Chromosoma (Berl.) 36, 343--374 (1972) 9 by Springer-Verlag 1972

Centromere Localization at Meiosis and the Position of Chiasmata in the Male and Female Mouse P. E. POLANI Paediatric Research Unit, Guy's Hospital 51edical School, London Received December 6, 1971 / Accepted December 22, 1971 Abstract. Techniques for obtaining differential Giemsa staining of the paracentromeric (p. c.) regions of male and female mouse meiotic chromosomes (centromeric heterochromatin) were explored and standard procedures developed for the different meiotic cells in the two sexes. The best result followed the use of heat at controlled pH in SSrcnsen's phosphate buffer or in Standard Saline Citrate (SSC) solutions. With these techniques, morphological features of the p.c. regions and their variation were studied in normal animals (CFLP strain) and in a strain (AKR) homozygous for a centric fusion [T(ll;?)-I Ald] between chromosomes No. 6 and No. 15 (Miller et al., 1971). The Y chromosome was often found to show distinct p.c. staining at first and apparently at second meiotic metaphase, and the X and u chromosomes were found to associate as bivalents by their long arms. Autosomal p.c. regions showed variation in size which might indicate differences between non-homologous chromosomes but a tendency to similarity between homologues. Differences were found between males and females in respect to proportions and variation of bivalents with single and double chiasmata. The relative positions of chiasmata were different in the two sexes. The presence of the eentric fusion in the males did not seem to affect the pairing behaviour of the remaining autosomes or of those taking part in the eentrie fusion. The possibility is discussed that the p.e. regions, to which also other functions would seem to appertain, may be important for chromosome recognition and pairing, possibly on a quantitative basis.

Introduction A full a p p r e c i a t i o n of b i v a l e n t configuration a t diakinesis requires identification of the position of the eentromere. This is directly possible i n some organisms or i n a few favourable cells of others, or it can be i n d i r e c t l y achieved i n certain special circumstances, m a k i n g inferences from the k n o w n somatic m e t a p h a s e appearance of chromosomes. However, the inferences can be fallacious. Therefore generally special techniques are required to d e m o n s t r a t e the eentromere, or the h e t e r o c h r o m a t i n n e x t to it, a t meiosis, a n d indeed were devised some time ago for the s t u d y of a few suitable organisms (see, e.g., Schrader, 1936; Levan, 1946), i n t e r e s t i n g l y m o s t l y those with large a m o u n t s of nuclear DNA. However, t h o u g h cytologically less suitable, it was f o u n d possible to stain

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selectively the centromeres of spermatogonial anaphase chromosomes of the mouse (Schrader, 1936). Rationale and Background of the Experiments The application of Caesium Chloride equilibrium sedimentation density gradient methods to reveal base pair differences in high molecular weight D~qA (Meselson et al., 1957), the development of in vitro dissociation and reassociation techniques (Dory et al., 1960; Marmur and Lane, 1960; Marmur et al., 1963) and of DNA/DNA and DIqA/RNA specific hybridization procedures (Hall and Spiegelman, 1961; Schildkraut et al., 1961), and the advances that followed the various DNA immobilization techniques, for example on cellulose acetate or agar (Bolton andMcCarthy, 1962), have led to the demonstration of the existence in eukaryotes (Britten and Kohne, 1966; Waring and Britten, 1966) of variable amounts of highly repetitive species of nuclear DS~A, characterized by relatively short runs (Southern, 1970) of very similar or identical sequences of base pairs. The highly repetitive nature of the sequences was first discovered when the high speed of reassociation following dissociation of a DNA fraction of the genome could be monitored by physico-chemical measurements (Britten and Kohne, 1968), but it was also noticed that, in some species, e.g. in the mouse, the base-pair composition of this fraction of the DNA deviated from that of the bulk of the DNA. The name of satellite DNA (Kit, 1962) was used to describe these accessory or minor components identified as distinct peaks in buoyant density fractionation. I t was also found that, for example in the mouse, this satellite fraction, which was comparatively A-~T rich, represented 10 % or more of the nuclear D~qA (see, e.g., Walker, 1968), while in other organisms there was much less of it and of different base composition and sometimes hidden within the main band of DNA. However, the highly repetitive DNA was present in all of an impressive list of animals and plants in which it had been sought (Britten and Kohne, 1969). The development (Gall and Pardue, 1969; John et al., 1969; Pardue and Gall, 1969) of an in situ cytological alkali or heat DNA dissociation technique followed by specific hybridization with labelled RNA or DNA sequences--both procedures being carried out on fixed cells at prophase or metaphase-has allowed the detection of the cellular and chromosomal localization of the highly repetitive satellite nuclear DNA. In the mouse (Jones, 1970; Pardue and Gall, 1970), the satellite DNA was found to be localized at, or close to, the centromere, a region previously described as heterochromatic (Ohno et al., 1957 ; Church, 1965), and Pardue and Gall, (1970) noticed that the use of Giemsa's stain coloured intensely the eentromeric heterochromatin to which the all-labelled satellite nucleic

Centromeres and Chiasmata in the Mouse

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acids h a d annealed. F o ll o w in g these observations, Giemsa staining alone, after alkali or h e a t " d i s s o c i a t i o n " o f t h e D N A a t t e m p e r a t u r e s in excess of 8 5 ~ an d " r e a s s o c i a t i o n " at lower t e m p e r a t u r e s , has been used to i d e n t i f y t h e h u m a n h e t e r o c h r o m a t i n (Arrighi and Hsu, 1971) an d those regions of the h u m a n and mouse chromosomes where t h e satellite D N A was t h o u g h t to be c o n c e n t r a t e d (Yunis et al., 1971; see also Chen a n d R u d d l e , 1971 ; H s u et al., 1971). I t is obvious t h a t in principle the Giemsa staining technique, insofar as it relies on a c h r o m o s o m a l characteristic c o n s t a n t in t h e genome, could be used to m a r k t h e position of t h e e e n t r o m e r e a t m ei o t i c m et aphase, for e x a m p l e in t h e mouse. This would be i m p o r t a n t for t h e i n v e s t i g a t i o n of t h e b e h a v i o u r a n d m o d e of association of n o r m a l biv a l e n t s an d s t r u c t u r a l l y a b n o r m a l chromosomes at diakinesis. ?daterial, Techniques, Outline of E x p e r i m e n t s In line with these considerations and in an attempt to define a standard procedure, two sets of techniques were used: fixed and air-dried meiotic cells were treated either at relatively high temperature for a short time, followed by a more prolonged treatment of the slides at a lower temperature, or an NaOH solution was used on them. In either case, the cells were subsequently stained in a buffered Giemsa (1904) solution.

