Proc. Nadl. Acad. Sci. U S A Vol. 87 , p p . 2795-2799, April 1990 Neurobiology Rescue o f t h e learning defect i n dunce, a Drosophila learning mutant, b y a n allele o f rutabaga, a second learning mutant (memory/cyclic AMP/adenylate cydase) M E L B . FEANY Department o f Neurobiology, Harvard Medical School, Boston, MA 02115 Communicated by David H . Hubel, January 2, 1990 ABSTRACT rutabaga' (rut'), a Drosophila learning mu- tant, h a s adenylate cyclase (E C 4.6.1.1) with reduced basal activity a n d t he absence o f calcium/calmodulin-stimulated activity. A second learning mutant, dunce, i s defective i n cyclic A M P degradation d u e t o decreased o r absent phosphodi- esterase activity. These opposing biochemical defects allow rut, t o partially suppress t h e female sterility caused b y elevated cyclic AMP levels i n dunce flies. Selection of mutations that suppress dunce sterility h a s le d t o t h e isolation o f t w o rutabaga alleles. T h e alleles (rut2 a n d rut3) decrease basal adenylate cyclase activity [Bellen, H . J., Gregory, B. K . , Olsson, C. L. & Kiger, J . A . (1987) Dev. Biol. 121, 432-444] but, unlike t h e original rutabaga mutation, leave t he calcium/calmodulin- stimulated activity intact. Behaviorally, t h e t w o alleles also differ from rut'. O n e o f t he mutations partially rescues t he dunce learning defect, a n d flies bearing both alleles learn. Calcium responsiveness m a y thus b e thecrucial component o f adenylate cyclase activity required f o r associative learning. Neurophysiology h a s long provided the basis f o r exploring the molecular mechanisms underlying complex behavior. Genetics complements this analysis with t h e introduction o f single-gene mutations that provide a specific noninvasive method to inducebiochemicalchanges.Drosophila, with i t s rich history o f classical genetics a n d techniques available t o introduce a n d express altered genes, i s a n ideal organism i n which t o apply t he genetic approach t o behavioral biochem- istry. Happily, physiology a n d genetics have s o f ar implicated t h e same biochemical pathways a s underlying learning a n d memory. Investigation o f learning a n d memory i n t h e s e a snail Aplysia h a s resulted i n a possible model o f simple learning. Kandel e t a l. ( 1) propose that sensitization, a simple nonas- sociative form o f behavioral plasticity, relies o n presynaptic facilitation. A sensitizing stimulus causes a neurotransmitter, possibly serotonin, t o b e released onto t he sensory nerve terminal, activating i n turn adenylate cyclase a n d cyclic AMP-dependent protein kinase. T h e kinase then phosphor- ylates a n d closes K + channels, depolarizing t h e cell. Subse- quent stimulation thus induces enhanced neurotransmitter release from t h e sensory neuron, producing behavioral sen- sitization. Kandel e t a l . ( 1 ) have expanded their model t o include associative learning. A second stimulus [conditioned stimu- l u s (CS)] presented immediately before t h e sensitizing stim- ulus [unconditioned stimulus (US)] produces a greater r e - sponse than either t h e C S or U S alone. T h e additional biochemical change i n t h e sensory neuron seems t o b e a consequence o f neuronal activity, probably Ca2+ entry, immediately prior t o neurotransmitter release onto t he pre- synaptic terminal. Adenylate cyclase c a n produce cyclic A M P i n response to both Ca2l a n d monoamines a n d i s, therefore, a likely substrate for the synergism between C S a n d U S . Several Drosophila mutations that affect learning a n d memory disturbthe monoamine-activated adenylate cyclase cascade. Mutants a t the Dopa decarboxylase (Ddc) locus cannot synthesize dopamine or serotonin ( 2 ) a n they d o n o t learn (ref. 3 , b u t s e e ref. 4 ) . dunce (dnc) flies have elevated cyclic AM P levels d u e t o a defective structural gene fo r the cyclic AMP-dependent phosphodiesterase (5-8). dunce flies c a n learn b u t learn less well than wild-type controls a n d forget much more rapidly ( 9 , 10). Since dunce a n d other "learning" mutants as well do learn in some olfactory a s well as visual assays (11), they might b e more accurately charac- terized a s partial memory mutants. I retainthe term "learn" here a s a convenient shorthand. O n e Drosophila mutation affects t h e keyenzyme in cyclic A M P regulation: rutabaga1 (rut') flies lack the calcium/ calmodulin-sensitive component o f adenylatecyclase (12, 1 3 ) a n d show learning a n d memory defects comparable to those o f dunce (10, 13). Since t h e dunce and rutabaga mutations influence cyclic A M P metabolism i n opposite directions, t h e possibility arises that learning requires a certain range o f cyclic A M P levels. O n t h e other hand, t h e Aplysia model o f associative learning requires a specific calcium-induced c y - clic AMP stimulation. In rut' flies the absent calcium r e - sponse, rather than theoverall cyclic A M P decrease, m a y b e t h e critical defect. Double-mutant dunce, rut1 flies have cyclic A M P levels nearer t o normal than either strain alone, b u t these flies still d o n ot learn, castingfurther doubt on t h e importance o f cyclic AMP levels alone (13). Although double- mutant flies d o n o t learn, rutabaga does partially compensate f o r o n e dunce phenotype-female sterility. Th e isolation o f suppressors of dunce sterility h a s produced mutations with various amounts of cyclase activity (14). I have used these alleles to assay t h e relative contribution of basal versus calcium-stimulated adenylate cyclase activity t o learning. MATERIALS AND METHODS Fly Stocks. Wild-type flies were th e Canton-Special (Can- ton-S) wild-type strain. rut1 w a s originally isolated as a learning mutant, induced b y ethyl methanesulfonate (EMS) mutagenesis i n a Canton-S stock. rut2 a n d rut' were induced b y E M S mutagenesis a nd selected as suppressors of dunce sterility i n a y dncml4 e c f stock (14); these mutants were kindly provided b y J. A . Kiger, J r . (University o f California, Davis). T h e markerf wa s lost i n t h e course o f t h e mutagen- esis. y cv rut2 a n d y c v rut3 stocks were prepared b y recombination with y c v v f car. Presence o f rut2 a n d rut3 wa s confirmed b y suppression o f dunce sterility. T h e double- mutant y dncml4 e c rut1 chromosome w a s constructed b y recombination of t h e parental dunce chromosomes with rut1. Abbreviations: S o r US , conditioned o r unconditioned stimulus, respectively; EMS, ethyl methanesulfonate. 2795 Th e publication costs o f thisarticle were defrayed i n part b y page charge payment. This article must therefore b e hereby marked "advertisement" in accordance with 1 8 U.S.C. §1734 solelytoindicate thisfact.
