Chromosoma (Berl.) 52, 123--136 (1975) 9 by Springer-Verlag 1975
Causes and Consequences of Robertsonian Exchange B e r n a r d J o h n a n d Michael F r e e m a n Department of Population Biology, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra City, A.C.T 2601, Australia Abstract. At least two types of Robertsonian exchange are now known in the acrocentrie chromosomes of man. Both types involve breakage in the arms adjacent to the centromere. Evidence is presented for a third type of exchange, one involving breakage within the centromere itself, in the grasshopper Percassa rugi/rons. In this species, which is regularly homozygous for a single fusion metacentric, the eighteen rod autosomes have small but pronounced granules at the centric end of the chromosome. When C-banded these granules show differential Giemsa staining and appear to represent centromeric chromomeres; these chromomeres are lacking in the metacentric fusion product. Equivalent fusions may have occurred in some mammal species too and possible examples of this are discussed in sheep and mice. The Percassa fusion has led to a modification in both the frequency and the distribution of chiasmata as judged by a comparison of these properties in the metacentric relative to the two next smallest rod equivalents. Comparable modifications are known to occur in other naturally occurring fusions but these changes are certainly not automatic consequences of fusion since they are not shown in at least some newly produced fusion mutants.
1. The Nature of Fusion I t was Muller in 1940a who i n t r o d u c e d t h e t e r m " w h o l e a r m t r a n s f e r " to describe exchanges where whole, or n e a r l y whole, a r m s are t r a n s p o s e d or interchanged. Such whole a r m transfers include those exchanges c o m m o n l y referred to as R o b e r t s o n i a n r e a r r a n g e m e n t s which involve the fusion of two rod chromosomes to y i e l d one m e t a c e n t r i c . F u s i o n s of this t y p e h a v e c o m m o n l y been docum e n t e d b o t h in n a t u r a l p o p u l a t i o n s a n d in tissue culture lines. I n a m a j o r i t y of these cases i t has been a s s u m e d t h a t t h e r e a r r a n g e m e n t involves t h e union of t h e long a r m s of two a c r o c e n t r i e chromosomes following b r e a k a g e p r o x i m a l to t h e c e n t r o m c r e in t h e long a r m of one c h r o m o s o m e a n d s i m u l t a n e o u s b r e a k a g e in t h e s h o r t a r m of t h e o t h e r ( t y p e 1 exchange, Fig. 1). There is no d o u b t t h a t such a m e c h a n i s m of e x c h a n g e does occur. I t has been convincingly d o c u m e n t e d , for e x a m p l e , in t h e case of homologous fusions b e t w e e n t h e g e n u i n e l y a c r o c e n t r i c autosome-13 p a i r of m a n (Hsu et al., 1973). I t is e q u a l l y clear, however, t h a t similar fusions involving this same p a i r of homologues m a y arise following b r e a k a g e w i t h i n each of t h e s h o r t a r m s of t h e two chromosomes (Hsu et al., 1973). The m e t a c e n t r i c s so p r o d u c e d are t h u s dicentrie (type 2 exchange, Fig. 1) a n d similar dicentrics h a v e now been identified in several o t h e r fusions involving t h e acrocentric c h r o m o s o m e s of m a n ( F e r g u s o n - S m i t h , 1972; Niebuhr, 1972; O'Neil a n d Miles, 1974). Such dicentrics are e v i d e n t l y quite stable since t h e y are t r a n s m i t t e d b e t w e e n generations. P r e s u m a b l y t h e close p r o x i m i t y of the two centromeres leads to p r e d o m i n a n t l y parallel s e p a r a t i o n w i t h t h e two c e n t r o m e r e s belonging to t h e s a m e c h r o m a t i d passing to t h e s a m e pole a t a n a p h a s e . To w h a t e x t e n t
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Rearrangement
Type
Associated loss
T~
t:i
IJ
89
Fig. 1. Possible modes of Robertsonian exchange leading to whole arm fusion. It is assumed that no direct adhesion of chromosome ends is possible so that fusion must depend on prior breakage. Break points are indicated by arrow heads. The blocks adjacent to the centromere represent constitutive heterochromatin
the metacentrics in other species m a y also prove to be " c l o s e " dicentrics is n o t known. Helwig (1941, 1958) believed t h a t the metacentrics of grasshoppers were regularly dicentric h u t did n o t offer a n y s u b s t a n t i v e evidence i n support
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of his belief. More recently Moens and lZothfels (unpublished) have evidence from EM studies t h a t the metaeentries of the grasshopper Chloealtis conspersa includes two structures identified by them as eentromeres which are in the same synaptonemal complex and 1 nm apart. Additionally in the blackflies Eusimulium aureum and Cnephia lapponica where the haploid number has been reduced by fusion from n = 3 to n = 2, the dicentrie nature of the large fusion chromosome has been inferred from the pattern of eetopic pairing (Dunbar, 1959; Rothfels and Mason, unpublished). Since, as the human data show so convincingly, morphologically similar fusions m a y arise in different ways it is apparent t h a t the rather loose term "Robertsonian fusion" is in need of dearer definition. This is particularly important in view of the fact that different modes of exchange involve the loss of more or less chromosome material (Fig. 1) and this, in turn, m a y be expected to lead to different consequences in terms of the effect of the fusion on viability or fertility (Brugre et al., 1974). Fundamental to any consideration of the nature of Robertsonian exchange is the problem of the organisation of the eentromere and the extent to which exchanges m a y occur within it. Rather surprisingly our most definitive knowledge of the structure of the eentromere still comes not from the electron microscope but from studies made with the light microscope. This m a y well stem from the fact that most electron microscope studies have used oriented meta-anaphase stages only whereas light microscope studies have employed a wider range of stages including e-mitotic chromosomes and paehytene-diakinesis stages. Such studies reveal that in metaeentrie chromosomes the region conventionally referred to as the eentromere includes one or more chromomeres joined to the main chromosome arms and, where applicable, to each other by extended fibrillae (Lima-de-Faria, 1956; John and Hewitt, 1966). The precise number of chromomeres per eentromere varies. In part this m a y reflect genuine differences in specific eentromere organisation. I n part, however, it also appears to reflect some tendency of these ehromomeres to fuse in more condensed stages of division or following prolonged eolchicine treatment. From the illustrations provided b y Lima-de-Faria it is clear t h a t in metaeentries of Hyacinthus and Traclescantia it is the central fibrilla zone which forms the site of attachment of halfspindle fibres (see Fig. 10, pg. 96 and Fig. 26 pg. 106 of Lima-de-Faria, 1956). W h a t remains to be established in these eases is the nature and function of the organelles referred to as eentromeric ehromomeres. I n at least some rod-chromosomes the fibrillae to which tile spindle fibres attach are terminal as in the ease of the grasshopper Stethophyma grossum (see Figs. 15 and 16, pg. 97 of Lima-de-Faria, 1956 and compare with Fig. 2 of this paper). The same is true of a second grasshopper species Bryodema tuberculata (Kl~tersk~ et al., 1974). The chromosomes of both these species are regarded by White (1973) as aeroeentrie but by all known criteria appear to be teloeentrie.
2. The P e v c a s s a Fusion
Chromosome complements regularly homozygous for one or more metacentrics have been described in several acridid species. Since a majority of male members
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Fig. 2a and b. The chromosomes of Stethophyma grossum at first metaphase of male meiosis: (a) represents the entire complement (2n = 23) while (b) is a single enlarged bivalent
of this family have 23 rod chromosomes it is natural to conclude that these metacentrics have been produced secondarily by fusion. The nature of this fusion has not been rigorously defined but, on the assumption that the rods involved are acrocentric, White (for detailed references see White, 1973) has consistently maintained that they must arise by type 1 exchange (Fig. 1). The Australian catantopinid Percassa rugiJrons, a montane and subalpine brachypterous species living on the low shrub stratum, has one fixed metaeentric pair. Here there are 21 chromosomes in the male complement (XO) anct 22 in the female (XX). If the 18 rod autosomes are arranged in decreasing size order it can be shown that the arms of the metacentric correspond to chromosomes 4 and 6 of an originally ll-membered autosome set (Fig. 3d). At both mitosis and meiosis the spindle attachment sites of the rods appear to be strictly terminal (Fig. 3 a--c). Especially clear small bodies are sometimes, though not invariably, visible at the eentric ends of the rod bivalents at diakinesis (Fig. 4b--f). These usually appear as single entities but can occasionally be seen to be double (Fig. 4 e). Equivalent structures can also be found in spermatogonial mitoses (Fig. 3d). No such bodies have ever been seen in the metacentries where the centromeres invariably appear as fine fibrillar regions (Fig. 4a). C-banding leads to intense staining of two small spherical bodies, which sometimes merge into one, at the ends of each rod both in mitotic (Fig. 4g) and in meiotic chromosomes (Fig. 4h). Again no equivalent structures occur in the metacentrics.