a) Animals We used male and female mice of the CFLP Carworth, Europe, strain (derived from I.C.I., Alderley Park, strain 1, mice), aged two to four months. We also used a few AKR males and females, homozygous for a centric fusion translocation (2n = 38 i.e., T(11; ?)-1Ald; Leonard and Deknudt, 1969; Lyon, 1971), referred to here as AKR T-1.

b) Preparation o/Slides Mitotic preparations from mouse bone marrow and from a fibroblast L-cell line [B82 TK(-)] were used as controls. Male meiotic slides were prepared by the ceilsuspension air-drying procedure of Evans et al. (1964), modified by adding to each 10 ml of the tubule suspension in 2.2 % Na citrate solution, 0.2 ml of Pancrex V (Paines and Burn; contains trypsin, lipase and amylase) stock solution (0.1 g in 20 mt of glass-distilled water) freshly prepared every week. The fixative was ethanol/ glacial acetic acid 3:1 and we "post-spread" the cells with three large drops of fixative, each before final drying of the previous drop, and post-fixed the quickly dried slides for 30 minutes. We also compared the results from this method with Meredith's (1969) processing technique and the latter proved inferior. Experiments, both with NaOH and heat, were done also on slides soaked beforehand for different times in 45 % glacial acetic acid, again with inferior results. Female meiotic ceils from young animals' ovarian follicles were prepared according to the technique developed in this Unit by Dr. G. Jagiello and based on Edwards' (1962) method, using culture medium TC 199 with 40 % calf serum for the eollection and incubation of the ova (about 41/2 hours for first and 141/2 hours for second meiotic metaphases) in 5 % C02 in air at 37 ~ After incubation we used hypotonic 0.7 % Na Citrate for t0 12 minutes, followed by a quick rinse of the ova in glass24

C h r o m o s o m a (Berl.), Bd. 36

346

P . E . Polani:

distilled water before fixing them on a slide with the 3:1 fixative under visual control, and air-drying. As in males we " p o s t - s p r e a d ' a n d then post-fixed for 30 minutes.

c) Alkali Procedures Molar concentrations of 0.01, 0.07 and 0.1 NaOH in either water or 0.15 M NaC1 were used for from 30 seconds to 4 minutes. Some of the experiments were concluded b y keeping the well-washed slides in phosphate buffer at p H 6.8 (v. i.) for from 30 minutes to 48 hours.

d) Acid Hydrolysis The effect was investigated of 5 N HC1 at room temperature for 5 to 60 minutes or of 1 N HC1 at 60~ for 4 to 1 0 m i n u t e s in steps of one minute, prior to standard Giemsa staining, with essentially unsatisfactory results.

e) Heat Procedures (i) Temperatures and Times. The heat experiments were done at high or low temperature or at both sequentially. Slides were heated a t one of the following temperatures in a water b a t h with mild agitation and in freshly prepared and bacteriafiltered solutions: 100 ~ 95 ~ 87 ~ 85 ~ 84 ~ 82 ~ 80 ~ 79 ~ 75 ~ a n d 70~ (high temperature); the slides were kept at high (variation of 0.5 ~ C) temperature for 3, 4, 5, or, on occasions, 10 or 20 minutes. For 100 ~ and 84 ~ or 85 ~ C, half the slides were kcpi, prior to staining, at 62 ~ C (i.e. 60-62 ~ C: low temperature) for varying tines from ten minutes to 48 hours, while the rest were dried and stained immediately. After the other high temperatures, slides were stained either without further t r e a t m e n t or after a fixed period of 30 or 60 minutes at 62 ~ C. I n some heat experiments, slides were immersed in ice-cold solution (v. i.) before drying a n d subsequent staining or before t r e a t m e n t at 62 ~ C prior to staining. A n u m b e r of experiments were concluded b y allowing the slides slowly to cool to room temperature in their heating solution (v.i.) before drying t h e m and staining them. Finally, some of the heat experiments were done only at 62 ~ C for from one to 48 hours. (ii) Solutions. We used SSrcnsen's phosphate buffer (phosphate buffers are usedin DNA reassociation work; see also Yunis et al., 1971) or in standard saline-citrate solution (SSC) (Marmur and Lane, 1960). The buffer was tried a t p H 5.0 (which proved unsatisfactory), 6.8 and 8.0, and SSC a t concentrations of 0.1 • ( J o h n et al., 1969), 1 x and 2 • (Corneo et al, 1968) and at p H t h a t varied from 7.5 to 8.5. Control over and constancy of hydrogen-ion concentration proved more import a n t t h a n ionic strength for the detection of the para-centromeric Giemsa staining. However, ionic strength seems relevant to the maintenance of (isolated) chromosome structure, particularly at high pH (Cantor and Hearst, 1970), and for female meiotic cells more consistent results were obtained with the SSC solutions.

f) Staining and Examination After experimenting with time and concentration of dye, the air-dried slides were stained at room temperature, inverted in jars of freshly-prepared filtered 1:20 Giemsa solution (Gurr's P~ 66; p H 6.8 in buffer made from Gurr's tablets) for 20 minutes, washed in glass-distilled water, dried, cleared in two changes of Xylene and mounted in Gurr's neutral mountant. All preparations were studied b y b o t h transmitted light and phase contrast microscopy, and a Zeiss B G 65 fluorescence barrier filter was used.