P r oc . N a d l .A c a d .S c i .USAVo l .8 7 ,p p .2 7 9 5 - 2 7 9 9 ,A p r i l1 9 9 0N e u r o b i o l o g y
R e s c u eo f t h e l e a r n i n gd e f e c t i n d u n c e ,a D r o s o p h i l al e a r n i n gm u t a n t ,b y a n a l l e l eo f r u t a b a g a ,a s e c o n dl e a r n i n gm u t a n t
( m e m o r y / c y c l i cAMP / a d e n y l a t ec y d a s e )
MELB . FEANYD e p a r t m e n to f N e u r o b i o l o g y ,H a r v a r dM e d i c a lS c ho ol , B o s t o n ,MA 0 2 1 1 5
C o m m u n i c a t e db yD a v i dH . H u b e l ,J a n u a r y2 ,1 9 9 0
ABSTRACT r u t a b a g a '( r u t ' ) ,a D r o s o p h i l al e a r n i n gmu-t a n t ,h a s a d e n y l a t e c yc l a s e( E C4 . 6 . 1 . 1 )w i t h r e d u c e db a s a la c t i v i t ya n d t h e a b s e n c eo f c a l c i u m / c a l m o d u l i n - s t i m u l a t e da c t i v i t y .A s e c o n dl e a r n i n gm u t a n t ,d u n c e ,i sd e f e c t i v ei nc y c l i cAMP d e g r a d a t i o nd u e t o d e c r e a s e do r a b s e n tp h o s p h o d i -e s t e r a s ea c t i v i t y .T h e s eo p p o s i n gb i o c h e m i c a ld e f e c t sa l l o wr u t ,t o p a r t i a l l ys u p p r e s s t h ef e m a l es t e r i l i t yc a u s e d b y e l e v a t e dc y c l i cAMP l e v e l si n d u n c ef l i e s .S e l e c t i o no fmu t a t i o n st h a ts u p p r e s sd u n c es t e r i l i t yh a s l e dt ot h ei s o l a t i o no ft w or u t a b a g aa l l e l e s .T h e a l l e l e s( r u t 2a n d r u t 3 )d e c r e a s eb a s a la d e n y l a t ec y c l a s ea c t i v i t y[ B e l l e n ,H . J . ,G r e g o r y ,B . K . ,O l s s o n ,C . L .& K i g e r ,J . A . ( 1 9 8 7 )D e v .B i o l .1 2 1 ,4 3 2 - 4 4 4 ]b u t ,u n l i k et h eo r i g i n a lr u t a b a g am u t a t i o n ,l e a v et h ec a l c i u m / c a l m o d u l i n -s t i m u l a t e da c t i v i t yi n t a c t .B e h a v i o r a l l y ,t h et w o a l l e l e sa l s od i f f e rf r o mr u t ' .O n eo f t h emu t a t i o n sp a r t i a l l yr e s c u e s t h ed u n c el e a r n i n gd e f e c t ,a n d f l i e sb e a r i n gb o t h a l l e l e sl e a r n .C a l c i u mr e s p o n s i v e n e s smay t h u sb e t h e c r u c i a lc o m p o n e n to fa d e n y l a t ec y c l a s ea c t i v i t yr e q u i r e df o ra s s o c i a t i v el e a r n i n g .
N e u r o p h y s i o l o g yh a s l o n gp r o v i d e dt h e b a s i sf o re x p l o r i n gt h emo l e c u l a rm e c h a n i s m su n d e r l y i n gc o m p l e xb e h a v i o r .G e n e t i c sc o m p l e m e n t st h i sa n a l y s i sw i t ht h ei n t r o d u c t i o no fs i n g l e - g e n em u t a t i o n st h a tp r o v i d ea s p e c i f i cn o n i n v a s i v em e t h o d
t o i n d u c e b i o c h e m i c a l c h a n g e s . D r o s o p h i l a ,w i t hi t s
r i c hh i s t o r yo fc l a s s i c a lg e n e t i c sa n d t e c h n i q u e sa v a i l a b l et oi n t r o d u c ea n d e x p r e s sa l t e r e dg e n e s ,i s a n i d e a lo r g a n i s mi nw h i c ht oa p p l yt h eg e n e t i ca p p r o a c ht ob e h a v i o r a lb i o c h e m -i s t r y .H a p p i l y ,p h y s i o l o g ya n dg e n e t i c sh a v es o f a ri m p l i c a t e dt h es a m eb i o c h e m i c a lp a t h w a y sa s u n d e r l y i n gl e a r n i n ga n dm e m o r y .I n v e s t i g a t i o no f l e a r n i n ga n d m e m o r yi n t h e s e a s n a i l
A p l y s i ah a s r e s u l t e di na p o s s i b l em o d e lo fs i m p l el e a r n i n g .K a n d e le t a l .( 1 )p r o p o s et h a ts e n s i t i z a t i o n ,a s i m p l en o n a s -s o c i a t i v ef o r mo f b e h a v i o r a lp l a s t i c i t y ,r e l i e so n p r e s y n a p t i cf a c i l i t a t i o n .A s e n s i t i z i n gs t i m u l u sc a us es an e u r o t r a n s m i t t e r ,p o s s i b l ys e r o t o n i n ,t o b e r e l e a s e do n t o t h es e n s o r y n e r v et e r m i n a l ,a c t i v a t i n gi n t u r n a d e n y l a t ec y c l a s ea n d c y c l i cAM P - d e p e n d e n tp r o t e i nk i n a s e .T h ek i n a s et h e n p h o s p h o r -
y l a t e sa n dc l o s e sK+ c h a n n e l s ,d e p o l a r i z i n gt h ec e l l .S u b s e -q u e n t s t i m u l a t i o n t h u si n d u c e se n h a n c e dn e u r o t r a n s m i t t e rr e l e a s ef r o mt h es e n s o r yn e u r o n ,p r o d u c i n gb e h a v i o r a ls e n -s i t i z a t i o n .K a n d e le t a l . ( 1 )h a v e e x p a n d e dt h e i rm o d e lt o i n c l u d e
a s s o c i a t i v el e a r n i n g .A s e c o n ds t i m u l u s[ c o n d i t i o n e ds t i m u -l u s( C S ) ]p r e s e n t e di m m e d i a t e l yb e f o r et h es e n s i t i z i ng s t i m -u l u s[ u n c o n d i t i o n e ds t i m u l u s( U S ) ]p r o d u c e sa g r e a t e rr e -s p o n s e t h a n e i t h e rt h eCS o r US a l o n e .T h e a d d i t i o n a lb i o c h em i c a lc h a n g ei n t h es e n s o r y n e u r o n s e e m s t o b e ac o n s e q u e n c eo f n e u r o n a l a c t i v i t y ,p r o b a b l yC a 2 +e n t r y ,i mm e d i a t e l yp r i o rt o n e u r o t r a n s m i t t e rr e l e a s eo n t o t h ep r e -s y n a p t i ct e r m i n a l .A d e n y l a t ec y c l a s ec a n p r o d u c ec y c l i c
