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The occurrence and role of DNA synthesis during meiosis in wheat and rye
Copyright All rights
8 1973 by Academic Press, Inc. of reproduction in any form reserved
Experimental
Cell Research 77 (1973) 15-24
THE OCCURRENCE AND ROLE OF DNA SYNTHESIS DURING MEIOSIS IN WHEAT AND RYE R. B. FLAVELL
and G. W. R. WALKER
Cytogenetics Department, Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, UK
SUMMARY The incorporation of 3H-thymidine into the DNA of rye meiocytes at zygotene, pachytenediplotene and metaphase I to telouhase II stages has been studied. Low levels of SH were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far. The DNA synthesis inhibitor 2’-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.
The synthesis of small amounts of chromosomal DNA during certain stages of meiosis has been demonstrated in several organisms. The incorporation of exogenously supplied tritiated thymidine into meiotic prophase chromosomes has been detected autoradiographically in wheat, [I], newt [2], man [3, 41, and mouse [5, 61. In the Liliucae two distinct periods of DNA synthesis occur during prophase [7, 81. During the first, at early to mid-zygotene when chromosome pairing occurs, semi-conservative replication of about 0.3 “b of the genome is characteristic. During the second at late zygotene and pachytene when recombination is assumed to occur, most of the DNA synthesis is of the nonsemi-conservative repair type. The two types of DNA products also have very different buoyant density distributions. Because the early-zygotene replication occurs when chro3 -721817
mosome pairing begins and because chromosome pairing and synaptonemal complex formation is prevented by inhibitors of early zygotene DNA synthesis, Tto, Hotta & Stern, [9] have concluded that the early zygotene DNA synthesis is essential for, and involved in, chromosome pairing. The importance of this finding, for an understanding of meiosis, prompted a similar biochemical and cytological investigation in cereals. METHODS In vitro culture of wheat anthers Plants of hexaploid wheat, variety Chinese Spring, and Chinese Spring x Aegilops mutica hybrids were grown under continuous illumination at approx. 20°C. Tillers at the appropriate stage were transferred to the laboratory and maintained in water at room temperature during anther isolation. One anther from each floret was fixed immediately in alcohol : acetic acid (3 : 1). The two remaining anthers were incubated in the dark at 23°C in separate 0.25 ml aliquots of Exptl Cell Res 77 (1973)
16 R. B. Flavell & G. W. R. Walker sterilised modified White medium pH 6.0, containing 0.15 M sucrose [IO]. After a predetermined period of incubation both anthers were fixed and all 3 anthers were stained with fuchsin and examined cytologically. In experiments where the effect of 2’-deoxyadenosine was investigated, one anther of each floret was fixed immediately, a second was incubated in White modified medium as above and the third in the same medium plus 0.015 M 2’-deoxyadenosine for the same period. Both incubated anthers were fixed, stained and observed at the same time.
Labelling and isolation of rye sporogenous tissuefor biochemical analysis Plants of Secab cereale, var. ‘Petkus’ were grown under continuous illumination at 20” i2”C. Tillers at the appropriate stage were transferred to the laboratory and maintained in water at room temperature during anther isolation. The time taken for this never exceeded 2.5 h. One anther from each floret was squashed in a&o-carmine stain and its meiotic stage estimated. The remaining two anthers, which develop essentially synchronously with the third anther [ll] were pooled with other anthers of a similar stage in 0.25 ml of the sterilised modified White solution containing 0.15 M sucrose pH 6.0 as described above. 100 ,& of sterilised aH-thymidine (3H-methvl 26 Cilmmol) were added and the cultures incubated at 25°C for 4 h. This time was considered the maximum suitable for labelling the prophase stages zygotene or pachytene which last approx. 14 and 9 h respectively under these conditions (unpublished results). The cultures were then stored at - 20°C.
Samples of sporogenous tissue, free from contamination by anther wall and tapetal tissue were prepared as follows. The frozen anthers were thawed slowly on crushed ice and the anthers washed by serial transfer through two batches of chilled, distilled water. Three or four anthers at a time were removed on to cellulose acetate paper, their ends removed and the columns of pollen mother cells forced out into a drop of water. Anther debris was removed and the columns were washed several times with drops of distilled water. This removed almost all of-the adhering tapetal cells. Subsequent absorption of the water into the cellulose acetate paper allowed the columns to be seen under oblique lighting. They were picked up on a blade point and transferred to a small volume of chilled water. The sporogenous tissue so obtained was stored frozen.
Purification of carrier DNA Material from mature plants of Secale cereale var. ‘Petkus’ was diced and stored frozen. The frozen material was blended for approx. 15 set in a high speed coffee blender and then ground with sand in chilled mortars in 1 vol/g tissue of 1 % sodium lauryl sulphate containing 0.1 M ethylenediamine tetraacetate and 0.3 M sodium chloride (SEN medium) to a fairly smooth paste. It was vigorously shaken with 2 vol of chloroform containing 1 % octanol (CO) Exptl
Cell Res 77 (1973)
and centrifuged for 10 min at 10 000 rpm. The aqueous phase was reextracted twice with CO, heated to 70°C for 5 min, cooled and sodium perchlorate was added to a final concentration of 1 M. After extraction with CO the nucleic acid was spooled out of the aaueous laver following ethanol urecioitation [12]. The nucleic- acid was dissolved in 0.i x SSC (SSC = 0.15 M sodium chloride 0.015 sodium citrate). re-extracted with CO and the aqueous phase treate’d with pancreatic ribonuclease A (0.5 mg/ml) either alone or together with ribonuclease r, for 2 h at 37°C. Two volumes of CO were then added and the preparation left overnight at 5°C. DNA was spooled out of the aqueous layer following ethanol precipitation and further purified by isopropanol precipitations as described by Marmur [13].
Preparation of DNA from sporogenoustissue The frozen columns of pollen mother cells were slowly thawed and transferred to small grinding bottles (Konte’s Glass Company, Vineland, N.J.). The columns readily sedimented and this enabled them to be easily separated from any floating debris and single cells. The material was ground for about 2 min with a few grains of sand in 0.2 ml of SEN medium. Approx. 0.5 mg of purified carrier DNA in 0.5 ml SSC was added and a purification procedure. essentially similar to that described for carrier DNA with the omission of the isopropanol precipitations, was followed.
Caesium chloride density gradient analysis This was performed exactly as described by Flamm, Burr & Bond [14] in an angle head rotor. After centrifugation for 4&48 h at 4&45 000 rpm at 20°C the bottom of each centrifuge tube was punctured and each 4.5 ml gradient fractionated into approx. 80 fractions. 0.3 ml of 0.1 M Tris-HCl buffer, pH 8.2 was added to each fraction and the OD,,, determined. A 0.3 ml aliquot of each fraction was then added to 10 ml of scintillation fluid consisting of 667 ml toluene, 333 ml Triton X-100, 0.2 g POPOP and 6 g butyl PBD/l and counted after 24 h in a liquid scintillation counter.
