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Chromosome Research 1994, 2, 405-41 0

T h e o r ig in o f h u m a n c h r o m o s o m e 2 a n a l y z e d b yc o m p a r a t iv e c h r o m o s o m e m a p p i n g w i th a D N Am i c r o l i b r a r y

J . Wienberg , A . Jauch , H . -J . L0decke, G . Senger ,B . Horsthem ke, U . C lausse n , T. Crem er , N . Arno ld& C . L e n g a u e rRec eived 7 March 1994; rece ived in rev ised form 5 May 1994;A ccep ted f o r pub li ca t ion by M. S chmid 5 M ay 1994

F l u o r e s c e n c e in s itu h y b r i d i z a t i o n ( F I S H ) o fm i c r o l i b r a r i e s e s t a b l i s h e d f r o m d i s t i n c t c h r o m o -s o m e s u b r e g i o n s c a n t e s t th e e v o l u t io n a r y c o n s e r v a -t io n o f c h r o m o s o m e b a n d s a s w e ll a s c h r o m o s o m a lr e a r r a n g e m e n t s t h a t o c c u r r e d d u r i n g p r i m a t e e v o l u -t i o n a n d w i ll h e l p t o c l a r if y p h y l o g e n e t i c r e l a ti o n -s h i p s . W e u s e d a D N A l ib r a r y e s t a b l i s h e d b ym i c r o d i s s e c t i o n a n d m i c r o c l o n i n g f r o m t h e e n t i r el o n g ar m o f h u m a n c h r o m o s o m e 2 fo r f l u o r e s c e n c e insitu h y b r i d i z a t io n a n d c o m p a r a t i v e m a p p i n g o f t h ec h r o m o s o m e s o f h u m a n , g r e a t a p e s (Pan t roglo-dytes , P an paniscus , G or i l la gor i l la, P ongopygmaeus) a n d O l d W o r l d m o n k e y s (Macaca fusc ataa n d Cercopi thecus aeth iops) . I n v e r s i o n s w e r e f o u n di n t h e p e r i c e n t r ic r e g i o n o f th e p r i m a t e c h r o m o s o m e2 p h o m o l o g s i n g r e a t a p e s , a n d t h e h y b r i d i z a t i o np a t te r n d e m o n s t r a t e s t h e k n o w n p h y l o g e n e t i c a l l yd e r i v e d t e l o m e r e f u s i o n i n t h e l i n e th a t l e a d s t o h u -m a n c h r o m o s o m e 2 . T h e h y b r i d i z a ti o n o f t h e 2 qm i c r o li b ra r y t o c h r o m o s o m e s o f O l d W o r ld m o n k e y sg a v e a d i f fe r e n t p a t t e r n f r o m t h a t i n th e g o r i l l a a n dt h e o r a n g - u t a n , b u t a p a t t e r n s i m i l a r to th a t o f c h i m -p a n z e e s . T h is s u g g e s t s c o n v e r g e n c e o f c h r o m o -s o m a l r e a r r a n g e m e n t s i n d i f fe r e n t p h y l o g e n e t i cl i n e s .Key words: evolut ion , f luorescent i n s i t u hybr idizat ion,microdissect ion, phylogeny, pr imates

I n t r o d u c t i o nC o m p a r a t i v e b a n d i n g a n a ly s is b e tw e e n h u m a n c h ro -m o s o m e s a n d t h e k a r y o t y p e o f g r e a t a p e s su g g e s ts

t h a t h u m a n c h r o m o s o m e 2 w a s d e r i v e d b y a fu s i o n oft w o p r i m a t e h o m o l o g s ( D u t r il l au x 1 97 9, Y u n i s &P r a k a s h 1 98 2). T h i s h a s b e e n c o n f i r m e d b y f l u o r e s -c e n c e i n s i t u h y b r i d i z a t i o n ( F IS H ) o f a h u m a n c h r o m o -s o m e 2 - sp e c if ic D N A l i b r a r y to p r i m a t e c h r o m o s o m e s( W i e n b e r g e t a l . 1 9 9 0 , 1 9 9 2 , J a u c h e t a l . 1 99 2). H o w e v e r ,t h e e x a c t p h y l o g e n e t i c i n t e r p r e t a t io n o f th e c h r o m o -s o m e r e a r r a n g e m e n t s n o t e d b e t w e e n t h e s e s p e c i e s i ss t i l l n o t c l e a r . O w i n g t o t h e p r e s e n c e o fp e r i c e n t r o m e r i c h e t e r o c h r o m a t i n i n g r e a t a p e c h r o -m o s o m e s , i t i s d i f f i c u l t t o m a t c h t h e h o m o l o g o u sb a n d s c l os e to t h e c e n t r o m e r e s b y c la s si ca l b a n d i n gs t u d ie s . F r o m h i g h - re s o l u t i o n c h r o m o s o m e b a n d i n g ,Y u n i s & P r a k a s h ( 19 82 ) p r o p o s e d a c o m m o n d e r i v e d( s y n a p o m o r p h i c ) p e r ic e n t r ic i n v e r s i o n in t h e h o m o l o gt o h u m a n c h r o m o s o m e 2 p i n a s u g g e s t e d c h i m p a n -z e e / h u m a n p h y l o g e n e ti c li n ea g e, w h i c h w a s f o l l o w e db y a f u s io n e v e n t in t h e h u m a n k a r y o t y p e .

