Abstract We are investigating which kinds of genes are related to diethylether anesthesia using many resistant and sensitive mutants of Drosophila melanogaster to diethylether. A resistant mutant strain EthAR223 and a sensitive mutant strain ethas319 were made artificially by inserting a P-element, one of transposon, into the chromosome. The strains were out-crossed by white strains that have normal sensitivity to diethylether, to replace most of the EthAR223 or ethas319 chromosome by the chromosome of the white strain, except the P-element insertion region. Both outcross lines showed the properties of EthAR223 or ethas319 rather than the white strains, suggesting the altered sensitivities of the EthAR223 and ethas319 were caused by the gene that were inserted by the P-element, not by mutation of other genes. To isolate and identify the gene inserted by the P-element, the genome DNA of the ethas319 was prepared and the 3 kb sized DNA region was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was sequenced and the sequence was found to have a homology with a part of a genomic clone AC019820 that was one of DNA sequence data provided by DNA Data Bank of Japan (DDBJ). The sequence has a gene that expands approximately 30 kb, has at least 14 exon sequences, and supposed to encode at least 413 amino acid sequence. This gene was supposed as the Drosophila counter part of the casein kinase 1, gamma 3 subunit (CK1- γ3) gene because it had a high homology with the rat CK1- γ3 by the sequence comparison. With the further analysis of the DNA sequence, the altered sensitivity of the ethas319 was caused by the P-element insertion at the non-coded region of the CK1- γ 3 gene that probably related to the nervous functions.
The P-element, one of the transposable gene (transposon) is able to insert into chromosomes of Drosophila melanogaster at random, sometimes causing mutations on a variety of genes. By screening the objective mutant and by the transposon tagging method that is to clone the gene related to the phenotype easily using the P-element as a scaffold, the genes related to diethylether anesthesia would be found to help revealing of anesthesia mechanisms. By this method we obtained and analyzed 19 P-element inserted strains having altered sensitivities to diethylether anesthesia, suggesting many genes would relate to the sensitivity to diethylether anesthesia. Among them, the para locus that encodes a voltage-dependent sodium channel gene and the crc locus that encodes a calcium storage protein calreticulin gene are closely involved in the hypersensitivity to diethylether anesthesia. In this study, we analyze other two mutant strains EthAR223 and ethas319 to understand the mechanisms of anesthesia in detail by clarifying more genes related to sensitivity to anesthesia.
In this study, we analyze other two mutant strains EthAR223 and ethas319 to understand the mechanisms of anesthesia in detail by clarifying more genes related to sensitivity to anesthesia.
Materials and Methods
A resistant mutant strain EthAR223 and a sensitive mutant strain ethas319 to diethylether anesthesia were made by inserting P-lwB, a kind of P-element transposon into the chromosome. Structure of the P-lwB is shown in Figure 1. This was made from original P-element by deleting the internal region, adding P-lacZ gene, heat shock-mini-white+ fusion gene (hs-white+ ), and Bluescript M13-KS vector. The hs-white+ gene makes the eyes of the white mutant fly red, thus this gene can be a marker of the presence of the P-lwB in the chromosome and utilized for making outcross lines. The Bluescript part can be used for cloning the DNA sequence flanking the P-lwB.
Figure 1. Structure of the P-lwB that was inserted in the two mutants EthAR223 and ethas319.
Two arrowheads at the ends are the inverted repeat derived from the original P-element. Regions indicated by PL1/2, 3, 4 are the cleavage sites by the restriction enzymes. Especially, the Sal I cleaving sites are important for the cloning. The locations and directions of the RV primer and FW primer are indicated at the top line. Directions are the start sites of base sequencing of the genome DNA surrounding the Bluescript. The P-lac Z is described in Tanaka et al. . The hs-white+ , and Bluescript M13-KS are explained in the text The EthAR223 was found to have the P-lwB at 55C of the second-right arm of chromosomes and the resistant property to diethylether anesthesia was genetically dominant, while the ethas319 was found to have theP-lwB at 90A of the third-right arm of chromosomes and the sensitive property was genetically recessive . The eye color mutant strains wi135 and w1 were used for making outcross lines.
