Patterns of hobo elements and their effects in natural populations of Drosophila melanogaster in Japan.

We studied the dynamics of hobo elements of Drosophila melanogaster in Japan with the goal of better understanding the invasion and evolution of transposons in natural populations. One hundred and twenty-six isofemale lines and 11 older stocks were tested for the presence and genetic phenotype of hobo elements. The oldest H strain, containing complete and deleted hobo elements, is Hikone-H (1957), but Hikone-R (1952) has no hobo-homologous sequences. The findings suggest that the hobo element invaded Japanese populations in the mid-1950s, at about the same time as the P element invasion in Japan. This chronology is consistent with the hypothesis of a recent worldwide hobo element invasion into D. melanogaster in the mid-1950s. In recently collected populations, H degrees strains (low hobo activity and high repression potency) are predominant, whereas H+ strains (high hobo activity and high repression potency) are predominant in the Sakishima Islands, the most southwestern islands of the Japanese archipelago. H' strains (high hobo activity and low repression potency) were first found in limited island populations. Japanese populations have not only full-size hobo elements and 1.5 kb Th elements but also characteristic deletion derivatives (1.6 and 1.8 kb XhoI fragments) that we have named Jh elements. These results are consistent with transgenic experiments with complete hobo elements, in which populations evolved to H+ or H degrees via H', and in which 1.8 kb fragments appeared. We conclude that hobo elements invaded the central region of Japan, spread to the far islands, and that the invasion is currently at an intermediate, nonequilibrium stage.

Sensitivity to diethylether anesthesia of fruit flies primarily depends on the genotypes of the sodium channel gene rather than the states of the membranes and the mechanisms might be different from heat-induced paralysis.

The para locus of Drosophila melanogaster encodes the alpha subunit of a voltage-sensitive sodium channel. Many of the mutants develop paralysis at the high temperature (37 degrees C) and are hypersensitive to diethylether anesthesia. We examined whether the two aspects of the phenotype are mediated by a same mechanism that involves the sodium channel molecule by investigating properties of the three para alleles (para(hd838), para(ts1) and para(ts3)). Larvae of the all para strains showed almost normal sensitivities to diethylether anesthesia while adult flies of them showed hypersensitivities to that in the following manner: para(hd838)<==para(ts1)

Repression of the wing vein development in Drosophila by the nuclear matrix protein plexus.

The wing of Drosophila is separated into several sectors by the wing veins. Vein primordia are specified by the positional information provided by hedgehog and decapentaplegic in the wing imaginal disc and express the key regulatory gene rhomboid. One model of this process is that boundaries of gene expression regulated by hedgehog or decapentaplegic provide reference points where rhomboid transcription is activated. We present an analysis of the gene plexus, whose loss of function causes an excess vein phenotype. Molecular cloning revealed that plexus encodes a novel 1990-amino acid protein with cysteine-rich motifs. Plexus protein was ubiquitously expressed and was tightly associated with the nuclear matrix. In plexus mutant wing imaginal discs, an anteroposterior positional coordinate was established normally as revealed by the wild-type pattern of spalt major and knirps expression. However, the expression of several vein-specific and intervein-specific genes was misregulated, as if they had neglected the positional coordinate. These results suggest that Plexus is an essential component of a global repressor of vein differentiation. Although Plexus protein was expressed in vein primordia of the wing disc, it does not appear to interfere with vein differentiation in the normal position. A genetic epistasis test between px and knirps suggests that plexus acts downstream of knirps. We propose that the vein differentiation takes place by inactivation of the plexus-mediated repression by prepattern genes such as knirps. Plexus may regulate transcription of vein-and intervein-specific genes by tethering transcriptional regulators to specific locations in the nucleus.

Fast atom bombardment tandem mass spectrometric analysis of phospholipids in Drosophila melanogaster.

Direct determination of the phospholipid components in adult Drosophila melanogaster was carried out by using fast atom bombardment tandem mass spectrometry (FAB-MS/MS) of both the positive and negative ions. Approximately 50 molecular species were detected, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI). Eight PE, one PC and three PS molecular species were identified. Some variations with age and a few differences among the D. melanogaster strains in the PE and PC molecular species were found. There was a difference in the fatty acid structure of a 741 Da PE molecular species between the wild-types and a mutant strain (EthAR201) which requires a higher concentration of diethylether for anesthesia than the wild-types; in the mutant sn-1-oleoyl-2-linoleoyl (18:1/18:2) but in the wild-types sn-1-linoleoyl-2-oleoyl (18:2/18:1) were speculated. This suggests that this technique will be useful for the screening of phospholipid molecular species mutation.

