bash, J. C. Hall, Neuron 1,141(1988); D. M. Zerr,J. C. Hall, M. Rosbash, K. K. Siwicki, J. Neurosci.10, 2749 (1990); I. Edery, L J. Zwiebel, M. E.Dembinska, M. Rosbash, Proc. Natl. Acad. Sci.U.S.A. 91, 2260 (1994).

7. S. T. Crews, J. B. Thomas, C. S. Goodman, Cell52, 143 (1988); E. C. Hoffman et al., Science252, 954 (1991); K. M. Burbach, A. Poland, C. A.Bradfield, Proc. Natl. Acad. Sci. U.S.A. 89, 8185(1992); H. Reyes, S. Reisz-Porszasz, O. Hankin-son, Science 256, 1193 (1992).

8. Z. J. Huang et al., Nature 364, 259 (1993).9. P. E. Hardin, J. C. Hall, M. Rosbash, ibid. 343, 536

(1990); Proc. Natl. Acad. Sci. U.S.A. 89, 11711(1992).

10. L. Cooley et al., Science 239, 1121 (1988).11. H. M. Robertson et al., Genetics 118, 461 (1988).12. A. C. Spradling and G. M. Rubin, Cell 34, 47

(1983).13. A. Sehgal, J. L. Price, B. Man, M. W. Young,

unpublished results.14. C. Helfrich and W. Engelmann, Z Naturforsch. 42C,

1335 (1987); M. J. Hamblen-Coyle et al., J. Insect.Behav. 5,417(1992); D. A. Wheeler, M. J. Hamblen-Coyle, M. S. Dushay, J. C. Hall, J. Biol. Rhythms 8,67 (1993).

15. M. Hamblen et al., J. Neurogenet. 3, 249 (1986);H. B. Dowse, J. C. Hall, J. M. Ringo, Behav.Genet. 17, 19 (1987).

16. Hybrid dysgenesis can induce P element excisionmutations that ultimately have few or no P elementsequences. See M. G. Kidwell, in Drosophila: APracticalApproach, D. B. Roberts, Ed. (IRL Press,Oxford, 1986), chap. 3; S. B. Daniels and A.Chovnick, Genetics 133, 623 (1993); and W. R.Engels, D. M. Johnson-Schlitz, W. B. Eggleston, J.Sved, Cell62, 515 (1990).

17. Wild-type (Canton S), per', and tim flies werecontemporaneously entrained as follows: 0- to5-day-old adult flies were transferred to freshbottles and maintained in LD 12:12 at 25°C for 3days. Beginning on the fourth day, collectionswere made at ZT2, 6, 10, 14, 18, and 22 (ZTO =lights on). Each collection consisted of shakingapproximately 1500 flies into a 15-ml conicalpolypropylene tube kept in dry ice. Total RNAwasextracted from the heads, which we separatedfrom the bodies by shaking frozen flies in sieves attemperatures below -20°C. A 32P-labeled ribo-probe that protects 316 nucleotides (nt) of permRNA (nt 123 to 438 of the per mRNA codingsequence) (3) was transcribed from cloned perDNA. A tubulin riboprobe also was produced thatprotects 86 nt (nt 57 to 142 from the start oftranscription) of al tubulin mRNA (19). Ribonu-clease (RNase) protection experiments were car-ried out as described (20) with minor variations.Ten micrograms of head RNA was used for eachtime point, and digestions with RNase were for 30min at 25°C. Hybridizations and RNase digestionswere performed contemporaneously for wild-type,per0, and tim RNA samples representing the first4 days. For days 5 and 6 of the tim record, RNAhybridizations and digestions involved newly la-beled riboprobes, so RNA from day 4 was againprocessed to allow direct comparison of per RNAamounts measured in the two sets of experiments.Digested samples were separated by electropho-resis on 5% polyacrylamide-8 M urea gels. Driedgels were exposed to x-ray film and also analyzedwith a phosphorimager (Molecular Dynamics) forquantitation.