Culturing the fly strains and determination of anesthetic properties of the flies.
The fly strains were fed with the standard medium, composed of 5% dry yeast, 7% glucose, 5% cornmeal, 1.2% agar, and 0.35% propionic acid at 24° as described in Tanaka et al. . The process of determining sensitivity to diethylether anesthesia and calculation of 50 % Effective Concentration (EC50) are described in Tanaka et al. [1,5], Tanaka and Gamo , Tomida et al. , and Tamai et al. . Twenty male or female adult flies two days after eclosion were put into a milk bottle and exposed to a given concentration of diethylether gas mixed with oxygen for 30 min. The anesthetizing apparatus was drawn in Figure 2. Diethylether was purchased from Nacarai Tesque (Nara, Japan) and the vaporation of diethylether was performed by ANESTHESIA APPARATUS, MODEL PH-2 (Akoma Ika Kogyo, Tokyo, Japan).Diethyether was vaporized by a Copper Kettle of the apparatus at 24±0.5°, and diluted by oxygen gas (2 liter per min flow) to gain a given concentration of diethylether.
Figure 2. Construction of anesthetizing apparatus. Oxygen gas (most right side) was flown by 2 liter per min and mixed with the diethylether gas vaporized by the mixer of the Copper Kettle at 24±0.5°. The ratio of the oxygen gas and diethylether gas can be adjusted by the dial under the indicator of the cylinder of the Kettle to make the desired concentration of the diethylether. The diethylether gas diluted by the oxygen gas was send to the milk bottle (the most left side) for 30 min. The exhaust gas was flown out. Sometimes the bottle was shaken lightly to let the flies absorb diethylether evenly.
The ether-oxygen mixed gas was flowed into the 200 mL milk bottle for 30 min. Then the flies were took out from the bottle and put on a plate, and stimulated lightly with a brush. The flies that had lost the avoidance reaction as walking or flying were recognized as anesthetized. The assay was performed with different concentrations of diethyl ether using an additional batch of the flies of the same strain, and the ED50 and the 95% confidence limits were calculated up to the three significant digit from dose-response curves with logarithm-probit transformation. In order to compare sensitivities between the original, mutant, and outcorss strains, an analysis of covariance were performed using Microsoft Excel version 5.0 .
Preparing of outcross lines
To confirm that the altered sensitivities of the EthAR223 and ethas319 were caused only by the P-lwB insertion, not by mutation of other genes, outcross lines were prepared and the lines were compared their sensitivities to diethylether anesthesia with the original mutant strains and white strains. The outcross lines were made by mating female virgin flies of the mutant strain with males of the white strains for ten generations. After that, the homozygous flies as for the the P-lwB insertion were measured the sensitivities to diethylether anesthesia, along with the original mutant flies and white flies, and calculated the EC50(% atm.). If the outcross lines showed sensitivities similar with the original mutant strains, the altered sensitivities were confirmed to be caused only by the P-lwB insertion, while if the outcross lines showed sensitivities similar with the white strains, the altered sensitivities were supposed to be caused by mutation of other gene, not be related to the P-lwB insertion.
Another mutant stain ms(3)89B (provided by Bloomington Stock Center) that has the P-element insertion at near the 90A site that the ethas319 has P-lwB insertion among the third-right arm of chromosomes was crossed with the ethas319 and the first filial generation (F1) flies were measured their EC50.