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In many eucaryote species the occurrence of constitutive heterochromatin in the arm material immediately adjacent to the centromere may lead to intense pro-centrie C-banding which tends to obscure details of the centromere itself. Even so there are good grounds for concluding that the proeentric heterochromatin and the centromere itself are quite distinct structures though they may be located contiguously on the chromosome. For example in the polytene chromosomes of blackflies (Simuliidae) the centromere can be identified as an expanded region marked in many species by a heavy band which undergoes specific ectopic pairing. Using C-banding techniques, Bedo (1975) has recently shown that in Simulium ornatipes the centromere band itself stains deeply but the surrounding proeentric heterochromatin does not. In Simulium melatum, on the other hand, the eentromere band is C-negative and appears as a non-staining gap with deeply staining flanking heterochromatic structures. Similarly, differentiation between pro-eentric C-bands and a more localised centromeric staining has been obtained in Me~ocricetus newtoni by using a modified C-banding technique involving pretreatment with N a O H prior to incubation with S SC (Voieulescu et al., 1972). Moreover contrary to the claim of Yunis and u (1971), constitutive heterochromatin is not universally found around the eentromere. Thus in Microtus agrestis the autosomes show only one or two deeply stained granules in the eentromere region. Similar granules have been reported by Stack et al. (1974) in the meta- and acro-centric chromosomes of Allium cepa and Ornithogalum virens and they have concluded that these granules represent fused centromerie chromomeres. This interpretation is supported by the fact that the spindle fibre remnants can be seen directly attached to them (see Fig. 11, pg. 368 of Stack et al., 1974). Like,rise in Bryodema tuberculata the site of spindle fibre attachment is terminal and lies distal to the C-band (see Fig. 15, pg. 399 of Kls et al., 1974). We are of the opinion that in Percassa the granules which stain with Giemsa on C-banding and the bodies which are present at diakinesis are one and the same structure and represent the centromeric chromomeres. This implies that the rods in Percassa are telocentric. This interpretation is supported by a comparison with the acridine grasshopper Gastrimargus musieus. In this species (Fig. 5a) a complete range of chromosome types exists from genuinely acrocentric to genuinely telocentrie. Moreover chromosomes which appear as acrocentrie in mitotic chromosomes can be shown to have two clear arms at second division of meiosis too whereas autosomes 9, 10 and 11 which appear as telocentrics in mitotic tissues never show a second arm at meiosis (Fig. 5b). When C-banded the acrocentrics show a clear short arm, which also stains differentially, beyond the C-positive granules of the centromere. Telocentrics, on the other hand, like the rods of Percassa, are terminated by the C-positive granules (Fig. 5e). The variation in the size of the short arms found in the acrocentrics of Gastrimargus is especially interesting and suggests that the centric regions of rod chromosomes may sometimes "grow" by the addition of heterochromatin so leading to the development of new arms. Thus the short arms of Gastrimargus acrocentrics show a distinctive appearance in C-banded preparations and appear intermediate between the non-staining long arms and the deeper staining centric chromomeres. Indeed in overstained preparations it is not always possible to distinguish the two. We conclude that the metacentrics of Percassa are the product of fusion between elements 4 and 6 of an originally ll-membered telocentric set. This implies
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Fig. 3a--d. The chromosomes of male Percassa rugi/rons to show centric activity at spermatogonial metaphase (a and b) and at first metaphase of male meiosis (c). The metacentric is designated as M and arrowheads indicate sites of eentric activity. (d) shows the haploid complement at spermatogonial metaphase
t h a t t h e exchange m u s t have occurred following b r e a k a g e in t h e fibrfllar comp o n e n t of two telocentrics ]eading to t h e loss of t h e centrie chromomeres. This suggests a t h i r d t y p e of R o b e r t s o n i a n fusion (type 3, Fig. 1), a t y p e which involves
Causes and Consequences of i~obertsonian Exchange
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Fig. 4 a ~ . Centric organisation of male Percassa bivalents at diakinesis of meiosis (a--f) and o5 C-banded chromosomes at e-mitosis (g) and diakinesis (h) resl0eetively. Note absence of eentromerie chromomeres in the metaeentrie (a and g) compared to their consistent presence (arrows) in the rods (b--h)
a m i n i m u m loss of chromosome material. A n e q u i v a l e n t fusion is, of course, also possible b e t w e e n acroeentrics (type 4, Fig'. 1). F u s i o n s of this t y p e , involving b r e a k a g e within t h e eentromere, m a y p r o v e to be m o r e widespread. I n d e e d t h e r e are several suggestive eases a l r e a d y in the l i t e r a t u r e in m a m m a l s . F o r e x a m p l e , sheep (Ovis aries) h a v e 54 chromosomes. Six of t h e 52 a u t o s o m e s are m e t a c e n t r i e , t h e r e m a i n d e r are rods. These rods h a v e s u b s t a n t i a l blocks of p r o c e n t r i e h e t e r o c h r o m a t i n b u t t h e eentromeres themselves do n o t stain with either Giemsa or with quinacrine. E v a n s et al. (1973) r e m a r k t h a t " i f we define t h e e e n t r o m e r e itself as t h e n a r r o w e s t p o i n t of the c h r o m o s o m e t h e n in some eases t h e r e is in some chromosomes a suggestion of a m i n u t e short a r m which does n o t stain i n t e n s e l y " a n d on this s o m e w h a t t e n u o u s g r o u n d a c c e p t t h a t t h e rods in sheep are aeroeentrie. A u t o s o m a l fusions between t h r e e d i s t i n c t pairs of rods h a v e been r e p o r t e d in elite breeds of sheep from New Zealand. I n two of these t h e m e t a c e n t r i c s show s u b s t a n t i a l blocks of h e t e r o c h r o m a t i n which are d i s t r i b u t e d e v e n l y on either side of t h e centromere. I n t h e t h i r d ease there is a single h e t e r o e h r o m a t i e block which is e x o c e n t r i e a l l y l o c a t e d in one of t h e a r m s of t h e m e t a c e n t r i e (Brugre et al., 1974). Bru6re also assumes t h a t t h e rods are acroeentric a n d suggests t h a t t h e t h i r d ease results from centric fusion (type 6, Fig. 1) whereas t h e fh'st a n d second eases are dieentries (type 2, Fig. 1). The a l t e r n a t i v e i n t e r p r e t a t i o n is t h a t t h e rods are telocentrie a n d t h a t t h e exchanges
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Fig. 5 a - - c . The chromosomes of male Gastrimargus musicus at c-mitosis (a) and their structure at second anaphase of meiosis (b) and following C-banding a t c-mitosis (c). Note t h a t where short arms are present these can be dectected b o t h at c-mitosis, where they stain differentially, and at second anaphase, where they either show flexure (I and 2) or else are drawn straight in the spindle (X, 5 and 8)
Causes and Consequences of Robertsonian Exchange
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are a consequence o f breakage within the eentromere (types 3 and 5, Fig. 1, respectively). A somewhat similar problem exists in the genus Mus. The house mouse M u s musculus, has 40 rods which are variously described as acro- or telocentric according to the preference of individual authors. Fusions are not uncommon in mouse cell cultures and here two C-positive blocks are often found one on each side of the eentromere; this could be accommodated by at least four types of exchange (types 1, 2, 3 and 4, Fig. 1). The 38 autosomal rods all show substantial blocks of C-positive material but nothing that can be construed as a short arm. If a short arm does exist then C-banding obscures it. The early studies of Tjio and Levan (1954, see for example their Fig. 146, pg. 32) suggests the rods are teloeentric. Using whole mount electron-microscopy of surface-spread colehieinetreated chromosomes Comings and Okada (1970) find that rods of mouse, sheep and goat show single areas of chromatid association. These are located at a terminal position with no evidence for short arms. At the light microscope level these areas correspond to the proximal arm regions, immediately adjacent to the centromere, where sister chromatids remain in contact at first anaphase of meiosis and at c-mitosis. Metacentric chromosomes of man, mouse L-cells, sheep and Chinese hamster, however, have two distinct areas of chromatid association separated by a central zone of attenuated fibres, as too do acrocentric chromosomes from mouse L-cells. Comings and Okada conclude that the rods in mouse, sheep and goat are telocentric and that the metacentrics in them have arisen as the result of a simple breakage reunion event within the fibres of the centromere itself. Fusions between rods have also occurred in natural populations of Mus. In the extreme case this has led to the development of a geographical isolate which is homozygous for seven distinct fusions and which is known by the specific name M u s poschiavinus, the tobacco mouse. Comings and Avelino (1972) have shown that there is no detectable loss of satellite DNA in this total of 14 fusions which implies that either the constitutive heterochromatic regions have been preserved intact during the fusion process or else that any loss consequent upon fusion has been subsequently rectified. This again is consistent with the exchange occurring either within the short arms, if such exist (types 1, 2, Fig. 1) or else within the eentromere (types 3 or 4, Fig. 1). 3. The Effects of Fusion on Chiasma Frequency and Distribution In acridids with rod complements and a distributed system of ehiasmata the ehiasma frequency of a particular chromosome is a function of the length of that chromosome (see for example Table 2, pg. 118 in John and Henderson, 1962). Table 1. A comparison of chiasma frequency in the fusion metacentric of Percassa and the two next smallest telocentric equivalents BivMents
46 5d-7
No. of Xta 1
2
3
4
5
17
570 87
108 328
5 283
-2
Total cells
Mean Xa freq.