A m o r e s e c u re d e m o n s t r a ti o n o f t he h o m o l o g y b e -t w e e n i n d i v i d u a l b a n d s i s p r o v i d e d b y f l u o r e s c e n c e i ns i t u h y b r i d iz a t io n o f c h r o m o s o m e b a n d - s p e ci f ic D N Al i b r a r i e s ( L e n g a u e r e t a l . 1 9 9 1 a , b ; T r a u t m a n n e t a l .1 99 1) . T h e c o n s t r u c t i o n o f s u c h l i b r a ri e s i s p o s s i b l e b ym i c r o d i s s e c t i o n a n d m i c r o c l o n i n g ( L f i d e c k e e t a l .1 98 9) . W e h a v e u s e d a m i c r o l i b r a r y e s t a b l i s h e d f r o mt h e lo n g a r m o f h u m a n c h r o m o s o m e 2 f o r c o m p a r a t iv ec h r o m o s o m e m a p p i n g o f h u m a n a n d p r i m a te s p ec ie s .T h e c h r o m o s o m e s o f O l d W o r l d m o n k e y s w e r eu s e d a s a n ' o u t g r o u p ' t o a n a l y z e t h e d i r e c t i o n o fc h r o m o s o m e c h a n g e s t h a t o cc u r r ed d u r i n g h o m i n o i de v o l u t i o n .

J . Wienberg (corresponding author) and N. Arn old are at the Ins t i t ut fa r Anthropologie un d Hum angene tik , Univers i tdt M iinchen , R ichard-WagnerS t r as s e lO f t , D-80333 M unic h , Ge r many . F ax : (+ 49) 89 5203 286 . A . J auc h is a t the Ins t i tu t fa r Hum ange ne t ik u nd A n thr opo log ie, Un iv er s it~ tHeide lberg, Im N euenhe imer Fe ld 328, D-69120 Heide lberg, Germany . H .-J . Uidecke and B . H ors themke are at the Ins t i tut fa r H umangene tik ,Univers i t~tsk l inikum Essen, Hufe landstr . 55, D-45122 Essen, Germany . G . Senger and U. Claussen are at the Ins t i tu t fa r Hu ma ngen e tik undAnthropologie , U niversi t4t Jena, Kollegiengasse 10, D-07740 Jena, Germany . C. Lengauer is at the Ins t i tut far Hu ma ngen e tik un d An thropologie,Univers i t~t H eide lberg, Im Neuenhe imer Fe ld 328, D-69120, Germany , an d is curren tly at the Research Ins t i tu te of Molecular Pathology , Dr . BohrGasse 7, A-10 30 Vienna, Austr ia.

~) 1994 Rapid Communications of Oxford Ltd Chromosome Research Vol 2 199 4 4 0 5


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J . W i e n b e r g et al.M a t e r i a l a n d m e t h o d sChromosome preparationsT h e p r e s e n t s t u d y i n c lu d e s c h r o m o s o m e p r e p a r a t io nf r o m h u m a n ( H S A ), P a n t r o g l o d y t e s ( P T R ) , P . p a n i s c u s( P P A ) , Gori l la gor i l la ( G G O ) , P o n g o p y g m a e u s( P P Y) , M a c a c a f u s c a t a ( M F U ) a n d C e r c o p i t h e c u sa e t h i o p s (C A E) . M e t a p h a s e c h r o m o s o m e s w e r e m a d ef r o m s t a n d a r d p r e p a r a ti o n s o f p h y t o h e m a g g l u t i n i n( P H A ) - s t im u l a t e d p e r i p h e r a l l y m p h o c y t e s o r f r o ml y m p h o b l a s t o i d c e ll li n e s e s t a b li s h e d f r o m b l o o ds a m p l e s k i n d l y p r o v i d e d b y t h e M u n i c h Z o o' T i e r g a r t e n H e l l a b r u n n ' o r f r o m D r s W . S c h e m p p ,F r e i b u rg , G e r m a n y , M . S c h m i d , W / i r z b u r g , G e r m a n y ,a n d R . S t a n y o n , G e n o a , I t a l y . M i c r o s c o p i c s l i d e sw e r e p r e p a r e d b y s t a n d a r d t e c h n iq u e s . B e f or eu s i n g t h e l y m p h o b l a s t o i d c e l l l i n e s i n t h i s s t u d y ,c h r o m o s o m e s w e r e c h e c k e d f o r a b e r r a t io n s a n dw e r e f o u n d t o b e n o r m a l d i p l o i d , e x c e p t f o r o n eo f t h e g o r i l l a c el l l in e s , w h i c h s h o w e d a t r a n s l o c a t i o ni n t h e c h r o m o s o m e h o m o l o g o u s t o h u m a nc h r o m o s o m e 2 q .