AMP i n r e s p o n s e t o b o t h C a 2 la n d m o n o a m i n e sa n d i s ,t h e r e f o r e ,a l i k e l ys u b s t r a t ef o r t h es y n e r g i s mb e t w e e nCSa n dU S .S e v e r a lD r o s o p h i l am u t a t i o n st h a ta f f e c tl e a r n i n ga n d
m e m o r yd i s t u r b t h emo n o a m i n e - a c t i v a t e da d e n y l a t ec y c l a s ec a s c a d e .M u t a n t sa t t h eD o p ad e c a r b o x y l a s e( D d c ) l o c u sc a n n o ts y n t h e s i z ed o p a m i n eo r s e r o t o n i n( 2 )a n dt h e yd o n o tl e a r n( r e f .3 ,b u ts e er e f .4 ) .d u n c e( d n c )f l i e sh a v ee l e v a t e dc y c l i cAMPl e v e l sd u e t o a d e f e c t i v es t r u c t u r a lg e n ef o rt h ec y c l i cAM P - d e p e n d e n tp h o s p h o d i e s t e r a s e( 5 - 8 ) .d u n c ef l i e sc a n l e a r nb u t l e a r nl e s sw e l lt h a n w i l d - t y p ec o n t r o l sa n df o r g e tm u c hm o r er a p i d l y( 9 , 1 0 ) .S i n c ed u n c ea n d o t h e r" l e a r n i n g "m u t a n t sa sw e l ld o l e a r ni ns o m eo l f a c t o r ya sw e l la sv i s u a la s s a y s ( 11 ) ,t h e ym i g h tb e m o r ea c c u r a t e l yc h a r a c -t e r i z e da sp a r t i a lm e m o r ymu t a n t s .I r e t a i n t h et e r m" l e a r n "h e r ea s a c o n v e n i e n ts h o r t h a n d .O n eD r o s o p h i l am u t a t i o na f f e c t st h ek ey e n z ym ei nc y c l i c
AMP r e g u l a t i o n :r u t a b a g a 1( r u t ' )f l i e sl a c kt h ec a l c i u m /c a l m o d u l i n - s e n s i t i v ec o m p o n e n to fa d e n y l a t e c y c l a s e( 1 2 ,1 3 )a n d s h o wl e a r n i n ga n dmemory d e f e c t sc o m p a r a b l et o t h o s eo fd u n c e( 1 0 ,1 3 ) .S i n c et h ed un c e a n dr u t a b a g amu t a t i o n si n f l u e n c ec y c l i cAMPm e t a b o l i s mi no p p o s i t ed i r e c t i o n s ,t h ep o s s i b i l i t ya r i s e st h a tl e a r n i n gr e q u i r e sa c e r t a i nr a n g e o fc y c l i cAMPl e v e l s .On t h eo t h e rh a n d ,t h eA p l y s i am o d e lo f
a s s o c i a t i v el e a r n i n g r e q u i r e sa s p e c i f i cc a l c i u m - i n d u c e dc y -c l i cAMP s t i mu l a t i o n . I nr u t 'f l i e st h ea b s e n t c a l c i u mr e -s p o n s e ,r a t h e rt h a nt h e o v e r a l lc y c l i cAMPd e c r e a s e ,may b et h ec r i t i c a ld e f e c t .D o u b l e - m u t a n td u n c e , r u t 1f l i e sh a v ec y c l i cAMPl e v e l sn e a r e r t o n o r m a lt h a ne i t h e rs t r a i na l o n e ,b u tt h e s ef l i e ss t i l ld o n o t l e a r n ,c a s t i n g f u r t h e rd o u b to n t h ei m p o r t a n c eo fc y c l i cAMP l e v e l sa l o n e( 1 3 ) .A l t h o u g hd o u b l e -m u t a n tf l i e sd o n o t l e a r n ,r u t a b a g ad o e sp a r t i a l l yc o m p e n s a t ef o ro n e d u n c ep h e n o t y p e - f e m a l es t e r i l i t y .T h e i s o l a t i o no fs u p p r e s s o r so fd u n c es t e r i l i t yh a s p r o d u c e dm u t a t i o n sw i t hv a r i o u sa m o u n t so f c y c l a s ea c t i v i t y( 1 4 ) .I h a v eu s e d t h e s ea l l e l e st o a s s a y t h e r e l a t i v ec o n t r i b u t i o no f b a s a lv e r s u sc a l c i u m - s t i m u l a t e da d e n y l a t ec y c l a s ea c t i v i t yt o l e a r n i n g .
MATERIALSANDMETHODSF l yS t o c k s .Wi l d - t y p ef l i e sw e r et h eC a n t o n - S p e c i a l( C a n -
t o n - S )w i l d - t y p es t r a i n .r u t 1w a s o r i g i n a l l yi s o l a t e da s al e a r n i n gmu t a n t ,i n d u c e db y e t h y lm e t h a n e s u l f o n a t e( E M S )mu t a g e n e s i si na C a n t o n - Ss t o c k .r u t 2a n dr u t 'w e r ei n d u c e db y EMSmu t a g e n e s i sa n d s e l e c t e d a ss up p r es so r s o fd u n c es t e r i l i t yi n a y d n c m l 4e c f s t o c k( 1 4 ) ;t h e s em u t a n t sw e r ek i n d l yp r o v i d e db y J . A . K i g e r ,J r . ( U n i v e r s i t yo fC a l i f o r n i a ,D a v i s ) .T h em a r k e r fw a sl o s ti n t h ec o u r s eo f t h em u t a g e n -e s i s .y c v r u t 2a n d y c v r u t 3s t o c k sw e r e p r e p a r e db yr e co m bi n a t io n w i t hy c v v f c a r .P r e s e n c eo f r u t 2a n dr u t 3w a sc o n f i r m e db y s u p p r e s s i o no f d u n c e s t e r i l i t y .T h e d o u b l e -m u t a n ty d n c m l 4e c r u t 1c h r o m o s o m ew a s c o n s t r u c t e db yr e co m bi n a t i on o ft h ep a r e n t a ld u n c ec h r o m o s o m e sw i t hr u t 1 .
A b b r e v i a t i o n s :CS o r U S , c o n d i t i o n e do r u n c o n d i t i o n e ds t i m u l u s ,r e s p e c t i v e l y ;EMS, e t h y lm e t h a n e s u l f o n a t e .
2 7 9 5
T h ep u b l i c a t i o nc o s t so f t h i s a r ti c l ew e r ed e f r a y e di np a r tb y p a g ec h a r g ep a y m e n t .T h i sa r t i c l em u s tt h e r e f o r eb e h e r e b ym a r k e d" a d v e r t i s e m e n t "i na c c o r d a n c ew i t h 1 8U . S . C .§ 1 7 3 4s o l e l y t o i n d i c a t et h i s f a ct .