RESULTS For the work described in this paper, the rapid uptake of 3H-thymidine and 2’-deoxyadenosine was essential. Thus it was first necessary to study the development of meiocytes cultured in a defined medium in order to establish an in vitro system which would be much more favourable to the rapid uptake of high levels of exogenously supplied substances than anthers in situ. In wheat and rye the meiocytes in the 3 anthers of a floret progress through meiosis more or less syn-
DNA synthesis during meiosis in wheat and rye chronously and any asynchrony is small compared with the duration of zygotene or pachytene [ll]. This enables the stage of development of the meiocytes in two anthers to be estimated by cytological examination of the third anther. Comparisons of several hundred wheat and rye anthers, fixed at various meiotic stages immediately after excision, with anthers taken from the same floret and incubated in culture for different time periods from 3 to 24 h, allowed the order of the stages of meiosis to be unambiguously determined and the duration of each stage to be estimated [l l] (fig. 4). Anthers of all the wheat and rye stocks used, continued to develop cytologically normally with excellent synchrony and viability for at least 12 h if explanted from midleptotene onwards. If explanted at early leptotene approx. 501,, failed to develop normally. This is most likely a result of mechanical damage inflicted during removal of anthers. After 15 h in culture approx. 30 ?b of the anthers possessed some meiocytes which showed signs of death and abnormal internal structure. However, in almost all anthers incubated for 24 h, some meiocytes remained viable and had apparently progressed through meiosis at a normal rate. From the appearance of wheat and rye meiocytes after many hours in culture and the close agreement in the duration of the meiotic stages between cultured anthers and anthers in situ, we have concluded that the culture of anthers introduces little disturbance into the meiotic process, at least during the first 12 h, which represents a considerable proportion of meiosis in cereals [l l] (fig. 4). For a biochemical analysis of the DNA synthesised in meiocytes during prophase we chose rye, since the anthers are larger, more easily handled and most important of all, the columns of meiocytes can be more easily extruded intact from whole anthers. The
01
I7
I 20
40
60
fraction number; ordinate: (lefr) counts per 30 min \ IOV. n , OD; 7, radioactivity. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from zygotene meiocytes exposed to 3Hthymidine, using unlabelled whole plant DNA as a carrier during purification. Fig. I. Abscissa: (right) 3H OD,m;
method developed for isolating rye sporogenous tissue yielded preparations of meiocytes uncontaminated with other cell types as far as could be judged by cytological examination after Feulgen staining. Anther wall tissue was readily removed as an intact mass and any adhering or free cells were easily washed from the columns. DNA was isolated from 3H-thymidine latelled meiocytes as described in Methods and centrifuged with carrier DNA to equilibrium in caesium chloride density gradients. The position of the whole plant DNA, shown by the optical density profile, and that of the labelled meiocyte DNA shown by the 3H-profile are presented in figs 1, 2 and 3 for DNA preparations from meiocytes cultured at zygotene, pachytene-diplotene and metaphase I-telophase II respectively. From the 3H-profiles of these gradients, estimates were made of the DNA synthesis per anther correcting for loss of DNA during purification from carriers DNA recoveries Exptl
Cell Res 77 (1973)
18 R. B. Flavell & G. W. R. Walker
20
40
60
SO
I
Fig. 2. Abscissa: fraction number; ordinate: (left) OD,,,; (right) *H counts per 30 min x 1O-2. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from pachytene-diplotene meiocytes exposed to 8Hthymidine, using unlabelled whole plant DNA as a carrier during purification.
and assuming (1) equal uptake and equal internal pools of thymidine at all stages; (2) equal average recoveries of meiocytes per anther at all stages. On this basis the 3H incorporated into DNA per anther in the zygotene, pachytene-diplotene and metaphase I-telophase II groups was 4:s: 1 respectively. The zygotene and pachytenediplotene estimates are probably reasonable but the loss of a relatively large proportion of columns of meiocytes in the metaphase I-telophase II group owing to their fragility, makes the estimate of incorporation into this group too low; the true value should, almost certainly, be closer to the zygotene value. The effect of inhibitors of DNA synthesis on meiotic development were investigated concurrently since such studies were likely to help in ascribing a functional role to any DNA synthesis detected. For some of these studies, anthers of hexaploid wheat were Exptl Cell Res 77 (1973)
used since wheat meiosis is shorter than that of rye. In other studies conducted with Mr G. A. Dover anthers of selected Chinese Spring x Aegilops mutica hybrids were used since they have the advantageous property of partial synapsis [15] and thus at late prophase normally contain both paired and occasional unpaired chromosomes making the former easier to distinguish. This was considered of importance in view of the fact that inhibitors of DNA synthesis prevent chromosome pairing in Lilium [16]. The DNA synthesis inhibitor chosen for most of the experiments was 2’-deoxyadenosine. Deoxyadenosine has been used extensively by other workers [17-201 including Ito, Hotta & Stern on Lilium [9], and is a relatively specific inhibitor of DNA synthesis.
I 17
20
40
60
30
Fig. 3. Abscissa: fraction number; ordinate (left) ODzao; (right) 3H counts per 60 min x lo-*. n , OD; 7, radioactivity. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from metaphase I-telophase II meiocytes exposed to SH-thymidine, using unlabelled whole plant DNA as a carrier during purification.
DNA synthesis during meiosis in waheatand rye LEPTOTENE
--
ZYG
-----------
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T,
PACH DD M, -
DYADS
TETRADS
M2-T2
I9
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Fig. 4. Abscissa: time (hours). Each horizontal line indicates the meiotic stage when the anther was explanted into culture and the stage reached after incubation in vitro. --, anther incubated with deoxyadenosine; , anther incubated without deoxyadenosine. . ., dotted extension indicates asynchrony of abnormal meiotic stages. Results with anthers from the same floret are shown in pairs. ZYG, zygotene; PACH, pachytene; DD, diplotene and diakinesis; M,-T,, metaphase I to telophase I; Mz-r,, metaphase 2 to telophase 2. Effect of 2’-deoxyadenosine on meiotic development and the duration of meiosis in anthers of hexaploid wheat var. Chinese Spring in vitro. Anthers containing any synapsed chromosomes were recorded as early zygotene rather than leptotene even though most of the chromosomes appeared to be still leptotene. The developmental stage within a named stage, for example zygotene, was estimated from the appearance of the cells and from the time taken for control anthers to reach a well-defined short-stage, for example telophase II.