18. L B. Vosshall, J. L Price, A. Sehgal, L. Saez, M.W. Young, Science 263, 1606 (1994).

19. W. E. Theurkauf, H. Baum, J. Bo, P. C. Wensink,Proc. Natl. Acad. Sci. U.S.A. 83, 8477 (1986).

20. K. Zinn, D. DiMaio, T. Maniatis, Cell 34, 865(1983).

21. A. Sehgal, J. Price, M. W. Young, Proc. Natl.Acad. Sci. U.S.A. 89, 1423 (1992).

22. We gratefully acknowledge the contributions ofmany single P element insertion lines obtainedfrom the laboratories of L. Y. Jan and Y. N. Janand C. Montell, J. Carlson, and A. Spradling, aswell as the contribution of a Drosophila tubulinprobe from V. Corbin. We would like to acknowl-

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edge L. Vosshall, L. Saez, C. Wesley, M. Myers,T. Lieber, and S. Kidd for fruitful discussions andcritical reading of the manuscript. Excellenttechnical assistance has been provided by M.Markowitz, H. Radziewicz, E. Alcamo, and T.Evering. M. Baylies graciously provided techni-

cal advice on RNase protections. Supported bythe Howard Hughes Medical Institute and theNational Science Foundation Science and Tech-nology Center for Biological Timing.12 November 1993; accepted 27 January 1994

Block in Nuclear Localization of period Proteinby a Second Clock Mutation, timeless

Leslie B. Vosshall,* Jeffrey L. Price, Amita Sehgal,tLino Saez, Michael W. Youngt

In wild-type Drosophila, the period protein (PER) is found in nuclei of the eyes and brain,and PER immunoreactivity oscillates with a circadian rhythm. The studies described hereindicate that the nuclear localization of PER is blocked by timeless (tim), a second chro-mosome mutation that, like per null mutations, abolishes circadian rhythms. PER fusionproteins without a conserved domain (PAS) and some flanking sequences are nuclear intim mutants. This suggests that a segment of PER inhibits nuclear localization in timmutants. The tim gene may have a role in establishing rhythms of PER abundance andnuclear localization in wild-type flies.

Mutations in the Drosophila period (per)gene disrupt circadian rhythms of pupaleclosion and adult locomotor behavior (1).Although per has been cloned and se-quenced and its pattern of expression hasbeen analyzed (2, 3), the biochemical func-tion of the PER protein is unknown. PERshares some homology with a family oftranscription factors (4-6) that possess acommon sequence motif called the PASdomain. The PAS domain consists of tworepeats of approximately 50 amino acidswithin a homology region of 258 to 308amino acids (7).

Immunocytochemical experiments dem-onstrated that PER is a nuclear protein in avariety of Drosophila tissues (8, 9). In cellsof the adult fly visual and nervous systems,the amount of PER protein fluctuates with acircadian rhythm (10), the protein is phos-phorylated with a circadian rhythm (10),and PER is observed in nuclei at night butnot late in the day (8). The expression ofper RNA is also cyclic. However, peakmRNA amounts are present late in the day,and the smallest amounts are present late atnight (11, 12). Three mutant alleles-per°,pers, and perL-cause arrhythmic behavioror shorten or lengthen periods, respectively(1). These mutations also produce corre-

Howard Hughes Medical Institute, National ScienceFoundation Science and Technology Center for Bio-logical Timing, and Laboratory of Genetics, The Rock-efeller University, New York, NY 10021, USA.*Present address: Center for Neurobiology and Be-havior and Howard Hughes Medical Institute, Colum-bia University College of Physicians and Surgeons,New York, NY 10032, USA.fPresent address: Department of Neuroscience, Uni-versity of Pennsylvania School of Medicine, Philadel-phia, PA 19104, USA.:To whom correspondence should be addressed.

SCIENCE * VOL. 263 · 18 MARCH 1994

sponding changes in the rhythms of perRNA and protein amounts (10-12) andPER immunoreactivity in nuclei (8). Thissuggests a possible role for molecular oscil-lations ofper in the establishment ofbehav-ioral rhythms (11). Recently, a new muta-tion, timeless (tim), was isolated that pro-duces arrhythmic behavior and suppressesthe circadian oscillation of per RNA (12).Here, we examine the effect of tim on theexpression and localization of PER protein.We compared PER protein expression in

wild-type, per0 mutant, and tim mutant fliesby staining head sections with PER anti-body. Because the amounts of PER proteinstaining in eye and brain nuclei fluctuatedaily (8), sections were prepared at fourtime points. Nuclear staining in wild-typephotoreceptor cells was most prominent atZeitgeber times 2 and 20 (ZT2 and 20)(13), intermediate at ZT7, and absent atZT13 (Fig. 1A). Because the per0 mutationintroduces a stop codon in the PER readingframe (2, 3), PER antibody specificity wasdemonstrated by the absence of staining insections from this null mutant at each timepoint (Fig. 1C). In tim mutants, nuclearstaining was not seen at any time point(Fig. 1B).