P r o c .N a t l .A c a d .S c i .USA8 7 ( 1 9 9 0 )
T h e p r es en c e o fr u t 'w a s c o n f i r m e db i o c h e m i c a l l ya n d b ys u p p r e s s i o no fd u n c es t e r i l i t y .A d e n y l a t eC y c l a s eA s s a y s .T e n h e a d so r a b d o m e n sf r o m
f e m a l eo r m a l ef l i e sw e r eh o m o g e n i z e db y h a n di n a g l a s s -g l a s sh om o g e n i z e r .M e m b r a n e sw e r ep r e p a r e db y c e n t r i f u -g a t i o na t 1 7 8 , 0 0 0x g f o r1 0 m i n .A d e n y l a t ec y c l a s ew a sa s s a y e db y t h em e t h o do f S a l o m o n( 1 5 ) .F r e e c a l c i u mw a sv a r i e db y u s i n ga 1 mM E G TA / C a C l 2b u f f e r( 1 6 ) .A l lc o m -p a r i s o n sa r em a d ei n t h es a m ee x p e r i m en t , b u te a c h r e s u l tr e p r e s e n t sd a t af r o m a t l e a s tt h r e e s e p a r a t ee x p e r i m e n t s .P r o t e i nw a s a s s a y e d u s i n gt h em e t h o do f B r a d f o r d( 1 7 ) .A d e n y l a t ec y c l a s ea c t i v i t yi n f e m a l ea n d m a l e h e a d s a n da b d o m e n si sq u a l i t a t i v e l yt h es a m e ,b u tt h ec a l c i u m - s e n s i t i v ec o m p o n e n tc om p r i s e sa m u c hg r e a t e rp r o po r t i on o ft o t a la c t i v i t yi na b d o m e n s( 1 3 ) .A b d o m e n sa r et h e r e f o r ea s s a y e dw h e n e v e rp o s s i b l e .U n f o r t u n a t e l y ,t h e c a l c i u m - b u f f e r i n gs y s t e mf u n c t i o n su n r e l i a b l yi n a s s a y s o f f em a l ea b d o m e n s ,p r o d u c i n gv a r i a t i o n si nt h ep e a kc a l c i u ms e n s i t i v i t y ,s oh e a d sa r eu s e d i n t h ec o m p l e m e n t a t i o na n a l y s e s .L e a r n i n g .L e a r n i n ga b i l i t yw a s m ea su r e di n a n o l f a c t o r y
d i s c r i m i n a t i o n t a s kw i t he l e c t r i cs h o c k r e i n f o r c e m e n t ,e x -a c t l ya sd e s c r i b e db y Q u i n ne t a l .( 1 8 ) .T h en um e r i c a li n d e xo f l e a r n i n gp e r f o r m a n c ei s d e f i n e da s t h e f r a c t i o no f f l i e sa v o i d i n gt h es h o c k - a s s o c i a t e do d o rm i n u st h ef r a c t i o no ff l i e sa v o i d i n gt h en o n r e i n f o r c e dc o n t r o lo d o r ,a v e r a g e df o rr e c i p -r o c a lh a l v e so ft h ee x p e r i m e n t .R e s u l t sa r er e p o r t e da sm e a n s± S E M .
RESULTSB e l l e ne t a l .( 1 4 )i s o l a t e dt w or u t a b a g aa l l e l e s ,r u t 2a n dr u t 3 ,a n d s h o w e dt h a tt h e yd e c r e a s e a d e ny l a t e c y c l a s ea c t i v i t ya p p r o x i m a t e l y3 0 a n d 1 0 % ,r e s p e c t i v e l y .S i n c et h eo r i g i n a lr u t a b a g a m u t a t i o n ,r u t 1 ,i s r e m a r k a b l ef o ra c om p l e t ea b -s e n c eo fme a s u r a b l ec a l c i u m / c a l m o d u l i n - s e n s i t i v ea d e n y l a t e
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c y c l a s ea c t i v i t y( 1 2 ,1 3 ) ,I a s s a y e dm u t a n t sr u t 2a n dr u t 3f o rt h e i rc y c l a s er e s p o n s ea t v a r i o u sc a l c i u mc o n c e n t r a t i o n s .L i k ea d e n y l a t e c y c l a s ea c t i v i t yf r o m m o sto t h e ro r g a n i s m s ,D r o s o p h i l ac y c l a s es h o w e da b i p h a s i cr e s p o n s et o c a l c i u m .Low ( 1 x 1 0 - 7M) c o n c e n t r a t i o n so f c a l c i u ma c t i v a t e dt h ee n zy m e, w h e r e a sh i g h e rc o n c e n t r a t i o n si n h i b i t e di t ( F i g s .1a n d 2 ) .H em i z y g o u sr u t 2a n d r u t 3f l i e ss h o w e de s s e n t i a l l yw i l d - t y p ea c t i v a t i o na n d i n h i b i t i o n ,d e s p i t e t h ed e c r e a s ei nb a s a la c t i v i t y( F i g .1 ) .T h e r u t 1m u t a t i o nd e f i n e st w o c om -p o n en t s o fa d e n y l a t ec y c l a s ea c t i v i t yi nD r o s o p h i l ah o m o g e -n a t e s :c a l c i u ms e n s i t i v ea n dc a l c i u mi n s e n s i t i v e( 1 2 ,1 3 ,1 9 ) .r u t 1r e m o v e st h ee n z y m ea c t i v i t yt h a tr e s p o n d st o t h e d i v a -l e n tc a t i o n( 1 2 ,1 3 ,1 9 ) .r u t 2a n d r u t 3a p p e a r e dt o b e h y p o -m o r p h st h a tp r o d u c el e s so f t h i sc a l c i u m - s e n s i t i v ea d e n y l a t ec y c l a s e .F i g .1 s h o w st h a ta l t h o u g ht h es i z eo ft h ec a l c i u ma c t i v a t i o np e a kd e c r e a s e di nr u t 2f l i e s ,t h ef o l ds t im u l a t i on a sa p r o p o r t i o no f c a l c i u m - s e n s i t i v ea c t i v i t yr e m a i n i n gw a sn e a r l yc o n s t a n t .T h ed e c r e a s em e r e l yr e f l e c t st h a tc a l c i u m -i n s e n s i t i v ea d e n y l a t ec y c l a s ec o m p r i s e da g r e a t e rp r o p o r t i o no ft o t a le n z y m ea c t i v i t yi nr u t 2h o m o g e n a t e st h a ni nw i l d - t y p ep r e p a r a t i o n s ,w h e r e a st h ec o n s t a n tf o l ds t i m u l a t i o ni n d i c a t e st h a tt h ec a l c i u m / c a l m o d u l i n - s e n s i t i v ea c t i v i t yr e m a i n i n gi nr u t 2f l i e sh a dw i l d - t y p ea c t i v a t i o n .T h e s er e s u l t sa r ec o n s i s -t e n tw i t hs e l e c t i v ee l i m i n a t i o no fc a l c i u m - s e n s i t i v ea d e n y l a t ec y c l a s ea c t i v i t yr a t h e rt h a nl o s so fc a l c i u m / c a l m o d u l i nr e g -u l a t i o no ft h ee n z y m e .C o m p l e m e n t a t i o nt e s t sa l s os u p p o r tt h i si n t e r p r e t a t i o n .T h eo r i g i n a lr u t a b a g aa l l e l e ,r u t 1 ,f a i l e d t oc o m p l e m e n tt h e b a s a lr e d u c t i o n so fm u t a t i o n sr u t 2a n d r u t 3( r e f .1 4 ;F i g .2 ) ,b u tc a l c i ums t i m u l a t i o ni n r u t 2 / r u t Ia n dr u t 3 / r u t 1h e t e r o z y g o t e sr e m a i n e dr o b u s t .G i v e nt h e i rb i o c h e m i s t r y ,I w o n d e r e di fr u t 2a n dr u t 3f l i e s
w o u l dl e a r n .T h e t w o a l l e l e sw e r ei s o l a t e da s s t e r i l i t ys u p -p r e s s o r so f t h ed u n c ea l l e l ed n c m l 4, w h i c h h a dt ob e r e m o v e db y r e c o m b i n a t i o nb e f o r et h e b e h a v i o r a la s s a y c o u l db ep e r f o r m e d .S i n c eg e n e t i cb a c k g r o u n dd o e si n f l u e n c el e a r n i n g
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i nm e m b r a n e sf r o ma b d o m e n so f m a l ew i l d - t y p e ,
P r o c .N a t l .A c a d .S c i .USA 8 7 ( 1 9 9 0 ) 2 7 9 7
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F I G .2 . A d e n y l a t ec y c l a s ea c t i v i t yi nm e m b r a n e sf r o mh e a d so f f e m a l ew i l d - t y p e ,r u t ' ,w i l d - t y p e / r u t ' ,r u t 2 / r u t i ,a n dr u t 3 / r u t 1f l i e s .A c t i v i t yi nh e a d sa n d a b d o m e n si s q u a l i t a t i v e l ys i m i l a r .