Typical results showing the effects of deoxyadenosine on meiotic development are shown in fig. 4 for Chinese spring wheat. Results using anthers of Chinese spring x Aegilops mutica hybrids were very similar although meiosiswas longer in these hybrids. Meiotic development was only inhibited when anthers were incubated with deoxyadenosine during late zygotene or pachytene (fig. 4), while in the absenceof the inhibitor, meiotic development proceeded at a rate similar to that in anthers in situ. No inhibition of meiot-
ic development or extensive chromosome fragmentation occurred during early or midzygotene, nor was chromosome synapsis inhibited (fig. 5). . . . Those anthers in fig. 4 whose development was inhibited by deoxyadenosine at late zygotene showed very extensive chromosome pairing and their classification as being at late zygotene rather than pachytene was made on the basis of chromosome condensation rather than chromosome pairing. Cells in anthers inhibited with deoxyadenoExptl
Cell Res 77 (1973)
20 R. B. Flavell & G. W. R. Walker sine at late zygotene were usually of normal appearance but cells inhibited at pachytene, diplotene or diakinesis always showed abnormalities, especially in chromosome condensation (fig. 5). Most cells first incubated with deoxyadenosine during pachytene remained at pachytene, but in a small proportion of the cells chromosome condensation continued, giving rise to abnormal diplotene or diakinesis configurations. Occasionally a few cells progressed further to metaphase or anaphase but their chromosomes were always grossly abnormal; chromosome clumping and the presence of anaphase fragments and bridges were common (fig. 5). No inhibition of development was observed in cells first incubated with deoxyadenosine after diakinesis (fig. 4). DISCUSSION The caesium chloride gradient analyses of purified DNA provide strong evidence for the synthesis of a small amount of DNA during the prophase and division phases of meiosis in rye. It is most unlikely that the observed 3H incorporation was due to contamination with microbial DNA since sterile techniques were used during exposure of the cells to 3H-thymidine and the pollen mother cells were extracted from within the thawed anthers after they had been removed from the 3H-thymidine solution and washed. The buoyant density distributions also support
Fig. 5. muticu
the conclusion that the labelled DNA was of rye and not microbial origin. It is also unlikely that the labelled DNA came from tapetal or anther wall cells since our cytological control preparations were free from such cells. In any case the major S phase replication of tapetal cell DNA does not occur during the meiotic stages studied here although some incorporation of 3H into tapetal nuclei is regularly observed on autoradiographs during these meiotic stages (R Riley & M D Bennett. Personal communication). We are, therefore, led to conclude that as in other organisms [l-8] some form of DNA synthesis can occur in the sporogenous tissue during some or all of the stages of meiosis beyond leptotene. Purified rye DNA banded symmetrically in caesium chloride gradients with no satellites. However the 3H profile indicated that a considerable proportion of the DNA synthesised in zygotene and pachytene-diplotene meiocytes, had a greater average buoyant density than whole plant rye DNA (figs 1, 2). This fraction of higher-than-average buoyant density DNA is unlikely to be chloroplast DNA which in wheat [21] has a lower G-C ratio than most nuclear DNA, but we cannot rule out the possibility that the DNA is of mitochondrial origin or that it represents a small amplification of the ribosomal RNA genes, as occurs during prophase in amphibians [22], since mitochondrial and ribosomal DNA’s are of slightly higher average
Effects of 2’-deoxyadenosine on meiotic development. (a) Chromosomes of Chinese spring x Aegilops hybrid at early zygotene; (6) chromosomes in a meiocyte of an anther from the same floret as (a) incubated for 24 h in the presence of deoxyadenosine. This cell was inhibited at early pachytene. The control anther from the floret developed to anaphase II in the absence of deoxyadenosine; (c) pachytene chromosomes in Chinese spring x Aegilops mutica anther incubated in the presence of deoxyadenosine from early pachytene for 24 h. The aggregated chromatin is characteristic of cells inhibited at pachytene. The control anther from the same floret incubated without deoxyadenosine developed to dyads; (d) abnormally condensed diakinesis chromosomes in an anther incubated in the presence of deoxyadenosine for 24 h from late-pachytene-early diplotene. The control anther from the same floret developed to tetrads; (e) a meiocyte of Chinese Spring wheat inhibited at metaphase I showing abnormal chromosome condensation and clumping after incubation at pachytene in the presence of deoxyadenosine for 24 h; (f) a meiocyte from the same anther as (e) showing chromosome fragments at telophase I. Exptl
Cell Res 77 (1973)
DNA synthesis during meiosis in wheat anu’ r~~c 2 1
* Sb
5d
Exptl
Cell
Res 77 ( 1973)
22
R. B. Flaaell h G. W. R. Walker
buoyant density than most nuclear DNA in wheat [21-231 and most probably in rye. Wheat also contains a relatively high percentage of 5’-methylcytosine [24] which tends to decrease the buoyant density and, if rye is similar to wheat, it could represent DNA synthesis of sequences relatively low in 5’methylcytosine. Irrespective of the cellular origin of the DNA synthesised during meiotic prophase, an important conclusion is that the DNA synthesised in the meiocytes classified as being at zygotene is indistinguishable by buoyant density measurements from that synthesised in meiocytes at pachytene-diplotene. Although our prophase anthers were separated into two groups, zygotene and pachytene-diplotene, it is highly likely that the zygotene group contained some cells at pachytene because of the diffuseness of the boundary between zygotene and pachytene, and because the zygotene classified anthers were incubated in 3H-thymidine for 4 h after classification. This point, together with the similar buoyant density distribution of the DNAs synthesised in the meiocytes of the zygotene and pachytene-diplotene groups prevents us from concluding that there is any meiotic DNA synthesis peculiar to zygotene in rye as in Lilium [8]. If zygotene-specific DNA synthesis does occur in rye, it must be considerably less than 50% of the amount that occurs in pachytene cells especially if the rate of DNA synthesis at diplotene is less than that at pachytene. In Lilium the zygotene DNA synthesis is three to five times that which occurs at pachytene [8]. Our studies with 2’-deoxyadenosine also point to the absence of extensive DNA synthesis at zygotene. Deoxyadenosine at a concentration which completely inhibited cell development at late zygotene and pachytene did not inhibit meiotic development during most of zygotene (fig. 4). Neither did Exptl
Cell Res 77 (1973)