700 700
2.1 3.2
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Fig. 6 a--c. Chiasma differences in the fusion metacentric (4 6) of Percctssaand its next smallest telocentrie equivalents (autosomes 5 and 7). The 4 6 bivalent has one interstitial (a), two distal-distal (b) and three distal-interstitial-distal (c) chiasmata respectively. By contrast bivalents 5 and 7 respectively have two proximal-distal/two proximal-distal (a) and two proximal-distal/one interstitial (b and c) ehiasmata
I n Percassa a u t o s o m e s 5 a n d 7 r e p r e s e n t t h e chromosomes n e x t smallest in size to those i n v o l v e d in t h e fusion a n d in t e r m s of t h e i r length are e x p e c t e d to h a v e a e h i a s m a f r e q u e n c y no g r e a t e r t h a n t h a t of 4 a n d 6. I f one compares b o t h t h e f r e q u e n c y a n d t h e d i s t r i b u t i o n of e h i a s m a t a in the fusion m e t a e e n t r i e with t h a t of autosomes 5 a n d 7 it is clear t h a t : (a) The m e a n ehiasma frequency of t h e 4 6 e l e m e n t is significantly below t h e c o m b i n e d value for 5 a n d 7 (Table 1, Fig. 6). This results from t h e fact t h a t t h e m o s t c o m m o n class in t h e 4 6 b i v a l e n t is t h a t with two e h i a s m a t a whereas in t h e c o m b i n e d 5 a n d 7 groups it is t h e three a n d four ehiasma classes which p r e d o m i n a t e . I n d e e d in e x t r e m e situations p a e h y t e n e p a i r i n g m a y fail a t t h e e e n t r o m e r e region of t h e 4 6 m e t a e e n t r i e a n d this failure m a y even e x t e n d to t h e entire 6-arm. (b) The d i s t r i b u t i o n of e h i a s m a t a is also significantly different. This results p r e d o m i n a n t l y from t h e fact t h a t t h e r e are m a n y m o r e p r o x i m a l a n d interstitial e h i a s m a t a in b i v a l e n t s 5 a n d 7 b y comparison w i t h t h e 4 6 b i v a l e n t (Table 2). C o m p a r a b l e p a t t e r n s of c h i a s m a m o d i f i c a t i o n are k n o w n in three other eases of fusion. Chloe~t4e conspersa, a n o r t h A m e r i c a n grasshopper, has t h r e e long
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Table 2. A comparison o4 chiasma distribution in ~he fusion metacentric of Percassa and the two next smallest telocentric equivalents. D = distal, I -- interstitial and P -- proximal chiasma siting BiN No. and type of Xta valents 1
D 4~6 5 7 5-~7
Total cells
2
I
16 1 91 33 253 44 344 77
3
P
DD
DI
-19 60 79
523 45 1 75 -4 1 79
PD
II
1 1 477 2 301 778 2
4
DPI
DPD DID
IID DDD
DPPD DIID
1 2
24 -1 1
2
4 ----
2
59 ---
22
1 ----
700 700 700 1400
m e t a c e n t r i c pairs which p r o b a b l y r e p r e s e n t fusions between elements 1-5,2-'8 a n d 3 7 of a n originally e l e v e n - m e m b e r e d set of r o d autosomes. The two largest of t h e five r e m a i n i n g rods (autosomes 4 a n d 6), which a p p r o a c h m o s t closely t h e size of the i n d i v i d u a l arms of t h e m e t a c e n t r i e s , show a p r e d o m i n a n c e of p r o x i m a l l y localised c h i a s m a t a . B y c o n t r a s t in t h e m e t a c e n t r i e p a i r all c h i a s m a t a are d i s t a l l y localised (l~othfels a n d Wrigley, unpublished). Similarly, in t h e L~ X fusion which characterises n e o - X u p o p u l a t i o n s of Podisma pedestris ( J o h n a n d H e w i t t , 1970; H e w i t t a n d J o h n , 1972) t h e L 3 p a i r form fewer c h i a s m a t a when conjoined to t h e X t h a n when t h e y occur as free elements in XO populations. There are also significantly m o r e d i s t a l l y sited c h i a s m a t a in the n e o - X Y state. F i n a l l y in exp e r i m e n t a l l y r e a r e d F l - i n d i v i d u a l s h e t e r o z y g o u s for a tobacco mouse m e t a c e n t r i c a n d t h e two e q u i v a l e n t p r o g e n i t o r house mouse telocentrics there is genetic evidence for a suppression of crossing-over in t h e c e n t r o m e r e region between the m e t a c e n t r i c a n d its homologous telocentries ( C a t t a n a c h a n d Moseley, 1973). A r e a d j u s t m e n t of chiasma p a t t e r n is not, however, an a u t o m a t i c consequence of fusion. F o r e x a m p l e , p o p u l a t i o n s of the g r a s s h o p p e r Oedaleonotus enigma are p o l y m o r p h i c for a 4 5 fusion. C h i a s m a t a are p r e d o m i n a n t l y d i s t a l in the chromosomes concerned in t h e fusion w h e t h e r these are p r e s e n t as t h e basic h o m o z y g o t e , t h e fusion h e t e r o z y g o t e or t h e fusion homozygote. Here, as one m i g h t expect, t h e absence of a n y effect d e p e n d s on prior distal localisation ( H e w i t t a n d Schroeter, 1968). Moreover in two n e w l y arisen fusion m u t a n t s which have n o t become e s t a b l i s h e d in n a t u r a l p o p u l a t i o n s no change in chiasma p a t t e r n has occurred. This is t r u e of the h e t e r o z y g o u s Ma M 5 fusion r e p o r t e d b y S o u t h e r n (1967) in the g r a s s h o p p e r Myrmeleotettix maeulatus. I t is also t r u e of A K R T-1 male mice h o m o z y g o u s for a fusion b e t w e e n a u t o s o m e s 6 a n d 15 (Polani, 1972). 4. The Possibilities for Fission
Three a r g u m e n t s are c o m m o n l y raised a g a i n s t the existence of stable telocentrics in n a t u r a l p l a n t a n d a n i m a l p o p u l a t i o n s :
(a) Experimentally produced telocentrics are unstable, consequently all teloeentrics are unstable (White, 1973). I t is w o r t h recalling t h a t m a n y i n d u c e d t r a n s l o c a t i o n s a n d inversions are similarly unstable, y e t b o t h these categories of exchange h a v e u n d o u b t e d l y p l a y e d a n i m p o r t a n t role in c h r o m o s o m e evolution. Clearly t h e i n s t a b i l i t y of i n d u c e d s t r u c t u r a l changes c a n n o t be used to p r e d i c t t h e beh a v i o u r of s p o n t a n e o u s changes of the same category.
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(b) There is no unequivocal evidence that centromeres do /use or cleave (Hsu and Mead, 1969), there are no known cases o/spontaneous/usion in vivo or in vitro and there is no satis/actory explanation o] the source o/ new centromeres needed to create new elements after /ission (Wurster and Benirschke, 1968, 1970). In fact at the time these assertions were made there was already substantive evidence for the occurrence of fission in vivo (Southern, 1969) and additional cases have been added since that time both in vivo (Hewitt and John, 1972) and in vitro (Kato et el., 1973). What is open to dispute is not the occurrence of fission, which is a fact, but the mechanism of fission. I t was Muller (1940b) who first clearly enunciated the rules of structural rearrangement. According to these rules : (1) All viable structural changes require at least two intercalary breaks, and (2) reconstructed chromosomes can only function mechanically if they are monocentric and dite]ic. That is, one centromere and two telomeres are necessary and permanent chromosome organelles. The assumption that all rod chromosomes must possess a second arm stems directly from these rules. To maintain them in cases of fission one has to interpose a donor chromosome which provides both a second intercalary site for breakage and a source of stable telomeres (White, 1957). I n all three cases of fission referred to above, however, it is clear that no donor chromosome has existed to provide for the "dissociation" of the metacentric. The human data show that, contrary to Muller's rules, a chromosome does not have to be monocentric to survive. Provided the two centromeres are close, dicentrics can function both in mitosis and in meiosis. Likewise fission can be accommodated without the necessity of a donor ff breakage can occur within the centromere and the fracture sites so produced can stabilise without the necessity of added telomeres. That the centromere has a capacity for engaging in exchange even when terminally located is suggested by observations relating to the occurrence of neocentric activity in the X-univalent of male trimerotropine grasshoppers. In one group of these, conventionally referred to as section B, some of the chromosomes, including the X, are metacentric. Since species with such metacentrics retain the diploid number of 23c~ 24 ~ it would appear that the metacentrics have arisen by intra-chromosomal rearrangement. Where the X is metacentric it frequently shows terminal neo-centric activity when present as a univalent at male meiosis (see Fig. 8, pg. 133 in John, 1973). One explanation which can account for such behaviour is that the rearrangement in question is a pericentric inversion in which one of the break points has been in a terminally located centromere. This would be expected to leave a portion of the original centromere at one end while transferring the bulk of it to an interstitial location and this, in turn, would explain the consistent neocentric activity at the original centric end of the now metacentric X. The occurrence of iso-chromosomes also confirms a capacity for breakage within the centromcre itself. Indeed the production and survival of iso-chromosomes must imply that in at least some metacentrics the centromere has an essentially duplicate nature. What remains to be qualified is whether this duplicate nature is itself a consequence of dicentricity or whether, as Lima-de-Faria ~ssumes, it stems from an inherently reverse repeat organisation. Indeed, the occurrence of both fusion and fission mechanisms raises the interesting possibility that meta-,
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acro- a n d telocentric entities m a y exist in either p r i m a r y or s e c o n d a r y states d e p e n d i n g on their m o d e of origin; states, moreover, with d i s t i n c t o r g a n i s a t i o n a l a n d e v o l u t i o n a r y potentialities.