( 10 , 2 0 ) ,t h ec h r o m o s o m eu s e d i n t h er e c o m b i n a t i o n ,y c v vf c a r ,w a sa s s a y e da sa c o n t r o l .T h e s ef l i e sl e a r n e da s w e l la s
w i l d - t y p ef l i e s( Ta b l e1 ) .B o t hr u t 2a n dr u t 3l e a r n e d( Ta b l e1 ) .L e a r n i n gi nr u t 3f l i e sw a sn o t d i s t i n g u i s h a b l ef r o mc o n t r o l s .r u t 2f l i e sl e a r n e da b o u t h a l fa s w e l la sw i l d - t y p ec o n t r o l s .L e a r n i n gs c o r e s o b t a i n e df o r t h ey c v r u t 2s t o c kw e r es i g n i f i c a n t l yd i f f e r e n tf r o mw i l d - t y p es c o r e so r r u t 'v a l u e s ,s u g g e s t i n gt h a tt h er u t 2b i o c h e m i c a ll e s i o nr e s u l t si n a b e -h a v i o r a ld e f e c t .T h ed e c r e a s ew a sn o t ,h o w e v e r ,d r a m a t i c .A l t h o u g ht h ec o n t r o lc h r o m o s o m el e a r n i n gv a l u e sw e r en o ts i g n i f i c a n t l yd i f f e r e n tf r o mC a n t o n - Sc o n t r o l s ,a n e f f e c to fg e n e t i cb a c k g r o u n dc o u l dn o t b e d i s m i s s e d .S i n c et h el e a r n -i n gp h e n o t y p e so fb o t h r u t 'a n dr u t 2w e r er e c e s s i v e( r e f .1 3 ;Ta b l e1 ) ,c o m p l e m e n t a t i o na n a l y s i ss h o u l dr e f l e c ta n a d d i t i v ee f f e c to f t h et w o a l l e l e s .U n e x p e c t e d l y ,t h ed a t ai nTa b l e1s h o w st h a tr u t 'd i dc o m p l e m e n tt h eb e h a v i o r a ld e f e c ts e e n i nr u t 2f l i e s .T h er e s c u ew a sd o u b l ys u r p r i s i n ga sr u t 'f a i l e dt oc o m p l e m e n tt h eb i o c h e m i c a ld e f e c t( r e f .1 4 ;F i g .2 ) .D o u b l e - m u t a n td n cm l 4 4 r u t 2f l i e sl e a r n e d( Ta b l e2 ) .T h e
l e a r n i n gs c o r e s o b t a i n e da v e r a g eo n l ya b o u t o n e - h a l fo f
Ta b l e 1 . L e a r n i n gi nm u t a n ta n dw i l d - t y p ef l i e s
G e n o t y p e L e a r n i n gi n d e x n
C S / C S 0 . 3 5± 0 . 0 2 1 2r u t ' / r u t ] 0 . 0 4± 0 . 0 4 6y c v v f c a r / yc v v f c a r 0 . 3 5± 0 . 0 3 4y c v r u t 2 / yc v r u t 2 0 . 1 9± 0 . 0 2 * 1 8y c v r u t 3 / yc v r u t 3 0 . 3 5± 0 . 0 2 4r u t / y c v r u t 2 0 . 3 6± 0 . 0 3 7r u t ' / yc v r u t 3 0 . 3 8± 0 . 0 3 5
L e a r n i n gi n d e xi sp r e s e n t e da sm e a n± S E M .n ,N u m b e ro f t r i a l s .
* S t a t i s t i c a l l ys i g n i f i c a n td i f f e r e n c e( P<0 . 0 1 ,o n e - w a ya n a l y s i so fv a r i a n c ew i t h s u p p l e m e n t a r yN e w m a n - K e u l st e s t ) .
w i l d - t y p e b u tr e m a i n e ds i g n i f i c a n t l yh i g h e rt h a n a n y o t h e rd u n c eo r d u n c e ,s u p p r e s s o rl e a r n i ng i n d e x .I t s e e m su n l i k e l y
t h a tb a c k g r o u n dd i f f e r e n c e sa r e r e s p o n s i b l ef o rt h ep a r t i a lr e s c u e .U n l i k ef l i e sw i t ht h ey c v r u t 2a n dy c v r u t 3c h r o m o -s om e s ,n o r e c o m b i n a t i o nw a sn e c e s s a r yt o a s s a y t h ed o u b l emu t a n t s ,a sb o t h r u t 2a n d r u t 3w e r ei n d u c e dd i r e c t l yo n t h ed n c m l 4c h r o m o s o m ed u r i n ga n EMSm u t a g e n e s i s .B o t ht h ep a r e n t a ls t r a i na n d t h e d o u b l e m u t a n td n c m l 4 , r u t 3w e r ea v a i l a b l e f o rc o m p a r i s o n ,a n dn e i t h e rd i s p l a y e da n y l e a r n i n g( Ta b l e2 ) .
DISCUSSION
Two s u p p r e s s o r so fd u n c es t e r i l i t yh a v eb e e nb i o c h e m i c a l l ya n d b e h a v i o r a l l yc h a r a c t e r i z e d .B e l l e ne t a l . ( 1 4 )i n d u c e dt h e s et w om u t a n t so n a d n c m4 c h r o m o s o m eb y u s i n gEMSa st h em u t a g e n .B a s e do n c o n s i s t e n tr e d u c t i o n si n a d e n y l a t ec y c l a s ea c t i v i t y,c o m p l e m e n t a t i o na n a l y s i so f e n z ym a t i ca c t i v i t ya n d o f a b i l i t yt o s u p p r e s sd u n c es t e r i l i t y( 1 4 ) ,a n dm e i o t i cm a p p i n g( H .J .B e l l e na n dJ . A . K i g e r ,J r . ,p e r s o n a lc o m m u n i c a t i o n ) ,t h et w om u t a t i o n sw e r ed e s i g n a t e dr u t 2a n dr u t 3 .I f o l l o wt h e i rt e r m i n o l o g yb u t a c k n o w l e d g et h a ta d e -n y l a t ec y c l a s ea c t i v i t yd e p e n d so n a n u m b e ro f p r o t e i n sa n d
Ta b l e2 . L e a r n i n gi nd u n c ea n d d u n c e , r u t a b a g af l i e sG e n o t y p e L e a r n i n gi n d e x n
d n c m l 4 / d n c m l 4 0 . 0 1+ 0 . 0 2 4d n c m l 4 , r u t l / d n c m l 4 , r u t l0 . 0 0 ± 0 . 0 3 3d n c m l 4 , r u t 2 / d n c m l 4 , r u t 20 . 1 7 ± 0 . 0 2 * 1 5d n c m l 4 , r u t 3 / d n c m l 4 , r u t 3- 0 . 0 2 ± 0 . 0 4 4
L e a r n i n gi n d e xi sp r e s e n t e da sm e a n± SEM. n ,N u m b e ro f t r i a l s .
* S t a t i s ti c a l l y s i g ni f i c an td i f f e r e n c e( P<0 . 0 1 ,o n e - w a ya n a l y s i so fv a r i a n c ew i t h s u p p l e m e n t a r yN e w m a n - K e u l st e s t ) .