it prevent chromosome pairing as defined by the very close alignment of matched chromosome threads, nor induce chromosome fragmentation at mid-zygotene (fig. 5) as it does in Lilium [9]. The apparent difference in zygotene DNA synthesis between the cereals and Lilium cannot be easily interpreted at this stage. However, since meiosis in rye (51 h) is considerably shorter than in Lifium (170 h at 20°C) [25] and there is mounting evidence that in cereals chromosome pairing may be organised in a premeiotic phase (M W Bayliss, G A Dover, M D Bennett & R Riley. Personal communication), variation in the process of chromosome pairing is not perhaps too surprising. Our results do not rule out the possibility that in the meiotic division of cereals replication of part of the genome is also delayed since, for example, the semiconservative DNA synthesis necessary for completion of genome replication could occur at some stage other than zygotene. The occurrence of DNA synthesis at pachytene we report here for rye is consistent with similar reports for other organisms, although in all except Lilium, the evidence has come from autoradiography [2-61. It is also consistent with current models of recombination [26-271, the occurrence of crossing over at pachytene and the effects of deoxyadenosine on recombination at pachytene [20]. The recombination models, the biochemical analysis of DNA synthesised at pachytene in Lilium [7, 81 and the enhanced DNA synthesis at this stage in man and mouse in response to irradiation [4, 61 all imply that the late zygotene-pachytene DNA synthesis is concerned with the non-semiconservative repair of chromosome lesions. In the recombination models it is postulated that at late zygotene numerous breaks are introduced into the DNA strands by nuclease action and during their subsequent repair after limited DNA
DNA degradation, hybrid DNA formation may occur between non-sister chromatid DNA strands to lead to a recombinational event. In support of these models, Howell & Stern [28] have recently described the appearance of an endonuclease, a polynucleotide kinase and a polynucleotide ligase activity during zygotene in Lilium. Our observations that deoxyadenosine inhibits cells from late zygotene to pachytene, but not earlier or later, also supports these models and indicates that the only DNA synthesis occurring after leptotene which is essential for meiotic development is confined to the late zygotenepachytene period. The inhibition of meiotic development by deoxyadenosine at these stages also suggests that recombinational repair is essential for meiosis to proceed. However, in other experiments with deoxyadenosine, we have found that it causes meiotic inhibition at a stage of chromosomal condensation characteristic of that at late zygotene or pachytene in an asynaptic mutant of wheat in which neither chromosome pairing nor recombination occur. Thus we cannot rule out the possibility that late prophase DNA synthesis is essential for some meiotic developmental process in addition to recombination. We did detect some DNA synthesis in anthers at metaphase I to telophase II (fig. 3), but deoxyadenosine did not inhibit meiotic development or produce any obvious cytological abnormalities after diplotene. Assuming a recovery of the more fragile columns of meiocytes at metaphase I to telophase II to be about 25 “; that of the zygotene and pachytene-diplotene columns, the amount of 3H-thymidine incorporated into the DNA of post-prophase meiocytes was similar to that incorporated into the DNA of cells of the zygotene group. This level of synthesis relative to that at pachytene is considerably higher than that found in Lilium, (assuming
synthesis during meiosis in wheat and r!‘c
23
that the labelled rye DNA was nuclear. was all from sporogenous tissue and that the level of pachytene DNA synthesis per pg nuclear DNA is not much greater in Lilium than in rye). However, in rye, with its shorter meiotic duration [25] it is conceivable that unless mechanisms exist to degrade quickly the enzymes involved in the breakage. excision and repair processesof recombination, these enzymes would be at a higher concentration in post-prophase cells and could therefore lead to residual breakage and repair in post-prophase stages. Alternatively it is conceivable that all recombination repair is not complete at diakinesis but is completed after metaphase I. Such events as these might be responsible for the site-specific incorporation of 3H into post-metaphase wheat chromosomes reported by Riley & Bennett [l]. It is also possible that the level of thymidine incorporation during metaphase I to telophase II reflects a low level of repair continuing throughout the cell cycle of rye. We have not investigated. but do recognise, the possibility that part of the DNA synthesis measured in this work may be the repair of radiation damage incurred from the relatively high levels of “H-thymidine used.
The chromosome abnormalities induced by deoxyadenosine in the cells which escaped total pachytene arrest are similar to those observed in Lilium [9], viz; abnormal chromosome clumping. fragmentation and ktnaphase bridge formation (fig. 54). It is difficult to interpret all these abnormalities in terms of specific events occurring during late prophase. However, in general, it is not difficult to imagine how chromosomes poasessingnumerous unrepaired single or double strand breaks might well condense abnormally, clump and fragment during the chromosomal changes associated with nuclear division. Exptl
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R. B. Flavell & G. W. R. Walker
We wish to thank Professor Ralph Riley for his stimulating interest in this work, Mr Gabriel Dover for valuable experimental help in part of this work and Dr Colin Law for help in the preparation of the manuscript. G. W. R. W. is on sabbatical leave from the Department of Genetics, University of Alberta, Edmonton, Alberta, Canada.
REFERENCES 1. Riley, R & Bennett, M D, Nature 230 (1971) 182. 2. Wimber, D E & Prensky, W, Genetics 48 (1963) 1731. 3. Lima-de-Faria, A, German, J, Ghatneker, M, McGovern, J & Anderson, L, Hereditas 60 (1968) 249. 4. Chandlev, A C & Kofman-Alfaro. _.S. Exntl - cell res 69 (1971) 45. 5. Mukheriee. ” ’ A B & Ghen. , M M. , Nature 219 (1968) . , 489. 6. Kofman-Alfaro, S & Chandley, A C, Exptl cell res 69 (1971) 33. 7. Hotta, Y, Ito, M & Stern, H, Proc natl acad sci US 56 (1966) 1184. 8. Hotta, Y & Stern, H, J mol biol 55 (1971) 337. 9. Ito, M, Hotta, Y & Stern, H, Dev biol 6 (1967) 6. 10. Ito, M & Stem, H, Dev biol 16 (1967) 36. 11. Bennett, M D, Chapman, V & Riley, R, Proc roy sot B 178 (1971) 259.
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12. Bolton, E T, Britten, R J, Cowrie, D B, Roberts, R B, Szafranski, P &Waring, M J, Carnegie inst year book (1964) 314. 13. Marmur, J, J mol biol 3 (1961) 208. 14. Flamm, W G, Burr, H E & Bond, S B, Biochim biophys acta 129 (1966) 310. 15. Dover, G A & Riley, R, Nature (1972). 16. Stern, H & Hotta, Y, Control of nuclear activity (ed L Goldstein) p. 47. Prentice-Hall, Englewood Cliffs, N.J. (1967). 17. Kihlman, B A & Hartley, B, Mut res 4 (1967) 765. 18. Kihlman, B A & Odmark, G, Hereditas 56 (1966) 71. 19. Reichland, P, Canellakis, Z N & Canellakis, E N, J biol them 236 (1961) 2514. 20. Davies, D R & Lawrence, C W, Mut res 4 (1961) 147. 21. Wells, R & Ingle, J, Plant physiol 46 (1970) 178. 22. Perkowska, E, MacGregor, H C & Birnstiel, M L, Nature 217 (1968) 649. 23. Ingle, J, Possingham, J V, Wells R, Leaver, C J & Loening, U E, Symp sot exptl biol 24 (1970) 303. 24. Schildkraut, C L, Marmur, J & Doty, P, J mol biol 4 (1962) 795. 25. Bennett, M D, Proc roy sot London B 178 (1971) 277. 26. Whitehouse, H L K, Nature 199 (1963) 1034. 27. Holliday, R, Genet res 5 (1964) 282. 28. Howell, J & Stern, H, J mol biol 55 (1971) 357. Received June 26, 1972 Revised version received August 30, 1972
8 1973 by Academic Press, Inc. of reproduction in any form reserved
Experimental
Cell Research 77 (1973) 15-24
THE OCCURRENCE AND ROLE OF DNA SYNTHESIS DURING MEIOSIS IN WHEAT AND RYE R. B. FLAVELL
and G. W. R. WALKER
Cytogenetics Department, Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, UK
SUMMARY The incorporation of 3H-thymidine into the DNA of rye meiocytes at zygotene, pachytenediplotene and metaphase I to telouhase II stages has been studied. Low levels of SH were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far. The DNA synthesis inhibitor 2’-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.