D N A probeT h e m i c r o l i b r a ry w a s e s t a b l is h e d f r o m t h e e n t i r el o n g a r m o f t h e h u m a n c h r o m o s o m e 2 .M i c r o d i s s e c t i o n w a s p e r f o r m e d e s s e n t i a l l y a sd e s c r i b e d b y L f i d e c k e et al . ( 1 9 8 9 ) e x c e p t t h a t t h ec h r o m o s o m e s w e r e n o t G T G b a n d e d . T w e n t y c h r o m o -s o m e f r a g m e n t s w e r e p o o l e d i n t o t h e c o l l e c t i o nd r o p a n d m i c r o re a c ti o n s w e r e p e r f o r m e d a s p r e v i -o u s l y d e s c r i b e d ( L ~ d e c k e e t a l . 1 9 8 9 ) . A n a l i q u o t(1 00 n g ) o f th e p r i m a r y a m p l i f i c a ti o n p r o d u c t s w a sb i o t i n l a b e l e d i n a 1 0 0 - ~ t l a s s a y c o n t a i n i n g 1 ~ tMu n i v e rs a l M 1 3 / p U C s e q u e n c i n g a n d r e v e r ses e q u e n c in g p r i m e r , 1 0 m M T r i s - H C L ( p H 8 .4 ),50 m M KC1, 1 .5 ~ tM M gC12, 0 .001% ge l a t i n , 200 ~ tMd A T P , d G T P a n d d C T P , 1 0 0 ~ t M d T T P , 1 5 0~ tMb i o - 1 1 - d U T P a n d 2 . 5 u n i t s o f T h e r m u s a q u a t i c u sD N A p o l y m e r a s e . A f t e r i n i t i a l d e n a t u r a t i o n a t 9 6 Cf o r 3 m i n , 2 5 c y c l e s o f p o l y m e r a s e c h a i n r e a c t i o n( P C R ) w e r e c a r r i e d o u t w i t h d e n a t u r a t i o n a t 9 6 C f o rI m i n , a n n e a l i n g a t 4 7 C f o r i m i n a n d e x t e n s i o n a t7 2 C f o r 2 m i n , 1 0 s .

C h r o m o s o m e b a n d i n gC h r o m o s o m e b a n d i n g p r io r to F IS H w a sa c h i e v e d a c c o r d i n g t o K l e v e r e t a l . (1991) . Br ief ly,a f te r r o u t i n e G T G b a n d i n g , m e t a p h a s e s w e r ep h o t o g r a p h e d ( A g f a - O r t h o 25 ), d e s t a i n e d w i t hf i x a t i v e ( m e t h a n o l - a c e t i c a c i d 3 : 1 ) a n d p o s t - f i x e dw i t h 4 % f o r m a l d e h y d e i n p h o s p h a t e - b u f f e r e d s a l in e( PB S ) f o r 1 5 m i n . P o s t - f i x a t i o n w a s e s s e n t i a l t o p r e -s e rv e c h r o m o s o m e m o r p h o l o g y i n s u b s eq u e n t F I S He x p e r i m e n t s .

Fluorescence i n s i t u h y b r i d i z a t i o nA b o u t 2 0 0 n g o f P C R a m p l i f ie d b i o t i n -l a b e le d p r o b ew a s c o - p r e c i p it a t e d w i t h 1 0 ~tg o f s a l m o n s p e r m D N Aa n d 3 0 ~ tg o f C o t- 1 D N A a n d t h e p e ll e t w a sr e d i s s o l v e d i n 5 0 % f o r m a m i d e , l x S S C a n d 1 0 %d e x t r a n s u l f at e . T h e b i o t in - l a b e l e d p r o b e w a s h y b r i d -i z e d u n d e r s u p p r e s s i o n c o n d i t io n s ( C r e m e r e t a l . 1988,L i c h t e r e t a l . 1988 , P i nke l e t a l . 1 98 8) a n d d e t e c t e d w i t hf l u o r e s c e i n - c o n j u g a t e d a v i d i n . A n a l y s i s w a s p e r -f o r m e d u s i n g a Z e i ss A x i o p h o t m i c r o s c o p e e q u i p p e dw i t h e p i f l u o r e s c e n c e a n d t h e f il t er c o m b i n a t i o n f o rf l u o r e s c ei n , r h o d a m i n e a n d D A P I (4 " , 6 -d i a m i n id i n o - 2 -p h e n y fi n d o le ) , C h r o m o s o m e s w e r e p h o t o g r a p h e dw i t h A g f a c h r o m e ( 1 00 0 A S A ) c o l o r s l id e f i l m s o rK o d a k T - M A X b l a c k a n d w h i t e (4 00 A S A ) f il m s .

T h e p h o t o g r a p h e d h y b r id i z a ti o n s i g n al s w e r es u p e r i m p o s e d o v e r t he p r e v io u s p h o t o g r a p h e d G -b a n d e d c h r o m o s o m e s t o id e n ti fy t h e m a r k e d c h ro -m o s o m e s a n d t h e p o s i ti o n o f s ig n a l s a lo n g t h ec h r o m o s o m e s .

R e s u l t sF l u o r e s c e n c e i n s i t u h y b r i d i z a t i o n ( F IS H ) o f t h e e s t a b -f is h e d 2 q m i c r o f i b r a r y t o h u m a n l y m p h o c y t em e t a p h a s e s p r e a d s r e s u l t e d a s e x p e c t e d i n a s p e c i f i cd e l in e a ti o n o f t h e lo n g a r m o f h u m a n c h r o m o s o m e 2( F ig u r e l a ). I n m e t a p h a s e s p r e a d s f r o m g r e a t a p e s ,g i b b o n s a n d O l d W o r l d m o n k e y s d i ff e r e n t h y b r i d i z a -t io n p a t t e r n s w e r e o b s e r v e d .