PER-P-galactosidase (PER-P3-gal) fusionproteins have been used extensively to studypatterns of per locus expression (9, 14, 15).Such fusion proteins have also allowed thefunctional dissection of the gene and theencoded protein (9, 14, 15). To explore theeffect of tim on PER protein expression andto map elements of the per locus respondingto tim, we compared patterns of expression ofa PER-P-gal fusion protein (PER-SG) (9) intransgenic flies with a wild-type or tim mu-tant genetic background. PER-SG contains

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the NH2-terminal half of PER (amino acids1 to 636) (2) fused to P-gal. The expressionof PER-SG in transgenic flies in vivo (9)closely matches that of the endogenous pro-tein (8). Whereas PER-SG localized to nu-clei in wild-type flies, we found that nuclearlocalization was blocked in tim mutants.Although no nuclear staining was observedin tim mutants, PER-SG accumulated in thesame tissues in both tim and wild-type flies(Fig. 2, A and D, and Fig. 3B). Specificity ofthe P-gal antibody used in these studies wasindicated by the complete absence of stain-ing in sections derived from wild-type or timflies lacking the transgene that encodes PER-SG (16).

To determine whether a single mutationcauses both aberrant intracellular localiza-tion of PER and the arrhythmic behavior oftim flies, we generated recombinants be-tween the second chromosome bearing thePER-SG construct and the tim mutantchromosome. Head sections prepared fromeach of 36 homozygous tim+ or tim recom-binant lines (17 tim+ and 19 tim) werestained with 3-gal antibody. The PER-SGfusion protein was nuclear in all 17 tim+,PER-SG recombinant lines. Nuclear local-ization was not observed in any of the 19 tim,PER-SG recombinants (16, 17). Therefore,mutant behavioral and localization pheno-types are likely to be the result of the samemutation. Representative sections fromthree independent tim+, PER-SG recombi-nants (with nuclear labeling in the photore-ceptor, cone, and pigment cells in the eyeand in the lamina, medulla, and centralbrain) are shown in Fig. 2, A through C.Representative sections from tim, PER-SGrecombinants show that nuclear localizationof the PER-SG protein was not observed inany of these cells (Fig. 2, D through F).We examined the intracellular localiza-

tion of the PER-SG fusion protein in tim/tim+ heterozygotes to learn whether theaberrant localization phenotype is a domi-nant or a recessive trait. PER-SG fusionproteins localize to nuclei in the heterozy-gotes (17). Therefore, like the behavioraldefect (12), the nuclear localization defectof tim is recessive. The nuclear localizationof PER-SG in tim' flies also is not depen-dent on the presence of endogenous PERprotein because its localization was nuclearin a per0 genetic background (17). Thus,neither functional PER protein nor behav-ioral rhythmicity is required to generatecorrect nuclear localization of the PER-SGfusion protein in tim+ flies. As well, the timmutant localization phenotype was inde-pendent of endogenous PER, because PER-SG fusion protein did not localize to nucleiin per0; tim double mutants (17).

To search for possible differences in therate of synthesis, stability, or structure ofPER-SG proteins isolated from wild-type

and tim mutant flies, we prepared proteinimmunoblots from tim+, PER-SG and tim,PER-SG head extracts. PER-3-gal fusionproteins of the same apparent size andabundance were detected in the two geneticbackgrounds (Fig. 2G). Thus, effects onnuclear localization observed immunocy-tochemically in tim flies are not the result ofreduced expression or increased proteolysisof the fusion protein.

To explore the specificity of the block innuclear localization in tim mutants, weexamined the expression in tim mutants oftwo P3-gal marker proteins (containing noPER sequences) that had been modified bythe addition of heterologous nuclear local-ization signals and are expressed in thenuclei of the eye and brain. The addition ofsuch nuclear localization signals is requiredfor nuclear localization of P-gal in wild-typeflies (16-18). Nuclear localization of thesemarker proteins was unaffected by tim (Fig.3, D and E). The tim mutation affectedneither the morphology nor the number ofnuclei visualized by these markers.We also examined the localization of

four additional PER-P-gal fusion proteinsin tim mutants and wild-type flies. Fusionproteins contained the first 23 amino acidsof PER (PER1-231p-gal), PER amino acids1 to 95 and 530 to 636 (PER-SGA96-529),the first 95 amnino acids ofPER (PER1-951-gal), or th~ NH2-terminal half of PERwithout a putative nuclear localization sig-nal (PER1-636ANLS[66-79]p-gal) (2).