The synthesis of small amounts of chromosomal DNA during certain stages of meiosis has been demonstrated in several organisms. The incorporation of exogenously supplied tritiated thymidine into meiotic prophase chromosomes has been detected autoradiographically in wheat, [I], newt [2], man [3, 41, and mouse [5, 61. In the Liliucae two distinct periods of DNA synthesis occur during prophase [7, 81. During the first, at early to mid-zygotene when chromosome pairing occurs, semi-conservative replication of about 0.3 “b of the genome is characteristic. During the second at late zygotene and pachytene when recombination is assumed to occur, most of the DNA synthesis is of the nonsemi-conservative repair type. The two types of DNA products also have very different buoyant density distributions. Because the early-zygotene replication occurs when chro3 -721817
mosome pairing begins and because chromosome pairing and synaptonemal complex formation is prevented by inhibitors of early zygotene DNA synthesis, Tto, Hotta & Stern, [9] have concluded that the early zygotene DNA synthesis is essential for, and involved in, chromosome pairing. The importance of this finding, for an understanding of meiosis, prompted a similar biochemical and cytological investigation in cereals. METHODS In vitro culture of wheat anthers Plants of hexaploid wheat, variety Chinese Spring, and Chinese Spring x Aegilops mutica hybrids were grown under continuous illumination at approx. 20°C. Tillers at the appropriate stage were transferred to the laboratory and maintained in water at room temperature during anther isolation. One anther from each floret was fixed immediately in alcohol : acetic acid (3 : 1). The two remaining anthers were incubated in the dark at 23°C in separate 0.25 ml aliquots of Exptl Cell Res 77 (1973)
16 R. B. Flavell & G. W. R. Walker sterilised modified White medium pH 6.0, containing 0.15 M sucrose [IO]. After a predetermined period of incubation both anthers were fixed and all 3 anthers were stained with fuchsin and examined cytologically. In experiments where the effect of 2’-deoxyadenosine was investigated, one anther of each floret was fixed immediately, a second was incubated in White modified medium as above and the third in the same medium plus 0.015 M 2’-deoxyadenosine for the same period. Both incubated anthers were fixed, stained and observed at the same time.
Labelling and isolation of rye sporogenous tissuefor biochemical analysis Plants of Secab cereale, var. ‘Petkus’ were grown under continuous illumination at 20” i2”C. Tillers at the appropriate stage were transferred to the laboratory and maintained in water at room temperature during anther isolation. The time taken for this never exceeded 2.5 h. One anther from each floret was squashed in a&o-carmine stain and its meiotic stage estimated. The remaining two anthers, which develop essentially synchronously with the third anther [ll] were pooled with other anthers of a similar stage in 0.25 ml of the sterilised modified White solution containing 0.15 M sucrose pH 6.0 as described above. 100 ,& of sterilised aH-thymidine (3H-methvl 26 Cilmmol) were added and the cultures incubated at 25°C for 4 h. This time was considered the maximum suitable for labelling the prophase stages zygotene or pachytene which last approx. 14 and 9 h respectively under these conditions (unpublished results). The cultures were then stored at - 20°C.
Samples of sporogenous tissue, free from contamination by anther wall and tapetal tissue were prepared as follows. The frozen anthers were thawed slowly on crushed ice and the anthers washed by serial transfer through two batches of chilled, distilled water. Three or four anthers at a time were removed on to cellulose acetate paper, their ends removed and the columns of pollen mother cells forced out into a drop of water. Anther debris was removed and the columns were washed several times with drops of distilled water. This removed almost all of-the adhering tapetal cells. Subsequent absorption of the water into the cellulose acetate paper allowed the columns to be seen under oblique lighting. They were picked up on a blade point and transferred to a small volume of chilled water. The sporogenous tissue so obtained was stored frozen.
Purification of carrier DNA Material from mature plants of Secale cereale var. ‘Petkus’ was diced and stored frozen. The frozen material was blended for approx. 15 set in a high speed coffee blender and then ground with sand in chilled mortars in 1 vol/g tissue of 1 % sodium lauryl sulphate containing 0.1 M ethylenediamine tetraacetate and 0.3 M sodium chloride (SEN medium) to a fairly smooth paste. It was vigorously shaken with 2 vol of chloroform containing 1 % octanol (CO) Exptl
Cell Res 77 (1973)
and centrifuged for 10 min at 10 000 rpm. The aqueous phase was reextracted twice with CO, heated to 70°C for 5 min, cooled and sodium perchlorate was added to a final concentration of 1 M. After extraction with CO the nucleic acid was spooled out of the aaueous laver following ethanol urecioitation [12]. The nucleic- acid was dissolved in 0.i x SSC (SSC = 0.15 M sodium chloride 0.015 sodium citrate). re-extracted with CO and the aqueous phase treate’d with pancreatic ribonuclease A (0.5 mg/ml) either alone or together with ribonuclease r, for 2 h at 37°C. Two volumes of CO were then added and the preparation left overnight at 5°C. DNA was spooled out of the aqueous layer following ethanol precipitation and further purified by isopropanol precipitations as described by Marmur [13].
Preparation of DNA from sporogenoustissue The frozen columns of pollen mother cells were slowly thawed and transferred to small grinding bottles (Konte’s Glass Company, Vineland, N.J.). The columns readily sedimented and this enabled them to be easily separated from any floating debris and single cells. The material was ground for about 2 min with a few grains of sand in 0.2 ml of SEN medium. Approx. 0.5 mg of purified carrier DNA in 0.5 ml SSC was added and a purification procedure. essentially similar to that described for carrier DNA with the omission of the isopropanol precipitations, was followed.
Caesium chloride density gradient analysis This was performed exactly as described by Flamm, Burr & Bond [14] in an angle head rotor. After centrifugation for 4&48 h at 4&45 000 rpm at 20°C the bottom of each centrifuge tube was punctured and each 4.5 ml gradient fractionated into approx. 80 fractions. 0.3 ml of 0.1 M Tris-HCl buffer, pH 8.2 was added to each fraction and the OD,,, determined. A 0.3 ml aliquot of each fraction was then added to 10 ml of scintillation fluid consisting of 667 ml toluene, 333 ml Triton X-100, 0.2 g POPOP and 6 g butyl PBD/l and counted after 24 h in a liquid scintillation counter.