P r o c .N a t l .A c a d .S c i .USA8 7 ( 1 9 9 0 )
t h a tt h e two s u p p r e s s o r s c o u l d a c t u a l l yb e m u t a t i o n si nt i g h t l yl i n k e dl o c ie n c o d i n gs t o i c h i o m e t r i c a l l yl i m i t i n gm o d -u l a t o r yf a c t o r s .d u n c e f l i e sc a r r y i n gt h e r u t 2 m u t a t i o n l e a r n .D o u b l e -
m u t a n tf l i e sp e r fo r m a b ou th a l fa s w e l la s w i l d - t y p ef l i e si nt h i s p a r a d i g m .Any l e a r n i n gi n d u n c e , r u t a b a g a f l i e si ss u r p r i s i n g s i n c ed n cm i l , r u t la n dd n c m l 4 , r u t lf l i e sd o n o t l e a r n( r e f .1 3 ;Ta b l e2 ) .d n c m ' l r u t 'f l i e sp e r f o r me v e n more p o o r l yt h a n e i t h e rm u t a n ta l o n ei n a n a s s o c i a t i v el e a r n i n gp a r a d i g m( 1 0 ) .S i n c e t h e d u n c e a n d r u t a b a g amu t a t i o n si n f l u e n c eb i o c h e m i c a l l yo p p o s i n genzyme a c t i v i t i e s( p h o s p h o d i -e s t e r a s e a n d a d e n y l a t ec y c l a s e ) ,o n e m i g h te x p e c t t h a tt h etwo mu t a t i o n sw o u l d c o m p l em e n te a c ho t h e rw h e np r e s e n ti nt h e s a m e f l y .I n d e e d ,d n c m l l , r u t lf l i e sh a v e n e a r n o r m a lc y c l i cAMP l e v e l s ,a n d r u t 1 d o e s p a r t i a l l ysupp re s s d n c m l ls t e r i l i ty ( 13 ) .T h e i n f e r i o rb e h a v i o r a lp e r f o r m a n c eo f t h ed o u b l e m u t a n t h a s t h u s b e e n p u z z l i n g( 1 0 ) .T h e A p l y s i am o d e lp r o v i d e sa p o s s i b l ea n swe r : a s s o c i a t i v el e a r n i n gmay
r e q u i r ea s y n e r g i s t i cr e s p o n s e b e t w e e nt h en e u r o t r a n s m i t t e rr e l e a s e dt o t h eUS a n d t h er e s u l to fn e u r o n a la c t i v i t yt r i g -g e r e d b y t h eCS . I f , as p o s t u l a t e d ,c a l c i u mi n f l u xm e d i a t e st h eCS s i g n a la n d a d e n y l a t ec y c l a s ep r o v i d e st h emo l e c u l a rc o n v e r g e n c e p o i n tf o rt h et w o s i g n a l s ,t h e n r u t 1 f l i e sw o u l dl a c kt h em a c h i n e r yf o r a s s o c i a t i v el e a r n i n ge v e n i f b a s a lc y c l i cAMP l e v e l sw e r e c om p l e t e l yc o m p en s a t e d b ya d u n c em u t a t i o n .L e a r n i n gi n d n c m l 4 , r u t 2f l i e sb u t n o t d n c m l 4r u t lf l i e ss u p p o r t s t h i sm o d e l . r u t 2 f l i e ss h o wa s i g n i f i c a n td e -c r ea se i n b a s a la d e n y l a t ec y c l a s ea c t i v i t y( 3 0 % )b u t h a v en o r m a ll e v e l so f c a l c i u m / c a l m o d u l i ns t i m u l a t i o n .I t i s t h e r e -f o r ep o s s i b l et h a tt h i sa l l e l ed e c r e a s e s a d e n y l a t ec y c l a s ea c t i v i t ye n o u g ht o c o m p e n s a t ef o rt h e e l e v a t e dc y c l i cAMPl e v e l si n d n c m l 4f l i e sw i t h o u t a b o l i s h i n gt h e c a l c i u m /c a l m o d u l i ns t i m u l a t i o nr e q u i r e df o ra s s o c i a t i v el e a r n i n g .A l t h o u g ht h i sd e m o n s t r a t i o no f t h e i n t e r a c t i o no f d u n c e
a n d r u t a b a g aa l l e l e so n b e h a v i o r a la n d b i o c h e m i c a ll e v e l sp r o v i d e sf u r t h e re v i d e n c et h a tb o t hm u t a t i o n sa f f e c tl e a r n i n gb y a l t e r i n gt h e s a m e m o l ec u l a r c a s c a d ei n t h e s a m e c e l l s ,i tl e a v e sa n i m p o r t a n tq u e s t i o nu n a n s w e r e d :Why d o r u t 1 f l i e sd i s p l a ya n y l e a r n i n gi n o t h e r o l f a c t o r ya n d v i s u a lp a r a d i g m s( 1 0 ,2 1 ,2 2 ) ?P e r h a p sa d d i t i o n a lb i o c h e m i c a lp r o c e s s e s ca np a r t i a l l yc o m p e n s a t ef o rt h e a b s e n c eo f c a l c i u m / c a l m o d u l i nc y c l a s ea c t i v i t y .r u t 1 c y c l a s em a y h a v e s o m e c a l c i u m /c a l m o d u l i ns t i m u l a t e da c t i v i t yi nv i v o .S e p a r a t es e c o n dme s -
senge r s y s t e m sm i g h tb e i n v o l v e di n f l i e s( 4 ) .T h e p r e s e n tr e s u l tc a n n e i t h e r s u p p o r tno r r e f u t et h e s e p o s s i b i l i t i e sb u tme r e l ye m p h a s i z e st h e i m p o r t a n c eo f c a l c i u m / c a l m o d u l i n -s t i m u l a t e dc y c l a s ea c t i v i t yt o l e a r n i n gas m e a s u r e db y t h eo l f a c t o r ya v o i d a n c ep a r a d i g mu s e d h e r e .T h ef a c tt h a tf l i e sb e a r i n ge i t h e rr u t a b a g am u t a t i o nr u t 2 o r
r u t 3 l e a r nd e s p i t ed e f i c i e n c i e si n t o t a le n z y m e a c t i v i t ya l s oh i g h l i g h t st h e i m p o r t a n c eo f t h e c a l c i u m - s t i m u l a t e dc ompo -n e n t o f a d e n y l a t ec y c l a s ea c t i v i t yt o t h e l e a r n i n gm e a s u r e dh e r e .r u t 2 d o e s l e a r nl e s sw e l l t h a nw i l dt y p e ,s u g g e s t i n gt h a ta 30% r e d u c t i o n i n b a s a l a d e n y l a t ec y c l a s ea c t i v i t ym a yp r o d u c ea l a r g ee n o u g hr e d u c t i o n i n t h e c o n c e n t r a t i o no fc y c l i cAMP t o i n f l u e n c et h e m o l e c u l a rl e a r n i n g a p p a r a t u s .Ad e c r e a s ei n t h e t o t a lamoun t o f a d e n y l a t ec y c l a s ec o m p e t e n tt o r e s p o n dt o c a l c i u m / c a l m o d u l i na c t i v a t i o nw o u l da l s o l e a dt o a d e c r e a s e i n p e a k c y c l i cAMP c o n c e n t r a t i o n sd u r i n gc a l c i u me n t r y ( 2 2 ) .T h u s , a d e f e c t i n t h e m e d i a t i o no f t h ec a l c i u ms i g n a lc o u l d a l s o e x p l a i nt h e l e a r n i n g d e f i c i e n c y .A l t e r n a t i v e l y ,t h e d e c r e m e n ti nl e a r n i n gs co r e s m a y b e d u e t oa n a l t e r a t i o no f g e n e t i cb a c k g r o u n dt h a t was i n t r o d u c e dd u r i n gt h e r e c o m b i n a t i o na n d i s u n r e l a t e dt o t h e b i o c h e m i c a ld e f i c i e n c y .S u p p o r tf o r t h i sp o s i t i o ni s p r o v i d e db y t h ec o m p l e m e n t a t i o nd a t a .r u t 2 ! r u t ' h e t e r o z y g o t e sl e a r n as w e l la s w i l d - t y p ef l i e s( T a b l e1 ) .S i n c ep r e v i o u sg e n e t i ce v i d e n c ei n d i c a t e st h a ta d e n y l a t ec y c l a s ea c t i v i t yi sp r o p o r t i o n a lt o t h en u m b e ro f c o p i e s o f t h e gene ( 1 3 )( i . e . ,t h a tr u t a b a g ai s a
