2 4 7

T HE BIT H OR AX C O MPLEX: T HE FI RST FIFTY YE A RS

Nobel Lect ure, Dece mber 8, 1995 b y

E D WA R D B. L E W I S

Divisio n of Biology, T he Califor nia I nstit ute of Tec h nology, Pasa de na, C A 9 1 1 2 5, U S A.

“ T he po wer of usi ng abstractio ns is t he esse nce of i ntellect, a n d wit h every i ncre ase i n abstractio n t he i ntellect ual tri u mp hs of scie nce are e n ha nce d. ” Bertra n d R ussell

I NTRODUCTIO N

Ge netics is a disci pli ne t hat has s uccessf ull y use d a bstractio ns to attac k ma n y of t he most i m porta nt proble ms of biology, i ncl u di ng t he st u dy of evol utio n a n d ho w a ni mals a n d pla nts develo p. T he po wer of ge netics to be nefit ma n- ki n d was first recog nize d by t he a war d of t he Nobel Prize i n p hysiology or me dici ne i n 1933 to T. H. Morga n. I n t he 23 years t hat ha d i nter ve ne d be- t ween the ti me Morgan intro d uce d Droso p hil a as a ne w orga nis m for t he st u d y of ge netics a n d t he a war d of t he Prize, he a n d his st u de nts, es peciall y, A. H. St urt e v a nt, C. B. Bri d g es a n d H. J. M ull er, h a d v astl y e xt e n d e d t h e l a ws of Me n del as t he res ult of a h ost of disc o veries, t o me nti o n o nl y a fe w: t hat t he ge nes ( Me n del’s factors) are arra nge d i n a li near or der a n d ca n be place d o n ge netic ma ps, t hat t hey m utate i n for war d a n d reverse directio ns, t hat t he y ca n exist i n ma n y for ms, or alleles, a n d t hat t heir f u nctio ni n g ca n de pe n d u po n t heir positio n. P urely o n t he basis of bree di ng ex peri- me nts, t hese early workers were able to de d uce t he existe nce of i nversio ns an d d u plications, for exa m ple, before it beca me possible to de monstrate t he m cytologically. T he list of t heir ac hie ve me nts is a lo ng o ne a n d o ne t hat has bee n p ut i nto historical pers pective by St urteva nt i n A History of Ge netics (1). All of t hese disc o veries were ma de wit h Droso p hil a by taki ng a d va ntage of its s mall size, ease of c ult uri n g, hi g h fec u n dit y, s h ort life c ycle, s mall c hr o- moso me nu mber, wealth of spontaneous and induced mutations, and, after their discovery in 1935, its giant salivary glan d chro moso mes. Of i m mense i m porta nce also was t he existe nce of sta n dar d or “ wil d-ty pe” strai ns. T hat Morga n’s co ntrib utio ns satisfie d t he criterio n of bei ng of be nefit to ma nki n d was evi de nt by t he re markable exte nt to w hic h t he ne w discoveries wit h Droso p hil a ha d direct a p plicatio n to t he u n dersta n di ng of t he i n heri- 2 4 8 P hysiology or Me dici ne 1995

ta nce of many h u man traits. For exa m ple, the inheritance of colorblin dness a n d he mo p hilia i n h u ma n bei ngs co ul d be u n derstoo d for t he first ti me. T h e s e c o n d N o b el Pri z e f or w or k i n t h e g e n eti cs of Droso p hil a was a war de d i n 1946 to H. J. M uller for his disco very i n 1928 t hat X-rays pro d uce ge ne m utati o ns a n d d o s o i n direct pr o p orti o n t o t he d ose (2) . M uller calle d atte ntio n to t he ge netic risks to t he h u ma n race pose d by i n discri mi nate use of ionizing ra diations, an d, pro phetically, he arg ue d that s uch uses wo ul d als o i ncrease t he ris k of ca ncers, if ca ncer is t he res ult of s o matic m utati o ns. T he i m plicatio ns of M uller’s work were not o verlooke d wit h t he a d ve nt of t he ato mic a ge. As a res ult, exte nsi ve ge netic st u dies were carrie d o ut i n Droso p hil a a n d mi c e t o ass ess t h e r el ati v e r at es of m ut ati o n i n t h es e or g a nis ms as a mea ns of assessi ng t he ge netic risks to h u ma n bei ngs fro m t he use of ato mic energy. The a war d of the Prize in 1995 for work with Droso p hil a recog nizes t he gro w- i ng i m porta nce of a fiel d t hat has co me to be calle d de velo p me ntal ge netics. The work of my co- winners, Eric Wiescha us an d Christiane N usslein- Volhar d, has i de ntifie d cr ucial ste ps i n t he earl y de velo p me nt of t he or ga nis m. S pecificall y, t he y ha ve i de ntifie d maj or ge nes i n v ol ve d i n setti n g u p t he i niti- al axes of t he e mbr yo a n d its ger m la yers (3) t hereb y setti n g t he sta ge for gro u ps of master co ntrol ge nes t hat t he n progra m t he fi nal bo dy pla n of t he or ga nis m. It is t his latter gr o u p of ge nes wit h which we will be concerne d h er e: w h at t h e y d o a n d h o w t h e y c a m e t o b e dis c o v er e d. M y p art i n t his st or y b e g a n i n t h e l at e 1 9 3 0s a n d it will b e first e x a mi n e d i n r el ati o n t o t h e c o n- c e pt of t h e g e n e at t h at ti m e.

T HE GE NE C O NCEPT

Jo ha n nse n coi ne d t he ter m, “ge ne,” in 1909 an d it q uickly re place d Me n del’s “factor” (4). T he co nce pt of t he ge ne is o ne of t he most po werf ul a bstractio ns i n biolo g y a n d o ne of great utilit y. For ma n y years t he ge ne co ul d be satisfactorily defi ne d as a u nit wit hi n w hic h ge netic reco mbi natio n, or crossi n g o ver, does not occ ur. T he u nit defi ne d i n t his wa y te n de d to cor- res po n d to a u nit of f u nctio n, as defi ne d b y t he sta n dar d p he not y pic test for all elis m, or t h e “co mple mentation” test, t o be disc usse d bel o w. I n 1925, St urteva nt ma de t wo i m porta nt discoveries t hat were eve nt ually to lea d to a reexa mi natio n of t he ge ne co nce pt i n ter ms of t he ge ne’s f u nctio n ( 5) . In analyzing the progeny of fe males ho mozygo us for the unstable eye m ut ati o n, B a r ( B), he pre dicte d t hat a rare m utati o n, do uble- Bar ( B B), w a s a tan de m d u plication that arose in the progeny of ho mozygo us B fe males as t he res ult of “ u neq ual crossi ng o ver.” H e t h e n s h o w e d t h at t h e e y es of B B/+ f e m al e s ar e sli g htl y s m all er t h a n t h o s e of B / B a n d de d uce d t hat t he f u nctio n of a ge ne ca n de pe n d u p o n its p ositi o n wit h res pect t o is nei g h b ors, t he first exa m ple of t he “ positio n effect,” as he na me d it. Eleve n years later, usi ng t he gia nt salivary gla n d c hro moso mes of t he Droso p hil a l ar v a, Bri d g e s ( 6) a n d M ull er a n d Pr o k of y e v a ( 7) r e p ort e d t h at t h e Ed ward B. Le wis 2 4 9

B m uta nt was act ually a ta n de m d u plicatio n of 7 ba n ds i n t he X c hro moso- m e a n d t h at B B w as a tri pli c ati o n f or t h at r e gi o n. H e n c e B B was arisi n g fro m uneq ually paire d d uplicate d regions acco mpanie d by nor mal rather than “ u neq ual” crossi ng o ver. I nteresti ngly, Wrig ht ha d pre dicte d t hat B itself wo ul d be a d u plicatio n before it was de mo nstrate d cytologically (8). Bri d ges ha d earlier calle d atte nti o n t o d u plicati o n-li ke str uct ures i n t he salivary gla n d c hro moso mes of wil d-ty pe larvae (9). I n partic ular, he i nter- prete d n u mero us do uble ban de d str uct ures, or “ do ublets,” as t wo d u plicat- e d ba n ds f use d alo ng t heir e dges. T heir str uct ure s uggests t hat t hey are re verse ( A B B A), rat her t ha n direct ( AB AB) , re peats of si n gle ba n ds ( Fi g. 1). Bri dges’ cytological evi de nce for s uc h re peats co mbi ne d wit h St urteva nt’s de mo nstratio n of positio n effect s u g geste d t hat m ulti ple alleles of a gi ve n ge ne mig ht i n so me cases be resol vable i nto t wo or more re peate d ge nes t hat acte d li ke o ne beca use of a p ositi o n effect. E vi de nce t hat m ulti ple alleles mi g ht b e r es ol v a bl e i nt o s e p ar a bl e l o ci b e g a n t o b e o bt ai n e d i n t h e l at e 1 9 3 0s b y C. P. Oliver at t he U niversity of Mi n nesota. He fo u n d a lo w freq ue ncy of r e v ert a nts t o wil d t y p e i n t h e offs pri n g of f e m al es h et er o z y g o us f or t w o r e c es- si v e loze nge (l z) eye m utatio ns. Alt ho ug h t he reverta nts were i nvariably asso- ciate d wit h crossi n g o ver i n t he re gio n, he was u na ble to detect a reci procal crossover having both m utants in the sa me chro moso me. He therefore co ul d o nly s uggest t hat t he re verta nts co ul d be ex plai ne d as t he res ult of “ u neq ual crossi ng over or crossi ng over bet wee n ‘re peats.’ ” ( 1 0).