RESULTS For the work described in this paper, the rapid uptake of 3H-thymidine and 2’-deoxyadenosine was essential. Thus it was first necessary to study the development of meiocytes cultured in a defined medium in order to establish an in vitro system which would be much more favourable to the rapid uptake of high levels of exogenously supplied substances than anthers in situ. In wheat and rye the meiocytes in the 3 anthers of a floret progress through meiosis more or less syn-
DNA synthesis during meiosis in wheat and rye chronously and any asynchrony is small compared with the duration of zygotene or pachytene [ll]. This enables the stage of development of the meiocytes in two anthers to be estimated by cytological examination of the third anther. Comparisons of several hundred wheat and rye anthers, fixed at various meiotic stages immediately after excision, with anthers taken from the same floret and incubated in culture for different time periods from 3 to 24 h, allowed the order of the stages of meiosis to be unambiguously determined and the duration of each stage to be estimated [l l] (fig. 4). Anthers of all the wheat and rye stocks used, continued to develop cytologically normally with excellent synchrony and viability for at least 12 h if explanted from midleptotene onwards. If explanted at early leptotene approx. 501,, failed to develop normally. This is most likely a result of mechanical damage inflicted during removal of anthers. After 15 h in culture approx. 30 ?b of the anthers possessed some meiocytes which showed signs of death and abnormal internal structure. However, in almost all anthers incubated for 24 h, some meiocytes remained viable and had apparently progressed through meiosis at a normal rate. From the appearance of wheat and rye meiocytes after many hours in culture and the close agreement in the duration of the meiotic stages between cultured anthers and anthers in situ, we have concluded that the culture of anthers introduces little disturbance into the meiotic process, at least during the first 12 h, which represents a considerable proportion of meiosis in cereals [l l] (fig. 4). For a biochemical analysis of the DNA synthesised in meiocytes during prophase we chose rye, since the anthers are larger, more easily handled and most important of all, the columns of meiocytes can be more easily extruded intact from whole anthers. The
01
I7
I 20
40
60
fraction number; ordinate: (lefr) counts per 30 min \ IOV. n , OD; 7, radioactivity. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from zygotene meiocytes exposed to 3Hthymidine, using unlabelled whole plant DNA as a carrier during purification. Fig. I. Abscissa: (right) 3H OD,m;
method developed for isolating rye sporogenous tissue yielded preparations of meiocytes uncontaminated with other cell types as far as could be judged by cytological examination after Feulgen staining. Anther wall tissue was readily removed as an intact mass and any adhering or free cells were easily washed from the columns. DNA was isolated from 3H-thymidine latelled meiocytes as described in Methods and centrifuged with carrier DNA to equilibrium in caesium chloride density gradients. The position of the whole plant DNA, shown by the optical density profile, and that of the labelled meiocyte DNA shown by the 3H-profile are presented in figs 1, 2 and 3 for DNA preparations from meiocytes cultured at zygotene, pachytene-diplotene and metaphase I-telophase II respectively. From the 3H-profiles of these gradients, estimates were made of the DNA synthesis per anther correcting for loss of DNA during purification from carriers DNA recoveries Exptl
Cell Res 77 (1973)
18 R. B. Flavell & G. W. R. Walker
20
40
60
SO
I
Fig. 2. Abscissa: fraction number; ordinate: (left) OD,,,; (right) *H counts per 30 min x 1O-2. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from pachytene-diplotene meiocytes exposed to 8Hthymidine, using unlabelled whole plant DNA as a carrier during purification.
and assuming (1) equal uptake and equal internal pools of thymidine at all stages; (2) equal average recoveries of meiocytes per anther at all stages. On this basis the 3H incorporated into DNA per anther in the zygotene, pachytene-diplotene and metaphase I-telophase II groups was 4:s: 1 respectively. The zygotene and pachytenediplotene estimates are probably reasonable but the loss of a relatively large proportion of columns of meiocytes in the metaphase I-telophase II group owing to their fragility, makes the estimate of incorporation into this group too low; the true value should, almost certainly, be closer to the zygotene value. The effect of inhibitors of DNA synthesis on meiotic development were investigated concurrently since such studies were likely to help in ascribing a functional role to any DNA synthesis detected. For some of these studies, anthers of hexaploid wheat were Exptl Cell Res 77 (1973)
used since wheat meiosis is shorter than that of rye. In other studies conducted with Mr G. A. Dover anthers of selected Chinese Spring x Aegilops mutica hybrids were used since they have the advantageous property of partial synapsis [15] and thus at late prophase normally contain both paired and occasional unpaired chromosomes making the former easier to distinguish. This was considered of importance in view of the fact that inhibitors of DNA synthesis prevent chromosome pairing in Lilium [16]. The DNA synthesis inhibitor chosen for most of the experiments was 2’-deoxyadenosine. Deoxyadenosine has been used extensively by other workers [17-201 including Ito, Hotta & Stern on Lilium [9], and is a relatively specific inhibitor of DNA synthesis.
I 17
20
40
60
30
Fig. 3. Abscissa: fraction number; ordinate (left) ODzao; (right) 3H counts per 60 min x lo-*. n , OD; 7, radioactivity. Fraction 1 = bottom of gradient. Caesium chloride gradient analysis of DNA isolated from metaphase I-telophase II meiocytes exposed to SH-thymidine, using unlabelled whole plant DNA as a carrier during purification.
DNA synthesis during meiosis in waheatand rye LEPTOTENE
--
ZYG
-----------
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-------__ ----
T,
PACH DD M, -
DYADS
TETRADS
M2-T2
I9
--
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Fig. 4. Abscissa: time (hours). Each horizontal line indicates the meiotic stage when the anther was explanted into culture and the stage reached after incubation in vitro. --, anther incubated with deoxyadenosine; , anther incubated without deoxyadenosine. . ., dotted extension indicates asynchrony of abnormal meiotic stages. Results with anthers from the same floret are shown in pairs. ZYG, zygotene; PACH, pachytene; DD, diplotene and diakinesis; M,-T,, metaphase I to telophase I; Mz-r,, metaphase 2 to telophase 2. Effect of 2’-deoxyadenosine on meiotic development and the duration of meiosis in anthers of hexaploid wheat var. Chinese Spring in vitro. Anthers containing any synapsed chromosomes were recorded as early zygotene rather than leptotene even though most of the chromosomes appeared to be still leptotene. The developmental stage within a named stage, for example zygotene, was estimated from the appearance of the cells and from the time taken for control anthers to reach a well-defined short-stage, for example telophase II.