Centromeres and Chiasmata in the ~ouse

347

Results and Discussion

A. Technical 1. Results The Mm was to obtain selective staining of the so-cMled constitutive heterochromatin in, or in the neighbourhood of, the centromere (eentromeric heterochromatin) and for descriptive purposes I shall refer to the proximal chromosomal region and to its Giemsa staining as paracentromeric (p.c.) regardless of its relationship to the centromere. I t could well be t h a t the selectively Giemsa-stMned region is, indeed, part of the centromere itself, or t h a t the centromere contributes to the region, or t h a t the region is outside the centromere but is responsible for its formation or otherwise influences its behaviour (v.i.). I t is possible to observe p.c. staining in otherwise untreated Giemsastained slides (see also Chen and Falek, 1969), but this is only seen in exceptional late diplotene or early diakinetic male cells, and furthermore in some bivalents both the centromeric and telomeric regions are stained (Fig. 11 e). Of the special techniques, the use of N a O H solution, especially 0.07 M, gave good p. c. staining, but poor chromosome morphology, particularly diakinesis and first meiotic metaphases (MI's) (Fig. t a, c and d). The addition of NaC1 often gave good chromosome morphology with good p. c. staining (Fig. 1 b). Some SSC procedures gave reasonable chromosome morphology and fairly reliable p. c. staining and examination was often possible with transmitted light (Figs. l e, 3e and f, 8f). Following heat treatment at 82 ~ C and higher, the p. c. regions were almost invariably distinctively stained and it was immaterial whether or not the higher temperature treatment was followed b y a period at a lower temperature. Nor did it matter whether the heated preparations were plunged into icecold buffer prior to staining (with or without previously heating at 62 o C) or whether they were either rapidly or slowly cooled to room temperature and then stained. Invariably, after using a temperature above 82 ~ C, the chromosomes had a grossly diminished general affinity for the Giemsa stain, and the p.c. regions, though selectively stained, were fainter than non-heat-treated preparations. Taking all heat experiments together the bivalent morphology was often quite altered, though even then familiarity with the MI configurations in the mouse mostly allowed plain recognition of the type of bivalent association in relation to ehiasmata, and their position with reference to the centromeres. However, variability was minimal and quality of metaphases o/ all three types, including p.c. staining and chromosome morphology, were best with temperatures of 84 ~ or 85~ in SSrensen's buffer at p H 6.8, for four or five minutes, followed by immersion in the same buffer, at 62 ~ C, for 30 to 60 minutes 24*

Fig. 11. CFLP males, a MI, n - - 2 1 , X and u univalents; 0.07M NaOH, 1 min (Ph). b MI, n = 2 0 , X Y bivalent; 0 . 0 7 ~ NaOH in 0.15M NaCl, l min (Ph). c M I I , ?Y (arrowed); as per (a) (Ph). d SPM; as per (a) (Ph). e M I ; SSC 8 hours (pH 8.0) (Trs). f M I ; SBS. (See text) 1 For footnote see opposite page.

349

P. E. Polani: Centromeres and Chiasmata in the Mouse Table 1. Results ]rom CIVLP ]emale meiotic cells Temperature C~

84~176 620-80 ~ 84 ~ 84~

~

85~

~

Totals

~

Ani- Cells* mals

Solution and pH

S6rensen S6rensen S6rensen S6rensen SSC

6.8 6.8 6.8 8.0

Results after heat experiments Cells Iost

MI's p.o. staining

MII's p.o. staining

Nil

Nil

Some All biv. biv.

33 a 1

---

---

---

9

- -

- -

- -

5 14

12 6

23 8

19

18

31 a

Some All biv. biv.

32 12 4 14 14

301 125 61 155 104

42 10 24 27 0

166 103 25 42 12

60 11

76

746

103

348

122

3

12 36

341b~ 28J 105 c

* Excluding 4% of hypoploid cells, all considered to be artefacts. Percentages (95% C.L.); a 13% (9-18)ofMI's; b 38% (30 46) ofMI's; e 18% (15-22) of MI's; d 46% (33-58) of MII's.

(Figs. 1 i, 2 a - c , 7 e, e a n d f, 8 a - e ) . T h e s e w e r e t h e m o d a l i t i e s u s e d in t h e f i n a l e e n t r o m e r e l o c a t i o n e x p e r i m e n t s in t h e m a l e s (v. i,). P r o l o n g e d h e a t ing a t t h e h i g h e r t e m p e r a t u r e s r e s u l t e d in gross c h r o m o s o m a l d i s t o r t i o n a n d d e f i c i e n t a n d u n e v e n p . e . s t a i n i n g , w h i l e i m m e r s i o n b e y o n d 30 to 60 m i n u t e s a t 62 ~ C, a f t e r t h e i n i t i a l h i g h e r t e m p e r a t u r e , c a u s e d loss of d i s t i n c t i v e n e s s of c h r o m o s o m e s a n d g e n e r a l p o o r q u a l i t y a n d l a c k of crispness of p . e . s t a i n i n g . C o n v e r s e l y , t h e use of l o w e r t e m p e r a t u r e s (e.g. 70 ~ C for 20 m i n u t e s : Figs. 2 d a n d e, 7b) a l l o w e d t h e p . e . r e g i o n s to be d i s t i n c t l y s t a i n e d w i t h g o o d c h r o m o s o m e m o r p h o l o g y a n d a g e n e r a l l e v e l of s t a i n i n g t h a t m a d e possible e x a m i n a t i o n in t r a n s m i t t e d light. H o w e v e r , o n l y r e l a t i v e l y few M I ' s h a d p. e. s t a i n i n g of all b i v a l e n t s . E v e n l o w e r t e m p e r a t u r e s (such as h a v e b e e n u s e d for D N A r e a s s o e i a t i o n ) c a n y i e l d a p r o p o r t i o n of d i f f e r e n t i a l l y well s t a i n e d p . c . r e g i o n s ( 6 2 ~ for one or t w o h o u r s : Figs. 2 f, 3 a - e or 62 ~ C for 24 h o u r s in 2 • S SC : Fig. 3 e). I t is of i n t e r e s t t h a t , w i t h f a i r l y low t e m p e r a t u r e s , o c c a s i o n a l a u t o s o m a l b i v a l e n t s will s h o w t e l o m e r i e as well as p. e. s t a i n i n g (e. g. Fig. 3 d). 1 All photographs in Figs. 1-12 were taken with a Carl Zeiss, Oberkochen, photomicroscope, in either phase contrast (Ph) or transmitted light (Trs) mode, on Gevaert Scientia 45-C-62 film, with • 100 oil immersion objectives. Abbreviations: (i) Treatment in S6rensen's buffer (SB) at 84 ~ or 85~ for 4 or 5 rain, followed by 62 ~ C for 30 min or 1 hour, at pH 6.8 (unless otherwise stated) and photograph in contrast of phase -- SBS. (ii) Treatment in 1 • Standard Saline Citrate (SSC) at 84 ~ or 85 o C for 4 or 5 min, followed by 2 • SSC for 30 rain or longer = SSC 30 rain etc. S P M SpermatogoniM metaphase. M I Diakinesis or first meiotic metaphase; M I I Second meiotic metaphase. All temperatures in ~

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P.E. Polani:

Seventy-six CFLP females (Table 1) were used and all the good ova from each animal were fixed on the same slide and identified, located and scored, and often photographed, with phase contrast optics prior to treating and staining (Fig. 4a and b). Four per cent of the cells were hypoploid and, after staining, 17 % could not be located or were so faintly stained as to be practically invisible. Selective p.c. staining was obtained in about 13 % of cells at p H 6.8 (Figs. 4a and b, 5a), but the most consistent results were with 84 ~ or 85 ~ C for 5 or 4 minutes followed by 62 ~ C for 30 minutes at either p H 8.0 in phosphate buffer (Fig. 5 b - d ) or in 1 x SSC and 2 X SSC for the higher and lower temperatures, respectively (Fig. 5 e). With these two methods, the p. c. regions of every bivalent were stained in 38 % of MI's. Only these two methods were tried on M I I ' s (Fig. 10a, b and d) and gave crisp positive results. Metaphases identified as polar body complements were generally of poor quality (but see Fig. 10b) and only some p.c. regions of some cells were stained. 2. Discussion The somewhat different response of male and female metaphases to ionic strength and p H m a y be due to differences in preparation technique or m a y possibly reflect a different type, or quality, of association of the chromosomal proteins with DNA at meiosis, in the two sexes. Technically, meiotic cells, especially of males (and some mitotic cells as well: Fig. 11 a and b) gave consistent and repeatable results. However, female meiotic preparations were capricious. There was variation between slides and also frequently, next to cells with well displayed p. c. regions, there were cells with barely discernible bivalents and, conversely, others whose chromosomes were heavily and uniformly stained. Yet I could not perceive any other obvious differences between these cells, such as degree of contraction of bivalents, or of their crowding, or the persistence of cytoplasmic remnants. I n the original in situ experiments on mouse cells, Pardue and Gall (1970) observed selective Giemsa staining of, as well as nucleotide hybridization with, the p.e. region of the chromosome where the highly repetitive murine satellite DNA is localized. However, it cannot be automatically assumed that the Giemsa stain picks out this DNA specifically, either in its dissociated or partly or completely reassociated forms, though some workers (Arrighi and tIsu, 1971; Sumner et al., 1971) but not all (Rowley and Bodmer, 1971) think that the dye m a y bind preferentially to undenatured DNA. I n our experiments, the higher temperature used first might have produced dissociation, followed by low temperature reassociation favoured by the highly repetitive nature of short sequences of the DNA in the p.e. regions (Yunis et al., 1971), However, first we must question whether the temperatures and times used could separate the

Centromeres and Chiasmata in the Mouse

351

Fig. 2a-f. CFLP males, a c MI; SBS. d and e MI; SB 70 ~ 20min (pH 6.8) (Trs). f MI; SB 62 ~ I hour (pH 6.8) (Trs). (See texb) D N A strands in situ in view of the stabihzing effect of histones on D~NA in vitro ( J o h n et al., 1969). A d m i t t e d l y , the low ionic s t r e n g t h t h a t we used m i g h t help dissociation and, of course, the biochemical circumstances

352

P . E . Polani:

Fig. 3a-f. CFLP males, a M I ; SB 62 ~ 1 hour (pH 6.8) (Trs). b and c M I ; SB 62 ~ 2 hours (pH 6.8) (Trs). d M I ; SB 79 ~ 20 min (pit 6.8) (Ph); bivalents with telomeres and eentromeres stained (arrowed). e SPM; 2 • SSC 62 ~ 24 hours (pH 8.0) (Trs). f SPM; SSC 8 hours (pH 8.0-8.5) (Trs). (See text)

Centromeres and Chiasmata in the Mouse

353

of meiotic metaphase chromosomes should be quite different from those

of mitotic ones, as are the preparatory techniques. Secondly, we do not know whether the highly repetitive DNA m a y reassociate selectively, even partially, in the conditions of temperature, time and sodium ionic strength (Britten and Kohne, 1968) that we mostly used. Thirdly, there is doubt whether the dye would bind specifically to the reassociated DNA. Finally, especially on female MI's, at temperatures .between 62 ~ and 70 ~ C, at neutral and slightly alkaline p H and at low ionic strength we observed the release from the bivalents of droplets (Fig. 12a) or larger aggregates (Fig. 12 b) of strongly Giemsa-positive material. As a result the chromatids were left very faintly stained and the p. c. regions often differentially, though rather faintly, stained (Fig. 12c). The material released, which influences staining by Giemsa, might represent an acidic protein relatively absent from the p.c. regions, or some other acidic moiety. This observation m a y be related to another phenomenon, noticed by MMo and Schildkraut (1969) in isolated mouse chromosomes: unlike the bulk of the DNA sequences, highley repetitive sequences remain bound to their chromosomal protein in 2 M NaC1. Therefore it is entirely possible that the protein coating of mouse chromosomes in the p.c. region, with its highly repetitive DNA, differs from that of the rest of the chromatid. Hence its staining affinities m a y be different, but this need not be only because different proteins or protein amounts or type of binding are involved. I t is equally possible t h a t differences in protein coat m a y permit differential access to DNA-specific dyes.

B. Applications o/the Technique 1. Paracentromeric Staining At MI the X u bivalent often showed p.c. staining of the sex chromosomes at their non-associated ends (Fig. 6). In general the p.c. region of the Y chromosome was smaller and less intensely and consistently stained compared with the X. However, the p.c. staining of the Y was detected both following heat techniques and N a O H treatment, both in bivalent association and as a univalent (Fig. i a and b) and bore, to some extent, an inverse relationship to its positive heteropycnosis (Table 2), which was present in about half the u chromosomes. While this manuscript was in preparation, Hsu et al. (1971) published their findings on mouse cells, ineluding male meiotic ceils. Their work suggested a similar distal end-to-end association of the X and Y chromosomes, as previously hypothesized by a number of workers. This type of association is supported by autoradiographic evidence (Kofman-Alfaro and Chandley, 1970). However, Hsu et al. (1971) failed to detect a distinctively staining p.e. region of the Y. Neither did they detect this in second meiotic metaphases (MII's), while

354

P. E. Polani:

Fig. 4a and b. CFLP female; lYlI. a (Ph). b SBS. (See text) Table 2. Cells with paracentromeric (p. c.) staining oI the Y chromosome Y

p.c.

CFLP males XY bivalent u univMent (88 lYlI's) (12 MI's) (%)

(%)

AKR T-1 males XY bivalent (69 MI's) (%)

CFLP end AKR T-I males XY bivalent (157 Mrs) (%)

heteropycnotie

stained unstained

18 (20) 22 (25)

-5 (42)

12 (17) 29 (43)

30 (19) 51 (33)

isopyenotic

stained unstained

33 (38) 15 (17)

6 (50) 1 (8)

25 (36) 3 (4)

58 (37) 18 (11)

we think t h a t the p.c. region of the Y chomosome m a y be stained also in these metaphases (for example Fig. 8a). Because the X and the Y cannot be positively identified in all or most such cells this point cannot be conclusively settled, b u t a diploid M I I displaying p.c. staining of all 80 chromatids must be accepted as evidence t h a t the p.c. region of the Y can stain selectively (Fig. 8b). I n spermatogonial metaphases one of the smaller chromosomes, sometimes somewhat heteropycnotic, interpreted as the Y chromosome, generally did not show p.e. staining (Fig. 7a, b and f), b u t in some of these cells faint p. e. staining was present (Fig. 7 e, e and particularly d). Chen and Ruddle (1971) and Hsu et el. (1971) observed t h a t the p.e. region of the u chromosome failed to stain (see also Hsu and Arrighi, 1971; Sehnedl, 1971) and, in their in situ hybridization experiments, P a r d u e and Gall (1970) failed to label it.