The pattern of PER1-23p-gal expressionin wild-type and tim genetic backgroundswas identical to that of PER-SG in timmutants (16, 17). Because no sequencesresembling a nuclear localization signal arefound in this segment of the PER protein,cytoplasmic accumulation of this fusion

ZT2 Z17

protein was predicted for both genetic back-grounds (16-18). PER-SGA96-529 was nu-clear in wild-type and tim flies (17). Thelocalization of PER1-95p-gal also was nu-clear in wild-type (Fig. 3A, left) and timbackgrounds (Fig. 3A, right), which indi-cates that sequences in this NH2-terminalregion ofPER would promote nuclear local-ization of a full-length PER protein but areunable to do so in tim mutant flies unlessanother, more centrally located region ofPER is deleted. PER1-636ANLS[66-79]lS-gal localized to nuclei in wild-type tissue(Fig. 3C, left), which indicates that PERcontains additional nuclear localization sig-nals. PER1-636ANLS[66-79]pf-gal failed toenter nuclei in a tim background (Fig. 3C,right). Together, these results suggest thatwild-type tim activity is required for nuclearlocalization of proteins that contain a spe-cific region of PER. Because both PER1-95p-gal and PER-SGA96-529 are nuclearin tim flies, the tim-sensitive region of PERmust be located between amino acids 96and 529. This interval includes the site ofthe per1- mutation and the PAS domain (2,3), but would not include the per' domain,a region in which mutations predominantlyconfer short-period phenotypes (19).We have drawn four principal conclu-

sions from this work. (i) The tim gene isrequired for nuclear localization of PER. (ii)Aberrant behavioral and localization phe-notypes appear to be a result of the samemutation. (iii) Regulation of nuclear local-ization by tim may be specific for PER. (iv)PER contains sequences that somehow in-hibit PER nuclear localization in the ab-sence of tim. A protein encoded by timmight interact with PER to facilitate nucle-ar entry, or tim may regulate factors respon-sible for cytoplasmic retention of PER pro-

ZT13 ZT20

Fig. 1. Expression of PER protein in (A) wild-type, (B) tim, and (C) per0 mutant backgrounds at fourtime points. The nuclear layer is marked with open arrows. Structures underlying the lens frequentlystain nonspecifically and do not reflect PER antibody staining in nuclei (black arrowheads). Flieswere raised at 25°C in a 12-hour light:12-hour dark cycle (LD 12:12). Frozen sections (14 p.m) fromadult fly heads collected at the indicated times were incubated with PER antibody (1:30 dilution ofimmune serum with PER antibody 19-06 raised against full-length PER protein and preabsorbedagainst per0 head lysate) for 16 hours at room temperature. Antibody staining was continued asdescribed (8) with the use of the Vectastain ABC kit (Vector Labs).SCIENCE * VOL. 263 * 18 MARCH 1994

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tein. The arrhythmic behavior and lack ofper RNA rhythms observed in tim mutants(12) are likely to be a direct consequenceof the failure of PER to enter the nucleus.Thus, PER must be present in the nucleusat some time of day for expression ofcircadian rhythms. Our detection of largeamounts of cytoplasmic PER-3-gal fusionprotein, but not full-length cytoplasmicPER protein, in tim mutants may alsoindicate that PER is unstable in the cyto-plasm and stabilized by transport to thenucleus. Thus, the observed cycling ofPER protein abundance (10) might reflect

cycling of PER nuclear localization.The region implicated in tim control of

nuclear localization of PER includes thePAS domain, nonconserved sequences up-stream of PAS (amino acids 96 to 232)(20), and 33 amino acids downstream ofPAS (amino acids 497 to 529) (4, 14).Regulation of nuclear localization has beenobserved for two other proteins containingthe PAS domain. The dioxin receptor is aligand-activated transcription factor (21)that accumulates in the cytoplasm untildioxin ligand interacts with the PAS domainof the AHR subunit (6). This induces the

formation of a complex with the ARNTsubunit and promotes nuclear localization ofthe receptor complex (22). In the absence ofeither ligand or ARNT, the receptor fails toenter nuclei and has no transcriptional ac-tivity (5, 23). No ligands or binding partners