S b b x d M C

I ,

Fi g. li c genes studied f or cis-tr a ns effects. At t he l eft ar e t h e correspondences found n e ar t he e xtre me left e n d of the second chro moso me; at the right is sho wn a section fro m the middle of the right ar m of t he t hir d c hr o- moso me. The sy mhots ss a n d Mc r ef er t o t h e l o ci s pi neless a n d Microcephalus ,r es p e cti v el y; o t h e r s y m b o l s a r e drscrihed in the text. Reprinted fro m (11).

ST AR A N D ASTER OI D

I was a n u n der gra d uate at t hat ti me a n d Oli ver ge nero usl y ga ve me a des k i n his laboratory a n d allo we d me to work o n a ne w ro ug h-eye d m uta nt t hat ha d b e e n gi v e n t o m e b y E. N o vits ki, w h o w as t h e n at P ur d u e U ni v ersit y. [ N o vits ki 2 5 0 Physiology or Medicine 1995 a n d I ha d beg u n o ur work wit h Drosophila i n hig h sc hool aro u n d 1935]. Bri d ges ha d s u g geste d t hat it be calle d Star-recessive ( S γ), si n c e it a ct e d as a n allele of a weakly do mi na nt ro ug h eye m uta nt, St ar ( S). T h us, S / + fli es h a v e sli g htl y s m all er e y e s t h at ar e sli g htl y r o u g h e n e d; S γ/ S γfli es ha ve eyes re d uce d to a bo ut half t heir nor mal size a n d wit h a ver y ro u g he ne d s urface; w hile S / S γ fli es ar e n e arl y e y el ess [fi g ur e d i n ( 1 I)]. Alt h o u g h i n a pr eli mi n ar y t est, I h a d fo u n d a re verta nt of S γ or of S i n 3,235 offs pri n g of S / S γ fe males, when flanking markers were intro d uce d I obtaine d no more wil d-ty pe pro d ucts a mo ng 9,294 offs pri ng (12). I n s pite of t hese i nc o ncl usi ve res ults, I c o nti n ue d t he st u d y of S a n d S γ a s o ne of St urte va nt’s gra d uate st u de nts at C alt e c h, co m me nci ng i n 1939. I n the tra dition of Morgan, St urtevant allo we d his st u dents consi derable free- do m to c hoose t heir t hesis researc h projects. Q uite a risk was i nvolve d i n choosing to work on S an d its “alleles.” Crossovers bet ween the m wo ul d be r ar e if t h e y w er e t o o c c ur at all. E v e n if t h e wil d-t y p e cr oss o v er c o ul d b e r e c o v- ere d, it was ex pecte d t hat it w o ul d be ver y diffic ult t o detect t he reci pr ocal, or do uble m uta nt, crossover. To i ncrease t he resol vi n g po wer of t he a nal ysis, I ma de use of t he i nter- chro moso mal effect of rearrange ments on crossing over. Intro d uction of heterozygosity for i nversio ns i n c hro moso me ar ms ot her t ha n t he left ar m of t he seco n d c hro moso me, i n w hic h S is locate d, res ulte d i n a n a p proxi mately f o ur-f ol d i n cr e as e i n t h e fr e q u e n c y of cr ossi n g o v er i n t h e vi ci nit y of S. As i n Oli v er’s w or k o n l z , the revertants were invariably associate d with crossing o ver bet wee n S a n d S γ. I r e n a m e d t h e l att er “ all el e, ” ast er oi d ( ast). A ta n de m d u plicatio n for t he S regio n w hic h I ha d fo u n d as a n x-ray i n d uce d reverta nt of ast ( 1 3) l e nt its elf t o t h e r e c o v er y of t h e S a st do uble m uta nt c hro moso me (14). A stri ki n g p ositi o n effect was i n e vi de nce: w hereas, S + / + ast is nearly eyeless, t he co m ple me ntary ge noty pe, S ast / + +, is n e arl y wil d t y pe, exce pt for a sli g htl y s maller a n d sli g htl y ro u g he ne d e ye i n disti n g- uis h a bl e fr o m t h at of S / + ( 1 4), fi g ur e d i n ( 1 1). S a n d ast pro ve d to be localize d to t he 21 E l-2 do u blet of t he sali var y glan d chro moso mes (Fig. I), the do ublet which Bri dges ha d single d o ut as bei ng a re prese ntative exa m ple (9). T hese cytoge netic st u dies of S a n d a st for me d my doctor’s t hesis (15) p ublis he d i n part i n 1945 (16). Co m pariso n of t he differe nce i n p he noty pe bet wee n cis vs tr a ns genoty pes is us u all y r ef err e d t o as t h e cis-tra ns t est, a n d t h e p ositi o n eff e ct, if pr es e nt, as t h e cis-tr a ns effect. F or a hist or y of t his ter mi n ol o g y see Ha yes (1’7).

E ARLY ST U DIES OF T HE BIT H OR AX M UT A NTS

I n 1 9 4 5, t h e ti m e s e e m e d ri p e t o l o o k f or m or e e x a m pl es of t h e Star-asteroid t y pe in the geno me. An intrig uing region of the thir d chro moso me incl u de d t hr e e l o ci wit hi n l ess t h a n o n e centi Morgan; na mely, t he bristle m utatio ns, St u b bl e ( S b), a n d s pi neless (ss) an d a ho meotic m utation, bit h or a x ( bx) ( Fi g. 1). Certain usef ul co mbinations of these m utants ha d alrea dy been synthesize d E d war d B. L e wi s 2 5 1 by Bri dges a n d mai ntai ne d i n t he Caltec h stoc k collectio n. T he recessi ve all el es of S b pr o v e d t o b e at a s e p ar at e l o c us, t h at I n a m e d st u b bl oi d (s b d), l e s s t h a n 0. 1 centi Morgan t o t h e l eft of t h e S b loc us. A n es pecially striki ng posi- ti o n effect occ urs: s b d 2 + / + S b flies ha ve extre mel y s h ort bl u nt bristles, w hile s b d 2 S b / + + flies are wil d-ty pe wit h no trace of t he do mi na nt s hort-bristle p he noty pe of S b/ + fli es. It soo n beca me e vi de nt t hat t he di verse array of existi ng m utatio ns of t he bit horax ty pe hel d co nsi derable pro mise of bei ng a cl uster of ge nes rat her t h a n a m ulti pl e all eli c s eri es. It w as f or t his r e as o n t h at t h e y w er e c h os e n f or st u d y r at h er t h a n wit h a n y b eli ef t h at t h e y w o ul d t ell us s o m et hi n g a b o ut h o w genes control develo p ment. T h e ori gi n al b x m uta nt ha d bee n fo u n d by Bri dges i n 1915 as a tra nsfor- matio n of t he t hir d t horacic seg me nt ( T3) to war d t he seco n d ( T 2), nota bl y ca usi ng t he halteres to beco me partially wi ng-like. Bo dy seg me nts a n d str uc- t ures of t he wil d-ty pe a d ult are correlate d wit h t hose of t he late e mbryo i n Fi g 2. b x was t he first exa m ple of a m uta nt t hat ex hibite d ho meosis, a ter m Bateso n ha d first coi ne d for co nversio n of o ne str uct ure i nto a n ho mologo us o n e [ dis c uss e d i n ( 1 8)]. In 1919, Bri dges fo un d a so me what si milar m utant that f ully co m ple mente d b x, s o h e n a m e d it bithoraxoid ( b x d); i. e., bx/bxd i s

Fig. 2 Co m pariso n of t he ve ntral c utic ular patter n of t he late e m bryo nic stage wit h t hat of t he a d ult stage. M H = ma n di b ular hoo ks; M V T = mi d-ve ntral t uft; V P = ve ntral pits; K O = Keili n’s orga n; V S B = ve ntral s et al belts; D L T = dorsal lo ngit u di nal (trac heal) tr u n k; P S P = posterior s piracle; H = h e a d; T = t h or a ci c; A = a b d o- mi n al; L = l e g; W = wi n g; H = h alt er; C = c o x a; C C = c ostal cell (of wi ng); V = &i n; W O = W heeler’s orga n; S S = se nsill u m (o n seg me nts Al to A 7, i ncl usive); S T = ster nite; A P = a nal plate. Mo difie d fro m (103). 2 5 2 Physiology or Medicine 1995 wil d ty pe i n p he noty pe. Ho wever, he later s ho we d t hat b x D w hi c h W. F. Hollan der ha d fo un d, faile d to co m ple ment either b x or b x d ( 1 9). Alt h o u g h t h e ori gi n al b x m uta nt has 100 % pe netra nce, it is a hi g hl y varia- ble tra nsf or mati o n of, as it t ur ns o ut, o nl y t he a nteri or p orti o n of T3 t o war d a nterior T2. Fort u natel y, t wo ot her b x -li k e m utants, b x 3 4 e ( J. Sc h ultz) a n d b x 3 ( C. Ster n) ha d also bee n save d by Bri dges (19). T hese have 100 % pe ne- tra nce a n d no n-variable weak a n d stro ng tra nsfor matio ns, res pectively, of a nterior T3 to war d a nterior T2. T he wi ng-like halter of t he b x 3 ho mozy- g ot e is s h o w n i n Fi g. 3. B C D