Centromeres and Chiasmata in the Mouse

355

Finally, in favourable instances both the X and Y chromosomes showed some telomerie staining (Fig. 1 b). Sex chromosomes apart, the appearance of the p.c. regions of the mitotic cells in meiotic preparations, which I accept as being of the germinal line, is of interest. First, in some of the cells which perhaps could be interpreted as prophases of a spermatogonial or, more doubtfully, of the first meiotic division, the selectively stained regions tend to be paired (see lower cell, Fig. 9a) and relies of this m a y be observed between some chromosome pairs also in metaphase plates (e.g. Fig. 7 e). Indeed, p.e. association m a y be seen even between the X and the Y, in early diakinesis, though possibly this m a y be a residual effect of the sex bivalent being in the sex vesicle (Fig. 3 a). Secondly, inspection of the spermatogonial metaphases (SPM) suggests the presence of quantitative differences in the p. c. regions, regardless of whether or not there are qualitative changes as well. The absolute size of the regions is not correlated with the size of the chromosomes. Small and large p.e. regions are seen on what could be homologous pairs, and sometimes even without apparent homology, suggesting in the latter case the existence of heteromorphic variation (Fig.7 b, c, e and f). There are also organizational differences between chromosomes in these regions. Some of the p.c. regions appear subdivided into two and even four elements (Fig. 7a). Variation in the appearance of eentromeres of teloeentrie chromosomes, such as multiplicity, has been described (Brinkley and Stubblefield, 1966). Some variability of size of the p.c. regions is also seen in SPM's of the A K R T-1 males (Fig. 9e) and in both MI's and M I I ' s (Figs. le, 2a, 8e-f). Similar variation has been noticed recently by Chen and I{uddle (1971) in mitotic cells, using Arrighi and Itsu's (1971) technique, and by Schnedl (1971) with his N a O I t denaturation method. I n the AKI~ T-1 males the two sub-metaeentric homologues clearly have a large eentromerie region (Fig. 9 c) which sometimes is longitudinally double (Fig. 9 b), a feature observed also by Chen and Ruddle (1971) in other metaeentrie mouse chromosomes (Fig. l l a and b) and, apparently, by Gim6nez-Martfn et al. (1965) in their study of eentromeres. Often the doubleness, and always its size, could be appreciated also in MI's (Fig. 9d, e and f), but in M I I ' s the centromeric region of the metaeentrie chromosome appears as a single block with a finer thread usually joining the homologous regions of the two ehromatids (Fig. 8d and e). I t is clear t h a t the T-1 chromosome is a centrie fusion element, which has preserved both p. c. regions. I t results from the fusion of chromosome No. 6, which forms the longer arm and is linkage group XI, and chromosome No. 15 (Lyon, personM communication, 1971; Miller etal., 1971). CFLP female MI's, like those of males, display variability of size of the p.e. regions (Figs. 4b, 5d and e). Also, clearly, as already stated, an

Fig. 5 a-f

P. E. Polani: Centromeres and Chiasmata in the Mouse

357

Fig. 6. CFLP and AKR T-1 males. A series of XY bivalents showing p. c. staining of the X and often of the Y and/or its heteropycnosis (see text and Table 3). Heat experiments: SB 84~ ~ for 3-5 min, 62 ~ 30 min or 1 hour or 4 hours (pH 6.8) (Ph)

e a r l y p o l a r b o d y c o m p l e m e n t will show p.e. staining (Fig. 10b) which is well visible in lV[II's a n d g e n e r a l l y d e m o n s t r a t e s v a r i a b i l i t y in size (Fig. 10a, b a n d d). A t a n a p h a s e of t h e first meiotic division, t h e p.e. regions become p a r t i c u l a r l y evident, s t r i k i n g l y v a r i a b l e in size a n d elong a t e d (Fig. 10e) a n d this can a c c o u n t for t h e o b s e r v a t i o n of n e g a t i v e l y h e t e r o p y e n o t i e p. e. regions in some e a r l y anaphases, otherwise u n t r e a t e d a n d s t a i n e d w i t h orcein (Fig. 10 e). This is a c o u n t e r p a r t to t h e observations of L i m a - d e - F a r i a (1958) on t h e lengthening a n d staining b e h a v i o n r of t h e eentromeres a t a n a p h a s e (see also Sehrader, 1936) in c o n t r a s t to w h a t h a p p e n s a t m i t o t i c division. Also in o t h e r ways, t h e b e h a v i o u r of t h e p.e. regions mimics closely t h e b e h a v i o u r of centromeres (kinetoehores) (see, e.g., Schrader, 1936; L i m a - d e - F a r i a , 1958; L u y k x , 1965; B r i n k l e y a n d Stubblefield, 1966, 1970) to which t h e regions t h e m s e l v e s m a y be i n t i m a t e l y r e l a t e d (v.8.). Fig. 5a-f. CFLP females; MI's. a SBS. b - d SBS (pH 8.0) (Ph). e SSC 8 hours (pH 8.0-8.5) (Ph) (inset is a bivalent from same ovum at a distance of two fields). f AKR T-1 female, MI, n = 21 (univalents arrowed); SSC 30 rain (pH 8.0-8.5) (Ph) (insets-bivalent of sub-metacentrics from an incomplete cell). (See text)

358

P.E. Polani:

Fig. 7 a f. C F L P m a l e s ; S P M ' s (b a n d c, e a n d f are f r o m two litter-mates). a, c - f SBS. b SB 70 ~ 20 rain, 62 ~ 1 h o u r (pH 6.8) (Trs). (See t e x t )

Centromeres and Chi~smatu in the Mouse

359

Fig. 8a-f. CFLP and A K R T-1 males; MII's. a CFLP, Y (arrowed); SBS. CFLPb diploid, X and Y (arrowed) ; SBS. e CI~LP, X (arrowed) ; SBS. d and e AKI~ T-1 ; sub-metacentric chromosomes arrowed; SBS. f CLFP, X (arrowed); SSC 8 hours (pH 8.0) (Ph). (See text)

Fig. 9 a-f.