Molecular genetical analysis of Drosophila ether sensitive mutants.

1. For a genetical approach to the molecular mechanisms of general anesthesia, we isolated 14 Drosophila transposon tagging mutants which showed altered sensitivities to diethylether anesthesia. Two mutated genes were identified and analyzed for their structures and functions. 2. One sensitive mutant, para(hd838), has a transposon, P-element, insertion in the 2nd intron of para locus (14C) which encodes an alpha subunit of a sodium channel. 3. Another sensitive mutant, eth(as)311. has a P-element in the 5'-untranslated region of Drosophila calreticulin gene (86BC). Calreticulin is known as a multifunctional calcium-binding protein in the endoplasmic reticulum of non-muscle cells. 4. We obtained their revertants to show that the P-element insertion in each gene induced the hypersensitivity to ether anesthesia. We describe here that the sodium channel and calreticulin might be targets for the ether anesthesia.

Induction of leg-shaking, knock-down and killing responses by gamma-ray irradiation in Shaker mutants of Drosophila melanogaster.

Induction of membrane-associated responses, a leg-shaking, a knock-down and a killing, by gamma-ray irradiation was investigated in Shaker (Sh) mutants of Drosophila melanogaster in which the gene cords for the A-current K+ channel. Sh mutants were more sensitive in the knock-down response after gamma-ray irradiation than wild types. There were a great amount of sex difference in the knock-down response, males being more sensitive than females, but not in the killing response. The sex difference was larger than gene dosage effect on X chromosome in females. Genetical analysis revealed that the sensitivity of the knock-down response is an incompletely dominant character without maternal effects. The leg-shaking response, which had previously been reported to be induced by ether treatment, was demonstrated in the head-removed flies of Sh mutants. It was found to be the most sensitive among the responses tested, and may involve changes in K+ channel. The knock-down response may be related to expansion of the leg-shaking response. The killing response should have causes different from the leg-shaking and the knock-down responses judging from the lack of correlation with them.

Protective effects of ether, oxygen and their mixture for radiation in Drosophila melanogaster.

Protective effects of ether mixed with air or oxygen against ionizing radiation damages were demonstrated in adult flies of Drosophila melanogaster. The protective effects against knock-down on the 2nd day and lethality on the 8th day after irradiation were not affected by the radiation sensitivity and DNA repair capacity of the strains. Ether (4.2%) in oxygen was more effective than ether in air for both endpoints. The protective effects may be due to damages not involving cell division, since no mitotic cells are observed in adult flies except in gonadal glands. A change in the orderliness of the cell membrane by ether is suggested to be the cause of the protective effects.

Temporal distribution of P elements in Drosophila melanogaster strains from natural populations in Japan.

Genetic and molecular investigations were carried out with 10 Japanese Drosophila melanogaster strains on P-M system of hybrid dysgenesis. The strains used here were collected in the years from 1952 to 1984 from various natural populations, and have been maintained in our laboratory. The whole genomic Southern hybridization was performed by using the 2.9-kb P element and the internal fragments as probes. Five strains possessed no P element copy and the other 5 strains possessed mainly incomplete P elements which had internal deletions. The former 5 strains were M, 2 of the latter were Q, and the remaining 3 were M' strains. Hikone-R, collected in 1952, had no P element copy, while Hikone-H, collected in 1957, was the earliest observed to possess multicopies of an incomplete P element. This revealed that P elements in Drosophila melanogaster were present more than 30 years ago in Japan, as already shown to have been the case on the American continent.

Sensitivity to ether anesthesia and to gamma-rays in mutagen-sensitive strains of Drosophila melanogaster.