(c) Forms of structure and organisation may exist in chromosomes which are not revealed by ordinary light microscopy after conventional cytological techniques. I t is in these t e r m s , for e x a m p l e , t h a t W h i t e (1973) defends his belief t h a t the r o d chromosomes of all animals, a n d b y i m p l i c a t i o n all p l a n t s too, are acrocentric a n d i n v a r i a b l y c o n t a i n a m i n u t e second arm. The a l t e r n a t i v e interp r e t a t i o n t h a t t h e m i n u t e s t r u c t u r e s r e g a r d e d b y h i m as a r m s are in r e a l i t y p a r t of t h e s t r u c t u r e of t h e c e n t r o m e r e is dismissed because " s u c h bodies are a l r e a d y split a t p r o p h a s e " a n d " t h e i r o r i e n t a t i o n a t e a r l y a n a p h a s e i n d i c a t e s t h a t it is t h e n o n - s t a i n i n g region which is t h e organ of a t t a c h m e n t to t h e s p i n d l e " . The visible doubleness of these "tiny b o d i e s " in fact has no bearing whatsoever on t h e issue since the eentromere, like t h e rest of t h e c h r o m a t i d , is k n o w n to be double a t all stages of m i t o t i c a n d meiotic prophase. Moreover, while i t is agreed t h a t t h e short a r m s of genuine acroeentrics often do n o t a p p e a r to flex on t h e first meiotic spindle, because t h e y either fold b a c k onto t h e m a i n a r m or else are d r a w n s t r a i g h t w i t h i n t h e spindle system, it is e v i d e n t t h a t in a t least some rods t h e spindle a t t a c h m e n t sites do a p p e a r to be genuinely t e r m i n a l
(Stethoph yma, Br yodema ). The objections a g a i n s t t h e occurrence of stable teloeentrics are t h u s all open to question, which implies t h a t t h e m e c h a n i s m s of R o b e r t s o n i a n exchange, w h e t h e r b y fusion or b y fission, are also open issues. E l e c t r o n m i c r o s c o p y m a y well y e t resolve this p e r p l e x i n g p r o b l e m b u t it c e r t a i n l y has n o t done so yet.
Aclcnowledgements. We are grateful to our colleague Graham Webb for his assistance with the C-banding preparations used in this study and to Professor Klaus Rothfels for constructive criticism of the manuscript.
References Bcdo, D.: C-banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera:Simuliidae). Chromosoma (Berl.) 51, 291-300 (1975) Bru~re, A. N., Zartman, D. L., Chapman, H. M. : The significance of the G-bands and C-bands of three different Robertsonian translocations of domestic sheep (Ovis aries). Cytogenet. Cell Genet. 13, 479488 (1974) Cattanach, B. M., Moseley, H.: Nondisjunction and reduced fertility caused by the tobacco mouse metacentric chromosomes. Cytogen. Cell Genet. 12, 264-287 (1973) Comings, D. E., Avelino, E. : DNA loss during Robertsonian fusion in studies of the tobacco mouse. Nature (Lond.) New Biol. 237, 199 (1972) Comings, D. E., Okada, T. A. : Whole mount electron microscopy of the centromere region of metacentric and telocentric mammalian chromosomes. Cytogenetics 9, 436449 (1970) Dunbar, R. W. : The salivary gland chromosomes of seven forms of blackflies included in Eusimulium aureum Fries. Canad. J. Zool. 37, 495-525 (1959) Evans, H. J., Buckland, 1~. A., Sumner, A. T. : Chromosome homology and heterochromatin in goat, sheep and ox studied by banding techniques. Chromosoma (Bert.) 42, 383402 (1973) Ferguson-Smith, M.A.: Human chromosomes in meiosis. Proc. IVth Int. Cong. Human Genet. chapter 4, 195-210 (1972) Helwig, E . R . : Multiple chromosomes in Philocleon anomalous (0rthoptera:Acrididae). J. Morph. 69, 317-327 (1941) Helwig, E. 1~. : Cytology and taxonomy. Bios 29, 59-71 (1958)
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Hewitt, G. M., John, B. : Inter-population sex-chromosome polymorphism in the grasshopper Podisma pedestris. II. Population parameters. Chromosoma (Berl.) ]7, 23-42 (1972) Hsu, L. Y. F., Hyon, J . K . , Sujansky, E., Kousseff, B., Hirschhorn, K.: Reciprocal translocation versus centric fusion between two No. 13 chromosomes. A case of 46, XX,-13,+t (13 ;13) (p12 ;q13) and a case of 46, XY,-13,+t (13 ;13) (p12 ;p12). Cytogenet. Cell Genet. 12, 235-244 (1973) Hsu, T.C., Mead, R . A . : Mechanisms of chromosomal changes in mammalian speciation. In: Comparative mammalian eytogenetics, pp. 8-17. Berlin-Heidelberg-New York: Springer 1969 John, B. : The cytogenetic system of grasshoppers and locusts. II. The origin and evolution of supernumerary segments. Chromosoma (Berl.) 44, 123-146 (1973) John, B., Henderson, S. A. : Asynapsis and polyploidy in Schistocerca paranensis. Chromosoma (Berl.) 1], 111-147 (1962) John, B., Hcwitt, G. M.: Karyotype stability and DNA variability in the Acrididae. Chromosoma (Berl.) 20, 155-172 (1966) John, B., Hewitt, G. M. : Inter-population sex chromosome polymorphis in the grasshopper Podisma pedestris. I. Fundamental facts. Chromosoma (Berl.) ]1, 291-308 (1970) Kato, H., Sagai, T., u T . H . : Stable telocentrie chromosomes produced by centric fission in Chinese hamster cells in vitro. Chromosoma (Berl.) 40, 183-192 (1973) KlA~tersk~, I., Natarajan, A.T., Ramel, C.: Heterochromatin distribution and chiasma localisation in the grasshopper Bryodema tuberculata (Fabr.) (Acrididae). Chromosoma (Berl.) 44, 393404 (1974) Lima-de-Faria, A.: The role of the kinetochore in chromosome organisation. Hereditas (Lund) 42, 85-160 (1956) Muller, H . J . : Bearings of the Drosophila work on systcmatics. In: The new systematics, pp. 185-268. Oxford: Clarendon Press 1940a Muller, H. J. : An analysis of the process of structural change in chromosomes of Drosophila. J. Genet. 40, 1-66 (1940b) Niebuhr, E.: Dicentric and monocentric Robertsonian translocations in man. Hum. Genet. 16, 217-226 (1972) O'Neill, F. J., Miles, C. P. : Specific C band patterns in continuous human lymphoblastoid cell lines. Canad. J. Genet. Cytol. 16, 305-315 (1974) Polani, P. E. : Centromere localisation at meiosis and the position of chiasmata in the male and female mouse. Chromosoma (Berl.) ]6, 343-374 (1972) Southern, D. I. : Spontaneous chromosome mutations in the truxaline grasshoppers9 Chromosoma (Ber].) 22, 241-247 (1967) Southern, D . I . : Stable telocentrie chromosomes produced following centrie misdivision in Myrmeleotettix maculatus (Thunb.). Chromosoma (Berl.) 26, 140-147 (1969) Stack, S. M. : Differential Giemsa staining of kinetochores and nucleolus organiser heterochromatin in mitotic chromosomes of higher plants. Chromosoma (Berl.) 47, 361-378 (1974) Tjio, J . H . , Levan, A.: Chromosome analysis of three hyperdiploid ascites tumors of the mouse. Lunds. Univ. Arsskr. N.F. Avd. 2, 1-51 (1954) Voiculeseu, I., Vogel, W., Wolf, U. : Karyotyp und Heterochromatinmuster des rum~nischen Hamsters (Mcsocrice~us newtoni). Chromosoma (Berl.) 39, 215-224 (1972) White, M. J. D. : Some general problems of chromosomal evolution and speeiation in animals. Surv. biol. Progr. ], 109-147 (1957) White, M. J. D. : Animal cytology and evolution, 3rd ed. London: Cambridge University Press 1973 Wurster, D. H., Benirschke, K. : Chromosome studies in the superfami]y Bovoidea. Chromosoma (Berl.) 25, 152-171 (1968) Wurster, D . H . , Benirschke, K.: Indian muntjac, Muntiacus muntjak: A deer with a low diploid chromosome number. Science 168, 1364-1366 (1970) Yunis, J. J., u W. G.: Heterochromatin, satellite DNA and cell function. Science 174, 1200-1209 (1971) Received June 16-30, 1975/Accepted June 17, 1975 by H. Bauer Ready for press July 1, 1975
Causes and Consequences of Robertsonian Exchange B e r n a r d J o h n a n d Michael F r e e m a n Department of Population Biology, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra City, A.C.T 2601, Australia Abstract. At least two types of Robertsonian exchange are now known in the acrocentrie chromosomes of man. Both types involve breakage in the arms adjacent to the centromere. Evidence is presented for a third type of exchange, one involving breakage within the centromere itself, in the grasshopper Percassa rugi/rons. In this species, which is regularly homozygous for a single fusion metacentric, the eighteen rod autosomes have small but pronounced granules at the centric end of the chromosome. When C-banded these granules show differential Giemsa staining and appear to represent centromeric chromomeres; these chromomeres are lacking in the metacentric fusion product. Equivalent fusions may have occurred in some mammal species too and possible examples of this are discussed in sheep and mice. The Percassa fusion has led to a modification in both the frequency and the distribution of chiasmata as judged by a comparison of these properties in the metacentric relative to the two next smallest rod equivalents. Comparable modifications are known to occur in other naturally occurring fusions but these changes are certainly not automatic consequences of fusion since they are not shown in at least some newly produced fusion mutants.
