10(-2) M) induced a high level of chromatid aberrations in neural ganglia of third-instar larvae of Drosophila melanogaster only when it was added to cells in late G2 and mitotic prophase. No aberrations were observed after treatment in late S--middle G2 or C-mitosis. We observed that, in these stages, caffeine strongly increased X-ray-induced damage (500 R). This potentiation was quantitatively similar. But it involved all types of aberration after treatment in C-mitosis, and essentially isochromatid deletions and chromatid exchanges after treatment in S--G2. Some hypotheses are put forth to explain the possible mechanism of action of caffeine in the potentiation of X-ray-induced damage.     

Cytology of aberrations induced by X-rays in oocytes of Drosophila melanogaster.

200 first-division configurations were analyzed for cytological aberrations induced by X-rays in late meiotic prophase in oocytes of Drosophila melanogaster. For the 3000 and 6000 r doses, 38 and 66%, respectively, were classified as abnormal. The aberrant divisions included displacement of the chromosomes suggesting their non-disjunction, loss of a whole chromosome, fragments and heterologous exchanges and unidentifiable aberrations. Non-disjunctional chromosomes were free of heterologous exchanges. The concept that a majority of X-ray-induced dominant lethals is due to chromosomal breakage is supported by the findings of the present study.     

Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity

A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet.     

Comparative studies of the induction of somatic eye-color mutations in an unstable strain of Drosophila melanogaster by MMS and X-rays at different developmental stages

An unstable white locus in Drosophila melanogaster originally described by Rasmuson and Green (1974) and further by Rasmuson et al. (1978, 1980) contains an IS element. This constellation interacts with the zeste mutation and forms a mutationally unstable system that is sensitive to a variety of mutagens. Mutational shifts between zeste and wild-type eye color as well as deletions and transpositions of the white locus are frequently occurring in the unstable X-chromosome in germ line and in somatic tissue. Germinal mutations from zeste to wild-type eye color are associated with an insertion of a piece of DNA, proximal to the wsp site, and the shifts from red to zeste are caused by an excision of the same piece (Rasmuson, in preparation). Mutations to pigmentless phenotype are interpreted as deletions of the white locus, while they always are irreversible and show non-complementation with wsp. The somatic system can be used as a screening test for potential mutagens, described by Rasmuson et al. (1984). This survey is an attempt to correlate the size of the mutated area of the eyes with the age of the larvae at mutagen treatment. X-Rays and MMS were used to give an indication of the mechanism of the instability, according to the different kinds of DNA damage induced. The results show that the mean size of red spots decreased with increasing age of larvae at treatment, while the mutation frequencies were increased because of the multiplication of the cells in the eye anlage susceptible to the mutagens. This is contradictory to the hypothesis maintained by Fahmy and Fahmy (1980) that the somatic shifts are not mutagenic but epigenetic events, due to altered regulation of the gene expression. Red spots induced with MMS are smaller in size than X-ray-induced red spots, indicating a delay in the establishment of mutations from chemically-induced lesions compared to irradiation damage. White spots on the other hand were equally large in size, irrespective of inducing agent and about twice the size of the chemically-induced red spots, implying a faster and more direct action for fixation of deletions than for the production of MMS induced shifts in eye color from zeste to red.     

Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping

Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC's role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688bp linear DNA fragment shortens by the equivalent of approximately 130bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133bp of DNA around the ORC complex.     

DNA underreplication in intercalary heterochromatin regions in polytene chromosomes of Drosophila melanogaster correlates with the formation of partial chromosomal aberrations and ectopic pairing

We studied the influence of the Suppressor of Underreplication (SuUR) gene expression on the intercalary heterochromatin (IH) regions of Drosophila melanogaster polytene chromosomes. We observed a strong positive correlation between increased SuUR expression, underreplication extent, amount of DNA truncation, and formation of ectopic contacts in IH regions. SuUR overexpression from heat shock-driven transgene results in the formation of partial chromosomal aberrations whose breakpoints map exclusively to the regions of intercalary and pericentric heterochromatin. It is important to note that all these effects are seen only if SuUR overexpression is induced during early stages of chromosome polytenization. Therefore, we developed the idea that ectopic pairing results from the joining of free DNA ends, which are formed as a consequence of underreplication.     

The induction of chromosome aberrations in mouse dictyate oocytes by X-rays and chemical mutagens.

Oocytes were collected from female mice and matured in vitro to Metaphase I during the first or third week after treatment with a dose of 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). In week 1 the mean number of oocytes per female was similar for all treatments but in week 3 irradiated females yielded fewer oocytes than the chemically treated or control females. In week 1 the proportion of oocytes maturing was smaller in irradiated females than in the other groups but in week 3 was similar in all groups.Structural chromosome aberrations, scored in the Metaphase I oocytes, were of the chromatid or isochromatid type and involved gaps, breaks, fragments, intrachanges and interchanges. Aberration frequency did not increase with time after either of the chemical mutagens but after irradiation was higher in the third week than in the first week. The aberration yield from IPMS-treated females was similar at both sampling times, while a lower yield was recorded in week 3 following TEM treatment than in week 1.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.     

Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster.

We have identified several GABAergic-modulated behaviors in Drosophila melanogaster by employing a pharmacological approach to disrupt GABA transporter function in vivo. Systemic treatment of adult female flies with the GABA transport inhibitors DL-2,4-diaminobutyric acid (DABA) or R,S-nipecotic acid (NipA), resulted in diminished locomotor activity, deficits in geotaxis, and the induction of convulsive behaviors with a secondary loss of the righting reflex. Pharmacological evidence suggested that the observed behavioral phenotypes were specific to disruption of GABA transporter function and GABAergic activity. The effects of GABA reuptake inhibitors on locomotor activity were dose dependent, pharmacologically distinct, and paralleled their known effects in mammalian systems. Recovery of normal locomotor activity and the righting reflex in DABA- and NipA-treated flies was achieved by coadministration of bicuculline (BIC), a GABA receptor antagonist that supresses GABAergic activity in mammals. Recovery of these behaviors was also achieved by coadministration of gabapentin, an anticonvulsant agent that interacts with mammalian GABAergic systems. Finally, behavioral effects were selective because other specific behaviors such as feeding activity and female sexual receptivity were not affected. Related pharmacological analyses performed in vitro on isolated Drosophila synaptic plasma membrane vesicles demonstrated high affinity, saturable uptake mechanisms for [3H]-GABA; further competitive inhibition studies with DABA and NipA demonstrated their ability to inhibit [3H]-GABA transport. The existence of experimentally accessible GABA transporters in Drosophila that share conserved pharmacological properties with their mammalian counterparts has resulted in the identification of specific behaviors that are modulated by GABA.