d o s a g e - s e n s i t i v el o c u s )a n d s i n c e r u t 1 f a i l st o c o m p l e m e n t
r u t 2b i o c h e m i c a l l y( r e f .1 4 ;F i g .2 ) ,r u t 1s h o u l df a i lt o c o m -p l em e n ta n yl e a r n i n gd e f e c tt h a tr e s u l t e df r o ma d e f i c i e n c yi na d e n y l a t ec y c l a s ea c t i v i t y.T h ed a t ai nTa b l e1 s h o w,h o w -e v e r ,t h a tr u t 1d o e sc o m p l e m e n tr u t 2b e h a v i o r a l l y .G i v e nt h i si n c o n s i s t e n c ya n dt h ew e a k n e s so ft h el e a r n i n gd e f e c ti nr u t 2f l i e s ,p e r f o r m a n c ei n o t h e rl e a r n i n gp a r a d i g m sw o u l db ei m p o r t a n tt o c l a r i f yt h ee x t e n to ft h eb e h a v i o r a ld i s a b i l i t y .I ti s c o m f o r t i n gt h a tt h em o r es e v e r e b i o c h e m i c a ld e f e c t ,a sm e a s u r e d
i nv i t r o ,p r o d u c e st h eb e h a v i o r a ld e f e c t( F i g .1 a n dTa b l e1 ) .I na d d i t i o n ,r u t 2i st h em o r ee f f e c t i v es u p p r e s s o ro fd u n c es t e r i l i t y( 1 4 ) .B i o c h e m i c a la n a l y s i so ft h et w or u t a b a g aa l l e l e s ,r u t 2a n d
r u t 3 ,s u p p o r t st h eh y p o t h e s i st h a tt h el o c u se n c o d e sa c a l c i -u m - s e n s i t i v ea d e n y l a t ec y c l a s ec a t a l y t i cs u b u n i t .L i k er u t 1 ,b o t hm u t a t i o n sd e c r e a s ea d e n y l a t ec y c l a s ea c t i v i t yi n ad o s a g e - d e p e n d e n tm a n n e r( r e f .14 ;F i g .2 ) .I nc o n t r a s tt o t h eo r i g i n a lmu t a t i o n ,n e i t h e rr u t 2n o r r u t 3d e c r e a s e sa d e n y l a t ec y c l a s ea c t i v i t ya s m u c ha s r u t 1 ,a n db o t hl e a v et h ec a l c i u m /c a l m o d u l i n - s e n s i t i v ec y c l a s ea c t i v i t yi n t a c t .A s s e e n m o s tc l e a r l yi n f l i e sb e a r i n gt h er u t 2mu t a t i o n( F i g .1 ) ,t h et o t a la m o u n to fc a l c i u ms t i m u l a t i o nd e c r e a s e si np r o p o r t i o nt o t h er e d u c t i o ni n b a s a le n z y m ea c t i v i t y,bu t t h ef o l dr e s p o n s er e m a i n sr e l a t i v e l yu n a f f e c t e d .T h ed e c r e a s ei nt o t a ls t i m u l a -t i o nr e f l e c t s
t h ef ac t t ha ti n
h o m o g e n a t e so f r u t 2f l i e st h e
c a l c i u m - i n s e n s i t i v ec o m p o n e n to fa d e n y l a t ec y c l a s ea c t i v i t yr e p r e s e n t sa g r e a t e rp r o p o r t i o no f t o t a le n z y m ea c t i v i t y .T h ec o n s e r v a t i o no f f o l ds t i m u l a t i o nd em o n s t r a t e st h a tt h er e -m a i n i n gc a l c i u m / c a l m o d u l i n - s e n s i t i v ea d e n y l a t e c y c l a s ee x -h i b i t sa w i l d - t y p er e s p o n s et o s t i m u l a t i o n .I f ,a s s e e m sl i k e l y ,r u t 2a n dr u t 3r e p r e s e n tm u t a t i o n si nt h er u t a b a g al o c u s ,t h e s ed a t ap r o v i d ee v i d e n c et h a tt h e two c h a r a c t e r i s t i c so f t h eo r i g i n a lr u t a b a g aa l l e l e ,d e c r e a s ei nb a s a la c t i v i t ya n da b l a -t i o no fc a l c i u m / c a l m o d u l i na c t i v a t i o n ,a r e s e p a r a b l e .I t i sp o s s i b l et o r em o v e a p o r t i o no ft h ec y c l a s ea c t i v i t yr e s p o n d -i n gt o c a l c i u mw h i l es p a r i n gt h el i g a n ds e n s i t i v i t yo f t h er e m a i n i n gc a l c i u m - s e n s i t i v ea c t i v i t y.T h eg e n e t i cs e g r e g a t i o no f f e r e dh e r eh a s s ome i m p l i c a t i o n sf o r t h en a t u r eo f t h er u t a b a g ag e n e p r o d u c t .P r e v i o u sb i o c h e m i c a la n d g e n e t i ca n a l y s i ss u g g e s t e dt h a tt h er u t a b a g al o c u se n c o d e se i t h e ras t o i c h i om e t r i c a l l yl i m i t i n gc o f a c t o rc o n f e r r i n gc a l c i u m /c a l m o d u l i ns e n s i t i v i t yo r a n a d e n y l a t ec y c l a s ec a t a l y t i cs u b -u n i t( 1 3 , 1 9 ) .A l t h o u g ht h ee v i d e n c ep r o v i d e db y t h ea n a l y s i so fa l l e l e sr u t 2a n d r u t 3d o e s n o t r i g o r o u s l yd i s t i n g u i s hb e -t w e e n t h e t w o a l t e r n a t i v e s ,i t d o e s s u p p o r t t h e s e c o n dp o s s i b i l i t ya s t h em u t a t i o n sd e f i n e db y r u t 2a n d r u t 3s e l e c -t i v e l ya l t e ra d e n y l a t ec y c l a s ea c t i v i t yw i t h o u tc h a n g i n gc a l -c i u m / c a l m o d u l i ns t i m u l a t i o n .R e c e n t m o l e c u l a ra n a l y s i sa l s oi n d i c a t e st h a tr u t a b a g a
e n c o d e so n e f o r mo fa d e n y l a t ec y c l a s e .K r u p i n s k ie t a l .( 2 3 )h a v e i s o l a t e da n d c h a r a c t e r i z e dcDNAs e n c o d i n gb o v i n ea d e n y l a t ec y c l a s e .B yu s i n g c r os s - h y br i d i z a t i o nt o a b o v i n eb r a i nc D N A ,a D r o s o p h i l ag e n o m i cc l o n eh a s b e e n i s o l a t e d .I n s i t uh y b r i d i z a t i o nt o p o l y t e n ec h r o m o s o m e sr e v e a l sh y -b r i d i z a t i o nt o t h es ame r e g i o n( 1 2 E 1 - 1 3 A 5 )t h a tc o n t a i n st h er u t a b a g al o c u s( 20 , 2 3 ) .B i o c h e m i c a la n dp e r h a p sb e h a v i o r a lr e s c u e o f t h e p h e n o t y p e sm a p p i n gt o t h e l o c u sb y t h ew i l d - t y p ea d e n y l a t ec y c l a s eg e n ew o u l dc o n c l u s i v e l ye s t a b -l i s ht h ei d e n t i t yo f t h er u t a b a g ag e n ep r o d u c t .B i o c h e m i c a la n db e h a v i o r a lc h a r a c t e r i z a t i o no f two r u t a -
b a g aa l l e l e s ,r u t 2a n dr u t 3 ,p r o v i d e sf u r t h e rg e n e t i ce v i d e n c et h a tt h er u t a b a g al o c u sd o e s i n d ee de n c o d ean a d e n y l a t ec y c l a s ec a t a l y t i cs u b u n i t .T h i sa n a l y s i sa l s oe m p h a s i z e st h es im i l a r i t yo fc e l l u l a rb i o c h e m i s t r yt h o u g h tt o u n d e r l i ea s s o -c i a t i v el e a r n i n gi n two s i m p l e ,b u t d i s t i n c t ,o r g a n i s m s :A p -l y s i aa n dD r o s o p h i l a .I n b o t h s y s t em sc a l c i u m - d e p e n dmo d u l a t i o no fc y c l i cAMPl e v e l ss e ems a c r u c i a l ,a l b e i tn o te x c l u s i v e ,m e d i a t o ro fn e u r o n a lp l a s t i c i t y .