1. The Nature of Fusion I t was Muller in 1940a who i n t r o d u c e d t h e t e r m " w h o l e a r m t r a n s f e r " to describe exchanges where whole, or n e a r l y whole, a r m s are t r a n s p o s e d or interchanged. Such whole a r m transfers include those exchanges c o m m o n l y referred to as R o b e r t s o n i a n r e a r r a n g e m e n t s which involve the fusion of two rod chromosomes to y i e l d one m e t a c e n t r i c . F u s i o n s of this t y p e h a v e c o m m o n l y been docum e n t e d b o t h in n a t u r a l p o p u l a t i o n s a n d in tissue culture lines. I n a m a j o r i t y of these cases i t has been a s s u m e d t h a t t h e r e a r r a n g e m e n t involves t h e union of t h e long a r m s of two a c r o c e n t r i e chromosomes following b r e a k a g e p r o x i m a l to t h e c e n t r o m c r e in t h e long a r m of one c h r o m o s o m e a n d s i m u l t a n e o u s b r e a k a g e in t h e s h o r t a r m of t h e o t h e r ( t y p e 1 exchange, Fig. 1). There is no d o u b t t h a t such a m e c h a n i s m of e x c h a n g e does occur. I t has been convincingly d o c u m e n t e d , for e x a m p l e , in t h e case of homologous fusions b e t w e e n t h e g e n u i n e l y a c r o c e n t r i c autosome-13 p a i r of m a n (Hsu et al., 1973). I t is e q u a l l y clear, however, t h a t similar fusions involving this same p a i r of homologues m a y arise following b r e a k a g e w i t h i n each of t h e s h o r t a r m s of t h e two chromosomes (Hsu et al., 1973). The m e t a c e n t r i c s so p r o d u c e d are t h u s dicentrie (type 2 exchange, Fig. 1) a n d similar dicentrics h a v e now been identified in several o t h e r fusions involving t h e acrocentric c h r o m o s o m e s of m a n ( F e r g u s o n - S m i t h , 1972; Niebuhr, 1972; O'Neil a n d Miles, 1974). Such dicentrics are e v i d e n t l y quite stable since t h e y are t r a n s m i t t e d b e t w e e n generations. P r e s u m a b l y t h e close p r o x i m i t y of the two centromeres leads to p r e d o m i n a n t l y parallel s e p a r a t i o n w i t h t h e two c e n t r o m e r e s belonging to t h e s a m e c h r o m a t i d passing to t h e s a m e pole a t a n a p h a s e . To w h a t e x t e n t
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Rearrangement
Type
Associated loss
T~
t:i
IJ
89
Fig. 1. Possible modes of Robertsonian exchange leading to whole arm fusion. It is assumed that no direct adhesion of chromosome ends is possible so that fusion must depend on prior breakage. Break points are indicated by arrow heads. The blocks adjacent to the centromere represent constitutive heterochromatin
the metacentrics in other species m a y also prove to be " c l o s e " dicentrics is n o t known. Helwig (1941, 1958) believed t h a t the metacentrics of grasshoppers were regularly dicentric h u t did n o t offer a n y s u b s t a n t i v e evidence i n support
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of his belief. More recently Moens and lZothfels (unpublished) have evidence from EM studies t h a t the metaeentries of the grasshopper Chloealtis conspersa includes two structures identified by them as eentromeres which are in the same synaptonemal complex and 1 nm apart. Additionally in the blackflies Eusimulium aureum and Cnephia lapponica where the haploid number has been reduced by fusion from n = 3 to n = 2, the dicentrie nature of the large fusion chromosome has been inferred from the pattern of eetopic pairing (Dunbar, 1959; Rothfels and Mason, unpublished). Since, as the human data show so convincingly, morphologically similar fusions m a y arise in different ways it is apparent t h a t the rather loose term "Robertsonian fusion" is in need of dearer definition. This is particularly important in view of the fact that different modes of exchange involve the loss of more or less chromosome material (Fig. 1) and this, in turn, m a y be expected to lead to different consequences in terms of the effect of the fusion on viability or fertility (Brugre et al., 1974). Fundamental to any consideration of the nature of Robertsonian exchange is the problem of the organisation of the eentromere and the extent to which exchanges m a y occur within it. Rather surprisingly our most definitive knowledge of the structure of the eentromere still comes not from the electron microscope but from studies made with the light microscope. This m a y well stem from the fact that most electron microscope studies have used oriented meta-anaphase stages only whereas light microscope studies have employed a wider range of stages including e-mitotic chromosomes and paehytene-diakinesis stages. Such studies reveal that in metaeentrie chromosomes the region conventionally referred to as the eentromere includes one or more chromomeres joined to the main chromosome arms and, where applicable, to each other by extended fibrillae (Lima-de-Faria, 1956; John and Hewitt, 1966). The precise number of chromomeres per eentromere varies. In part this m a y reflect genuine differences in specific eentromere organisation. I n part, however, it also appears to reflect some tendency of these ehromomeres to fuse in more condensed stages of division or following prolonged eolchicine treatment. From the illustrations provided b y Lima-de-Faria it is clear t h a t in metaeentries of Hyacinthus and Traclescantia it is the central fibrilla zone which forms the site of attachment of halfspindle fibres (see Fig. 10, pg. 96 and Fig. 26 pg. 106 of Lima-de-Faria, 1956). W h a t remains to be established in these eases is the nature and function of the organelles referred to as eentromeric ehromomeres. I n at least some rod-chromosomes the fibrillae to which tile spindle fibres attach are terminal as in the ease of the grasshopper Stethophyma grossum (see Figs. 15 and 16, pg. 97 of Lima-de-Faria, 1956 and compare with Fig. 2 of this paper). The same is true of a second grasshopper species Bryodema tuberculata (Kl~tersk~ et al., 1974). The chromosomes of both these species are regarded by White (1973) as aeroeentrie but by all known criteria appear to be teloeentrie.
2. The P e v c a s s a Fusion
Chromosome complements regularly homozygous for one or more metacentrics have been described in several acridid species. Since a majority of male members
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Fig. 2a and b. The chromosomes of Stethophyma grossum at first metaphase of male meiosis: (a) represents the entire complement (2n = 23) while (b) is a single enlarged bivalent
of this family have 23 rod chromosomes it is natural to conclude that these metacentrics have been produced secondarily by fusion. The nature of this fusion has not been rigorously defined but, on the assumption that the rods involved are acrocentric, White (for detailed references see White, 1973) has consistently maintained that they must arise by type 1 exchange (Fig. 1). The Australian catantopinid Percassa rugiJrons, a montane and subalpine brachypterous species living on the low shrub stratum, has one fixed metaeentric pair. Here there are 21 chromosomes in the male complement (XO) anct 22 in the female (XX). If the 18 rod autosomes are arranged in decreasing size order it can be shown that the arms of the metacentric correspond to chromosomes 4 and 6 of an originally ll-membered autosome set (Fig. 3d). At both mitosis and meiosis the spindle attachment sites of the rods appear to be strictly terminal (Fig. 3 a--c). Especially clear small bodies are sometimes, though not invariably, visible at the eentric ends of the rod bivalents at diakinesis (Fig. 4b--f). These usually appear as single entities but can occasionally be seen to be double (Fig. 4 e). Equivalent structures can also be found in spermatogonial mitoses (Fig. 3d). No such bodies have ever been seen in the metacentries where the centromeres invariably appear as fine fibrillar regions (Fig. 4a). C-banding leads to intense staining of two small spherical bodies, which sometimes merge into one, at the ends of each rod both in mitotic (Fig. 4g) and in meiotic chromosomes (Fig. 4h). Again no equivalent structures occur in the metacentrics.