Fig. 3 Ca mera l uci da dra wi ngs of: ( A) t he wil d-ty pe ( T2) wi n g; ( B) t he corres po n di ng a p pe n dage o n T3 of a b x 3 ho mozygote; ( C) t he corres po n di ng a p pe n dage, o n T3 of a b x d 1 0 0 ho mozygote; ( D) t he wil d-ty pe T3 hal- t er. O nl y ( B) a n d ( C) ar e dr a w n t o t h e sa me scale. Re pri nte d fro m (11). Flies ho mozygo us for b x d s ho w 100 % pe netra nce for a partial tra nsfor ma- tio n of o nly t he posterior portio n of T3 to war d posterior T2. T he wi ng-like 1 0 0 halter of a ho mozygote for a n extre me b x d m ut ati o n, b x d , i s s h o w n i n Fi g 3. I n a d diti o n, b x d flies also ha ve t he first ab do mi nal ( Al) seg me nt tra nsfor- m e d t o w ar d T 3, occasionally pro d ucing tiny r u di mentary T 3-li k e le gs. A b x d he mizygote has a well develo pe d T 3-li k e l e g o n t h e tr a nsf or m e d Al ( Fi g 4 D). A crossi ng-o ver a nalysis s ho we d t hat b x D occ u pies a se parate loc us bet wee n t h e b x a n d b x d l o ci, a n d t h er ef or e it w as first r e n a m e d Bit hora x-like (B xl) ( Fi g. 1 ) , a n d l at er, Ultr a bit h or a x ( Ubx) (11). T his a nalysis pro vi de d a n u mber of cis a n d tra ns ge n ot y pes t hat ex hi bite d p ositi o n effects. Exa m ples are s h o w n i n Fi g. 5.

GE NE EV OL UTI O N BY T A N DE M D UPLIC ATI O N

T hese early st u dies were vie we d as s u p porti ng a si m ple hy pot hesis abo ut ho w ne w ge nes arise fro m pre-existi ng ge nes. Base d o n t he work of St urte va nt a n d E d w ar d B. L e wis 2 5 3

Fig. 4 Extre me seg me ntal tra nsfor matio ns. a) Wil d ty pe male. b) a bx b x 3 p b x ho mozygote, in which T 3 is tra ns- for med to ward T 2. c) Wil d t y p e f e m al e, v e ntr al vi e w. d) b x d / Df- P 2 f e m al e, ve ntral vie w having an extra pair of T 3-li k e l e gs o n Al ( un p ublishe d).

Bri dges, alrea dy cite d a bove, t he hy pot hesis pro pose d t hat ne w ge nes evolve fro m ol d ge nes by a t w o- st e p pr o c e s s: t a n d e m ge ne d u plicatio n follo we d by

b x

Fig. 5 Cis-tra ns effects i n v ol vi n g t he bit h or a x ( b x) a n d Ultr a bit h or a x ( here desig nate d B xl ) m uta nts, ill ustrate d by ca mera l u ci d a dra wi n gs of t he dorsal a n d lateral re gio n of T 3 of t he a d ult fl y. T he pair of ge n ot y pes i n eac h vertical col u m n are i de ntical exce pt for t he way i n w hic h t he alleles are distri h ute d bet wee n ho molo- gous chro moso mes. Reprinted fro m (11). 2 5 4 Physiology or Medicine 1995 o ne of t he res ulti ng d u plicates m utati ng to a ne w f u nctio n (11). T his “ ne w” ge ne wo ul d ge nerally not be easily establis he d i n t he po p ulatio n u nless t he ot her, or “ ol d” ge ne, was retai ne d t o carr y o ut t he ol d f u ncti o n. As a res ult t he ge no me wo ul d be ex pecte d to co ntai n cl usters of closely li nke d a n d f u nc- ti o n all y r el at e d g e n es t h at s u p erfi ci all y a ct li k e a si n gl e g e n e. At t h e C ol d S pri n g Har b or i n 1950, I re p orte d (11) o n t he e vi de nce i n s u p p ort of t his hy pothesis fro m three st u dies: of other organis ms; of the above mentione d S , S b a n d b x r e gi o ns; a n d of l z m utants by Green an d Green (20).

A N E ARLY M O DEL OF T HE CIS-TR A NS EFFE CT

A mo del ( Fi g. 6) was also prese nte d at t hat S y m posi u m to acco u nt for t he cis- tra ns effect (11). It was base d o n t he t he n ge nerally acce pte d bioc he mical dog ma t hat ge nes were protei ns, a n d t hat t hey co ul d catalyze e nzy matic reac- ti o ns. T h e wil d-t y p e all el es of a a n d b were ass u me d to co ntrol seq ue ntial st e ps i n a bi o c h e mi c al p at h w a y i n w hi c h a s u bstr at e, S, is c o n v ert e d i nt o t w o pro d ucts, A a n d B, t hat are pro d uce d at t he site of t he ge nes i n t he c hro- moso me. T he a a n d b m utants are ass u me d to lo wer pro d uction of A an d B, s y m b oli z e d as < A a n d < B, r es p e cti v el y ( Fi g 6 1 1). As a r es ult, a b / + + ( Fi g 6 I) is ex pecte d to pro d uce e no u g h B to be wil d t y pe, or nearl y so. B y co n- tr ast, a + / + b (Fig 6 II) wo ul d pro d uce i ns ufficie nt B, a n d t herefore be m uta nt i n p he not y pe (11). T he mo del co ul d t herefore also acco u nt for polarize d cis-tra ns effects. F or exa mple, when b x 3 is o p posite a n extre me x-ray i n d uce d b x d all el e, s u c h as b x d 1 0 0 , b x 3 + / + b x 1 0 0 flies ha ve a very slig ht wi ng-like tra nsfor matio n of t he posterior portio n of t he halteres. O n t he ot her ha n d, t hey ha ve no trace of t h e b x p he noty pe, e ve n t ho ug h t he latter p he noty pe is a more se nsiti ve o ne f or t he detecti o n of sli g ht effects t ha n is t he b x d phenoty pe. Hence b x 3 a p pears to weakly i nacti vate b x d + , b ut e ve n extre me b x d m uta nts d o n ot i nac- ti v at e b x + .

I. II. a t S B S - < A - < B

S A - < B a b b Fig. 6 A n early mo del t o ex plai n cis-trans effects. Paire d ho mologo us c hro moso mes are diagra m med b y t h e l o n g horizo ntal li nes. T wo a djace nt loci ar e s ho w n wit h eit her wil d-ty pe ( +) or m uta nt (a or b) alleles. T he ge nes at t hese loci are ass u me d to catal yze t he reactio n of t he s u bstrate S i nto pro d uct A, a n d pro d uct A to pro d uct B. T he A pro d uct is ass u me d t o re mai n i n t he vici nity of t he loc us w here it is pro d uce d. T he cis c o n- fig uratio n ( part I) pro d uces s ufficie nt B to give a nearly wil d-ty pe p he noty pe. T he tra ns co nfig uratio n ( part II) pro d uces i ns ufficie nt B res ulti ng i n a m uta nt p he noty pe. Re pri nte d fro m ( 1 1). E d w ar d B. L e wis 2 5 5

In retros pect the mo del is no longer co m patible with o ur present kno w- le dge of t he str uct ure a n d f u nctio n of t he ge ne. Ho wever, si nce no ass u m p- tio ns were ma de abo ut t he nat ure of t he pro d ucts S, A a n d B, t h e m o d el mi g ht still be te na ble if S, A a n d B corres po n d to no n-co di n g R N A tra n- s cri pts. T h e r e al v al u e of t his h y p ot h esis w as t h at it l e d t o a n e x p eri m e nt t h at reveale d a ne w pheno menon of “tra ns vectio n,” to be disc usse d belo w.

C O NTRABIT H ORAX-A GAI N OF FU NCTI O N MUTATI O N

I n 1 9 5 4 ( 2 1), an x-ray in d uce d m utation was fo un d that ha d T2 transfor me d to war d T3. This “gain-of-f unction” (22) phenoty pe was therefore the in- verse of t he T3 to T2 tra nsfor matio n c haracteristic of t he bx a n d bx d m uta- tions. S ur prisingly, ma p ping sho we d it to be a do uble m utation ma de u p of a gai n-of-f u nctio n m utatio n, Co ntrabit horax ( Cbx), t h e l o c us of w hi c h li es b e- t w e e n t h e b x a n d U b x loci, a n d a recessi ve loss-of-f u nctio n m utatio n, p ost- bit hor ax ( p b x), t h at o c c u pi es a n e w l o c us dist al t o t h at of t h e ori gi n al b x d m ut a- ti o n. T h us t h e m a p e x p a n d e d t o fi v e l o ci, at w hi c h t h er e w er e m ut ati o ns wit h effects o n o ne or more of t he se g me nts, T2, T3 a n d Al (23). T his cl uster of m uta nt l oci ca me t o be calle d t he U b x do mai n of a m uc h lar ger cl uster, t he bit horax co m plex ( B X- C) ( Fi g. 7). T he latter na me is d eri v-