I n b o t h c h i m p a n z e e s ( P . t ro g l o d y t e s a n d P . p a n i s c u s ) ,t h e e n t i r e c h r o m o s o m e s P T R 1 3 a n d P P A 1 3 w e r es t a i n e d , e x c e p t f o r a s m a l l p a r t o f th e s h o r t a r m t h a tr e m a i n e d u n l a b e l e d . O n t h e c h r o m o s o m e s P T R 1 2 a n dP P A 1 2 h y b r i d i z a t i o n s i g n a l s w e r e r e s t r i c t e d t o t h es h o r t a r m a l s o , l e a v i n g a s m a l l u n l a b e l e d b a n d ( F i g u r el c ), w h i c h m a y r e p r e s e n t t e l o m e r ic h e t e r o c h r o m a t i n .T h e h y b r i d i z a t i o n p a t t e r n o f t h e g o r i ll a c h r o m o -s o m e G G O 1 1 w a s s i m i l ar to t h a t f o u n d o n t h e c h i m -p a n z e e c h r o m o s o m e s P T R 13 a n d P P A 1 3 e x c e p t t h a tt h e u n l a b e l e d t e l o m e r i c b a n d w a s m o r e p r o n o u n c e d .T h e l a b e l in g o f th e g o r i ll a c h r o m o s o m e G G O 1 2 w a sd i ff e re n t f r o m t h a t o f b o t h c h i m p a n z e e c h r o m o s o m e sP T R 1 2 a n d P P A 1 2, w h i c h a r e s u p p o s e d t o b eh o m o l o g s . H y b r i d i z a t i o n s i g n a l s w e r e f o u n d o n t h el o n g a r m o f g o ri ll a c h r o m o s o m e G G O 1 2 c l os e to t h ec e n t r o m e r e , w h e r e a s t h e s h o r t a r m w a s n o t l a b e le d( F ig u r e l e ). A b r i g h t b a n d w i t h t h e c o u n t e r s t a in D A P Ii n d i ca t e s t h a t t h e s h o r t a r m o f G G O 1 2 m a y b e t o t a l lyh e t e r o c h r o m a t i c ( n o t s h o w n ) . T h e t w o o r a n g - u t a nh o m o l o g s ( P P Y 12 a n d P P Y 1 1) w e r e l a b e l e d a s i n th eg o r i l l a , e x c e p t f o r t h e t e l o m e r i c h e t e r o c h r o m a t i n ,w h i c h i s n o t p r e s e n t i n t h i s s p e c i e s ( F i g u r e l g ) .

H y b r i d i z a t i o n o f t h e 2 q m i c r o l i b r a ry t o O l d W o r l dm o n k e y c h r o m o s o m e s g a v e a s im i l a r p a t t e r n a s in t h et w o c h i m p a n z e e s . I n b o t h s p e c ie s a n a l y z e d o n es u b m e t a c e n t r i c c h r o m o s o m e p a i r ( M FU 1 5 , C A E 1 6 )

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w a s e n t i r e l y l a b e l e d i n c l u d i n g t h e s h o r t a r m s ( F i g u r el h a n d i ). O n a n o t h e r s u b m e t a c e n t r ic c h r o m o s o m ep a i r ( M F U 9 , C A E 1 3 ) l a b e l i n g w a s r e s t r i c t e d t o t h es h o r t a r m , a g a i n e x c e p t f o r t he h e t e r o c h r o m a t i c r e g i o ni n C A E . A s c h e m a t i c s u m m a r y o f t h e h y b r i d i z a t io np a t te r n s f o u n d i n h o m i n o i d a n d O l d W o r l d m o n k e yc h r o m o s o m e s i s s h o w n i n F i g u r e 2 .D i s c u s s i o nR e c e nt ly w e h a v e s h o w n t h a t F IS H o f D N Am i c r o l ib r a r ie s e s t a b l is h e d f r o m m i c r o d i s s e c te d h u -m a n c h r o m o s o m e r e g i o n s c an b e u s e d a s a t oo l toc o n f i r m t h e o r ig i n o f t h e b u l k D N A s e q u e n c e s f r o mt h e c o r r e s p o n d i n g r e g i o n ( L e n g a u e r et al . 1991a , b ,T r a u t m a n n et a l . 1 99 1). T h i s p r e s e n t p a p e r d e m o n -s t r a te s t h e sp e c i fi c v i s u a l i z a t i o n o f h u m a n c h r o m o -s o m e 2 q m a t e r ia l o n h o m o l o g o u s p r i m a t e c h r o m o -s o m e s u s i n g a m i c r o l i b r a r y e s t a b l i s h e d a f t e rm i c r o d i s s e c ti o n o f th e e n t i re l o n g a r m o f h u m a n c h r o -m o s o m e 2. T h e h y b r i d i z a t i o n p a t t e r n a l l o w s a d i r e c te v a l u a ti o n o f c h r o m o s o m e r e a r r a n g e m e n t s t h a t o c-c u r r e d d u r i n g h o m i n o i d e v o l u t i o n .I n al l g r e a t a p e a n d O l d W o r l d m o n k e y s p e c i esa n a l y z e d , t h e h u m a n 2 q m i c r o l i b r a r y ' p a i n t s ' o n ec h r o m o s o m e p a i r e n ti re l y . B y c o n v e n t i o n a l b a n d i n gp a t te r n s, t h e s e c h r o m o s o m e s w e r e a l r ea d y s u p p o s e dt o b e h o m o l o g o u s t o h u m a n c h r o m o s o m e 2 q . In a d d i-t i o n t o t h i s p a i n t i n g s i g n a l , a s m a l l s e g m e n t o f a n o t h e rg r e a t a p e a n d O l d W o r l d m o n k e y c h r o m o s o m e p a irw a s l a b e l e d . C o n v e n t i o n a l b a n d i n g p a t t e r n a n a l y s i sh a d a s s ig n e d t h is c h r o m o s o m e p a i r t o b e h o m o l o g o u st o h u m a n c h r o m o s o m e 2 p . H o w e v e r , t he p r e s e n t FI S Hr e s u l t s o f t h e 2 q m i c r o l i b r a r y s h o w t h a t t h i s c h r o -m o s o m e p a i r a ls o c on t ai n s a c h r o m o s o m e s e g m e n th o m o l o g o u s t o h u m a n c h r o m o s o m e 2 q . T h e re s u lt sc l e ar l y d e m o n s t r a t e t h a t t h e f u s i o n p o i n t o f t h e t w oa n c es t ra l c h r o m o s o m e s o f h u m a n c h r o m o s o m e 2 w a sb e y o n d t h e c e n t r o m e r e i n t h e l o n g a r m . F r o m t h el e n g t h o f th e l a b e l e d s e g m e n t w e s u g g e s t t h a t t h ef u s io n p o i n t w a s s o m e w h e r e c l os e t o h u m a n b a n d2q13 .