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In many eucaryote species the occurrence of constitutive heterochromatin in the arm material immediately adjacent to the centromere may lead to intense pro-centrie C-banding which tends to obscure details of the centromere itself. Even so there are good grounds for concluding that the proeentric heterochromatin and the centromere itself are quite distinct structures though they may be located contiguously on the chromosome. For example in the polytene chromosomes of blackflies (Simuliidae) the centromere can be identified as an expanded region marked in many species by a heavy band which undergoes specific ectopic pairing. Using C-banding techniques, Bedo (1975) has recently shown that in Simulium ornatipes the centromere band itself stains deeply but the surrounding proeentric heterochromatin does not. In Simulium melatum, on the other hand, the eentromere band is C-negative and appears as a non-staining gap with deeply staining flanking heterochromatic structures. Similarly, differentiation between pro-eentric C-bands and a more localised centromeric staining has been obtained in Me~ocricetus newtoni by using a modified C-banding technique involving pretreatment with N a O H prior to incubation with S SC (Voieulescu et al., 1972). Moreover contrary to the claim of Yunis and u (1971), constitutive heterochromatin is not universally found around the eentromere. Thus in Microtus agrestis the autosomes show only one or two deeply stained granules in the eentromere region. Similar granules have been reported by Stack et al. (1974) in the meta- and acro-centric chromosomes of Allium cepa and Ornithogalum virens and they have concluded that these granules represent fused centromerie chromomeres. This interpretation is supported by the fact that the spindle fibre remnants can be seen directly attached to them (see Fig. 11, pg. 368 of Stack et al., 1974). Like,rise in Bryodema tuberculata the site of spindle fibre attachment is terminal and lies distal to the C-band (see Fig. 15, pg. 399 of Kls et al., 1974). We are of the opinion that in Percassa the granules which stain with Giemsa on C-banding and the bodies which are present at diakinesis are one and the same structure and represent the centromeric chromomeres. This implies that the rods in Percassa are telocentric. This interpretation is supported by a comparison with the acridine grasshopper Gastrimargus musieus. In this species (Fig. 5a) a complete range of chromosome types exists from genuinely acrocentric to genuinely telocentrie. Moreover chromosomes which appear as acrocentrie in mitotic chromosomes can be shown to have two clear arms at second division of meiosis too whereas autosomes 9, 10 and 11 which appear as telocentrics in mitotic tissues never show a second arm at meiosis (Fig. 5b). When C-banded the acrocentrics show a clear short arm, which also stains differentially, beyond the C-positive granules of the centromere. Telocentrics, on the other hand, like the rods of Percassa, are terminated by the C-positive granules (Fig. 5e). The variation in the size of the short arms found in the acrocentrics of Gastrimargus is especially interesting and suggests that the centric regions of rod chromosomes may sometimes "grow" by the addition of heterochromatin so leading to the development of new arms. Thus the short arms of Gastrimargus acrocentrics show a distinctive appearance in C-banded preparations and appear intermediate between the non-staining long arms and the deeper staining centric chromomeres. Indeed in overstained preparations it is not always possible to distinguish the two. We conclude that the metacentrics of Percassa are the product of fusion between elements 4 and 6 of an originally ll-membered telocentric set. This implies
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Fig. 3a--d. The chromosomes of male Percassa rugi/rons to show centric activity at spermatogonial metaphase (a and b) and at first metaphase of male meiosis (c). The metacentric is designated as M and arrowheads indicate sites of eentric activity. (d) shows the haploid complement at spermatogonial metaphase
t h a t t h e exchange m u s t have occurred following b r e a k a g e in t h e fibrfllar comp o n e n t of two telocentrics ]eading to t h e loss of t h e centrie chromomeres. This suggests a t h i r d t y p e of R o b e r t s o n i a n fusion (type 3, Fig. 1), a t y p e which involves
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Fig. 4 a ~ . Centric organisation of male Percassa bivalents at diakinesis of meiosis (a--f) and o5 C-banded chromosomes at e-mitosis (g) and diakinesis (h) resl0eetively. Note absence of eentromerie chromomeres in the metaeentrie (a and g) compared to their consistent presence (arrows) in the rods (b--h)
a m i n i m u m loss of chromosome material. A n e q u i v a l e n t fusion is, of course, also possible b e t w e e n acroeentrics (type 4, Fig'. 1). F u s i o n s of this t y p e , involving b r e a k a g e within t h e eentromere, m a y p r o v e to be m o r e widespread. I n d e e d t h e r e are several suggestive eases a l r e a d y in the l i t e r a t u r e in m a m m a l s . F o r e x a m p l e , sheep (Ovis aries) h a v e 54 chromosomes. Six of t h e 52 a u t o s o m e s are m e t a c e n t r i e , t h e r e m a i n d e r are rods. These rods h a v e s u b s t a n t i a l blocks of p r o c e n t r i e h e t e r o c h r o m a t i n b u t t h e eentromeres themselves do n o t stain with either Giemsa or with quinacrine. E v a n s et al. (1973) r e m a r k t h a t " i f we define t h e e e n t r o m e r e itself as t h e n a r r o w e s t p o i n t of the c h r o m o s o m e t h e n in some eases t h e r e is in some chromosomes a suggestion of a m i n u t e short a r m which does n o t stain i n t e n s e l y " a n d on this s o m e w h a t t e n u o u s g r o u n d a c c e p t t h a t t h e rods in sheep are aeroeentrie. A u t o s o m a l fusions between t h r e e d i s t i n c t pairs of rods h a v e been r e p o r t e d in elite breeds of sheep from New Zealand. I n two of these t h e m e t a c e n t r i c s show s u b s t a n t i a l blocks of h e t e r o c h r o m a t i n which are d i s t r i b u t e d e v e n l y on either side of t h e centromere. I n t h e t h i r d ease there is a single h e t e r o e h r o m a t i e block which is e x o c e n t r i e a l l y l o c a t e d in one of t h e a r m s of t h e m e t a c e n t r i e (Brugre et al., 1974). Bru6re also assumes t h a t t h e rods are acroeentric a n d suggests t h a t t h e t h i r d ease results from centric fusion (type 6, Fig. 1) whereas t h e fh'st a n d second eases are dieentries (type 2, Fig. 1). The a l t e r n a t i v e i n t e r p r e t a t i o n is t h a t t h e rods are telocentrie a n d t h a t t h e exchanges
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Fig. 5 a - - c . The chromosomes of male Gastrimargus musicus at c-mitosis (a) and their structure at second anaphase of meiosis (b) and following C-banding a t c-mitosis (c). Note t h a t where short arms are present these can be dectected b o t h at c-mitosis, where they stain differentially, and at second anaphase, where they either show flexure (I and 2) or else are drawn straight in the spindle (X, 5 and 8)
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are a consequence o f breakage within the eentromere (types 3 and 5, Fig. 1, respectively). A somewhat similar problem exists in the genus Mus. The house mouse M u s musculus, has 40 rods which are variously described as acro- or telocentric according to the preference of individual authors. Fusions are not uncommon in mouse cell cultures and here two C-positive blocks are often found one on each side of the eentromere; this could be accommodated by at least four types of exchange (types 1, 2, 3 and 4, Fig. 1). The 38 autosomal rods all show substantial blocks of C-positive material but nothing that can be construed as a short arm. If a short arm does exist then C-banding obscures it. The early studies of Tjio and Levan (1954, see for example their Fig. 146, pg. 32) suggests the rods are teloeentric. Using whole mount electron-microscopy of surface-spread colehieinetreated chromosomes Comings and Okada (1970) find that rods of mouse, sheep and goat show single areas of chromatid association. These are located at a terminal position with no evidence for short arms. At the light microscope level these areas correspond to the proximal arm regions, immediately adjacent to the centromere, where sister chromatids remain in contact at first anaphase of meiosis and at c-mitosis. Metacentric chromosomes of man, mouse L-cells, sheep and Chinese hamster, however, have two distinct areas of chromatid association separated by a central zone of attenuated fibres, as too do acrocentric chromosomes from mouse L-cells. Comings and Okada conclude that the rods in mouse, sheep and goat are telocentric and that the metacentrics in them have arisen as the result of a simple breakage reunion event within the fibres of the centromere itself. Fusions between rods have also occurred in natural populations of Mus. In the extreme case this has led to the development of a geographical isolate which is homozygous for seven distinct fusions and which is known by the specific name M u s poschiavinus, the tobacco mouse. Comings and Avelino (1972) have shown that there is no detectable loss of satellite DNA in this total of 14 fusions which implies that either the constitutive heterochromatic regions have been preserved intact during the fusion process or else that any loss consequent upon fusion has been subsequently rectified. This again is consistent with the exchange occurring either within the short arms, if such exist (types 1, 2, Fig. 1) or else within the eentromere (types 3 or 4, Fig. 1). 3. The Effects of Fusion on Chiasma Frequency and Distribution In acridids with rod complements and a distributed system of ehiasmata the ehiasma frequency of a particular chromosome is a function of the length of that chromosome (see for example Table 2, pg. 118 in John and Henderson, 1962). Table 1. A comparison of chiasma frequency in the fusion metacentric of Percassa and the two next smallest telocentric equivalents BivMents
46 5d-7
No. of Xta 1
2
3
4
5
17
570 87
108 328
5 283
-2
Total cells
Mean Xa freq.