Fi g. 7 Ge netic a n d Molec ular ma ps of the R X- C. A.) A d ult fe male, sho wing the seg ments affecte d by R X- C m utati o ns. B.) Reg ulatory regio ns ali g n e d t o t h e D N. 4 m a p w hic h co vers 3 3 8 k b ( 1 0 1). C .) M uta nt lesio ns. I nsertio ns ar e i n dicate d by tria ngles, deletio ns by horizo ntal bars, a n d rearra nge me nt break poi nts b y v erti c al arro ws D.) Tra nscri ptio n u nits wit hi n t he t hree do mai ns, Abd B abd A a n d U bx. Alter nate pro moters a n d alt er- n at e s plici n g patter ns are i n dicate d. G L U mat-ks a seq ue nce pre dicte d t o enco de a ho molog of a ma m mali- a n gl uc ose tra ns p orter pr otei n; t he fl y sequence has no a p pare nt f u nctio n i n seg mental s pecificati o n ( 1 0 1). T h e iab- 4 a n d bx d tra nscri ptio n u nits do not enco de pr otei ns (s e e t e xt). T h e iab- 9 thro ugh iab- 5 reg ulatory regio ns co ntrol ex pressio n patter ns of A b d- B ; i a b- 4, ia b- 3 a n d i a b- A regio ns co ntrol a b d- A; a n d t h e b x d , b x, a n d a b x regio ns c o ntr ol U b x. 2 5 6 Physiology or Medicine 1995 e d fr o m “ ge ne c o m plex,” a ter m i nve nte d by Bri nk for a closely li nke d cl us- ter of ge nes t hat he pre dicte d wo ul d be closel y relate d i n f u nctio n (24). Kauf man and his co- workers defined the Antennapedia-co mplex ( A NT- C) t hat c o ntr ols t he i de ntit y of se g me nts a nteri or t o t h ose c o ntr olle d b y t he B X- C ( 2 5). U nli k e t h e b x d m uta nt, p b x has o nl y a tra nsfor matio n of t he posterior por- tio n of T3 to war d posterior T2. T he tra ns heterozygote, b x d + / + p b x , sho ws a p b x p he noty pe b ut no trace of t he tra nsfor matio n of Al to war d T3 t hat is t y pi c al of t h e b x d ho mozygote. Further more, b x 3 + / + p b x als o s h o ws, al beit w e a kl y, a p b x p he n ot y pe, b ut n o trace of a b x p he not y pe. I n bot h of t hese exa m ples t he cis- heterozygotes are wil d ty pe. T h us, polarize d i nacti vatio n of pbx + f u ncti o n ca n be effecte d i n ci s b y eit h er b x d or b x 3

T HE TRA NSVECTIO N P HE NO ME NO N.

O ne of t he pre dicti o ns of t he earl y m o del of t he cis-tra ns effect ( Fi g. 6) was t hat disr u ptio n of so matic pairi ng mig ht i nte nsify t he differe nce bet wee n cis a n d tr a ns t y p e s. S pecifically, heterozygosity for a chro moso mal rearrange- m e nt t h at w o ul d disr u pt p airi n g i n a n a + / + b individual would be expected to ca use a more extre me b phenoty pe. The pre diction was borne o ut, an d a po werful ne w method was discovered for detecting chro moso mal rearrange- me nts i n t he first ge nerati o n after t heir i n d ucti o n. T he met h o d was first use d to meas ure the freq uency of in d uction of s uch rearrange ments in the pro- ge ny of males ex pose d to ne utro ns fro m a n ato mic bo mb test (26). The metho d detects only the majority of rearrange ments having one brea ka ge poi nt i n a “critical” re gio n of so me 500 ba n ds of t he sali var y gla n d chro moso mes; na mely, the region bet ween the centro mere of the thir d chro- moso me a n d t he loc us of t he B X- C. Si milar fi n di ngs were later obtai ne d for t h e decape ntaplegic ( d p p) r e gi o n i n 2 L ( 2 7) a n d f or t h e eyes-abse nt (ey a) re gi o n i n t h at ar m ( 2 8). Alt h o u g h at first o nl y tr a ns genotypes sho we d the pheno menon, it was s o o n f o u n d t h at C b x U b x / + + was als o s u bject t o tra ns vecti o n (23). C b x i n t his ge noty pe was fo u n d to exert a slig ht gai n of f u nctio n of U b x + , c hiefl y ex presse d by s prea d wings an d a re d uce d al ula, when the chro moso mes are paire d. T hat effect is abolis he d ( wi ngs nor mal) w he n pairi ng is disr u pte d by transvection-s u p pressing rearrange ments. As a res ult, it beca me possible to m utagenize wil d ty pe an d to select rearrange ments that abolishe d the weak C b x effect of t he C b x Ubx / + + genotype. A mong the resultant rearrange- ments, so me, as ex pecte d, ha d breaks within the B X- C. These breaks were u nselecte d for a ny effect o n f u nctio n i n t he B X- C ot her t ha n s u p pressio n of transvection. Such rearrange ments, when subsequently teste d over deletions of t he B X- C, pro vi de d t he basis for disco veri ng a d ditio nal infra-abdo minal (i a b) regio ns a n d or deri ng all of t he k no w n regio ns fro m i a b- 2 t o iab- 8, i n cl u- si v e. T h e iab- 9 re gio n has bee n i de ntifie d by mea ns of break poi nts associate d E d war d B. Le wis 2 5 7 wit h gai n-of-f u nctio n m uta nts i n t hat regio n, na mely U a b ( 2 9) a n d T a b ( 3 0) ( Fi g. 7). I n t he process of isolati ng tra nsvectio n-s u p pressi ng rearra nge me nts, a sex- linke d m utant was recovere d in t wo in de pen dent cases, whose effect was to e n ha nce t he bit hor ax p he not y pe of b x 3 4 e / U b x. T his m uta nt, origi nally na me d, enhancer-bithorax (e- b x), pr o v e d t o b e a n all el e of t h e z e st ( z) ge ne (31) a n d t o b e li k e t h e z a , or n ull, all el es of Ga ns ( 3 2). It w as so o n fo u n d t hat z ae-b x a s it is no w sy mbolize d, s u p presses tra nsvectio n not o nly i n t he case of t he B X- C b ut als o d p p. T h e z protei n has bee n s ho w n to be a D N A-bi n di ng protei n t hat bi n d s i n vitr o t o t h e U b x ge ne as well as to ot her ge nes (33). Be nso n a n d Pirotta s uggest t hat “tra ns vectio n effects are a by- pro d uct of nor mal i ntrage- ni c z acti o n” (34). Re markably, ta n de m d u plicatio ns for t he B X- C regio n act as po werf ul s u p- pressors of tra nsvectio n, w he n place d o p posite t he C b x U b x chro moso me ( Le wis, u n p ublis he d). Evi de ntly, t he d u plicate regio ns pair intrachro mo- so mally wit h o ne a not her a n d preve nt t he C b x m uta nt fro m gai ni ng access to t h e U b x + regions. In organis ms which lack so matic pairing bet ween ho mol- ogo us chro moso mes, s uch as t h e vertebrates, i ntrac hro moso mal pairi ng of ta n- d e m r e p e ats m a y still o c c ur. I n t h at e v e nt, tr a ns v e cti o n m a y pr o v e t o b e a g e n e- ral pheno menon a p plicable to tan de mly re peate d regions in all organis ms.

M OBILE ELE ME NTS I N T HE BIT H OR AX C O MPLEX

I n 1932 Bri d ges re porte d (35) t he disco ver y of o ne of t he first s u p pressor m ut a nts i n Droso p hil a. H e n a m e d it s uppressor-of- Hairy wi n g [no w sy mbolized su( H w)] a n d f o u n d t hat it acte d as a recessi ve s u p press or of certai n alleles of a n u mber of ot her ge nes. Alt ho ug h t he b x 3 m utati o n ha d bee n sa ve d as a bal- a nce d stock, w he n I use d it i n 1946 t he ho mozygote a p peare d wil d ty pe i n p he noty pe, as if t he m uta nt ha d re verte d. I n fact, t he stock ha d ac q uire d a s u p pressor t hat ma p pe d to t he sa me loc us as t hat of Bri dges’ s u( H w). Hi s m utant ha d been lost, b ut the ne w occ urrence, na me d s u 2 - H w, suppressed t he sa me gr o u p of s pecific alleles as was re p orte d f or s u( H w). I n a d diti o n, w e fo u n d t hat it not o nly s u p presse d b x 3 ,b x 3 4 e a n d b x d, b ut als o s p e cifi c all el es of ma ny ot her ge nes (36). The mechanis m by which s u 2 - H w s u p presses s pecific alleles pro ve d el usi ve u ntil m a n y y e ars l at er, w h e n it w as s h o w n t h at s u c h all el es ar e t h e r es ult of a n i nsertio n of t he mobile ele me nt, gypsy , al most i n variably i n t he no n-co di ng portio n of t he ge ne (37). T he wil d-t y pe s u 2 - H w gene co des for a D N A-bin- di n g protei n (38) t hat is ass u me d to bi n d to s pecific se q ue nces i n t he gyspy ele me nt, t hereby lo weri ng t he rate of tra nscri ptio n of t he ge ne co ntai ni ng t h at el e m e nt ( 3 9). H e n c e, i n t h e s u 2 - H w ho mozygote, fail ure of t he m uta nt protei n to block tra nscri ptio n of t hat ge ne wo ul d restore t he wil d ty pe p he- n ot y pe. I n retros pect, it no w see ms extre mely fort u nate t hat t he early ma p pi ng of m uta nts i n t he Ubx do mai n was carrie d o ut usi ng m utatio ns t hat were i nser- 2 5 8 Physiology or Medicine 1995 ti o ns or deleti o ns. T h us, b x 3 a n d b x d ar e gy psy i nserti o ns ( 7 k b i n l e n gt h), U b x is a “ Doc " mobile ele me nt ( 4 0), a n d p b x a n d C b x are a deleti o n a n d i nsertio n, res pecti vely, of a 17 kb seg me nt of D N A (40). Ha d t hey bee n tr ue poi nt m utatio ns, t hey mig ht t he n ha ve bee n s ubject to ge ne co n versio n, a p he no me no n first discovere d i n f u ngi a n d c haracterize d by hig h negative i nterfere nce over s hort ma p regio ns a n d aberra nt segregatio n of alleles i n a m ei oti c t etr a d [r e vi e w e d b y H olli d a y ( 4 1)]. As a res ult, u na mbig uo us or der- i n g of m ut a nts i n t h e U b x do main wo ul d probably not have been possible.