WHd type #rmeles

Fig. 2. Immunocytochemistry and protein im- Gmunoblot analysis of a PER-3-gal fusion pro- " c

tein (PER-SG) (9) in tim+ and tim recombinantlines. Shown is expression of PER-SG in head - ^ . .X x xsections from tim+ (A through C) and tim mu-tant (D throughFl recombinant lines. For (A)through (C), staining of nuclei was observed inphotoreceptor cells (ret), lamina (lam), medulla 200-(med), and some cells of the central brain.Open arrows indicate nuclear labeling. In (D)through (F), staining of photoreceptor cells andcells of the lamina, medulla, and central brainwas also observed, but there is no indication of 07.4-nuclear localization of the stain. Frozen sec-tions were incubated with affinity-purified E3-galpolyclonal antibody (Cappel/Organon Tech-nika, Durham, North Carolina) (1:1000 dilutionpreabsorbed against adult fly acetone powder)and processed as described (Fig. 1). (G) Linesof tim +and tim mutant flies containing PER-SGor a control line (Canton-S) lacking the trans-gene, were raised as described (Fig. 1). Fies (6 to 12 days old) were collected from ZT18 to ZT22.Extracts were prepared from the heads of flies as described (15). Extract containing 1 x (10 I.g byBradford assay), or the indicated fraction of total protein, was electrophoresed in each lane of anSDS-polyacrylamide gel (24) and analyzed for 13-gal antigen levels as described by Saez andYoung (8). The primary antibody was a mouse 13-gal monoclonal antibody, and the secondaryantibody was goat antibody to mouse immunoglobulin G (heavy and light chains) alkalinephosphatase conjugate (both from Promega). Extracts from tim, PER-SG flies were loaded in thefirst four lanes; tim+, PER-SG extract in the fifth lane; and extract from a line that does not containthe PER-SG transgene (Canton-S) in the sixth lane. Molecular size markers (Amersham) areindicated to the left of the gel.

Fig. 3. Localization of PER-13-gal and 13-galmarker proteins in wild-type (left column) andtim mutant (right column) adult head sections.(A) PER1-9513-gal. (B) PER1-63613-gal (identi-cal in amino acid composition to PER-SG, butconstructed as described below). (C) PER1-636ANLS[66-79113-gal. (D) Enhancer trap lineS45 (expresses -gal modified by the additionof a nuclear localization signal in the eye and afew cells in the brain). (E) Eye protein kinaseC-P-gal (1-gal modified by the addition of theSV40 nuclear localization signal under the con-trol of the eye-specific protein kinase C promot-er) (18). Staining with antibodies to 13-gal wasas described in legend to Fig. 2. PER-p-galfusion constructs were made by inserting anXho I linker at nucleotide 3183 (PER1-9513-gal;Ace I site) or at nucleotide 4936 (PER1-63613-gal and PER1-636ANLS[66-79]13-gal; Sac Isite) (2). PER1-636ANLS[66-79]p-gal is iden-tical to PER1-63613-gal except that a fragmentencoding amino acids 65 to 95 has been de-leted in the former (2, 16). The Sph I-Xba I perpromoter fragment was added and the perfragment excised with Xho I. All constructswere cloned into pCaSpeR-13-gal (25), alteredto create three different frames with Xho I link-ers (16).

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analogous to ARNT have been identified forPER. However, it has recently been shownthat the PAS domain of the PER protein canfunction as a protein interaction domain(14). Further mapping of the tim-sensitiveregion of PER should determine whetherPAS is centrally involved in the control ofPER nuclear localization.

Peak amounts of per RNA expression(11, 12) precede the greatest amount ofPER protein staining in nuclei of the pho-toreceptors and brain (8) by about 8 hours.Thus, nuclear immunoreactivity of PERoccurs when the amount of per RNA issmall. As suggested above, the entry of PERprotein into the nucleus may be undertemporal control, so that some of the pre-viously observed oscillation in PER proteinamounts and nuclear staining (8, 10) mayreflect rhythmic movement between thecytoplasm and the nucleus, with a phasethat is distinct from that of per RNA syn-thesis. In light of these observations andour work with tim, we speculate that forma-tion of an intracellular circadian clock mayrequire nuclear localization of PER to belimited to a particular time of day.