I t h a n kMa r g a r e tL i v i n g s t o n ea n dR . S c o t tH a w l e yf o ra d v i c ea n d
e n c o u r a g e m e n t .T h a n k sa l s og o e s t o L e o n o r eD I u h yf o re x c e l l e n t
t e c h n i c a la s s i s t a n c e .T h i sw o r kwa s s u p p o r t e db yP r e s i d e n t i a lYo u n gI n v e s t i g a t o rAw a r d8 3 5 2 1 5 0f r o mt h eN a t i o n a lS c i e n c eF o u n d a t i o nt o M a r g a r e tL i v i n g s t o n ea n dG r a n tDC B8 8 1 5 7 4 9t o R . S c o t tH a w l e yf r o mt h eN a t i o n a lS c i e n c eF o u n d a t i o n .
1 . K a n d e l ,E . R . ,A b r a m s ,T . ,B e r n i e r ,T . ,C a r e w ,T . J . ,H a w k -i n s ,R. D . & S c h w a r t z ,J . H . ( 1 9 8 3 )C o l dS p r i n gH a r b o rS y m p .Q u a n t .B i o l .4 8 ,8 2 0 - 8 3 0 .
2 . L i v i n g s t o n e ,M . S . & Te m p e l ,B . L . ( 1 9 8 3 )N a t u r e( L o n d o n )3 0 3 ,6 7 - 7 0 .
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4 . T u l l y ,T . ( 1 9 8 7 )T r en d s N e ur o S c i .1 0 ,3 3 0 - 3 3 5 .5 . By e r s ,D . ,D a v i s ,R . L .& K i g e r ,J . A . ( 1 9 8 1 )N a t u r e( L o n d o n )
2 8 9 ,7 9 - 8 1 .6 . D a v i s ,R . L . & K i g e r ,J . A . ( 1 9 8 1 )J . C e l lB i o l .9 0 ,1 0 1 - 1 0 7 .7 . K a u v a r ,L .M . ( 1 9 8 2 )J . N e u r o S c i .2 , 1 3 4 7 - 1 3 5 8 .8 . C h e n ,C . - N . ,D e n o m e ,S . & D a v i s ,R. L . ( 1 9 8 6 )P r o c . N a t l .
A c a d .S c i .USA 8 3 ,9 3 1 3 - 9 3 1 7 .9 . D u d a i ,Y . ,J a n ,Y . N . ,B y e r s ,D . ,Q u i n n ,W. G . & B e n z e r ,S .
( 1 9 7 6 )P r o c . N a t l .A c a d .S c i .USA 7 3 ,1 6 8 4 - 1 6 8 8 .
P r o c . N a t ! .A c a d .S c i .USA8 7 ( 1 9 9 0 ) 2 7 9 9
1 0 . T u l ly , T .& Qu i n n ,W. G . ( 1 9 8 5 )J . C o m p .P h y s i o l .1 5 7 ,2 6 3 - 2 7 7 .
1 1 . F o l k e r s ,E . ( 1 9 8 2 )J . I n s e c tP h y s i o l .2 8 ,5 3 5 - 5 3 9 .1 2 . D ud a i , Y.& Z v i ,S . ( 1 9 8 4 )N e u r o s c i .L e t t . 4 7 ,1 1 9 - 1 2 4 .1 3 . L i v i n g s t o n e ,M . S . ,S z i b e r ,P . P .& Q u i n n ,W. G . ( 1 9 8 4 )C e l l
3 7 ,2 0 5 - 2 1 5 .1 4 . B e l l e n ,H . J . ,G r e g o r y ,B . K . , O l s s o n , C . L . & K i g e r ,J . A .
( 1 9 8 7 )D e v .B i o l .1 2 1 ,4 3 2 - 4 4 4 .1 5 . S a l o m o n ,Y . ( 1 9 7 9 )A d v .C y c l i cN u c l eo t i d e R es .1 0 ,3 5 - 5 5 .16 . C a l d w e l l ,P .C .( 1 9 7 0 )i nS y m p o s i u mo n C a l c i u ma n dC e l l u l a r
F u n c t i o n ,e d .C u t h b e r t ,A . W. ( S t .M a r t i n ' s ,N ewYo r k ) ,p p .1 0 - 1 6 .
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5 9 9 2 - 5 9 9 6 .2 0 . D u d a i ,Y . ( 1 9 7 7 )J . C o m p .P h y s i o l .11 4 ,6 9 - 8 9 .2 1 . D ud a i , Y.( 1 9 8 3 )P r o c . N a t d .A c a d .S c i .USA 8 0 ,5 4 4 5 - 5 4 4 8 .2 2 . K i g e r ,J . A .& S a l z ,H . ( 1 9 8 5 )A d v .I n s e c tP h y s i o l .1 8 ,1 7 1 - 1 7 2 .2 3 . K r u p i n s k e ,J . ,C o u s s e n ,F . ,B a k a l y a r ,H . A . ,Ta n g ,W . , F e i n -
s t e i n ,P .G . , O r t h ,K . ,S l a u g h t e r ,C . ,R e e d , R . R . & G i l m a n ,A . G . ( 1 9 8 9 )S c i e n c e2 4 4 ,1 5 5 8 - 1 5 6 4 .