H ALF-TETR A D M APPI N G OF T HE ULTR ABIT H OR AX D O M AI N

T he great diversity of p he noty pes re prese nte d by m uta nts at t he five k no w n l o ci of t h e U b x do mai n ma de it relatively easy to derive do uble m uta nts a n d, i n t ur n, hi g h er m ulti pl es, i n cl u di n g t h e q ui nt u pl e m ut a nt, b x 3 C b x U b x bx d p b x. Although flanking marker reco mbination provided una mbiguous order- i n g of t hese loci, t he possi bilit y of ge ne co n versio n was of s ufficie nt co ncer n t hat I u n dertoo k a half-tetra d a nal ysis of t hat do mai n. Attac he d a utoso mal ar ms ha d bee n sy nt hesize d, partly to be able to per- for m s uc h a n a nalysis, by I. Ras m usse n a n d E. Orias, worki ng i n my labora- tory (42). Fe males were constr ucte d with the q uint u ple m utant co mbination i n o ne of t he attac he d ar ms a n d t he corres po n di ng five wil d-ty pe alleles i n t he ot her ar m, alo ng wit h a p pro priate fla nki ng markers (Fig. 8); t heir p he- noty pe was i n disti ng uis hable fro m t hat of Ubx/+ (43). A mong approxi mate- ly 221,000 fe male offs ri n g, 19 were t he res ult of exc ha nges i n t he regio ns b et w e e n t h e l o ci of b x 3 a n d p b x. Reci procal crossovers were recovere d si m ult- a n e o usl y fr o m f o ur o ut of fi v e of t h e r e gi o ns a n d w er e e asil y d et e ct e d b y t h eir 539 58.2 5 8. 8 6 2 . 0 6 3 . 1 7 0 . 7

c e n t . I I

I I I I I I I + +

+ + + + + + + I I I I I

I I I I I I l - s + + + + + + + e - -

Fig. 8 Diagra m of the genetic constit ution of attache d 3 R chro moso mes heterozygo us f or a q ui nt u pl e bit h o- r a x m uta nt co mbination an d for closely linke d marker genes. The sy mbols are: cent., centro mere; c v-c, cr oss- vei nless-c ; +, wil d t y p e all el e; b x, t h e B X- C: a, bit hor ax- 3; B, Co ntr a bit hor ax; C, Ultr abit hor ax; d, bit hor axoi d; e, p ost- bit h o r a x; sr, stri pe; s b d 2 , st ubbbloi d-2; gl, gl ass; e s , ebo ny-sooty. The standard map locations are sho wn above t h e m uta nt sy mbols, i n centi Morgan u nits. Re pri nte d fro m (43). Ed ward B. Le wis 2 5 9 ha vi n g stro n g cis-tr a ns effects w he n co m pare d wit h t he mater nal U b x / + p he- noty pe. No ne of t he half-tetra ds s ho we d evi de nce of ge ne co nversio n. As o ne possible ex pla natio n it was s uggeste d t hat “o ne or more of t he m uta nts are associate d with minute rearrange ments which have preclu de d the occurren- ce of i ntrage nic reco mbi natio n” (43). I ha d earlier use d attac he d- X fe males to perfor m half-tetra d a nalyses of t h e w hit e ( w) e y e m ut a nt a n d it s “ all el e, ” w hite- a pticot ( w a ) ( 4 4). E x c h a n g e s bet wee n w a n d a pricot ( a pr), a s I r e n a m e d w a , were detecte d i n t he proge ny of w + / + a pr attac he d- X fe males carryi ng closely li nke d fla nki ng markers. Reci procal crossover pro d ucts of s uch exchanges were recovere d si m ultane- o usly i n several da ug hters. W hereas, w + / + a pr f e m al e fli e s h a v e a p al e pi n k e y e c ol or, w a pr / + + fe males ha ve t he re d e ye color of wil d t y pe. Fla n ki n g mar kers i n dicate d t hat a pr li es t o t h e ri g ht of w , t he ma p dista nce bei ng a b o ut 0. 0 1 centi Morgan. No evi dence of gene conversion was detecte d.

T HE BIT H OR AX C O MPLEX A N D ITS OR G A NIZ ATI O N

Duncan has provi de d a co mprehensive an d thorough revie w of the co mplex (45). I ha ve rece ntl y gi ve n a brief historical re vie w of wor k o n t he ho meotic cl usters i n a n u mber of orga nis ms (46). T he follo wi ng sectio ns will be co n- cer ne d c hiefly wit h t he orga nizatio n a n d f u nctio n of t he B X- C. By ge nerati ng so matic mosaics for t he b x p he noty pe, I was able to s ho w t hat t he effects of t he b x m utants are highly a utono mo us (47). Th us, when c ells m ut a nt f or t h e b x 3 f u nctio n arise fro m i n d uce d so matic crossi ng over i n b x 3 / + a ni mals, t he cells ex press t he ex pecte d m uta nt p he noty pe, na mely T 2- ty pe bristles o n T3, w hic h nor mally lacks a ny bristles. Morata a n d Garcia- Belli do provi de d a d ditional exa m ples an d sho we d that the m utant tiss ue c o ul d aris e fr o m e x c h a n g e e v e nts i n d u c e d as l at e as t h e l ast l ar v al i n st ar ( 4 8). T h us, t he wil d-t y pe pr o d ucts of at least t he U b x do mai n are not diff usi ble to any a p preciable extent, an d s uch pro d ucts contin ually reg ulate the develo p me nt of c utic ular str uct ures of T3 i nt o late lar val life. In 1964, borro wing fro m the then- prevailing bioche mical dog ma base d on t he o pero n mo del, I i nter prete d t he f u nctio n of t he ge nes of t he B X- C to be to “re press certai n syste ms of cell ular differe ntiatio n a n d t hereby allo w ot her s yste ms to co me i nto pla y” (49). Clearl y, t hat f u nctio n co ul d also be to acti- vate ot her s yste ms, as Garcia- Belli do later poi nte d o ut (50). Early st u dies of the B X- C ha d reache d an i m passe until ho mozygotes for deletio ns of parts, or of all, of t he co m plex were fo u n d to ha ve stri ki n g effects o n c utic ular str uct ures of t he late e mbr yo. Si m ple pre paratio ns of late e mbryos cleare d i n a dro p of lactic aci d per mitte d t he st u dy of ma ny e mbry- o nic let hal p he noty pes. It beca me evi dent that the B X- C incl u de d genetic material that progra m- me d t he de velo p me nt of not o nly T3 a n d Al, b ut also all of t he re mai ni ng ab do minal seg ments fro m A2 thro ugh A9, incl usive (29). Th us, ani mals lack- i n g t he e ntire B X- C, as t he res ult of bei n g ho moz y go us for deletio ns t hat 2 6 0 P hysiology or Me dici ne 1995 r e m o v e d all of t h e 8 9 El- 4 ba n ds, were fo u n d to die at t he e n d of e mbryo nic develo p me nt a n d to have a striki ng tra nsfor matio n of t he first seve n ab do- mi nal seg me nts to war d t he T2 seg me nt. T he c utic ular str uct ures i nvolve d i ncl u de a nterior s piracles, ve ntral pits, Keili n or ga ns a n d ot her se nse or ga ns. T he A8 a n d A9 seg me nts tra nsfor m eve n more a nteriorly to war d a hea d seg- me nt, base d o n t heir develo pi ng ti ny r u di me nts of t he ma n dib ular hooks ( Fi g. 2). It is al ways da ngero us to de d uce t he wil d-ty pe f u nctio n of a ge ne fro m a loss-of-f unction m utations, es pecially for genes which affect mor phology. T he wil d-t y pe f u nctio n of major re gio ns of t he B X- C co ul d be i nferre d b y a d d in g th e m to a h o m o z y g o u s d e le tio n o f th e B X -C (Df- P 9 ) ( 2 9). F or e x- a m pl e, a d u pli c ati o n, Dp(3)bxd 1 0 0 t hat i ncl u des a wil d-t y pe c o p y of t he U b x d o m ai n pr o xi m al t o t h e b x d regio n, restores t he lo ngit u di nal trac heal tr ucks i n all seg me nts fro m T2 to A8, i ncl usi ve. T he ge nes of t he B X- C co ntrol t he develo p ment of s pecific str uct ures an d organs of the seg ments rather than seg mentation per se . T he partic ular seg me nts i n w hic h a gi ve n B X- C ge ne is ex presse d is deter mi ne d by t he co mbi ne d actio n of tra ns- reg ulatory ge nes. T he a nal ysis of t he f u ncti o ns of cis -reg ulatory r e gi o ns l o c at e d dist al t o t h e U b x do main, made use of chro moso mal rearrange ments having breakpoints in those regions. S uch rearrange ments have a recessive loss-of-f unction ex presse d as a tra nsfor matio n of a posterior seg me nt to war d a more a nterior o ne; t h us rearra nge me nt break poi nts i n t he iab-2 cis-reg ulatory regio n ca use A2 to tra nsfor m to war d Al. B y 1978, t hree ia b regio ns ha d bee n i de ntifie d, iab- 2,- 3 , a n d - 8, a n d a f o urt h, ia b 5, was i nferre d fr o m a n a nal ysis of re verta nts of a do mi na nt gai n-of-f u nctio n m utatio n, Miscadastral pig me ntatio n ( Mcp) b y M. Crosby (29). S ubseq uently, the regions of the B X- C controlling ab do mi- nal develo p ment were divi de d into t wo do mains, abdo minal- A (abd- A) a n d Abdo minal- B ( Abd- B) , base d o n let hal co m ple me ntatio n st u dies (51, 52).