In such a model, constitutively cytoplas-mic (as in tim mutants) or constitutivelynuclear PER would fail to generate circa-dian rhythms, and tim' activity might playa role in temporal regulation of the access ofPER to the nucleus. It was proposed thatPER may directly or indirectly regulate itsown transcription, because the cycling ofper transcription is blocked in per0 mutants(11). Because the tim mutation also abol-ishes these RNA rhythms (12), a form offeedback regulation may exist in whichcycling nuclear localization of PER mightproduce rhythmic signals influencing pertranscription. We wish to emphasize thattim was recovered in a screen for clockmutations that was not biased to affordrecovery of new mutations interacting withper (12). Thus, the discovery of a function-al interaction between tim and per indicatesthat a single intracellular mechanism isprobably central to the generation of circa-dian rhythms in Drosophila. The effects oftim on per RNA oscillation and PER nucle-ar localization would presumably be compo-nents of this mechanism.

REFERENCES AND NOTES

1. R. J. Konopka and S. Benzer, Proc. Natl. Acad.Sci. U.S.A. 68, 2112 (1971).

2. M. K. Baylies, L. Weiner, L. B. Vosshall, L. Saez,M. W. Young, in Molecular Genetics of BiologicalRhythms, M. W. Young, Ed. (Dekker, New York,1993), pp. 123-153.

3. M. Rosbash and J. C. Hall, Neuron 3, 387 (1989).4. S. T. Crews, J. B. Thomas, C. S. Goodman, Cell

52, 143 (1988); J. R. Nambu, J. O. Lewis, K. A.Wharton Jr., S. T. Crews, ibid. 67, 1157 (1991).

5. H. Reyes, S. Reisz-Porszasz, O. Hankinson, Sci-ence 256, 1193 (1992); E. C. Hoffman et al., ibid.252, 954 (1991).

6. K. M. Burbach, A. Poland, C. A. Bradfield, Proc.Natl. Acad. Sci. U.S.A. 89, 8185 (1992).

7. Sequence similarity between PER and a proteinregulating aspects of Drosophila development,SIM (encoded by the gene single-minded), wasfirst recognized by Crews et al. (4). Subsequentstudies have shown that this homology region isalso found in ARNT (arylhydrocarbon receptornuclear translocator), a component of the multi-subunit mammalian dioxin receptor (5), and inAHR, the ligand-binding subunit of the dioxinreceptor (6). The region of homology is nowreferred to as the PAS domain, after the firstthree proteins in which it was identified: PER,ARNT, and SIM (14).

8. K. K. Siwicki, C. Eastman, G. Petersen, M. Rosbash,J. C. Hall, Neuron 1,141 (1988); L. Saez and M. W.Young, Mol. Cell. Biol. 8, 5378 (1988); D. M. Zerr, J.C. Hall, M. Rosbash, K. K. Siwicki, J. Neurosci. 10,2749 (1990).

9. X. Liu, L. Lorenz, Q. Yu, J. C. Hall, M. Rosbash,Genes Dev. 2,228 (1988); X. Liu etal., Neuron 6,753 (1991); X. Liu et al., J. Neurosci. 12, 2735(1992); J. Ewer, B. Frisch, M. J. Hamblen-Coyle,M. Rosbash, J. C. Hall, ibid., p. 3321.

10. I. Edery, L. J. Zwiebel, M. E. Dembinska, M.Rosbash, Proc. Natl. Acad. Sci. U.S.A. 91, 2260(1994).

11. P. E. Hardin, J. C. Hall, M. Rosbash, Nature 343,536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89,11711 (1992).

12. A. Sehgal, J. L. Price, B. Man, M. W. Young,Science 263, 1603 (1994).

13. Zeitgeber time (ZT) is an arbitrary standard formeasuring time; ZTO is defined as lights onor dawn, and ZT12 is defined as lights off or dusk.

14. Z. J. Huang, I. Edery, M. Rosbash, Nature 364,259 (1993).

15. L J. Zwiebel, P. E. Hardin, X. Liu, J. C. Hall, M.

Rosbash, Proc. Natl. Acad. Sci. U.S.A. 88, 3882(1991).