Fig. 10a-e Fig. 9a-f. CFLP and A K R T-1 males, a Probable spermatogonial or meiotic prophase, CFLP; SBS. The small mass at 3 o'clock in the upper cell is not a sex vesicle b u t an overlapping sperm head. b and c AKP~ T-1 SPM's; SBS. d - f AKP~ T-1 N I ' s ; SBS. (See text) Fig. 10a-e. CFLP females: a M I I SBS (pit 8.0) (Ph). b Two cells (MII and polar body probably: this can be considered a late Anaphase I) separated by about one field; as per a. Two separated ehromatids are arrowed, c Anapha.se I; as per a. d M I I SSC 30 rain (pH 8.0) (Ph). Schneider female: e Anaphase I, aeeto-lacto orcein (Ph). (See text) 25 Chromosoma(BerI.), Bd~36

362

P . E . Polani:

Fig. 11 a and b. Mouse L-cell line, B 82, TK(--) (fibrobl~st culture), a SBS. b SSC 30 rain (Trs). e CFLP male; early M I, Giemsa-stained but otherwise untreated (Trs). (See text)

P a r t i c u l a r l y a p p l i c a b l e seem to be t h e o b s e r v a t i o n s of S e h r a d e r (1936) on t h e a p p e a r a n c e , size a n d doubleness of t h e eentromeres in b i v a l e n t s of t h e Amphiuma male. There is also a t e c h n i c a l s i m i l a r i t y as he too h a d to "de-stain" his sections to reveal the centromeres. A t M I t h e homologous p.c. regions are generally well s e p a r a t e d e x c e p t for some of t h e b i v a . lents with p r o x i m a l / d i s t a l c h i a s m a t a (Figs. 2c, 5d). Most of t h e p.e.

Centromeres and Chiasmata in ghe Mouse

363

Fig. 12a-e. CFLP females; MI's. a SB 62 ~ 4 hours (pH 6.8) (Ph). b SB 62 ~ 4 hours (pI-I 8.0) (Ph). c SB 82 ~ 4 rain, 62 ~ 1 hour (10It 8.0) (Ph). (See text)

regions of t h e d i a d s are single a t diakinesis (Fig. 3a) a n d M I b u t t h e y are clearly bifid, a n d sometimes t h e y are double in b o t h males a n d females (Figs. 2 e, 5b). These findings correspond to the observations of K e z e r a n d M a c G r e g o r (1971) in t h e male s a l a m a n d e r a n d of L u y k x (1965) in t h e eggs of t h e m a r i n e worm, Urechi8 (see also L i m a - d e - F a r i a , 1958). 2. C h i a s m a t a F o r an analysis of t h e p o s i t i o n of e h i a s m a t a in r e l a t i o n to the centromere, i d e n t i f i e d b y p.e. staining, we h a v e used M I ' s from t h r e e sets of 25*

364

P.E. Polani:

animals. On slides prepared by the best heat techniques, ten MI's were selected from each of ten CFLP males, and 69 cells were scored from five A K R T-1 males. The ova from 76 CFLP females were examined (Table 1) but we obtained only few satisfactory MI's from the two available AKI{ T-1 females (Fig. 5f). I made no effort to select the earlier diakineses or metaphases but cells had to be complete, with overlap-free and distinct bivalents showing p. e. staining. The average minimum number of chiasmata per cell for CFLP males, A K R T-1 males and CFLP females was 22.58 ! 0.166 (S. D. = 1.904), 23.07 • 0.229 (S. D. = 1.659) and 27.53 • 0.225 (S. D. = 2.249) respectively. Both in males and females, early diakineses are seldom seen and I have arbitrarily assumed that there is no change within each sex in the relative position of ehiasmata observed in early or later stages of the first meiotic divisions. Bivalents with central (within the middle third or half of a bivalent), proximal and distal chiasmata could be distinguished; those with two ehiasmata in different positions could be well identified. The proportion of cells with one, two or more such bivalents per cell has been plotted for the three sets of MI's in the form of comparable histograms (Figs. 13 to 15). I n addition, the average number, per cell, of bivalents with different ehiasma types was determined per sex and strain and the variation estimated (Table 3). There are differences between the sexes: for example, there is in females a higher proportion of cells t h a t have no bivalents with proximal chiasmata and a different distribution, compared with males, of cells with bivalents with distal ebiasmata, shown also by a nearly twice as high frequency of distal ehiasmata in males and a significantly greater variation in females (Table 3). Conversely, there are on an average more central chiasmata in female than in male cells. However, when comparing relative chiasma positions in males and females (Figs. 13 and 14), it must be stressed t h a t the female and male results m a y differ simply for technical or for cytological reasons, though there m a y be true biological differences between sexes in miee in this respect. There are known to exist differences of localization of ehiasmata between sexes in animals, f o r example in newts (Watson and Callan, 1963), and such differences have been inferrred by genetic tests and are known to operate also in plants (Fogwill, 1958; Ved Brat, 1966). A simple correlation analysis shows that, in both sexes, an increase of bivalents with double proximal/distal chiasmata is strongly correlated with a decrease of bivalents with single distal ehiasmata, and it seems possible t h a t the control of bivalents with double ehiasmata occurs by an increase of bivalents with distal ehiasmata at the expense of other single chiasmata. I n the original interpretation of the sequential origin of ehiasmata, Mather (1937) had suggested that the first ehiasma arises at a

--

Triple

a See text.

Triple

-

-

Distal/Distal Distal/Central

Proximal/Distal Central/Distal Proximal/Central

Double

Centric fusion bivalent Double

Proximal Central Distal

Single

Chiasmata

--

---

--

2.83• 0.80 :k 0.092 --

1.96 :t: 0.103 1.76 :~ 0.096 11.65 ~= 0.180 1.44 0.92

1.04 0.97 1.81

--

__ __

0.02 ~: 0.014

5.04~0.183 2.15i0.125 0.22 ~ 0.044

1.23 ~: 0.108 5.11 ~= 0.225 6.23 • 0.315

0.14

1.83 1.25 0.44

1.08 2.25 3.15

S.D.

M e a n • S.E.

Mean:s S.E.

S.D.

CFLP, ~ (N = 100)

CFLP, ~ ( N : 100)

0.04~0.024

0.78 s 0.050 0.17 • 0.046

--

3.44• 0.55• 0.04 ~ 0.024

2.13 ~: 0.141 2.62 ~= 0.122 8.24 ~ 0.181

M e a n • S.E.