Typical results showing the effects of deoxyadenosine on meiotic development are shown in fig. 4 for Chinese spring wheat. Results using anthers of Chinese spring x Aegilops mutica hybrids were very similar although meiosiswas longer in these hybrids. Meiotic development was only inhibited when anthers were incubated with deoxyadenosine during late zygotene or pachytene (fig. 4), while in the absenceof the inhibitor, meiotic development proceeded at a rate similar to that in anthers in situ. No inhibition of meiot-
ic development or extensive chromosome fragmentation occurred during early or midzygotene, nor was chromosome synapsis inhibited (fig. 5). . . . Those anthers in fig. 4 whose development was inhibited by deoxyadenosine at late zygotene showed very extensive chromosome pairing and their classification as being at late zygotene rather than pachytene was made on the basis of chromosome condensation rather than chromosome pairing. Cells in anthers inhibited with deoxyadenoExptl
Cell Res 77 (1973)
20 R. B. Flavell & G. W. R. Walker sine at late zygotene were usually of normal appearance but cells inhibited at pachytene, diplotene or diakinesis always showed abnormalities, especially in chromosome condensation (fig. 5). Most cells first incubated with deoxyadenosine during pachytene remained at pachytene, but in a small proportion of the cells chromosome condensation continued, giving rise to abnormal diplotene or diakinesis configurations. Occasionally a few cells progressed further to metaphase or anaphase but their chromosomes were always grossly abnormal; chromosome clumping and the presence of anaphase fragments and bridges were common (fig. 5). No inhibition of development was observed in cells first incubated with deoxyadenosine after diakinesis (fig. 4). DISCUSSION The caesium chloride gradient analyses of purified DNA provide strong evidence for the synthesis of a small amount of DNA during the prophase and division phases of meiosis in rye. It is most unlikely that the observed 3H incorporation was due to contamination with microbial DNA since sterile techniques were used during exposure of the cells to 3H-thymidine and the pollen mother cells were extracted from within the thawed anthers after they had been removed from the 3H-thymidine solution and washed. The buoyant density distributions also support
Fig. 5. muticu
the conclusion that the labelled DNA was of rye and not microbial origin. It is also unlikely that the labelled DNA came from tapetal or anther wall cells since our cytological control preparations were free from such cells. In any case the major S phase replication of tapetal cell DNA does not occur during the meiotic stages studied here although some incorporation of 3H into tapetal nuclei is regularly observed on autoradiographs during these meiotic stages (R Riley & M D Bennett. Personal communication). We are, therefore, led to conclude that as in other organisms [l-8] some form of DNA synthesis can occur in the sporogenous tissue during some or all of the stages of meiosis beyond leptotene. Purified rye DNA banded symmetrically in caesium chloride gradients with no satellites. However the 3H profile indicated that a considerable proportion of the DNA synthesised in zygotene and pachytene-diplotene meiocytes, had a greater average buoyant density than whole plant rye DNA (figs 1, 2). This fraction of higher-than-average buoyant density DNA is unlikely to be chloroplast DNA which in wheat [21] has a lower G-C ratio than most nuclear DNA, but we cannot rule out the possibility that the DNA is of mitochondrial origin or that it represents a small amplification of the ribosomal RNA genes, as occurs during prophase in amphibians [22], since mitochondrial and ribosomal DNA’s are of slightly higher average
Effects of 2’-deoxyadenosine on meiotic development. (a) Chromosomes of Chinese spring x Aegilops hybrid at early zygotene; (6) chromosomes in a meiocyte of an anther from the same floret as (a) incubated for 24 h in the presence of deoxyadenosine. This cell was inhibited at early pachytene. The control anther from the floret developed to anaphase II in the absence of deoxyadenosine; (c) pachytene chromosomes in Chinese spring x Aegilops mutica anther incubated in the presence of deoxyadenosine from early pachytene for 24 h. The aggregated chromatin is characteristic of cells inhibited at pachytene. The control anther from the same floret incubated without deoxyadenosine developed to dyads; (d) abnormally condensed diakinesis chromosomes in an anther incubated in the presence of deoxyadenosine for 24 h from late-pachytene-early diplotene. The control anther from the same floret developed to tetrads; (e) a meiocyte of Chinese Spring wheat inhibited at metaphase I showing abnormal chromosome condensation and clumping after incubation at pachytene in the presence of deoxyadenosine for 24 h; (f) a meiocyte from the same anther as (e) showing chromosome fragments at telophase I. Exptl
Cell Res 77 (1973)
DNA synthesis during meiosis in wheat anu’ r~~c 2 1
* Sb
5d
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R. B. Flaaell h G. W. R. Walker
buoyant density than most nuclear DNA in wheat [21-231 and most probably in rye. Wheat also contains a relatively high percentage of 5’-methylcytosine [24] which tends to decrease the buoyant density and, if rye is similar to wheat, it could represent DNA synthesis of sequences relatively low in 5’methylcytosine. Irrespective of the cellular origin of the DNA synthesised during meiotic prophase, an important conclusion is that the DNA synthesised in the meiocytes classified as being at zygotene is indistinguishable by buoyant density measurements from that synthesised in meiocytes at pachytene-diplotene. Although our prophase anthers were separated into two groups, zygotene and pachytene-diplotene, it is highly likely that the zygotene group contained some cells at pachytene because of the diffuseness of the boundary between zygotene and pachytene, and because the zygotene classified anthers were incubated in 3H-thymidine for 4 h after classification. This point, together with the similar buoyant density distribution of the DNAs synthesised in the meiocytes of the zygotene and pachytene-diplotene groups prevents us from concluding that there is any meiotic DNA synthesis peculiar to zygotene in rye as in Lilium [8]. If zygotene-specific DNA synthesis does occur in rye, it must be considerably less than 50% of the amount that occurs in pachytene cells especially if the rate of DNA synthesis at diplotene is less than that at pachytene. In Lilium the zygotene DNA synthesis is three to five times that which occurs at pachytene [8]. Our studies with 2’-deoxyadenosine also point to the absence of extensive DNA synthesis at zygotene. Deoxyadenosine at a concentration which completely inhibited cell development at late zygotene and pachytene did not inhibit meiotic development during most of zygotene (fig. 4). Neither did Exptl
Cell Res 77 (1973)