R ULES G OVER NI N G CIS-RE G UL ATI O N OF T HE BX-C

T h e B X- C is r e g ul at e d i n cis a n d i n tra ns . R ules gover ni ng its ® ulatio n are consi dere d first an d were de d uce d fro m genetic analysis. Many of the r ul es ar e hi g hl y u n us u al a n d p ossi bl y u ni q u e. It s e e ms li k el y t h at t h eir m ol e- c ular analysis will reveal hitherto uns us pecte d reg ulatory mechanis ms. C oli n e arit y. T h e r ul e of c oli n e arit y ( C O L) states t hat t he or der of t he B X- C l oci i n t he c hr o m os o me parallels t he or der i n w hic h t he u nits at t h ose l oci are ex presse d alo ng t he a ntero- posterior axis of t he bo dy. T wo ty pes of gra- di e nts h a d b e e n i n v o k e d t o e x pl ai n t his r ul e: “a n a ntero- posterior gra die nt in re pressor concentration along the e mbryo an d a proxi mo- distal gra dient alo ng t he c hro moso me i n t he affi nities for re pressor of eac h ge ne’s cis- re g u- latory ele me nt” (29). Molec ular st u dies co nfir me d t he r ule a n d exte n de d it to all of t he ab do m- i n al cis -reg ulatory regio ns fro m iab- 2 t o iab- 8 , i ncl usive (53). Associate d wit h t he C O L r ule is t he stro ng te n de ncy for t he protei ns of t he B X- C, o nce Ed ward B. Le wis 2 6 1 ex presse d to contin ue to be ex presse d more posteriorly in the bo dy exce pt f or t h e t er mi n ali a. T his is el e g a ntl y s h o w n i n Fi g. 9, f or t h e U b x, a b d- A a n d A b d- B protei ns vis ualize d by t he use of i m m u nostai ni ng wit h a ntibo dies s pe- cifi c t o e a c h.

Fig. 9 E mbryos stai ne d wit h monoclonal a ntibo dies to t he protei n pro d ucts of t he B X- C. Pre paratio ns are of l0-12 hr e mbryos, s plit alo ng t heir dorsal mi dli ne a n d flatte ne d. Brackets i n dicate paraseg ments 5 a n d 12, which corres pon d a p proxi mately with the thir d thoracic an d seventh ab do minal seg ments, res pectively. U b x protei n a p pears i n paraseg ments 5- 1 3, ab d- A p r ot ei n i n 7- 1 3, a n d Ab d- B p r ot ei n i n l0-13 (fr o m W. Be n der, u n p u b l i s h e d )

Cis-i nacti vati o n. T he sec o n d r ule of cis -i nacti vati o n ( CI N) states t hat l oss- of-f u nctio n m utatio ns i n a gi ve n cis -re g ulator y re gio n te n d to i nacti vate t he next more distal region of the co m plex. Exa m ples have alrea dy been cite d f or t h e p ol ar i n a cti v ati o n of t h e p b x + f u n cti o n b y b x 3 a n d b y b x d. Ot her exa m- ples were later fo u n d i n a nalyzi ng rearra nge me nt break poi nts i n t he i a b re gio ns of t he B X- C (54). It has not bee n possi ble to esta blis h w het her t here are CI N effects bet wee n major do mai ns of t he B X- C. Cis-o verex pressio n. T he t hir d r ule of cis -o verex pressio n ( C O E) is a q uite s ur prisi n g o ne. T he r ule states t hat t he l oss of f u ncti o n ass ociate d wit h a gi v e n cis -reg ulatory regio n te n ds to be acco m pa nie d by a n overex pressio n of t he f u nctio n associate d wit h t he cis -reg ulatory regio n t hat lies i m me diately proxi mal to it. I n t he ab do mi nal do mai ns, rearra nge me nts wit h breaks i n t h e i a b- 3 regio n, for exa m ple, not o nly ha ve a loss-of-f u nctio n i a b- 3 p he no- ty pe ( A3 tra nsfor me d to war d A2) b ut a gai n-of-f u nctio n of t he iab-2 + r e gi o n t hat is ma nifeste d as a tra nsfor matio n of t he Al seg me nt to war d A2. Ot her exa m ples have bee n describe d (54). C O E effects are kno wn not only for break points of chro moso mal rearran- ge me nts b ut for gy psy i nsertio ns. A n i m porta nt o ne is a C O E effect of b x 3 . Flies ho mozygo us for b x 3 ha ve a re d uctio n i n t he extre me a nterior re gio n of T2. T his effect is do mi na nt si nce it is not s u p presse d by d u plicatio ns t hat totally s u p press t he recessi ve b x 3 tra nsfor matio n of T3 to war d T2. A n x-ray in d uce d m utant, anterobithorax ( a b x), was discovere d t hat ha d a weak bit h o- rax-li ke p he not y pe. It is locate d j ust proxi mal to b x, a n d a b x b x 3 do uble m uta nts lac k t he C O E effect o n T2 see n i n t he b x 3 si ngle m uta nt ge noty pe. 2 6 2 P h ysi ol o g y or Me dici ne 1995

U ntil a b x h a d b e e n f o u n d, it w as n ot p ossi bl e t o a c hi e v e a f ull tr a nsf or m ati o n of T 3 t o w ar d T 2; i. e. t h e b x 3 p b x d ou bl e m uta nt ho mozygote fails to tra ns- for m t he most a nterior portio n of T3. Flies ho mozygo us for t he tri ple m ut a nt, a b x b x 3 p b x , were constr ucte d an d prove d to have virt ual co m plete tra nsfor matio n of t he wi ng a n d c utic ular str uct ures of T3 tra nsfor me d t o w ar d t h os e of T 2, r es ulti n g i n a f o ur- wi n g e d fl y ( Fi g. 4).

NE G ATIVE TR A NS-RE G UL ATI O N OF T HE BIT H OR AX C O MPLEX.

In 1947, a re markable x-ray in d uce d do minant m utant, Polyco mb ( P C ), w a s f o u n d b y P. H. Le wis (55). It ha d sex c o m bs o n n ot o nl y o n t he first, b ut t he 2 n d a n d 3r d pair of legs, a n d r u di me ntary a nte n na1 to leg tra nsfor matio ns rese mbli ng t hose of Antennapedia ( A ntp) m uta nts. It als o ha d effects t hat were o nl y l at er r e ali z e d t o b e g ai n of f u n cti o n of g e n es i n t h e U b x do main; na me- l y, re d uctio n i n t he extre me a nterior re gio n of T2 a n d re d uctio n i n t he wi n g si mil ar t o t h at of w e a k C b x p h e n ot y p es, s u c h as i n C b x U b x / +. It w a s n e arl y 3 0 y e ars b ef or e it w as r e ali z e d t h at P c i s a m ut ati o n i n a g e n e t h at a ct s a s a n e g a- ti ve reg ulator of t he B X- C, a n d of t he A N T- C co m plex as well. T h us, t he ho mozygous P c e mbryo has t he t hree t horacic a n d t he first se ve n ab do mi nal seg me nts all tra nsfor me d to war d A8, pres u mably as the res ult of dere pres- si o n of t h e A b d- B do mai n (29) [fig ure d i n D u nca n ( 5 6)]. D u nca n fo u n d a seco n d m uta nt of t he Polyco mb t y p e, Polyco mb-like ( Pc-l) ( 5 6). P C a n d P cl ha ve pr o ve d t o be b ut t w o of a fa mil y of ge nes t hat act as ne gati ve re g ulators (57). T hat t he P c pr ot ei n is i n v ol v e d i n bi n di n g t o t h e B X- C a n d t he A N T- C regio ns (as well as to ot her regio ns) has bee n elega nt- ly sho wn by i m munostaining of salivary gland chro moso mes with an anti- b o d y t o t h at pr ot ei n ( 5 8). Si n c e t h e P c protein is a non-histone chro moso mal protei n, rat her t ha n a D N A-bi n di ng protei n (59) its bi n di ng s pecificity may r e si d e i n it s co mplexing wit h pr otei ns of ot her ge nes of t he P c fa mily, so me of w hi c h first bi n d s p e cifi c all y t o B X- C a n d A N T- C.