An ether-resistant strain of Drosophila melanogaster, Eth-29, has previously been found to be radiosensitive. Some mutagen-sensitive strains are known to be hypersensitive to X-rays in larvae. The correlation between sensitivities to ether anesthesia and to gamma-rays was examined in adult flies of 12 mutagen-sensitive strains and 6 control strains. A wide variation in sensitivities to ether anesthesia, gamma-ray knock-down and gamma-ray lethality was demonstrated. No correlation between DNA-repair capacity and ether sensitivity or gamma-ray knock-down sensitivity was shown. Only mei-9 and mus201, which are deficient in excision repair, as well as Eth-29 were found to be sensitive to gamma-ray lethality. These findings indicate that the targets for ether anesthesia, knock-down and lethality may be different. Lethality appears to be caused by DNA damage, while the other 2 endpoints appear not to be related to DNA damage.

Anesthetic-resistant spontaneous mutant of drosophila melanogaster: intensified response to 60Cobalt radiation damage.

Accumulating evidence suggests that the extent of acute damage by ionizing irradiation is closely related to the state of membrane orderliness. Decreased orderliness apparently protects organisms from ionizing irradiation. Because anesthetics decrease membrane orderliness, anesthesia is expected to affect damages caused by ionizing irradiation. The present study compared the effects of 60Co irradiation on Drosophila melanogaster between an anesthetic-resistant spontaneous mutant and an anesthetic-sensitive strain. We have previously established an anesthetic-resistant mutant strain, Eth-29, of Drosophila melanogaster. Eth-29 is resistant to diethylether, chloroform and halothane. The anesthetic-resistant strain was found to be radiosensitive when evaluated by survival at the eighth day after irradiation or by dyskinesia (knock-down) at the second day. The results indicate that anesthetic resistance may be related to an increase in orderliness. The findings in reciprocal crosses between Eth-29 and the control strain indicate that the mechanism of survival is different from that of knock-down. Presumably, knock-down is the direct sequela of irradiation, and the present result suggests that membrane damage may be involved in inducing knock-down.

Loss of high frequency of sister chromatid exchanges in Epstein-Barr virus-established lymphoblastoid cell lines from two patients with Bloom's syndrome.

Seven lymphoblastoid cell lines were established through transformation by Epstein-Barr virus of peripheral blood lymphocytes from two patients with Bloom's syndrome (BS), the parents of a patient, and normal controls. High baseline levels of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes of BS were reduced to about 10% of their initial value in BS lymphoblastoid cell lines, and the elevation of SCE frequencies induced by ethylmethanesulfonate was the same as in controls.

Alteration in molecular species of phosphatidylethanolamine between anesthetic resistant and sensitive strains of Drosophila melanogaster.

An analysis of lipid composition was carried out in resistant and sensitive strains of Drosophila melanogaster. Amount of total lipid and amount of phosphate of phospholipids were not different from each other in both strains. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) did not differ in amount between both strains. Determination of molecular species of PE using gas chromatography (GC) and GC-mass spectrometry showed that the resistant strain had increased 34:2 and decreased 36:2, 36:3 and 36:5 relative to the sensitive strain. The molecular species of PC did not differ between the two strains. Chromosomal analysis revealed that the alterations in 34:2 and 36:2 of PE were regulated by the X- and third chromosomes of the resistant strain. Therefore, the changes in PE may explain the mechanism of anesthetic resistance because genetic analyses indicate that these chromosomes have more influence on the anesthetic resistant traits of the resistant strain.

Strain differences in minimum anesthetic concentrations in Drosophila melanogaster.

An ether-resistant strain of Drosophila melanogaster has been maintained at this laboratory since the appearance of one female mutant in 1961. Sensitivity was defined using mortality as an endpoint when exposed to a high concentration of diethylether; this does not necessarily mean that anesthetic requirements are higher in the resistant strain. The present study was undertaken to determine the difference in anesthetic potency between the ether-resistant strain (Eth-29) and one of the sensitive strains (bw;st;svn). The median effective dose (ED50) for halothane was 0.0096 atm in females and 0.0091 atm in males of the bw;st;svn strain, while in the Eth-29 strain the ED50 was 0.0148 atm in both sexes. The ED50 values for chloroform anesthesia were 0.0051 atm in females and 0.0050 atm in males of the bw;st;svn strain and 0.0100 atm in the Eth-29 strain in both sexes. Strain differences in response to the two anesthetics were statistically significant. Thus the Eth-29 strain shows a cross-resistance to both halothane and chloroform anesthesia. Reciprocal crosses between the two strains revealed that the resistance to halothane anesthesia was a sex-linked recessive trait and that the resistance to chloroform anesthesia was an autosomal incompletely dominant trait.

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.