A Mechanism of Drug Action Revealed by Structural Studies of Enoyl Reductase

M. Livingstone, P. Sziber, W. Quinn, Cell 37, 205 (1984); P. Drain, E. Folkers, W. Quinn, Neuron 6, 71 (1991); Z. L. Wu et al., Proc. Natl. Acad. Sci. U.S.A. 92, 220 (1995); W. Li, T. Tully, D. Kalderon, Learn. Mem. 2, 320 (1996). 3. E. Skoulakis, D. Kalderon, R. Davis, Neuron 11, 197 (1993). 4. A. Nighorn, M. Healy, R. Davis, ibid. 6, 455 (1991); P. Han, L. Levin, R. Reed, R. Davis, ibid. 9, 619 (1992). 5. M. Heisenberg, A. Borst, S. Wagner, D. Byers, J. Neurogenet. 2, 1 (1985); J. S. de Belle and M. Heisenberg, Proc. Natl. Acad. Sci. U.S.A. 93, 9875 (1996). 6. J. S. de Belle and M. Heisenberg, Science 263, 692 (1994). 7. A. Brand and N. Perrimon, Development 118, 401 (1993). 8. F. Quan, W. Wolfgang, M. Forte, Proc. Natl. Acad. Sci. U.S.A. 86, 4321 (1989); W. Wolfgang et al., J. Neurosci. 10, 1014 (1990). 9. F. Quan, L. Thomas, M. Forte, Proc. Natl. Acad. Sci. U.S.A. 88, 1898 (1991). 10. M. Simon, M. Strathmann, N. Gautam, Science 252, 802 (1991); H. Bourne, D. Sanders, F. McCormick, Nature 349, 117 (1991); W. Tang and A. Gilman, Cell 70, 869 (1992); A. Spiegel et al., in G Proteins (Molecular Biology Intelligence Unit, R. G. Landes Co., Austin, TX, 1994), pp. 19–20; E. Neer, Cell 60, 249 (1995). 11. Complementary DNAs encoding the short forms of GasWT and Gas* (9) were subcloned into pUAST (7) and used to transform flies [A. Spradling, inDrosophila: A Practical Approach, D. Roberts, Ed. (IRL Press, Oxford, 1986), pp. 75–197]. The UAS-Gas lines were verified by Msc I digestion of polymerase chain reaction–amplified Gas transgenes to detect the presence of a site found in GasWT but lacking as a result of the Q215L mutation. 12. 238Y and 201Y are described in (17) and C232 in (18). C747 shows identical expression to that of C772 (17) (24). C309 is described by J. D. Armstrong [thesis, University of Glasgow, Scotland (1995)]. The O’Kane laboratory screened 500 P-GAL4 lines to identify OK66, OK86, OK62, OK107, OK348, and OK415. 13. T. Tully and W. Quinn, J. Comp. Physiol. 157, 263 (1985); T. Tully, T. Preat, S. Boynton, M. Del Vecchio, Cell 79, 35 (1994). 14. In total, we analyzed behaviorally 16 P-GAL4 insertions that expressed to some degree in MBs. Flies with four insertions (OK66, OK86, KL65, and KL107) showed reduced learning in the absence of expression of constitutively activated Gas*, most likely because of genetic background differences. These were not tested further. Flies with eight insertions showed defects in olfactory acuity (30Y, C35, OK62, OK107, C302, OK415, and C532) or shock reactivity (C772), as transheterozygotes with UAS-Gas*. These were eliminated from further behavioral testing. Flies with the remaining four P-GAL4 insertions (201Y, 238Y, C309, and C747) showed disrupted learning but normal olfactory acuity and shock reactivity, as transheterozygotes with at least one UASGas* insertion. We characterized a total of two PGAL4 insertions (C232 and OK348) that expressed to some degree in the CC. 15. We suggest that olfactory learning results from Gsdependent convergence of the conditional stimulus (CS) and unconditional stimulus (US) in theMBs. This belief is reinforced by the observation that expression of Gas* in the CC does not affect learning. Formally, however, we cannot exclude the possibility that other sites of convergence of the CS and US contribute to the conditioned behavior, as in honeybees [M. Hammer and R. Menzel, J. Neurosci. 15, 1617 (1995)]. 16. Direct immunolocalization of transgenic Gas forms proved impossible, as endogenous Gas is expressed throughout the central nervous system (8). We have not observed any significant differences in qualitative expression between UAS-regulated insertions [see also (18) and D. Lin and C. Goodman, Neuron 13, 507 (1994)]. Thus, P-GAL4–driven reporter gene expression should reflect genuine expression patterns of Gas* under UAS regulation. 17. M. Y. Yang, J. D. Armstrong, I. Vilinsky, N. Strausfield, K. Kaiser, Neuron 15, 45 (1995). 18. K. O’Dell, J. Armstrong, M. Y. Yang, K. Kaiser, ibid., p. 55. 19. Pan-neural expression was driven by P-GAL4 1407 [generated by J. Urban and G. Technau; described in L. Luo, Y. Liao, L. Jan, Y. Jan,Genes Dev. 8, 1787 (1994)]. 20. The P-GAL4 C309 line was crossed to UAS-lacZ and UAS-lacZ;UAS-Gas* lines. Brains from the resultant progeny were dissected, and b-Gal activity was detected as described [S. T. Sweeney, K. Broadie, J. Keane, H. Niemann, C. J. O’Kane, Neuron 14, 341 (1995)]. 21. L. Levin et al., Cell 68, 479 (1992). 22. D. Cooper, N. Mons, J. Karpen, Nature 374, 421 (1995). 23. D. Clapman, Annu. Rev. Neurosci. 17, 441 (1994); W. Schreibmayer et al., Nature 380, 624 (1996). 24. K. Kaiser, unpublished data. 25. S. Boynton and T. Tully, Genetics 131, 655 (1992). 26. R. Sokal and F. Rohlf, in Biometry (Freeman, New York, 1981), pp. 229–240. 27. E. Yeh, K. Gustafson, G. Boulianne, Proc. Natl. Acad. Sci. U.S.A. 92, 7036 (1995). 28. L. Luo, T. Tully, K. White, Neuron 9, 595 (1992). 29. We thank K. Moffat, J. Keane, K. Störtkuhl, R. Greenspan, M. Yang, G. Boulianne, and A. Brand for reagents. Supported by grants from the Wellcome Trust and the Science and Engineering Research Council (GR/F94989) (to C.J.O’K.), the Human Frontiers Science program (to C.J.O’K., K.K., and M.F.), and NIH grant HD 32245 (to T.T.).