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By Hitoshi Okamoto, Kuniyoshi Sakai, Satoshi Goto, Etsuko Takasu-Ishikawa, and Yoshiki Hotta Department of Physics, Faculty of Science, University of Tokyo, 113 Tokyo (Communicated by Setsuro Ebashi, m. j. a., Sept. 14, 1987) Molecular basis of the membrane excitability has recently Introduction. ' been elucidated by cloning the genes encoding the voltage sensitive ionic channels. 1)-3) We report in this paper an identification of the two Drosophila melanogaster genes which have homologies to the eel voltage sensitive sodium channel. Drosophila melanogaster is an ideal organism for the study of gene regulation as well as its fine structure analysis, since any gene once cloned can be properly manipulated and delivered again into the genome by the P-element mediated gene transfer method.4) Therefore, we now expect that the two tentative Drosophila ionic channel genes we cloned can be used as materials for the study of specific gene expression in excitable cells. Materials and methods. Genomic DNA was extracted from adult flies.5) The genomic library was made with EMBL4 as a cloning vector.63 The 64mer probe oligonucleotide was synthetized with an automatic DNA synthetizer (ABI Model 381A), and purified with Spectra-Physics HPLC system and Nucleosil 300-7C18 reverse phase column.  It was kinased with 32P and used for the library screening.7)  Hybridization was performed at low stringency (formamide 20%, 6xSSC, at 42°C). Nucleotide sequence was determined by the chain termination method8) using plasmids as templates (Takara 7-Deaza Sequencing Kit). To determine the sequence upstream from the 64mer hybridizing region in pNa16 (see the text of Results), a set of clones with various deletions from the 5'end of the insert were made. 9  Clones with the deletions up to near the 64mer region was used for sequencing.  Other recombinant DNA experiments were carried out by the standard methods.7) In situ hybridization to salivary gland chromosomes were performed according to Engels et al.10) The deficiency stocks Df(2R)M-c33a =Df (2R) 60E2-3 ;60E11-12 and Df (2L) H68=Df (2L) 36B1-2 ;37B1 were obtained from Umea Drosophila stock center and Dr. K. Mogami, respectively. These strains were crossed to the normal strain (Canton-S) and used for chromosomal preparations. Results. To isolate Drosophila homologs of the eel sodium channel gene, we screened the genomic library with a synthetic 64mer oligonucleotide probe This region corresponds to the transmembrane segment IVS6 which (Fig. 2). is well conserved through the repeat I to IV within the eel gene, 1 ' and is also conserved between the eel and rat gene.2)  To increase the detection sensitivity, we hybridized the probe at low stringency to the membranes on which phages had been amplified in situ after transfer from plates.7)  Out of 160,000 inde- pendent genomic clones, we obtained eight clones which showed positive hybridi- zation signals. They were named as λNa14,ｽNa16,ｽNa17,ｽNa20,ｽNa21,ｽNa24, 2Na30 and 2Na31. DNA of each clone was purified and digested with EcoRI. The EcoRI fragments which hybridized with the probe were subcloned into pUC18 (Fig. 1). The subclones were named as pNa14, pNa16, pNa20, pNa21, pNa24, pNa30 and pNa31, respectively. From the patterns of the restriction enzyme digestion and from the cross-hybridization experiments of these subclones, we classified the original phage clones into four groups; I) ¿Na16 and ¿Na30, II)ｽNa14,ｽNa17 andｽNa21, III)ｽNa24 andｽNa31, IV)ｽNa20. The EcoRI fragments in ANa16 and ANa30, which had been classified into the group I, showed far more intense hybridization signals than those from other groups of clones (Fig. 1). These fragments alone hybridized also to the eel sodium channel cDNA (pSCH50)1) at low stringency (data not shown). Therefore, we expected that these clones most likely encode a Drosophila sodium channel homolog. The EcoRI fragment subclones were digested with Sau3AI and further subcloned at random into pUC18. These clones were screened with the synthetic 64mer, and the nucleotide sequences of the positive clones were determined. The nucleotide sequences and the deduced amino acid sequences of the clones belonging to the group I and II turned out to have significant homologies to the eel IVS6 sequence. In the group I (2Na16 and 2Na30), the homology of the deduced amino acid sequence to that of the eel sodium channel was the most remarkable. We further determined the nucleotide sequence upstream from the 64mer hybridizing region in pNa16. The combined result is shown in Fig. 2. The amino acid sequence of this region coincides with that of the eel sodium channel at 60%. This suggests strongly that this region encodes a Drosophila sodium channel, and that the region corresponding to the eel VIS4 to VIS6 is not interrupted
