REFERENCES AND NOTES ‘2 Dels and K. Alder. Justus Liebus Ann. Chem. 460. 9s (1928). 0. Diels. J. H. Blom. W. Koll. {bid. 443. 242 (1925); 8. M. Trosl. I. Flemirg. L. A. Paquette. Comprehensive Organic Synthess (Fergamon. Oxford. 1991), vol. 5. pp. 316; F. Friyuelli and A. Talichi. Dienes in Ihe Die/s-A/der fieacfion(Wiley. New York, 1990). and references therein; W. Carruthers. Cyc/u~dditionReacfions in Crpwnic Synfhesis (Pergamon. Oxford. 1990). ancl references therein; G. Desimoni. G. Tmi. A. 3arco. G. P. Pollini. ACS Monograph No. 180 (hmican Chemicat Society. Washingloo. DC. 19.33). and references cited therein. Fcr recent reviews. see U. Rndur. G. LUQ. C. 0110. C~T. Rw. 93. 741 (1993); H. B. tbgm and 0. Rant. ibd 92. 1007 (1992). and references mein. 3. D. Hihrert. K. W. HiII..K. D:Nared. M-T. M.Auditor. J. An Chem. SOC. 111. 9261 (1989). 4. A. C. Braisfed and P. G. Schultl. ibid. 112. 7430 (1!=)1. 5. C. J. Suckling, M. C. Tedford. L. M. Bence. J. I. Imine. W. H. Slimson, Ebg. Med. Chem. Lett. 2, 49 (1992). 6. K. 0. Janda. C. G. Shevtin. R. A Lemer. Science 7. i E. Omman. G. F. Taylor, C. E. Petty. P. J. 259. 490 (1993). 8. For example: d/-pumiliotoxin. C. L E. Overman Jessup, 3. Org. Chem. 43. 2164 (1978). and P. J. Jessup. Terrahedfon Len. 1253 (197; J. Am. Chem. Soc. 100.5179 (1978); d-rc- gephyrotoxin. L E. Overman and C. Fukaya. ibd. 102 1454 (1980); dkisogabaculine, S. Danishef- sky and F. M. Hershenson. J. Ofg. Chem. 44. 1180 (1979);’lytidine. L E. Overman. C. B. Petty. R. J. Doedens. ibid.. p. 4183; arnaryirdaceae alkaloids. L E. Overman eta/., J. Am. CM. SOC. 103. 2816 (1981); gephirotoxine, L E. Overran, D. Lesuisse. M. Hashimoto, ibid. 105, 16. 5373 (1 983). 9. Diene 1 b was prepared by neutralizing the me- wing carboxylic acid suspended in PES (x+ dim phosphate buffer) with one equivalent of 10. All new adducts were characterized by NMR NaOH. 11. In the crude mixture of the cycloaddtion and mass spectrometry. could not detect any trace of the meta regioiso- .we ‘ mer by ‘H NMR (>98% orfhuregioselecjve). 12. M. J. Frisch el a/.. Gaussian 92 (Gaussian. Pms- 13. R. J. Loncharich. F. K. Brown, K. N. Houk J. Org. burgh. PA, 1932). Chem. 54. 1129 (1989). AI1 structJres were hlty optimized with the 321G basis set. Reactants were also fully optimized with the MlG‘ basis set. Wbrational frequencies for all trm sm- tures were calculated at the RHF/3-21G level and shorvn to have one imaginary trequency. In addk tion. the energies were evaluated by singlepoint caldalioas with the 631G‘ basis set m 3-21G 14. K N. Houk. R. J. Loncharich. J. Blake. W. Joc- geometries. 15. L E. Overman. G. F. Taylor, K. N. Ha I. N. gensen, J. Am. Chem: Sac. 111.9172 (1989). 16. M. I. Page and W. P. Jencks. Proc, NaU. Amd. Dometsmith, ibid. 100.31 82 (1 978). 17. F. Bemardi ef a!.. J. Am. Chern. Sot. 110. 3050 &i. USA 68. 1678 (1971). (1989); F. K. Brown and K. N. Houk. remedm Len. 25. 4609 (1984). 18. This strategy to avoid product inhibilion was first described by Braisled and SchulQ in (4). 19. The sterecchemishy was established on the basis of lH NMR analysis. For example: Suo. bH6 = 2.65 ppm and ‘Je.,, = 3.5 Hz; sendo. Sn, = 3.12 ppm. and &Je,,* = 0 Hz. 20. G. Kohler and C. Milstein. Nafure256.495 (1975): Ihe KW conjugate was prepared by hvly add- ing 2 mg of the hapten dissolved in 1133 pI of dimethyfiormamide Io 2 mg of KLH in 900 $1 of 0.01 M scdium phosphate buffer. pH 7.4. Fwr 8-week-old 129GIX+ mice each receked 2 h traperitoneal (IP) injaclims of 100 pg of the hap len conjugated lo KM and RIB1 actin* (Mh and TOM emulsion) 2 weoks apart. A .SO pg IP 208 injection of KLH ccniLyate in alum was gwen 2 weeks later. One menth aRer the second injeclion. the mouse with the hqhest liler was injected Intravenously with 50 pg of KLH-conjugate: 3 days later. the spleen \\as taken for the prepara- tion of hybridomas. Spleen cells (1.0 x lo8) were fused wlth 2.0 x 10- SPZO myeloma cells. Cells were plated into 30 Swell plates: each well conlamed I50 pl c1 likpoxanthine. aminopterin. lhymidine-Dulbecco’s niintmal essential medium (HAT-DMEM) contaming 1?6 nulridoma. and 2% fll;r,roace?ic acid)/acelonilrile. at 1.5 mlhin, 240 om. The !??ention time for Ihe frans isomer is 23. Vk Iharh J Ashley for lechnrcal assistance in 6 00 min and for the cis isomer is 6.60 min. iF.2 %mer% analysis: L. Ghosez. K. B. Sharpless, relpful dwssions and comments on the manu- K. C. FJiCclaou. €. Keinan. and 0. Boger for script: arcj 3. K. Chadha for the x-ray crystallo- graphic ;m. Supported in part by the National kance F>mdalion (CHE-9116377) and the A. P. Sloan %undation (K.D.J.). We also thank FLnCacih Ram6n-Areces (Spain) for a fellow- mp to 8.P.T 16 July 1%. accepted 3 September 1993 Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment Charles E. Lawrence, Stephen F. Akchul, Mark S. Boguski, Jun S. Liu, Andrew F. Neuwald, John C. Wootton A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier to deciphering these sequences and under- standing the relations among them is the difficulty of detecting subtle local residue patterns common to multiple sequences. Such patterns frequently reflet? similar molecular struc- tures and biological properties. A mathematical definition ofthis ’‘ ocal multiple alignment” problem suitable for full computer automation has been used 1 o develop a new and sensitive algorithm, based on the statistical method of itme sampling. This algorithm finds an optimized local alignment model for N sequences in Klinear time, requiring only seconds on current workstations, and allows the simultaneous detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix- turn-helix proteins, lipodins, and prenyltransferases. Patterns shared by multiple protein or nucleic acid sequences shed light on molec- ular structure, function, and evolution. The recognition of such patterns generally relies upon aligning many sequences, a complex, multifaceted research process whose difficulty has long been appreciated. This problem may be divided into “global multiple alignment” (I, 2), whose goal is to align complete sequences, and “local mul- tiple