T h e s m a l l la b e l e d s e g m e n t o n o n e o f th e c h r o m o -s o m e s h o m o l o g o u s t o h u m a n c h r o m o s o m e 2 is f o u n di n t h e s h o r t a r m i n c h i m p a n z e e s a n d i n t h e l o n g a r mo f g o r il la a n d o r a n g - u t a n c h r o m o s o m e s ( F i g u re 2 ).T h e h y b r i d i z a t i o n p a t t e r n i n d ic a t es t h a t t h i s s e g m e n tw a s m o v e d b y o n e o r m u l t i p l e p e r ic e n t ri c i n v e r si o n s .F I S H o f t h e 2 q m i c r o l ib r a r y o n g r e a t a p e c h r o m o s o m e ss u p p o r t s t h e s i m p l e s t e x p l a n a t i o n f o r t h e o r i g i n o fh u m a n c h r o m o s o m e 2 , w h i c h w o u l d i m p l ic a t e at e l o m e r e - t o - te l o m e r e f u s i o n o f t w o s u b m e t a c e n t r i cc h r o m o s o m e s s i m i l a r t o t h a t f o u n d i n t h e c h i m p a n -z e e s ( D u t r i l l a u x 1 9 7 9, Y u n i s & P r a k a s h 1 98 2 ). T h u s ,t h e c e n tr o m e r e o f h u m a n c h r o m o s o m e 2 s h o u ld h a v eb e e n d e r i v e d f r o m a c h r o m o s o m e s i m i l a r t o P T R 12 o rP P A 1 2, w h i l e t h e c e n t r o m e r e f r o m t h e c h r o m o s o m es i m i l a r t o P T R 1 3 o r P P A 1 3 w a s i n a c t i v a t e d o r l o s t( D u t r i l la u x 1 97 9). T h i s h y p o t h e s i s i s s u p p o r t e d b y