Fig. 3. The restriction map of the DIC60 gene. The arrangement of ¿Na30, ¿Na16 and ¿Na40 is shown below the map. The direction of transcription is found to be from left to right from the sequence data. Bar=2 kb, E=EcoRI, S=SacI.  The probe 64-mer hybridizes to the region indicated by an arrow head. by introns. A new clone λNa40 was obtained by screening the genomic library again with pNa16 and pNa30 as probes. The restriction map of the region covered by  λNa40, λNa16 aud λNa30 is shown in Fig. 3. In the group II (XNa14, XNa17 and XNa21), a stretch of fifteen nucleotides is completely matched with a part of the 64mer probe, followed by the additional seven amino acids homologous to the eel sodium channel (Fig. 2). In this clone, however, a stop codon appears in frame just 5' to the completely matched region, but no homology is found in the adjacent upstream sequence. We determined the chromosomal locations of the two distinct groups of clones by in situ hybridization to polytene chromosomes. The clones of the group I (λNa16, λNa30, λNa40) are located at 60E of chromosome 2R (Fig. 4 A, B). The clones of the group II (XNa14, XNa17, XNa21) are at 37B-D (Fig. 4 D, E). These results were further confirmed by using the deficiency strains (Fig. 4 C, F). No mutations assumed to have a defect in a sodium channel are located at these sites.11)  However, the first gene may be the same with that reported by Salkoff, L. et al. at t the Cold Spring Harbor meeting in 1986.12)  We call our two Drosophila ionic channel gene candidates as DIC60 and DIC37 according to their chromosomal locations. We identified two Drosophila genes (DIC60 and DIC37) which Discussion. have a homology with the eel sodium channel gene. DIC60 has a region where the amino acid sequence coincides with that of the eel sodium channel through IVS5 to IVS6 at 60%. DIC37 has a region of twelve amino acids with a high homology to the eel IVS6, but the homology is interrupted by the appearance of a stop codon in-frame just 5' to this region. Since it is unlikely that such a high homology occurs simply by chance, we expect that the homologous region in DIC37 is an exon isolated from the main part of a gene by an intron. It is not also excluded that it is a remnant of some evolutionary degraded pseudogene. We screened the oligo-dT primed cDNA library from pupae with pNa21 as a probe. Although we could get several positive clones, their inserts did not reach the 64mer hybridizing region. Therefore, we cannot yet determine whether the upstream stop codon in DIC37 is averted by splicing in the mature mRNA. The first thing that should be elucidated with respect to the functions of our tentative ionic channel genes is to identify their ionic specificity. Recently, a candidate for a calcium channel gene was cloned. 3)  It has a remarkably similar structure with sodium channels. Therefore, the genes we isolated cannot be con- sidered to encode a sodium channel simply from their structural homology. One way to answer this question is to isolate the full length cDNA and to express its product in cells which lack sodium channel such as in Xenopus oocyte.13) However, this system may not necessarily work for the Drosophila cDNA, since the eel sodium channel cDNA could not be expressed in the Xenopus oocyte membrane. We may be able to overcome this difficulty in an alternative way by exploiting sophisticated technology specific to Drosophila. As the chromosomal locations of the genes are already known, we can study flies with a deficiency of these region. From the electrophysiological defect of such flies, we can draw inference as to the ionic specificity of our tentative channel genes. We can confirm it, if the defect is rescued by introducing the normal gene into the genome of the deficiency strain. To examine the structure and function relationships into further details, it is necessary to accumulate information as to how the function is affected by various mutations. In the Xenopus oocyte system, mutations are introduced into the cDNA in vitro. This method can be extended in Drosophila to obtain trans- formed flies having in vitro mutagenized genes by using P-element mediated transformation method. We can also collect a number of mutant alleles system- atically by screening the alleles which do not complement the electrophysiological defects of the deficiency strains. They may be collected either as lethal or con¬ ditional mutants. Since many temperature sensitive paralytic mutants are known in Drosophila, the ionic channel function may also become temperature-dependent, if a proper mutation is introduced. Such a conditional mutant is useful since the gene function can be switched on and off at cellular or even at individual levels. Drosophila melanogaster is also the most suitable for studying the regulatory mechanism of the ionic channel gene expression. We are performing the P- element mediated transformation experiments to re-introduce the chimeric gene composed of an E. coli β-galactosidase and our tentative channel genes.  With this system, we can visualize the tissue specific expression pattern of our genes and identify the regulatory region necessary for their proper expression. We acknowledge Drs. S. Numa and M. Noda for Acknowledgements. generously providing us with the eel sodium channel cDNA clone (pSCH50), Drs. A. Rasmussen, K. Ekstroem and K. Mogami for the deficiency stocks, Dr. S. Ishiwa and Ms. K. Fukami for the instruction of the in situ hybridization technique, Ms. M. Akama, T. Naoi and K. Hotta for technical assistance. This work was supported by the Grants-in Aid to Y.H. from the Ministry of Edu- cation, Science and Culture of Japan, and in part by the fellowships given to H.O. from the Japan Society for the Promotion of Science for Japanese Junior Scientists.