0.21

0.42 / 0.38 /

1.62 0.76 0.21

1.17 1.02 1.51

S.D.

A K R T-l, ~ ( N : 69) uncorrected a

Table 3. Number o] bivalents o/the speci/ied chiasma types per cell

c~176

Divided for

---

3.48 ~ 0 . 2 0 5 0.55 ~: 0.091 0.04 ~ 0.024

2.13 -~ 0.141 2.79 • 0.136 10.01 • 0.193

M e a n • S.E.

1.70 0.76 0.21

1.17 1.14 1.60

S.D.

A K R T-l, 3 ( N = 6 9 ) corrected a

9

c~ tr

P. E. Polani:

366

~ 60

Cetls with proximal Xta ~

20

3

Cells with centrai Xta

~ 60

LO

40

20

20

Cells with distal Xtu

I [ I I F-4 ~ q I I I I I I I I I I [ I 0246810 2 4 6 8 10 121416 2 L 6 810 Ceils with proximal / Cells with central/ Ceils with proximal/ 80-distal double Xta 80 distal double Xta 100- centrcd doubte Xta 60

60

80

L0

40

60

20

~0-

20

pl~_.~q i i iIi~ 2

4 6 8 10

I~

III[I;

0246810 Bivalents

[ I 1 li

2

I I 1 1 I

46810

Fig. 13. Frequency distributions (per cent) of CFLP male MI's with autosomal bivalents with single and double chiasmata (Xta), whose position was determined by reference to the centromere. Based on 100 cells, n = 20. Each column gives the proportion of cells, per cent, with 0, 1, 2, etc. bivalents with the specified type of chiasma

variable differential distance from the centromere. However, Henderson (1963) in Schistocerca gregaria--a locust with distinct bivalents with centromeres marked b y heteroehromatic blocks and in which diplotene nuclei, especially favourable for chiasma work, could be easily obtained thought t h a t the differential distance was near zero and t h a t the best fit to his findings was if the first chiasma was distal. Nevertheless he t h o u g h t t h a t sometimes chiasmata could be formed at both centro- and telo-meres. I n the case of the A K R T-1 males there arises the question of the influence of the ecntric fusion on the other autosomes and of the chiasma behaviour of the chromosomes in the translocation itself. Therefore, first the chiasma behaviour of the centric fusion bivalent was recorded separately: each cell had a single " r i n g " with two chiasmata (e.g. Fig. 9d and f) with the exception of three cells (Fig. 9e) in which the bivalent had three. Secondly, I added to the rest of the autosomal complement, as appropriate, the two bivalents t h a t could be imagined to result from splitting at the centromere in each cell the two homologous sub-metaeentric chromosomes. B o t h the histograms (Figs. 13 and 15) and Table 3,

Centromeres and Chiasmata in the Mouse

%

Cells with .proximal Xta

20

%

Cells with central Xta

%

'

Cells with distal Xta

2O

20 [

367

I

r

I

0 2 4 6 8 10 0 2 4 6 8 10 12 0 2 L 6 8 10 12 1L Cells with proximu[/ Cells with central / Cells with proximal/ 80 distal double XtQ 80 distal double Xto 80 central double Xta

60

60

6O

40

40

40

20

2O [

2

4 6 8 10

I

[

[

I

0 2 h 6 8 10 Bivalents

~

I

I

I

'

I

0246810

Fig. i4. See legend of Fig. 13. Refers to bivalents of CI~LP females. Based on 100 ceils, n = 20. Two cells in 100 had a single bivalent each, with three chiasmata

as well as t h e figures for t o t a l e h i a s m a t a a n d cell v a r i a t i o n , show how little t h e presence of t h e eentrie fusion chromosome in t h e A K R T-1 males d i s t u r b s t h e overall r e g u l a t i o n in t h e cell of ehiasma f o r m a t i o n ; a n d t h e corrected results o b t a i n e d b y s p l i t t i n g t h e i n t e r c h a n g e bivalent, which are s t r i k i n g l y similar to a n o r m a l male d i s t r i b u t i o n of t y p e s of e h i a s m a t a , suggest t h a t c h i a s m a f o r m a t i o n is n o t a l t e r e d w i t h i n t h e chromosomes i n v o l v e d in t h e centric fusion. Considering t h e s u b - m e t a c e n t r i e chromosome, it can be seen t h a t in 17 % of cells t h e r e was a central ehiasma in one of t h e arms. This seemed to be a l w a y s t h e long arm, i.e. c h r o m o s o m e No. 6 of Miller et al. (1971). I n t h r e e cells in which t h e subm e t a e e n t r i c h a d three e h i a s m a t a , again i t seemed t h a t t h e longer a r m h a d two. Thus i t a p p e a r s t h a t , a t least in this s i t u a t i o n a n d in males, the chromosome n u m b e r e d 6 (linkage group X I ) has an average 0.79 freq u e n c y of distal, 0.17 of central a n d 0.04 of double chiasmata, whereas t h e s h o r t e r No. 15 m u s t h a v e seldom, if ever, a n y b u t single d i s t a l chiasmata. 3. P a r a c e n t r o m e r i c Regions a n d Meiosis W e should briefly consider t h e possible functions of the highly repetitive p.c. D N A a t meiosis. I t is curious t h a t in f a v o u r a b l e conditions t h e telomeric regions of t h e chromosomes show an affinity, a l b e i t smaller in e x t e n t a n d more variable, to t h e Giemsa stain, analogous qualita-

368

%

P.E. Polani:

Cells with proximal Xta

20

%

Celts with central Xta

[,-

F]

20 , [ [ I I I I

Cells with distal Xto ~

20 I i I I I

", IIII

I

0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 12 1[, Ceils with proximal / Ceils with centra[/ Celts with proximal / 8O distal double Xta 80 distal double Xta 100 :_ central double Xta 100