P OSITIVE TR A NS-RE G UL ATI O N OF T HE BIT H OR AX C O MPLEX

P ositi v e tra ns- reg ulators w er e als o f o u n d, s u c h as Re g ul ator of bit hor ax ( Rg-bx). A n a nal ysis of t his m uta nt, a n d of deficie ncies w hic h i ncl u de t he loc us, i n di- c at e t h at t h e wil d-t y pe ge ne is a positi ve re g ulator of t he B X- C (60). A par- ti al l oss- of-f u n cti o n all el e, trit hor ax (tr x), was t he n fo u n d by I ng ha m (61). T he t r x ge ne has bee n clo ne d a n d is a D N A-bi n di ng protei n of t he zi nc fi nger category (62, 63). More rece ntly, Ke n niso n a n d Ta mk u n have i de ntifie d a f a mil y of g e n es li k e t r x t hat act w he n m utate d as e n ha ncers of b x phenotypes ( 6 4). A d diti o n al cl ass es of tr u ns -re g ulators of t he B X- C ha ve co me fro m t he st u- dies of N usslei n- Vol har d a n d Wiesc ha us (3). For exa m ple, t he ga p ge ne, h u nc hback ( hb), is i nvolve d i n establis hi ng major s ub divisio ns of t he bo dy re gio ns. It e nco des a zi nc li n ger protei n a n d acts as a ne gati ve re g ulator of E d w ar d B. L e wi s 2 6 3 t he B X- C, kee pi ng t he co m plex t ur ne d off i n t he a nterior regio ns of t he bo dy, pres u mably by t he bi n di ng of t he h b pr otei n t o at least o ne s pecific m otif i n t h e U b x ge ne (65). A do mi na nt m uta nt, Reg ul ator of postbit hor ax ( Rg- p b x), is no w k no w n to be a gai n-of-f u nctio n m utatio n i n t he h b g e n e ( 6 6). It pro d uces variable pbx-like tra nsfor matio ns of t he halter (67). A not her exa m ple is t he Kr üpple ( Kr) ge ne of Gl o or (68). It is als o a ga p ge ne a n d e nco des a D N A-bi n di ng protei n (69, 70). O ne motif to w hic h it bi n ds is i n t h e i a b 2 region an d, on t wo in depen dent occasions, a m utation i n a si ngle s pecific base pair of t hat motif has res ulte d i n a do mi na nt Hyperabdo minal ( H a b) p he noty pe (71). T hese gai n-of-f u nctio n m uta nts have poor pe netra nce, b ut i n so me crosses H a b / + flies occasi o nall y ha ve o nl y fo ur legs a n d no halteres o wi ng to T3 bei ng tra nsfor me d to war d A2 (29).

M OLEC UL AR A N ALYSIS OF T HE BIT H OR AX C O MPLEX.

M olec ular a nal ysis of t he U b x do mai n of t he B X- C was i nitiate d by D. Hog ness a n d co- workers i n 1978 a n d t hey soo n i de ntifie d t he major feat ures of t h at r e gi o n. T h e b x m ut a nts, U b x, a n d s e v er al b x d m uta nts all pr o ve d t o be i nsertio ns of tra ns posable ele me nts (40). Molec ular st u dies reveale d a si ngle tra nscri ptio n u nit co di ng for protei ns i n t he U b x d o mai n (72, 73). T he e mbryonic distribution of the Ubx protein pro ducts was deter mine d by W hite a n d Wilcox (74) a n d by Beac hy et al., (75) see als o Fi g. 8. T he tra ns- cri ptio n u nit a n d protei n pro d uct of t he seco n d do mai n, abdo minal- A, w er e c haracterize d by Karc h et al. , ( 7 6). T h e t hir d d o m ai n, Abdo minal- B, pro d uces at least f o ur tra nscri pts (77-81) a n d t w o Ab d- B pr ot ei ns ( 8 0, 8 2, 8 3). S ur prisi n gl y, t he cis -re g ulat or y re gi o ns are tra nscri pti o nall y acti ve, as first s h o w n f or t h e b x d re gio n of t he B X- C (84). T his re gio n pro d uces a lar ge ( 2 6. 5 k b) pri m ar y tr a ns cri pt, t h at is t h e n s pli c e d t o yi el d a f a mil y of n o n- pr o- tei n c o di n g R N As (i.e., co ntai ni n g m ulti ple sto p co do ns). Si milar n o n- co di ng tra nscri ptio n u nits are k no w n for t he i a b- 4 r e gi o n ( 8 5).

T HE TRA NS-ABDO MI NAL MUTATIO N

Ki ng a n d Wilso n (86) calle d atte ntio n to t he possible i m porta nce i n evol u- tio n of creati ng novel p he noty pes solely by rearra nge me nts i nvolvi ng cis- reg ulatory seq ue nces. A striki ng exa m ple was o ur disco very of a n X-ray i n d uc- e d do minant m utation, Transabdo minal ( Tab). Tab / + fli es h a v e a s e x u all y di mor p hic patter n of pig me nte d ba n ds i n t he dorsal t horax of T2 (Fig. 10). U nlike t he great majority of do mi na nt gai n-of-f u nctio n p he noty pes, T u b / + has 100 % penetrance an d co m plete ex pressivity. Molec ular an d mor pholo- gical st u dies (30) i n dicate t hat t he pig me ntatio n patter n of t he ba n ds rese m- bles t hat nor mally fo u n d i n t he tergites of seg me nts A5 a n d A6. T h us, t he pig me nte d ba n ds i n t he T a b / + male d orsal t h orax are br oa d as i n t he A5 a n d A 6 m al e t er git e s; w h er e a s, i n t h e T a b / + f e m al e t h e y ar e n arr o w a s i n t h e corres pon ding fe male tergites. The T a b m uta nt is associate d wit h a n i n ver- 2 6 4 Physiology or Medicine 1995

Fi g. 1 0 Tra mab do mi nal, a sex ually di morphic mutant of tbe Abdo minal- B do main. a) Wild type male. b) T a b / + m al e. c) T a b / + f e m al e. T horacic tra nsfor matio ns are descri be d i n t h e text. ( U n p u blis he d). sion having o ne break poi nt i n the iab-9 cis -reg ulatory regio n (Fig. 7) a n d t he ot her near t he stri pe (sr) l o c us i n 9 0 D which co des for an early gro wth-res pon- se tra nscri pti o n fact or (87). I n sit u st u dies (88) of t he dorsal t horacic disc of T 2, w hi c h gi v es ris e t o t h e d ors al t h or a x, s h o w c ells i n Ta b / + a ni m als t h at ex press the Ab d- BII protein an d its R N A. These cells corres pon d to the sites of t h e b a n ds i n t h e T a b / + a d ult t horax a n d a p pear to be t he sites of attac h- ment for certain thoracic m uscles. O ur st u dies of the R N A an d protein dis- tri b uti o ns i n e m br y os a n d i m a gi n al dis cs i n di c at e t h at t h e Ta b m utation re presents a case in which ® ulatory regions of a gene involve d in defi- ning the develo p ment of m uscle attach ment sites is no w driving Abd- B pr o- tein ex pression (87). Other minor dist urbances in the ab do minal tergites of Ta b / + flies are believe d to i nvolve ecto pic ex pressio n of t he Abd- B pr otei n in s uch attach ment sites for ab do minal tergite m uscles.

CO NTROL OF SO MATIC GO NAD DEVELOP ME NT I N DROSOP HILA A N D B O MBYX. As early as 1943, Itika wa re porte d (89) on a m utant designate d E N w hose phenoty pe when ho mozygo us parallels closely that of the ho mozygo us defi- ciency for the B X- C in Droso phila ( Df- P 9 ). Iti k a w a’s dis c o v er y t h at c ert ai n m ut a nts of t h e “ E ” series lacke d go na ds le d me to exa mi ne a do mi na nt m ut a nt, Ultra-abdo mi nal 4 (Ua b 4 ), w hi c h is ass o ci at e d wit h a r e c essi v e iab- 3 p he noty pe. I nter nally, t he U a b 4 he mizygote was found to lack gonads (29). S u bs e q u e ntl y, I f o u n d t h at r e arr a n g e m e nts wit h br e a k p oi nts i n t h e iab- 4 region of the B X- C, when viable as ho mozygotes a p pear virt ually wil d ty pe, Ed ward B. Le wis 2 6 5 b ut i nt er n all y t h e y l a c k g o n a ds. ( 5 4). L oss of gona ds i n i a b- 2 a n d iab-3 m utant a ni m als r es ults fr o m cis-i n a cti v ati o n of t h e iab-4 r e gi o n [ L e wis, u n p u blis h e d]. Si nce t he g o na d is of mes o der mal ori gi n, its l oss was o ne of t he first i n dica- tio ns t hat t he B X- C p he noty pes were not li mite d to ecto der mal tiss ues. A co m parative molec ular a nalysis of t he i a b 4 cis-reg ulatory wit h regio ns co ntrolli ng go na d for matio n i n Bo mbyx a n d ot her a ni mals may s ho w ho w t he ho meotic ge nes co ntrol t he develo p me nt of a s pecific str uct ure. T h us, si nce so me of the more pri mitive non-seg mente d ani mals, s uch as the ne mato de, ha ve s o matic g o na ds, it is li kel y t hat c o ntr ol of t he i nitiati o n of t heir de ve- lo p me nt will ha ve co m mo n feat ures. Of great i nterest will be t he target ge nes i n Drosophila t hat acco m plis h s uc h i nitiatio n. A pro misi ng a p proac h to un derstan ding the process in h u man beings can be ex pecte d to co me fro m a nalyzi ng molec ularly t he basis of i n herite d defects i n t he h u ma n go na d.