Analysis of DNA
Methods of extraction of genomic DNA from flies and of ligating DNA fragments are described in Gamo et al. . The method of the polymerase chain reaction (PCR) is also described in the above reference. Briefly, the PCR was performed by using KOD dash (2.5U/ μL, TOYOBO, Osaka, Japan), with M13FW (5’-GTAAAACGACGGCCAGT-3’) and M13RV (5’-CAGGAACAGCTATGACCATG-3’) primers. The condition of amplification was 94°, 1 min, 30 cycles of (94°, 1 min → 57° , 1 min, → 72° , 3 min), and 72°, 2 min. PCR product was purified by Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Bio-Rad Laboratories, Berkeley, USA) and determination of DNA sequence was ordered to Bex Co. Ltd. using M13FW and M13RV primers as start points of sequencing. Published homologous genomic DNA sequences of the obtained sequence were searched using the Basic Local Alignment Search Tool-nucleotide (BLASTN) program provided by DNA Data Bank of Japan (DDBJ) and published homologous Expressed Sequence Tags (ESTs, partial sequences of the Drosophila cDNA clones) were searched using the database of Barkeley Drosophila Genome Project (BDGP) and DDBJ. Published homologous amino acid sequences of the obtained sequence were searched using the Basic Local Alignment Search Tool-protein (BLASTP) program provided by DDBJ.
Results and Discussion
Results of sensitivities of the outcross lines are shown in Table 1. Female flies are more resistant to diethylether anesthesia than male flies in the cases of original EthAR223 strain, white mutants (wi35 and w1 ), and most of outcross lines. The phenomenon could be observed in Drosophila mutant strains of voltage-dependent sodium channel (parahd838, and parats1)  and other anesthesia resistant mutants (EthAR315 and EthAR217) . These mutants might also cause the sexual differences of neural functions related to anesthesia. By this reason, comparing the resistance or senstivity to anesthesia should be performed among males and females, separately. In both sexes, outcross lines of the EthAR223 strain (223-wi35 ?~?, and 223-w1 ? and ?) had almost similar resistance with the original EthAR223 strain, significantly higher than the wi35 and w1 stains. Outcross lines of the ethas319 strain, 319-w1 ?~?, had almost similar sensitivity with the original ethas319 strain, significantly lower than the w1 stain, although outcross lines of the ethas319 strain, 319-wi35?~? had the intermediate sensitivity. These results confirm that both the resistance of the EthAR223 to diethylether anesthesia and the hypersensitivity of the ethas319 to diethylether anesthesia were caused by the P-element insertion into the chromosome.
By the complementation test, heterozygous F1 flies of the ethas319 and ms(3)89B that was found to have P-insertion at the close loci of the P-lwB insertion site of ethas319 had much lower EC50 than the wi35 strain, and almost similar EC50 with the ethas319 strain (data not shown). This fact indicates that the P-element of the ms(3)89B was inserted into the same or close gene as the P-lwB. Further analysis of the ms(3)89B would reveal the mechanisms of anesthesia in detail. The genomic sequences of the flanking region of the P-lwB of ethas319 determined by M13RV primer and M13FW primer (Figure 3) showed 97 % and 96 % homology, respectively, with a part of the sequence AC007760 (195,037bp), a Drosophila melanogaster genomic clone of region 89B, a loci of chromosome 3R. Next, ESTs sequences were searched by the homology with the sequence AC007760 to obtain the exon sequences that included amino acid translated regions. Approximately 30 ESTs sequences had homology with the AC007760. Among them three ESTs sequences, BT004871, LD31233, and BT001330 had regions of long homology with AC007760 (Figure 4), indicating the possibility that the AC007760 contains a genuine gene. A possible splicing pattern was also
Table 1. Sensitivities to diethylether anesthesia of the original strains and outcross lines by white mutants. The EC50 is the ether anesthesia concentration (% atm.) which half flies become anesthetized. In addition, the 95 % confidence limit is indicated by parentheses.
