Materials and methods Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue8-9.Sept2000:

776 N.E. Gruntenko et al. Insect Biochemistry and Molecular Biology 30 2000 775–783 In this work, we analysed the mutations ts403, Met, T bh and ebony e with respect to the response of JH- degradation system to stress. The recessive temperature sensitive lethal mutation llts403 results in the failure of heat shock protein HSP83 and HSP35 to be expressed, and a number of HSP70 proteins are only par- tially expressed Evgen’ev and Denisenko, 1990. Met 27 is a null allele of the Methoprene-tolerant gene that shows resistance to the toxic effects of both JH and a JH analog, methoprene. The mechanism of the resistance appears to be altered JH receptionWilson and Fabian, 1986; Shemshedini and Wilson, 1990. Met 27 completely lacks Met transcript and is clearly a null allele Wilson and Ashok, 1998. T bh nM18 is a null mutation at the Tyr- amine b-hydroxylase locus, which results in complete absence of the tyramine β -hydroxylase protein and blockage of octopamine biosynthesis Monastirioti et al., 1996. e is postulated to be the mutation of N- b-alanyl dopamine synthetase gene, based on the fact that e has twice as much DA as normal Hodgetts, 1972; Hodgetts and Konopka, 1973; Ramadan et al., 1993. Here we asked whether these mutations would affect the decrease in JH degradation occurring in D. mel- anogaster when stressed. In order to answer this ques- tion, we studied the JH degradation in individuals of ts403, n Met 27 , T bh nM18 and Ste strains carring llts403, Met 27 , T bh and e mutations, respectively, under normal and stress conditions, and compared their stress-reac- tivity calculated as percent change in JH hydrolysis under stress compared to hydrolysis under normal conditions with that in a number of wild type and lab- oratory strains. We demonstrated 1 that ts403 females respond to stress by a decrease in JH degradation, as occurs in wild type females, but that their stress-reactivity significantly differs from that of wild type; 2 that in young v Met 27 females, similar to wild type flies, JH hydrolysis is decreased upon stress, but their stress-reactivity is sig- nificantly lower than in wild type; 3 that JH degra- dation is unaffected in older v Met 27 females under stress; 4 that T bh nM18 females show a significantly higher JH-hydrolysis level and different stress-reactivity than does the wild type; and 5 that young Ste females demonstrate significantly lower JH-hydrolysis and stress-reactivity, compared to the wild type.

2. Materials and methods

2.1. Drosophila strains The following D. melanogaster strains were used: the wild type laboratory strain Canton S; wild type iso- female strain 921500 from a natural population of Gorno-Altaisk; laboratory balancer strain First Multiple Seven FM7; vermilion n strain from which the n Met 27 strain was derived; laboratory balancer strain In2LRCyL ; In3LRDSb , carrying morphological mutations with recessive lethal action Curly, Lobe chromosome 2 and Dichaete, Stubble chromosome 3; hereafter termed CyLDSb; strain ts403 carrying the recessive temperature sensitive lethal mutation llts403 Arking, 1975; strain n Met 27 carrying a null allele of the Methoprene-tolerant gene Wilson and Ashok, 1998; strain T bh nM18 carrying a null mutation at the Tyr- amine b-hydroxylase locus Monastirioti et al., 1996; and the laboratory Ste strain carrying the e mutation. Cultures were raised on standard medium Rauschenbach et al., 1987 at 25 ° C, and adults were synchronized by eclosion. Flies were subjected to stress at 38 ° C for 3 h, and were subsequently frozen in liquid nitrogen and stored at 220 ° C. 2.2. JH hydrolysis JH hydrolysis was measured by the assay of Ham- mock and Sparks, 1977. A fly was homogenized on ice in 30 µ l of 0.1 M Na-phosphate buffer, pH 7.4, contain- ing 0.5 mM phenylthiourea. The homogenates were cen- trifuged for 5 min at 12,000 rpm, and samples of the supernatant 10 µ l were utilized for the reaction. A mix- ture consisting of 0.1 µ g unlabeled JH-III Sigma and 12,500 dpm 3 H labeled JH-III 17.4 Cimmol at C-10, NEN Research Products, Germany was used as sub- strate. The reaction was carried out in siliconized tubes in 100 µ l of incubation mixture for 3 h, and it was stopped by the addition of 250 µ l heptane and 50 µ l of a solution containing 5 ammonia and 50 methanol VV. The tubes were shaken vigorously and centri- fuged at 12,000 rpm for 10 min. Samples 100 µ l of both aqueous and heptane phases were placed in vials containing dioxane scintillation fluid and counted. Con- trol experiments have shown a linear substrate–reaction relationship Gruntenko et al., 1999, as well as the fact that measured activity is proportional to homogenate i.e. enzyme concentration Rauschenbach, 1991; unpub- lished data. The significance of the differences between the data sets was tested by Student’s t-test. Sample size varied from 12 to 28 individuals for each measurement in all experiments.

3. Results