[ ] =distal/central/distal []= dis~al/ceatral

Fl=disfgl/disial

60

80-

80

Z,O

4.0

60

'60 -

20

20

40

40

~' 6o

C/F Bivalent

k !rll 0246810

~,,i fl,, I, ; h II1[[!111 4 6 810 2 4.6810 Bivalents

~ 0

1 1 f II

I I I i

h 6 8 10

Fig. 15. See legend of Fig. 13. Refers to autosomal bivalents of AKI~ T-t males. Based on 69 cells, n = 19. The centrie fusion (C/F) bivalent is considered separately in the last histogram. The traeted (---) columns of the first six histograms are obtained by splitting the C/F bivalent at the centromeres into two bivalents and adding them to the autosomal bivalents as is appropriate to their chiasma positions. (See text) tively to that shown by the p.e. regions. The analogy may be more than trivial and might be due to the presence of some, but relatively fewer (Pardue and Gall, 1970), repetitive sequences in the telomeres, irrespective of how the Giemsa stain is related to these sequences here or in the p.e. regions (v. s.). Thomas (1966), in discussing homologous pairing, had pointed out that synapsis "seems to be a feature of chromosomes, not of D N A " . I n the present state of ignorance of the true functions of the highly repetitive DNA, it is possible that it may have special meiotic functions in addition to other "house-keeping" functions to do with chromosome structural integrity, movement or folding which may pertain to it (Walker et al., 1969). Homologous chromosomes might have a eentromerie and telomeric affinity which could sort out homology, and it might be, perhaps, the quantity of repetitive DNA that may be used in some way for an approximate pairing off of the chromosomes and to differentiate north and south. Naturally, there may well exist qualitative variation as well in the p.c. and/or telomerie regions. At any rate, the relatively large size of chromosomes generally and the special "teloeentric" make-up of murine chromosomes may well require a large amount

Centromeres and Chiasmata in the Mouse

369

of p.c. material to ensure accurate recognition of homology. The process of recognition could be facilitated, or may be started, by the suggested attachment of the chromosomes to the nuclear membrane at meiotic prophase (Comings and Okada, 1970a), either at their telomeres or, in the mouse, at centromeres and telomeres (Woollam et al., 1966 ; see also Sved, 1966), so that perhaps the homologucs are in proximity (Comings, 1968; Monakhova and Kostcnko, 1969). In the mouse, this could be a proximity of ccntromeres, p. c. regions or "basal knobs" (Woollam and Ford, 1964), but the clustering of the ccntromeric ends of pachytene mouse chromosomes around the sex vesicle may reflect the special tendency of satellite DNA to attach to nucleoli (Schildkraut and Maio, 1968). Incidentally, phenomena such as affinity (Michie, 1953; Wallace, 1953; Lindegren et al., 1962) could be explained if at times non-homologous p.c. regions tended to be rather similar quantitatively, leading to a similarity of " c e n t r o t y p e s " (Wallace, 1961; see also Griffen, 1960; Walker et al., 1969). After first recognition of homology, setting in register of the homologues might be a function of repetitive DNA segments inserted as signals of homology along the axis of the chromatid, while the synaptonemal proteins might tie the homologues together, as proposed by Comings and Okada (1970b). I t is clear that, in the case of the X and Y chromosomes, para-centromeric similarity, either quantitative or qualitative, or of both, or synchrony of DNA synthesis (v.i.), might be avoided by action on the Y chromosomes, as indeed seems to be the case. Possibly, telomeres are more relevant in this case. However, a p. c. organization of the Y, similar to that of the other chromosomes, may be essential for other reasons. There is evidence that the para-centromeric heterochromatin influences chromosome activity during mitosis (Lindsley and Novitski, 1958; Crouse, 1960) and it has been proposed that the centromere itself acts as an organizer for spindle elements (Luykx, 1965) and that, "while the chromosome is inert in 'this stage' and remains highly condensed, the kinetochore remains active and persists in an extended s t a t e " (Brinkley and Stubblefield, 1966). We have seen changes in the p.c. regions at meiotic anaphase so that it would seem that these parts are implicated in meiotic chromosome movement and, if so, the Y chromosome would be expected to display p.c. staining, as is indeed the csse in the mouse. H o t t a and Stern (1971) suggest that in L i l i u m a small amount of G d- C rich DNA is synthesized at zytogene, differing from the bulk of nuclear DNA and from the fraction synthesized at pachytene which has charscteristics suggestive of repair replication. I t is remotely possible that DNA synthesis at zytogene might be relevant to pairing by highly repetitive DNA. Certainly there is evidence that during the mitotic cycle the p.c. (and satellite) DNA is synthesized late in the S phase (Evans, 1964;

370

P.E. Polani:

Church, 1965; Tobia et al., 1970; F l a m m et al., 1971) and t h a t late-synthesized DNA is replicated at the nuclear membrane (Williams and Oekey, 1970). I n the course of meiosis, p. c. DNA is replicated even l a t e r - though apparently asynchronously as between autosomes, the X and the Y during the last premeiotic replication in the mouse (Kofman-Alvaro and Chandley, 1970; Odartchenko and Pavillard, 1970). Holliday (1968) had proposed a model for chromosome pairing similar to the one suggested here, based on the presence of discontinuities in the DNA of the chromatids, which, however, would not be made of a " l i n k e r " material other than DNA but would consist of short sequences of purine and pyrimidine bases. These runs could be irregularly distributed but similarly placed in homologues and ensure stability of binding between them. The method of recognition and binding would be by the synthesis of a fibrillar contractile meiotic protein which would help form the synaptonemal complex. The intercalated sequences could also function as sites for recombination. Somewhat similar is the synaptomere-zygosome hypothesis of King (1970). Crick (1971) has suggested a general model for the chromosomes of higher organisms based on a very long mononemic chromatid in which most of the DNA is used for control purposes. This DNA has numerous recognition sites which would consist of mainly unpaired single-stranded stretches of double-stranded DNA (the Unpairing Postulate) contained within relatively large segments of globular, super-coiled DNA. I n addition to having controlling functions over adjoining regions of fibrous DNA, to which the coding DNA sequences arc mainly relegated, Crick postulates t h a t the single-stranded regions can mediate a very specific collimation of adjacent chromatids, as in the bands of polytene chromosomes, b y complementary base pairing, and t h a t similar interactions could occur at meiotic synapsis. If the conclusion is correct that only a small fraction of the total DNA of higher organisms is very narrowly controlled in function while a large portion is less strictly constrained by selection against mutability and is non-informationai (Southern, 1970 ; Ohta and Kimura, 1971), then the highly repetitive satellite DNA could belong possibly to the former class (Southern ]970). However, even so, it could subserve important functions and could, interestingly, have a multiplicity of responsibilities (see, e.g., Walker et al., 1969; Hearst and Botchan, 1970; Jones and Robertson, 1970 ; Maio, 1971), some of which, at meiosis, have been briefly touched upon. Acknowledgements. I warmly thank Mr. P. Bates and Miss J. Constable for invaluable technical help. I am grateful to Mr. L. Kelberman for help with photography, to Dr. A. Leonard for the AKt~ T-1 mice, to Dr. J. L. Hamerton for the mouse L-cell line maintained in this laboratory by Dr. F. Giannelli, to Dr. G. Jagiello for permission to use Fig. 10e, and to Miss D. A. Baker for help with

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