Figure 3. A Partial DNA sequence from the Sal I digested, ligated, and PCR-amplified genome DNA fragment of the ethas319. The former half is the sequence from the M13RV primer and the latter half is the complementary reversed sequence from the M13FW primer. Sequences surrounded by boxes are the uncertain ones by our methods. Intermediate dot region is the undetermined sequence, but approximately 190,000bp sequence is supposed to exist because the clone AC007760 homologous to our sequence has long sequence in the corresponding region. An underlined six base sequences show the Sal I cleavage site.
Figure 4. Three EST clones (ESTs) having homology with the DNA sequences from the ethas319 and a part of AC007760 genomic DNA sequence.
indicated in Figure 4. By the information of each EST sequences, this genuine gene are made of at least 14 exons, among them some regions would be the alternatively spliced because the regions are used as exons or introns depending on the kind of mRNA. Insertion site of the P-element in ethas319 was uncertain but it was supposed to be inserted upstream of the translation start site. By BLASTP searching, the three amino acid sequences from BT004871, LD31233, and BT001330 had 67.5 %, 84.1 %, and 79.5 % homology with the rat casein kinase 1, gamma subunit 3 (CK1-γ3), respectively, thus, we concluded the gene as the Drosophila counter part of the CK1-γ3. One example showing amino acid homology of BT004871 that is the EST clone having the longest homology with the U22321 (rat CK1-γ3 mRNA sequence from DDBJ) is shown in Figure 5. These facts suggest that the gene where the P-element insertion occurred in the ethas319 strain is the CK1-γ3 of Drosophila that expands approximately 30 kb, has at least 14 exons, and encodes at least 410 amino acids. These amino acid sequences include LLGPSLEDLF and EQSRRDD those are specific motifs of the casein kinase (CK) and high homology with the sequences of rat CKγ1, γ2, and γ3, confirming further that the gene is the Drosophila CK1- γ3 gene. The existence of the CK1- γ3 is not reported in Drosophila, thus, the gene was named as dckγ(Drosophila casein kinase gamma). Because in adult flies of the ethas319 strain, the expression of the reporter gene lacZ included in the P-lwB was observed in the brain and ganglia that closely relate with the general anesthesia , the innate CK1- γ is supported to express in the same organs. In rats, the CK1- γ3 were reported to express in brains . In addition, the protein kinase that is encoded by a double-time gene of Drosophila melanogaster and has a homology of approximately 73 % with the dck1-γ3 gene is also expressed in a Drosophila brain region concerned with a biological clock . As for the general anesthetic agent, it is principally known by loss consciousness or reflex inhibition, thus, the mutation by the P-element insertion into the Drosophila CK1-γ3 gene had influenced on the functions of central nervous systems causing the hypersensitivity to diethyl ether anesthesia and it is supposed that the Drosophila CK1- γ3 is one of the target of diethylether.
As for the hypersensitive ethas319 strain, analysis of the expression pattern from the gene inserted by the P-lwB such
Figure 5. Alignment of the translated amino acid sequences of BT004871 and rat CK-1γ3 mRNA (U22321, derived from DNA Databank of Japan) was performed by utilizing ClustaW. Asterisks under the amino acid sequences indicate the identical amino acids among the two sequences. BT004871 had 67.5 % homology (282 identical amino acids among 418), while LD31233 and BT001330 (not shown) had 84.1 (95 % identical amino acids among 113) and 79.5 % (248 identical amino acids among 312), respectively.
as the amount of the dck1-γ3 mRNA and alternatively spliced mRNA would help to reveal the detailed mechanism of diethylether anesthesia, like the following two genes. In Drosophila mutant strain of calreticulin, ethas311, mRNA pxpression of the calreticulin gene was greatly reduced compared with the wild type because of the P-element insertion in the gene . In Drosophila mutant strain of voltage-dependent sodium channel, parahd838, alternative splicing of mRNAs occurred from the sodium channel gene causing hypersensitivity to diethylether anesthesia . As for the resistant EthAR223 strain, cloning and analysis of the sequence around the P-lwB insertion site would be required because genes closely related with diethylether anesthesia may reside in the insertion site.
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