A n a l y s i s o f h u m a n c h r o m o s o m e 2 w i t h a D N A m i c ro l ib r a ryr e c e n t i n s i t u h y b r i d i z a t i o n e x p e r i m e n t s w i t hc e n t ro m e r e a n d t e lo m e r e D N A s e q u en c e s o f h u m a nc h r o m o s o m e 2 ( W e l l s et al . 1990, I jdo et al . 1991,A v a r e l l o et al . 1992 , Ba l d i n i et a l . 1993) . C l ose t o t hea s s u m e d f u s i o n p o i n t ( 2 q 13 ) a r e li c t o f a n a l p h o i dd o m a i n w a s f o u n d b y i n s i t u h y b r i d i z a t i o n o f a s a t e l-l i t e D N A c l o n e u n d e r l o w h y b r i d i z a t i o n s t r i n g e n c y( A v a r e l l o et a l . 1992 , Ba l d i n i et al . 1 9 9 3 ) . T h i s a l p h o i dd o m a i n m a y i n d i c a t e t h e p r e s e n c e o f t h e i n a c t iv a t e dc e n t r o m e r e i n h u m a n b a n d 2 q2 1 . I n a d d it i o n , p r o x i -m a l t o 2 q 21 , te l o m e r i c se q u e n c e s w e r e f o u n d i n b a n d2 q 1 3 b y i n s i t u h y b r i d iz a t io n o f D N A p r o b e s c o n t a in -i n g i n v e r t e d a r r a y s o f th e v e r t e b r a t e t e l o m e r i c r e p e a ti n a h e a d - t o - h e a d a r r a n g e m e n t ( W e l l s et a l . 1990 , I j doet al . 1 99 1) . T h i s b a n d s h o u l d t h e r e f o r e r e p r e s e n t t h ef u s i o n p o i n t o f t h e t e lo m e r e s o f t h e t w o p r i m a t eh o m o l o g s .T h e F I S H p a t t e r n s o f t h e 2 q m i c r o l i b r a r y i n d i c a tet h a t p e r ic e n t ri c i n v e r s io n ( s ) a l t e re d t h e m o r p h o l o g y o fg r e a t a p e c h r o m o s o m e s , h o m o l o g o u s t o h u m a n c h r o-m o s o m e 2 p . To s h o w t h e d ir e ct io n o f c h r o m o s o m ec h a n g e s d u r in g h u m a n a n d g r e a t a p e e v o l u ti o n w ea l so a n a l y z e d t h e F I S H p a t t e r n s f o r t h e 2 qm i c r o li b r ar y in O l d W o r l d m o n k e y s a s a n ' o u t g r o u p ' .I n p a r t i c u l a r , t h e h y b r i d i z a t i o n p a t t e r n o f th e s m a l ls e g m e n t i n re s p e c t to t h e c e n t r o m e r e i n th e ' o u t g r o u p 'w o u l d a l l o w t h e o r ig i n o f t h e a s s u m e d p e r i ce n t r ici n v e r s i o n t o b e d e f i n e d .

I f t h e F I S H p a t t e r n i n t h e ' o u t g r o u p ' h a d b e e n s i m i -l a r t o t h a t f o u n d i n o r a n g - u t a n a n d g o r i l l a , t h e n t h er e su l ts w o u l d h a v e s u p p o r t e d t h e a s su m e d e v o l u ti o n -a r y - d e r i v e d ( s y n a p o m o r p h i c ) i n v e r s i o n i n t h e c h r o -m o s o m e 2 p h o m o l o g s ( D u t r il la u x 1 97 9, Y u n i s &P r a k a s h 1 98 2). T h i s w o u l d h a v e p h y l o g e n e t i c a l l yl i n k e d h u m a n a n d c h i m p a n z e e s a f t e r t h e ir d i v e r g e n c ef r o m t h e g o r i l l a l in e .

H o w e v e r , t h e h y b r i d i z a t io n p a t t e r n s o f th e 2 qm i c r o li b r ar y f o u n d o n t h e c h r o m o s o m e s o f O l d W o r l dm o n k e y s ( M. f u s c a t a , C . ae t h iops ) w e r e s i m i l a r t o t h a tf o u n d i n t h e c h i m p a n z e e s . T h e s i g n a l o f t h e s m a l lb a n d o n t h e c h r o m o s o m e 2 p h o m o l o g w a s f o u n d i nt h e c o r r e s p o n d i n g s h o r t a r m s . T h e r e f o r e , t h e F I S Hp a t t e r n s i n t h e ' o u t g r o u p ' i n d i c a t e t h a t t h e c h r o m o -s o m e f o r m f o u n d i n th e c h i m p a n z e e s s h o u l d b e c o n -s i d e r e d a s t h e an c e s t r al f o r m , w h i l e t h e c h r o m o s o m e2 p h o m o l o g s i n g o r i l l a a n d o r a n g - u t a n e x p e r i e n c e dc o n v e r g e n t c h r o m o s o m a l m u t a t i o n s f o r th i s i n v e r s io n .

C o n v e r g e n t c h r o m o s o m a l re a r r a n g e m e n t s m a y b em o r e c o m m o n t h a n u s u a ll y a s s u m e d ( S t a n y on et a l .1 99 4) . S im i l a r r e s u l t s c o u l d b e o b t a i n e d r e c e n t l y b yc o m p a r a t i v e m a p p i n g o f D N A p r o b e s d e r i v e d f r o mt he V - k a p p a i m m u n o g l o b u l i n g e n e s, w h i c h m a p o nb o t h h u m a n c h r o m o s o m e 2 p a n d 2 q cl os e t o th ec e n t r o m e r e ( A r n o l d et a l . s u b m i t t e d ) . T h e r e f o r e th ep h y l o g e n y o f h u m a n c h r o m o s o m e 2 a n d it s h o m o l o g si n t h e g r e a t a p e s c a n n o t b e u s e d t o d i s c r i m i n a t e b e -t w e e n t h e r e c e n t m o s t f a v o r e d p h y l o g e n e t i c t r e e s li n k -i n g ei th e r h u m a n a n d c h i m p a n z e e o r c h i m p a n z e e a n dg o r i l l a .

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J. Wienberg et a l .