T HE H O ME OB OX A ND TA NDE M GE NE DUPLICATI O N

Molec ular s u p port for the ass u m ption that tan de m gene d u plication was res po nsible for at least t he co di ng portio ns of t he B X- C a n d t he A N T- C co m- plex fi nally ca me wit h t he discovery of t he ho meobox i n 1984, by Mc Gi n nis et al. ( 9 0) an d Scott an d Weiner (91) who in de pen dently sho we d that the protei ns e nco de d b y t he U b x a n d A nt p ge nes contain a re markably conserve d group of a mino acids, kno wn as the ho meodo main. The D N A sequence encoding the ho meodo main was na med the ho meobox (90). The ho meo- box seq ue nce is co nserve d to a re markably hig h degree t hro ug ho ut t he a ni- mal ki ng do m a n d it was use d to probe for ho mologs of t he B X- C a n d A N T- C in many other organis ms, incl u ding vertebrates as well as invertebrates (92). Most of t hese orga nis ms have t he ho mologs of bot h t he B X- C a n d t h e A N T- C in a single co mplex kno wn as the ho meotic co mplex ( H O M- C). In unseg mente d organis ms like Caenorhabditis ( 9 4) there are a p parently o nly a fe w H O M- C ge nes. I nsects s uc h as t he silk wor m, Bo mbyx ( 9 5) a n d t h e fl o ur beetle, Tri b oli u m ( 9 6) ha ve lar ger cl usters as i n Dr os o p hil a. T he most pri miti ve vertebrates re prese nte d by t he la ncelet, A mphioxus ( 9 7, 9 8) als o have a single large H O M- C. Ho wever, higher vertebrates have Four se mi- re d un dant co pies of the H O M- C. In the mo use an d h u man, each co py is on a differe nt c hro moso me. T his re d u n da ncy makes it diffic ult to dissect t he f u ncti o n of a gi v e n ge ne i n a n y o ne of t he sets. Re mar ka ble pro gress is bei n g ma de by usi ng ge ne k nock-o ut tec h niq ues i n mice, to st u dy t he role of t he H O M- C genes in develo p ment. H O M- C gene ex pression in the mo use, as in Dr os o p hil a, o b e ys t h e r ul e of c oli n e arit y [revie we d by Le wis ( 4 6)]. T heir se g- me ntal ex pressi o n li mits are als o re g ulate d i n tr a ns b y ge nes t hat are re mar k-

a bl y p ar all el t o t h os e of t h e P C Gro u p a n d tr x Group (revie wed by Si mon ( 9 9). H O M- C genes are no w regar de d as master control genes whose proteins bi n d t o t h e cis -re g ulator y re gio ns of tar get ge nes. T he latter t he n acti vate or re press syste ms of cell ular processes that acco m plish the final develo p ment 2 6 6 Physioloy or Medicine 1995 of t he orga nis m. Eve n mi nor m uta nt lesio ns i n H O M- C genes may be ex pec- te d to ha ve global effects o n s uc h syste ms. A n exa m ple is a targete d ge ne- disr u ptio n of t he mo use H O X A3 ge ne (for merly H O X 1.5) t hat lea ds to defects i n t he t hyroi d gla n ds a n d s urro u n di ng tiss ues (100). T he res ulta nt gro u p of defects rese m bles t hose see n i n t he co n ge nital Di George syndro me of h u man beings.

C O MPLETE SE Q UE NCE OF T HE BIT H OR AX C O MPLEX.

T he D N A seq ue nce of t he B X- C has no w been co m pletely deter mine d (101) a n d a preli mi nary a nalysis ma de of it (102). T he protei n co di ng regio ns co m prise o nly 2 % of t he e ntire seq ue nce. T he ot her 9 8 % is e x p e ct e d t o c o n- tai n a di verse gro u p of motifs to w hic h tra ns -reg ulatory protei ns bi n d, t he- reby co nferri ng t he s pecific s patial a n d te m poral ex pressio n of t he protei n pro d ucts of eac h do mai n. T here may also be a reg ulatory role for n o n- c o di n g R N A’s of t h e t y p e i d e ntifi e d i n t h e b x d a n d iab- 4 r e gi o ns.

T HE NEXT FIFTY YE ARS

O nly t hree of t he ma ny f ut ure c halle nges will be o utli ne d: (1) molec ular an d genetic approaches are nee de d to deter mine the i m me diate target ge nes t hat are t ur ne d o n or off by t he ge nes of t he H O M- C; (2) si nce t he g e n es of t h e H O M- C h a v e t e n d e d t o r e m ai n ti g htl y li n k e d a n d c oli n e ar wit h t heir ex pressio n patter ns alo n g t he bo d y axis, it will be exciti n g to disco ver the un derlying mechanis ms that have kept the m together an d; (3) co mpa- rative D N A sequence analysis of the H O M- C a mong many different orga- nis ms may provi de evi de nce t hat t he cis -reg ulatory regio ns have evolve d by tande m duplication. Ulti mately, co mparisons of the H O M- C throughout the a ni mal ki ng do m s ho ul d provi de a pict ure of ho w t he orga nis ms, as well as the genes of the H O M- C, have evolve d.

CO NCLUSIO NS

Basic research concerne d with testing a si m ple hy pothesis abo ut ho w ne w ge nes arise fro m ol d ge nes le d after ma n y circ uito us ro utes to t he disco ver y of t he hor n &tic co m plex ( H O M- C). T his cl uster of master co ntrol ge nes progra ms m uch of the develo p ment of all higher ani mal organis ms. Each of the genes contain a ho meobox, a re markably conserve d D N A sequence that provi des molec ular s u p port for t he hy pot hesis t hat t he co m plex itself arose b y a pr ocess of tan de m gene d u plication. The high degree of conservation of t he H O M- C, itself, bet wee n vertebrates a n d i n vertebrates i n dicates t hat it arose fro m a n a ncestral co m plex over 5 0 0 millio n years ago, t he esti mate d ti me of se paratio n of t hese t wo great gro u ps of orga nis ms. It is likely t hat m utatio ns wit hi n t he H O M- C’s of h u ma n bei ngs are t he ca use of certai n ge netically base d ab nor malities t hat arise at vario us stages E d w ar d B. L e wis 2 6 7

of hu man develop ment. So matic mutations in genes of the H O M- C may con- ceivably be i nvolve d i n t he ge neratio n of t u mors. Mea n w hile, f ut ure ge netic a n d molec ular st u dies of t he H O M- C i n lo wer creat ures t hat ha ve b ut o ne set of t he co m plex pro mise to a d va nce o ur u n dersta n di ng of its role as a master reg ulator of develo p me nt. M uc h has bee n lear ne d abo ut t he role of t he H O M- C i n develo p me nt, a n d abo ut its molec ular pro d ucts. Ne vert heless, we are still u nable to make se nse of m uc h of t he D N A seq ue nce of t he bit horax co m plex ( B X- C) or to ex plai n h o w t h e c o m pl e x is its elf r e g ul at e d. Pr o gr ess will still n e e d t o b e dri v e n b y t he lo gic of ge netics a n d b y f urt her i ncreases i n a bstractio n.

ACKNO WLEDG MENTS

Recent work on the B X- C has been s u p porte d by research grants fro m the Natio nal I nstit utes of Healt h, t he A CS a n d t he Marc h of Di mes. I thank Welco me Ben der, Ho war d Li pshitz, S usan Celniker an d Joanne To pol, for a critical rea di n g of t he ma n uscri pt, a n d Jo h n K nafels, Victor Hs u a n d Bet h T ur ner for assista nce i n t he pre paratio n of it. A nti bo dies were ki n d- l y pr o vi de d b y R. W hite, a gai nst U b x pr otei n a n d b y 1. D u nca n a gai nst a b d- A. I a m i n d e bt e d t o W. B e n d er f or pr o vi di n g Fi g. 9. W hil e at C alt e c h, a n u mber of colleag ues have directly co ntrib ute d to o dr researc h, na mely, Welco me Ben der, Marie- Paz Ca p devila, S usan Celniker, L ori n g Cray mer, Ma deline Crosby, Ian D uncan, Antonio Garcia- Belli do, Willia m Gelbart, Alain G hyse n, H a ns Gl o or, E. H. Gr ell, R h o d a Gr ell, Lil y J a n, B ur k e J u d d, H o w ar d Li ps hit z, Margit Lohs-Schar din, Rolf Nothiger, E d uar do Orias, Inge Ras m ussen an d S hi g e S a k o nj u. Fi n all y, I w a nt t o str ess t h e cl os e c o o p er ati o n t h at w e h a v e h a d over the years with Davi d Hogness an d colleag ues at Stanfor d University, Welco me Ben der at Harvar d Me dical School an d Ian Duncan at Washington U niversity. It was Davi d Hog ness’ foresig ht to la u nc h t he molec ular a nalysis of t he bit horax co m plex i n 1978 i n his la borator y.

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