16. L. B. Vosshall, thesis, Rockefeller University(1993).

17. L. B. Vosshall, J. Price, M. W. Young, unpublishedobservations.

18. D. P. Smith etal., Science 254, 1478 (1991).19. M. K. Baylies, L. B. Vosshall, A. Sehgal, M. W.

Young, Neuron 9, 575 (1992).20. H. V. Colot, J. C. Hall, M. Rosbash, EMBO J. 7,

3929 (1988).21. M. S. Denison, J. M. Fisher, J. P. Whitlock Jr.,

Proc. Natl. Acad. Sci. U.S.A. 85, 2528 (1988); J.Hapgood, S. Cuthill, M. Denis, L. Poellinger, J. A.Gustafsson, ibid. 86, 60 (1989).

22. A. Fujisawa-Sehara, M. Yamane, Y. Fujii-Kuriyama, ibid. 85, 5859 (1988); A. B. Okey et al.,J. Biol. Chem. 255, 11415 (1980).

23. S. O. Karenlampi, C. Legraverend, J. M. Gudas,N. Carramanzana, O. Hankinson, J. Biol. Chem.263, 10111 (1988).

24. U. K. Laemmli, Nature 227, 680 (1970).25. C. S. Thummel, A. M. Boulet, H. D. Lipshitz, Gene

74, 445 (1988).26. Fly stocks were kindly provided by the following

investigators: J. Treisman, Z. Lai, and G. M. Rubin(S45 enhancer trap line); D. P. Smith and C. S.Zuker (eye protein kinase C-NLS-p-gal trans-formant lines 5201 and 5202); and M. Rosbash(PER-3-gal transformant line PER-SG). We thank D.Sulzer, T. Lieber, M. Myers, M. E. Hatten, and R. Axelfor critically reading the manuscript. Supported bythe Howard Hughes Medical Institute and the Na-tional Science Foundation Science and TechnologyCenter for Biological Timing. The work of L.B.V. waspartially funded by National Research ServiceAwards training grant GM07982-09.

12 November 1993; accepted 27 January 1994

Activation of Phosphatidylinositol-3' Kinase bySrc-Family Kinase SH3 Binding to the p85 Subunit

Christopher M. Pleimian, W. Marc Hertz, John C. Cambier*Engagement of antigen receptor complexes induces rapid activation of Src-family kinasesand association with phosphatidylinositol-3' kinase (PI-3 kinase). Here it was found thatthe Src homology 3 (SH3) domain of Lyn and Fyn bound to a proline-rich region (residues84 to 99) within the 85-kilodalton subunit (p85) of PI-3 kinase. The binding of SH3 to thepurified kinase led to a five- to sevenfold increase in the specific activity of PI-3 kinase.Ligand-induced receptor stimulation activated PI-3 kinase, and this activation was blockedby a peptide containing residues 84 to 99 of p85. These data demonstrate a mechanismfor PI-3 kinase activation and show that binding of SH3 domains to proline-rich targetsequences can regulate enzymatic activity.

The response of lymphocytes to antigen ismediated through a multisubunit cell sur-face receptor complex. Antigen receptorengagement can initiate multiple intracel-lular signaling events that lead to activa-tion, differentiation, and tolerance induc-tion, depending on the nature of the stim-ulus and differentiative stage of the cell (1).The binding of ligand to B cell antigenDivision of Basic Sciences, Department of Pediatrics,National Jewish Center for Immunology and Respira-tory Medicine, 1400 Jackson Street, Denver, CO80206, USA, and Department of Microbiology andImmunology, University of Colorado Health ScienceCenter, Denver, CO 80206, USA.*To whom correspondence should be addressed.

SCIENCE · VOL. 263 * 18 MARCH 1994

receptors causes activation of the receptor-associated Src-family tyrosine kinases p55blk(Blk), p59hnf (Fyn), and p53/56'y" (Lyn)(2). Lyn associates with the noncatalytic85-kD subunit of PI-3 kinase in B cells afterantigen receptor ligation (3).

The PI-3 kinase is a heterodimeric pro-tein composed of a noncatalytic p85 sub-unit (4), catalytic 110-kD subunits (p1 10)(5), and phosphorylated inositol lipids onthe D-3 hydroxyl position (6). The exactroles of the phosphoinositide products gen-erated by PI-3 kinase in signaling pathwayshave not been determined, but they accu-mulate in cells activated by growth factors(7). PI-3 kinase interacts with SV40 middle

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Block in nuclear localization of period protein by a second clock mutation, timelessLB Vosshall, JL Price, A Sehgal, L Saez and MW Young

DOI: 10.1126/science.8128247 (5153), 1606-1609.263Science 

ARTICLE TOOLS http://science.sciencemag.org/content/263/5153/1606

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