700 700
2.1 3.2
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Fig. 6 a--c. Chiasma differences in the fusion metacentric (4 6) of Percctssaand its next smallest telocentrie equivalents (autosomes 5 and 7). The 4 6 bivalent has one interstitial (a), two distal-distal (b) and three distal-interstitial-distal (c) chiasmata respectively. By contrast bivalents 5 and 7 respectively have two proximal-distal/two proximal-distal (a) and two proximal-distal/one interstitial (b and c) ehiasmata
I n Percassa a u t o s o m e s 5 a n d 7 r e p r e s e n t t h e chromosomes n e x t smallest in size to those i n v o l v e d in t h e fusion a n d in t e r m s of t h e i r length are e x p e c t e d to h a v e a e h i a s m a f r e q u e n c y no g r e a t e r t h a n t h a t of 4 a n d 6. I f one compares b o t h t h e f r e q u e n c y a n d t h e d i s t r i b u t i o n of e h i a s m a t a in the fusion m e t a e e n t r i e with t h a t of autosomes 5 a n d 7 it is clear t h a t : (a) The m e a n ehiasma frequency of t h e 4 6 e l e m e n t is significantly below t h e c o m b i n e d value for 5 a n d 7 (Table 1, Fig. 6). This results from t h e fact t h a t t h e m o s t c o m m o n class in t h e 4 6 b i v a l e n t is t h a t with two e h i a s m a t a whereas in t h e c o m b i n e d 5 a n d 7 groups it is t h e three a n d four ehiasma classes which p r e d o m i n a t e . I n d e e d in e x t r e m e situations p a e h y t e n e p a i r i n g m a y fail a t t h e e e n t r o m e r e region of t h e 4 6 m e t a e e n t r i e a n d this failure m a y even e x t e n d to t h e entire 6-arm. (b) The d i s t r i b u t i o n of e h i a s m a t a is also significantly different. This results p r e d o m i n a n t l y from t h e fact t h a t t h e r e are m a n y m o r e p r o x i m a l a n d interstitial e h i a s m a t a in b i v a l e n t s 5 a n d 7 b y comparison w i t h t h e 4 6 b i v a l e n t (Table 2). C o m p a r a b l e p a t t e r n s of c h i a s m a m o d i f i c a t i o n are k n o w n in three other eases of fusion. Chloe~t4e conspersa, a n o r t h A m e r i c a n grasshopper, has t h r e e long
Causes and Consequences of Robertsonian Exchange
133
Table 2. A comparison o4 chiasma distribution in ~he fusion metacentric of Percassa and the two next smallest telocentric equivalents. D = distal, I -- interstitial and P -- proximal chiasma siting BiN No. and type of Xta valents 1
D 4~6 5 7 5-~7
Total cells
2
I
16 1 91 33 253 44 344 77
3
P
DD
DI
-19 60 79
523 45 1 75 -4 1 79
PD
II
1 1 477 2 301 778 2
4
DPI
DPD DID
IID DDD
DPPD DIID
1 2
24 -1 1
2
4 ----
2
59 ---
22
1 ----
700 700 700 1400
m e t a c e n t r i c pairs which p r o b a b l y r e p r e s e n t fusions between elements 1-5,2-'8 a n d 3 7 of a n originally e l e v e n - m e m b e r e d set of r o d autosomes. The two largest of t h e five r e m a i n i n g rods (autosomes 4 a n d 6), which a p p r o a c h m o s t closely t h e size of the i n d i v i d u a l arms of t h e m e t a c e n t r i e s , show a p r e d o m i n a n c e of p r o x i m a l l y localised c h i a s m a t a . B y c o n t r a s t in t h e m e t a c e n t r i e p a i r all c h i a s m a t a are d i s t a l l y localised (l~othfels a n d Wrigley, unpublished). Similarly, in t h e L~ X fusion which characterises n e o - X u p o p u l a t i o n s of Podisma pedestris ( J o h n a n d H e w i t t , 1970; H e w i t t a n d J o h n , 1972) t h e L 3 p a i r form fewer c h i a s m a t a when conjoined to t h e X t h a n when t h e y occur as free elements in XO populations. There are also significantly m o r e d i s t a l l y sited c h i a s m a t a in the n e o - X Y state. F i n a l l y in exp e r i m e n t a l l y r e a r e d F l - i n d i v i d u a l s h e t e r o z y g o u s for a tobacco mouse m e t a c e n t r i c a n d t h e two e q u i v a l e n t p r o g e n i t o r house mouse telocentrics there is genetic evidence for a suppression of crossing-over in t h e c e n t r o m e r e region between the m e t a c e n t r i c a n d its homologous telocentries ( C a t t a n a c h a n d Moseley, 1973). A r e a d j u s t m e n t of chiasma p a t t e r n is not, however, an a u t o m a t i c consequence of fusion. F o r e x a m p l e , p o p u l a t i o n s of the g r a s s h o p p e r Oedaleonotus enigma are p o l y m o r p h i c for a 4 5 fusion. C h i a s m a t a are p r e d o m i n a n t l y d i s t a l in the chromosomes concerned in t h e fusion w h e t h e r these are p r e s e n t as t h e basic h o m o z y g o t e , t h e fusion h e t e r o z y g o t e or t h e fusion homozygote. Here, as one m i g h t expect, t h e absence of a n y effect d e p e n d s on prior distal localisation ( H e w i t t a n d Schroeter, 1968). Moreover in two n e w l y arisen fusion m u t a n t s which have n o t become e s t a b l i s h e d in n a t u r a l p o p u l a t i o n s no change in chiasma p a t t e r n has occurred. This is t r u e of the h e t e r o z y g o u s Ma M 5 fusion r e p o r t e d b y S o u t h e r n (1967) in the g r a s s h o p p e r Myrmeleotettix maeulatus. I t is also t r u e of A K R T-1 male mice h o m o z y g o u s for a fusion b e t w e e n a u t o s o m e s 6 a n d 15 (Polani, 1972). 4. The Possibilities for Fission
Three a r g u m e n t s are c o m m o n l y raised a g a i n s t the existence of stable telocentrics in n a t u r a l p l a n t a n d a n i m a l p o p u l a t i o n s :
(a) Experimentally produced telocentrics are unstable, consequently all teloeentrics are unstable (White, 1973). I t is w o r t h recalling t h a t m a n y i n d u c e d t r a n s l o c a t i o n s a n d inversions are similarly unstable, y e t b o t h these categories of exchange h a v e u n d o u b t e d l y p l a y e d a n i m p o r t a n t role in c h r o m o s o m e evolution. Clearly t h e i n s t a b i l i t y of i n d u c e d s t r u c t u r a l changes c a n n o t be used to p r e d i c t t h e beh a v i o u r of s p o n t a n e o u s changes of the same category.