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An alternative hypothesis considers the possibilitythat ancestral populations could have been polymor-phic for these chromosome variants. Different chro-mosome variants may have become fixed in differentphylogenefic lines (Stanyon & Chiarelli 1983). Yet,random fixation of polymorphic chromosome formsin the different phylogenetic lines would not allow aparsimonious interpretation. This hypothesis obtains

Ana lysis o f hum an chromosome 2 wi th a DN A microlibrarysupport from chromosome polymorph isms still foundin human populations. In about 0.1% of humans apericentric inversion is found on chromosome 2 in-volving the segment 2pll-2q13, which is close to theassumed breakpoints of the pericentric inversion as-sumed in primate homologs (for review see Djalali e ta l. 1986). In Sephardic Jews this inversion has a fre-quenc y of 0.8% and even ho mozy gof ic carriers with-

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F i g u r e 2. Schematic summ ary of the hybridizat ion patterns a fter FISH with the human 2q microl ibra ry to great ape and OldWorld monkey chromosomes. The human chromosome 2 idiogram (left) and the schematic drawing of the hybridizat ionpatterns are shown for hum an (HSA) and primate homologs [Pan troglodytes (PTR), P. paniscus (PPA), Go rilla goril la (GGO),Pongo pygmaeus (PPY), Macaca fuscata (MFU) and Cercopithecus aethiops (CAE)], without indicating the chromosomebanding. The numbers beside the ape chromosomes are the chromosomal nomenclature within these species. The paintedchromosome regions are indicated by a hatched pattern. Telomeric C -banding in PTR, PP A, GGO and CAE is indicated byblack bands. Note that the hybridizat ion pattern on the 'classical human chromosome 2p homologs' in both chimpanzeespecies is more sim ilar to the pattern found in the 'outgreup' (MFU an d CA E) than that in the gori l la or in the orang-utan. Inthese species the signals are found in the corresponding long arm.F i g u r e 1. FISH with a 2q microl ibrary to human and primate chromosomes ( a - j ) . a Paint ing of the long arm of humanchromosome 2 shows the origin of the DNA microclone l ibrary, b & Hybridizat ion to chromosomes of Pan troglodytes ().Chromosom e pair 13 is painted entirely (arrows), demonstrat ing the hom ology to human chromosom e 2q as identif ied fromits G-banding pattern (b). In addit ion, a small segment (arrowheads) of chromosome pair 12 is also painted. Note that thelabeled segment spans the short arm of the chimpanzee chromosome. The posit ion of the centromere was identi f ied by G-banding (b) and DA PI staining (not shown), d & e Painted chromosome s of Go rilla goril la (e). Chromosom e pair 11 is paintedentirely (arrows), whereas a sm all labeled band can be detected in the long arm of the gori l la chromosom e 12 (arrowheads).Again, the centromere was identi fied by G-banding (d) and DAP I staining (not shown), f & g Painted chromosomes of Pongopygmaeus (g). Chromosome pair 11 is painted e ntirely (arrows), where as a small labeled band can be detected in the longarm of the pongo chromosome 12 (arrowheads). The posit ion of the centromere was identi f ied by G-banding (f) and DAPIstaining (not shown), h- j Hybridizat ion to chromosomes of Old World monkeys. Chromosomes of Macaca fuscata (h) andCercopithecus aethiops ( i & j) . B oth species sho w a sim ilar hybridizat ion pattern a s found in Pan troglodytes. O nechromosome pair (15 and 16 respectively) is entirely painted (arrows). A sma ll segment of the short arm of chromosom e 9and 13 respec tively s painted in both spec ies indicating i ts human chromosome 2q homology (arrowheads). The centromereswere identi f ied by G-banding ( i) and DAPI staining (not shown).

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J . W i e n b e r g e t a l .out a ny c l inical abnormal i t ies have been observed(Gelman-Kohan et al. 1993). Inversion polymo rphi smsin general are s til l foun d in homi noi d karyotypes. Theorang-utah is polymorphi c for invers ions betweenand within subspecies (de Boer & Seuanez 1982). Forexample , the g ibbons analyzed by in situ hybr id iza-tion of chromosome-specific DNA libraries could allbe d is t ingui shed on the basis of a chromosome 8polym orphis m (Jauch et al. 1992). All 44-chro moso megibbon species share th is polym orphis m (S tanyon et al1987), indicating that i t is not tr ansient but has sur-vived even speciation events .

The decision as to whether the inversions foun d inthe different phylogenetic l ines are the same or s implythe result of apparent convergence due to s imilarbreakpo ints can be made on the molecular level. Suchrear rangements can be def ined by FISH with DNAprobes that span the breakpoints (Ried et al. 1991,Lengauer et al. 1992). The mole cul ar analy sis of break -point regions will help to ascertain chromosomalrear rangements that occurred dur ing pr imate evolu-tion and will f inally clarify phylogene tic relationshipsat the molecular level.AcknowledgementsWe wo uld like to thank A. Wiegen stein for photo-graphic work and R. Stany on for suggestions and dis-cussions. This work was supported by a grant fromthe Dutsche Forschungsgemeinschaft.ReferencesArnold N, Wienberg J, Ermert K, Zachau H-G Comparative

mappi ng of hum an and great ape chromosomes by in situhybridization of DNA probes derived from the V-kappaimmunoglobulingenes. Genomics, submitted.Avarello R, Pedicini A, Caiulo A, Zuffardi O, Fraccaro M(1992) Evidence for an ancestral alphoid do main on the longarm of hum an chromosome 2. Hum Genet 89: 247-249.Baldini A, Ried T, Shridhar Vet al. (1993) An alphoid DNAsequence conserved in all human and great ape chromo-somes: evidence for ancient centromeric sequences at hu-man chromosomal regions 2q21 and 9q13. Hum Genet 9 0 :577-583.Boer de LEM, Seu~nez HN (1982) The chromosomes of theorangutan and their relevance to the conservation of thespezies. In: de Boer LEM, ed. The Orangutan. Its Biology andConservation. The Hague: Junk, pp 135-170.