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(b) There is no unequivocal evidence that centromeres do /use or cleave (Hsu and Mead, 1969), there are no known cases o/spontaneous/usion in vivo or in vitro and there is no satis/actory explanation o] the source o/ new centromeres needed to create new elements after /ission (Wurster and Benirschke, 1968, 1970). In fact at the time these assertions were made there was already substantive evidence for the occurrence of fission in vivo (Southern, 1969) and additional cases have been added since that time both in vivo (Hewitt and John, 1972) and in vitro (Kato et el., 1973). What is open to dispute is not the occurrence of fission, which is a fact, but the mechanism of fission. I t was Muller (1940b) who first clearly enunciated the rules of structural rearrangement. According to these rules : (1) All viable structural changes require at least two intercalary breaks, and (2) reconstructed chromosomes can only function mechanically if they are monocentric and dite]ic. That is, one centromere and two telomeres are necessary and permanent chromosome organelles. The assumption that all rod chromosomes must possess a second arm stems directly from these rules. To maintain them in cases of fission one has to interpose a donor chromosome which provides both a second intercalary site for breakage and a source of stable telomeres (White, 1957). I n all three cases of fission referred to above, however, it is clear that no donor chromosome has existed to provide for the "dissociation" of the metacentric. The human data show that, contrary to Muller's rules, a chromosome does not have to be monocentric to survive. Provided the two centromeres are close, dicentrics can function both in mitosis and in meiosis. Likewise fission can be accommodated without the necessity of a donor ff breakage can occur within the centromere and the fracture sites so produced can stabilise without the necessity of added telomeres. That the centromere has a capacity for engaging in exchange even when terminally located is suggested by observations relating to the occurrence of neocentric activity in the X-univalent of male trimerotropine grasshoppers. In one group of these, conventionally referred to as section B, some of the chromosomes, including the X, are metacentric. Since species with such metacentrics retain the diploid number of 23c~ 24 ~ it would appear that the metacentrics have arisen by intra-chromosomal rearrangement. Where the X is metacentric it frequently shows terminal neo-centric activity when present as a univalent at male meiosis (see Fig. 8, pg. 133 in John, 1973). One explanation which can account for such behaviour is that the rearrangement in question is a pericentric inversion in which one of the break points has been in a terminally located centromere. This would be expected to leave a portion of the original centromere at one end while transferring the bulk of it to an interstitial location and this, in turn, would explain the consistent neocentric activity at the original centric end of the now metacentric X. The occurrence of iso-chromosomes also confirms a capacity for breakage within the centromcre itself. Indeed the production and survival of iso-chromosomes must imply that in at least some metacentrics the centromere has an essentially duplicate nature. What remains to be qualified is whether this duplicate nature is itself a consequence of dicentricity or whether, as Lima-de-Faria ~ssumes, it stems from an inherently reverse repeat organisation. Indeed, the occurrence of both fusion and fission mechanisms raises the interesting possibility that meta-,
Causes and Consequences of gobertsonian Exchange
135
acro- a n d telocentric entities m a y exist in either p r i m a r y or s e c o n d a r y states d e p e n d i n g on their m o d e of origin; states, moreover, with d i s t i n c t o r g a n i s a t i o n a l a n d e v o l u t i o n a r y potentialities.
(c) Forms of structure and organisation may exist in chromosomes which are not revealed by ordinary light microscopy after conventional cytological techniques. I t is in these t e r m s , for e x a m p l e , t h a t W h i t e (1973) defends his belief t h a t the r o d chromosomes of all animals, a n d b y i m p l i c a t i o n all p l a n t s too, are acrocentric a n d i n v a r i a b l y c o n t a i n a m i n u t e second arm. The a l t e r n a t i v e interp r e t a t i o n t h a t t h e m i n u t e s t r u c t u r e s r e g a r d e d b y h i m as a r m s are in r e a l i t y p a r t of t h e s t r u c t u r e of t h e c e n t r o m e r e is dismissed because " s u c h bodies are a l r e a d y split a t p r o p h a s e " a n d " t h e i r o r i e n t a t i o n a t e a r l y a n a p h a s e i n d i c a t e s t h a t it is t h e n o n - s t a i n i n g region which is t h e organ of a t t a c h m e n t to t h e s p i n d l e " . The visible doubleness of these "tiny b o d i e s " in fact has no bearing whatsoever on t h e issue since the eentromere, like t h e rest of t h e c h r o m a t i d , is k n o w n to be double a t all stages of m i t o t i c a n d meiotic prophase. Moreover, while i t is agreed t h a t t h e short a r m s of genuine acroeentrics often do n o t a p p e a r to flex on t h e first meiotic spindle, because t h e y either fold b a c k onto t h e m a i n a r m or else are d r a w n s t r a i g h t w i t h i n t h e spindle system, it is e v i d e n t t h a t in a t least some rods t h e spindle a t t a c h m e n t sites do a p p e a r to be genuinely t e r m i n a l
(Stethoph yma, Br yodema ). The objections a g a i n s t t h e occurrence of stable teloeentrics are t h u s all open to question, which implies t h a t t h e m e c h a n i s m s of R o b e r t s o n i a n exchange, w h e t h e r b y fusion or b y fission, are also open issues. E l e c t r o n m i c r o s c o p y m a y well y e t resolve this p e r p l e x i n g p r o b l e m b u t it c e r t a i n l y has n o t done so yet.
Aclcnowledgements. We are grateful to our colleague Graham Webb for his assistance with the C-banding preparations used in this study and to Professor Klaus Rothfels for constructive criticism of the manuscript.
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Hewitt, G. M., John, B. : Inter-population sex-chromosome polymorphism in the grasshopper Podisma pedestris. II. Population parameters. Chromosoma (Berl.) ]7, 23-42 (1972) Hsu, L. Y. F., Hyon, J . K . , Sujansky, E., Kousseff, B., Hirschhorn, K.: Reciprocal translocation versus centric fusion between two No. 13 chromosomes. A case of 46, XX,-13,+t (13 ;13) (p12 ;q13) and a case of 46, XY,-13,+t (13 ;13) (p12 ;p12). Cytogenet. Cell Genet. 12, 235-244 (1973) Hsu, T.C., Mead, R . A . : Mechanisms of chromosomal changes in mammalian speciation. In: Comparative mammalian eytogenetics, pp. 8-17. Berlin-Heidelberg-New York: Springer 1969 John, B. : The cytogenetic system of grasshoppers and locusts. II. The origin and evolution of supernumerary segments. Chromosoma (Berl.) 44, 123-146 (1973) John, B., Henderson, S. A. : Asynapsis and polyploidy in Schistocerca paranensis. Chromosoma (Berl.) 1], 111-147 (1962) John, B., Hcwitt, G. M.: Karyotype stability and DNA variability in the Acrididae. Chromosoma (Berl.) 20, 155-172 (1966) John, B., Hewitt, G. M. : Inter-population sex chromosome polymorphis in the grasshopper Podisma pedestris. I. Fundamental facts. Chromosoma (Berl.) ]1, 291-308 (1970) Kato, H., Sagai, T., u T . H . : Stable telocentrie chromosomes produced by centric fission in Chinese hamster cells in vitro. Chromosoma (Berl.) 40, 183-192 (1973) KlA~tersk~, I., Natarajan, A.T., Ramel, C.: Heterochromatin distribution and chiasma localisation in the grasshopper Bryodema tuberculata (Fabr.) (Acrididae). Chromosoma (Berl.) 44, 393404 (1974) Lima-de-Faria, A.: The role of the kinetochore in chromosome organisation. Hereditas (Lund) 42, 85-160 (1956) Muller, H . J . : Bearings of the Drosophila work on systcmatics. In: The new systematics, pp. 185-268. Oxford: Clarendon Press 1940a Muller, H. J. : An analysis of the process of structural change in chromosomes of Drosophila. J. Genet. 40, 1-66 (1940b) Niebuhr, E.: Dicentric and monocentric Robertsonian translocations in man. Hum. Genet. 16, 217-226 (1972) O'Neill, F. J., Miles, C. P. : Specific C band patterns in continuous human lymphoblastoid cell lines. Canad. J. Genet. Cytol. 16, 305-315 (1974) Polani, P. E. : Centromere localisation at meiosis and the position of chiasmata in the male and female mouse. Chromosoma (Berl.) ]6, 343-374 (1972) Southern, D. I. : Spontaneous chromosome mutations in the truxaline grasshoppers9 Chromosoma (Ber].) 22, 241-247 (1967) Southern, D . I . : Stable telocentrie chromosomes produced following centrie misdivision in Myrmeleotettix maculatus (Thunb.). Chromosoma (Berl.) 26, 140-147 (1969) Stack, S. M. : Differential Giemsa staining of kinetochores and nucleolus organiser heterochromatin in mitotic chromosomes of higher plants. Chromosoma (Berl.) 47, 361-378 (1974) Tjio, J . H . , Levan, A.: Chromosome analysis of three hyperdiploid ascites tumors of the mouse. Lunds. Univ. Arsskr. N.F. Avd. 2, 1-51 (1954) Voiculeseu, I., Vogel, W., Wolf, U. : Karyotyp und Heterochromatinmuster des rum~nischen Hamsters (Mcsocrice~us newtoni). Chromosoma (Berl.) 39, 215-224 (1972) White, M. J. D. : Some general problems of chromosomal evolution and speeiation in animals. Surv. biol. Progr. ], 109-147 (1957) White, M. J. D. : Animal cytology and evolution, 3rd ed. London: Cambridge University Press 1973 Wurster, D. H., Benirschke, K. : Chromosome studies in the superfami]y Bovoidea. Chromosoma (Berl.) 25, 152-171 (1968) Wurster, D . H . , Benirschke, K.: Indian muntjac, Muntiacus muntjak: A deer with a low diploid chromosome number. Science 168, 1364-1366 (1970) Yunis, J. J., u W. G.: Heterochromatin, satellite DNA and cell function. Science 174, 1200-1209 (1971) Received June 16-30, 1975/Accepted June 17, 1975 by H. Bauer Ready for press July 1, 1975