Cremer T, Lichter P, Borden J, Ward DC, Manueli dis L (1988)Detection of chromosome aberrations in metaphase andinterphase turn out cells by in situ hybridization using chro-mosome specific l ibrary probes. Hum Genet 80: 235-246.Djalali M, Steinbach P, Bullerdiek J, Holmes-Siedle M,Verschraegen-Spae MR, Smith A (1986) The significance ofpericentric inversions of chromosome 2. Hum Genet 7 2 :32-36.Dutrillaux B (1979) Chromosomal evolu tion in primates. Ten-tative phylogeny from Microcebus murinus (prosimian toman. Hum Genet 48: 251-314.Gelman-K ohan Z, Rosensaft J, Nisani B-C, Chemke J (1993)Homozygosity for inversion (2) (p12q14). Hum Genet 9 2 :427.4 1 0 ChromosomeResearch Vol 2 1994

Ijdo JW, Baldini A, Ward DC, Reeders ST, Wells RA (1991)Origin of human chromosome 2: an ancestraltelomere-telomere fusion. Proc Natl Acad Sci USA 88:9051-9055.Jauch A, Wienberg J, Stanyon R et al. (1992) Reconstruction ofgenomic rearrangements in great apes an d gibbons by chro-mosome painting. Proc Natl Acad Sci USA 89: 8611-8615.Klever M, Grond- Ginsbach C, Scherthan H, Schroeder-KurthT (1991) Chromosomal in situ suppression hybr idizationafter Giemsa bandin g. Hum Genet 86: 484-486.Lengauer C, Liidecke H-J, Wienberg J, Cremer T, HorsthemkeB (1991a) Comparative chromosome band mappi ng in pri-mates by in situ suppression hybridi zation of ban d specificDNA microlibraries. Hum Evol 6: 67-71.Lengauer C, Eckelt A, Weith A et al. (1991b) Painti ng of definedchromosomal regions by in situ suppression hybridizationof libraries from laser-microdissected chromosomes.Cytogenet Cell Genet 56: 27-30.Lengauer C, Riethman HC, Speicher Met al. (1992) Metaphaseand interphase cytogenetics with Alu-PCR amplified YACclones contain ing the BCR-gene and the protooncogenes c-raf-1, c-fms, c-erbB-2. Cancer Res 52: 2590-2596.

Lichter P, Cremer T, Borden J, Manue lidi s L, Ward DC (1988)Delineation of individual human chromosomes inmetaphase an d interphase cells by in situ suppression hy-bridization using recombinantDNA libraries.Hum Genet 8 0 :224-234.Liidecke H-J, Senger G, Claussen U, Horsthemke B (1989)Cloning defined regions of the human genome bymicrodissection of banded chromosomes and enzymaticamplification. Nature 338: 348-350.Pinkel D, Landege nt J, Collins C et al. (1988) Fluorescence insitu hybridizat ion with hum an chromosome specific librar-ies: detection of trisomy 21 and translocation of chromo-some 4. Proc Natl Acad Sci USA 85: 9138-9142.Ried T, Lengauer C, Cremer T et al. (1991) Specific me taphaseand interphase detection of the breakpoint region in 8q24 ofBurkitt lymphoma cells by triple colour fluorescence in situhybridization. Genes, Chromosomes and Cancer 4: 1-6.Stanyon R, Chiarelli B (1983) Mode and tempo in primatechromosome evolution: implications for hylobatidphylogeny. J Hum Evol 12: 305-315.Stanyon R, Sineo L, Chiarelli B, Camperio-Ciani A, HaimoffAR, Mootnick EH (1987) Banded karyotypes of the 44-chro-mosome Gibbons. Folia Primatol 4 8 : 56-64.Stanyon R, Arnold N, Koehler U, Bigoni F, Wienberg J (1994)Chromosomal painting shows that ' marked chromosomes'in lesser apes and Old World monkeys are not homologousand evolved by convergence.Cytogenet Cell Genet, in press.Traut mann U, Leuteritz G, Senger G, Claussen U, BallhausenWG (1991) Detection of APC region-specific signals bynonisotopic chromosomal in situ suppression(CISS)-hybridization using a microdissection library as aprobe. Hum Genet 87: 495-497.Wells RA, Gem ino GG, Kr ishna S, Buckle VJ, Reeders ST (1990)Telomere-related sequences at interstitial sites in the hum angenome. Genomics 8: 699-704.Wienberg J, Jauch A, Stanyon R, Cremer T (1990) Molecularcytotaxonomy of primates by chromosomal in situ suppres-sion hybridization. Genomics 8: 347-350.Wienberg J, Stanyon R, Jauch A, Cremer T (1992) Homologiesin human and Maccacafuscata chromosomes revealed by insitu suppression hybridization with human chromosomespecific DNA libraries. Chromosoma 101: 265-270.Yunis JJ, Prakash O 1982 The origi n of man: a ch romosomalpictorial legacy. Science 215: 1525-1529.

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