it prevent chromosome pairing as defined by the very close alignment of matched chromosome threads, nor induce chromosome fragmentation at mid-zygotene (fig. 5) as it does in Lilium [9]. The apparent difference in zygotene DNA synthesis between the cereals and Lilium cannot be easily interpreted at this stage. However, since meiosis in rye (51 h) is considerably shorter than in Lifium (170 h at 20°C) [25] and there is mounting evidence that in cereals chromosome pairing may be organised in a premeiotic phase (M W Bayliss, G A Dover, M D Bennett & R Riley. Personal communication), variation in the process of chromosome pairing is not perhaps too surprising. Our results do not rule out the possibility that in the meiotic division of cereals replication of part of the genome is also delayed since, for example, the semiconservative DNA synthesis necessary for completion of genome replication could occur at some stage other than zygotene. The occurrence of DNA synthesis at pachytene we report here for rye is consistent with similar reports for other organisms, although in all except Lilium, the evidence has come from autoradiography [2-61. It is also consistent with current models of recombination [26-271, the occurrence of crossing over at pachytene and the effects of deoxyadenosine on recombination at pachytene [20]. The recombination models, the biochemical analysis of DNA synthesised at pachytene in Lilium [7, 81 and the enhanced DNA synthesis at this stage in man and mouse in response to irradiation [4, 61 all imply that the late zygotene-pachytene DNA synthesis is concerned with the non-semiconservative repair of chromosome lesions. In the recombination models it is postulated that at late zygotene numerous breaks are introduced into the DNA strands by nuclease action and during their subsequent repair after limited DNA
DNA degradation, hybrid DNA formation may occur between non-sister chromatid DNA strands to lead to a recombinational event. In support of these models, Howell & Stern [28] have recently described the appearance of an endonuclease, a polynucleotide kinase and a polynucleotide ligase activity during zygotene in Lilium. Our observations that deoxyadenosine inhibits cells from late zygotene to pachytene, but not earlier or later, also supports these models and indicates that the only DNA synthesis occurring after leptotene which is essential for meiotic development is confined to the late zygotenepachytene period. The inhibition of meiotic development by deoxyadenosine at these stages also suggests that recombinational repair is essential for meiosis to proceed. However, in other experiments with deoxyadenosine, we have found that it causes meiotic inhibition at a stage of chromosomal condensation characteristic of that at late zygotene or pachytene in an asynaptic mutant of wheat in which neither chromosome pairing nor recombination occur. Thus we cannot rule out the possibility that late prophase DNA synthesis is essential for some meiotic developmental process in addition to recombination. We did detect some DNA synthesis in anthers at metaphase I to telophase II (fig. 3), but deoxyadenosine did not inhibit meiotic development or produce any obvious cytological abnormalities after diplotene. Assuming a recovery of the more fragile columns of meiocytes at metaphase I to telophase II to be about 25 “; that of the zygotene and pachytene-diplotene columns, the amount of 3H-thymidine incorporated into the DNA of post-prophase meiocytes was similar to that incorporated into the DNA of cells of the zygotene group. This level of synthesis relative to that at pachytene is considerably higher than that found in Lilium, (assuming
synthesis during meiosis in wheat and r!‘c
23
that the labelled rye DNA was nuclear. was all from sporogenous tissue and that the level of pachytene DNA synthesis per pg nuclear DNA is not much greater in Lilium than in rye). However, in rye, with its shorter meiotic duration [25] it is conceivable that unless mechanisms exist to degrade quickly the enzymes involved in the breakage. excision and repair processesof recombination, these enzymes would be at a higher concentration in post-prophase cells and could therefore lead to residual breakage and repair in post-prophase stages. Alternatively it is conceivable that all recombination repair is not complete at diakinesis but is completed after metaphase I. Such events as these might be responsible for the site-specific incorporation of 3H into post-metaphase wheat chromosomes reported by Riley & Bennett [l]. It is also possible that the level of thymidine incorporation during metaphase I to telophase II reflects a low level of repair continuing throughout the cell cycle of rye. We have not investigated. but do recognise, the possibility that part of the DNA synthesis measured in this work may be the repair of radiation damage incurred from the relatively high levels of “H-thymidine used.
The chromosome abnormalities induced by deoxyadenosine in the cells which escaped total pachytene arrest are similar to those observed in Lilium [9], viz; abnormal chromosome clumping. fragmentation and ktnaphase bridge formation (fig. 54). It is difficult to interpret all these abnormalities in terms of specific events occurring during late prophase. However, in general, it is not difficult to imagine how chromosomes poasessingnumerous unrepaired single or double strand breaks might well condense abnormally, clump and fragment during the chromosomal changes associated with nuclear division. Exptl
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We wish to thank Professor Ralph Riley for his stimulating interest in this work, Mr Gabriel Dover for valuable experimental help in part of this work and Dr Colin Law for help in the preparation of the manuscript. G. W. R. W. is on sabbatical leave from the Department of Genetics, University of Alberta, Edmonton, Alberta, Canada.
REFERENCES 1. Riley, R & Bennett, M D, Nature 230 (1971) 182. 2. Wimber, D E & Prensky, W, Genetics 48 (1963) 1731. 3. Lima-de-Faria, A, German, J, Ghatneker, M, McGovern, J & Anderson, L, Hereditas 60 (1968) 249. 4. Chandlev, A C & Kofman-Alfaro. _.S. Exntl - cell res 69 (1971) 45. 5. Mukheriee. ” ’ A B & Ghen. , M M. , Nature 219 (1968) . , 489. 6. Kofman-Alfaro, S & Chandley, A C, Exptl cell res 69 (1971) 33. 7. Hotta, Y, Ito, M & Stern, H, Proc natl acad sci US 56 (1966) 1184. 8. Hotta, Y & Stern, H, J mol biol 55 (1971) 337. 9. Ito, M, Hotta, Y & Stern, H, Dev biol 6 (1967) 6. 10. Ito, M & Stem, H, Dev biol 16 (1967) 36. 11. Bennett, M D, Chapman, V & Riley, R, Proc roy sot B 178 (1971) 259.
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12. Bolton, E T, Britten, R J, Cowrie, D B, Roberts, R B, Szafranski, P &Waring, M J, Carnegie inst year book (1964) 314. 13. Marmur, J, J mol biol 3 (1961) 208. 14. Flamm, W G, Burr, H E & Bond, S B, Biochim biophys acta 129 (1966) 310. 15. Dover, G A & Riley, R, Nature (1972). 16. Stern, H & Hotta, Y, Control of nuclear activity (ed L Goldstein) p. 47. Prentice-Hall, Englewood Cliffs, N.J. (1967). 17. Kihlman, B A & Hartley, B, Mut res 4 (1967) 765. 18. Kihlman, B A & Odmark, G, Hereditas 56 (1966) 71. 19. Reichland, P, Canellakis, Z N & Canellakis, E N, J biol them 236 (1961) 2514. 20. Davies, D R & Lawrence, C W, Mut res 4 (1961) 147. 21. Wells, R & Ingle, J, Plant physiol 46 (1970) 178. 22. Perkowska, E, MacGregor, H C & Birnstiel, M L, Nature 217 (1968) 649. 23. Ingle, J, Possingham, J V, Wells R, Leaver, C J & Loening, U E, Symp sot exptl biol 24 (1970) 303. 24. Schildkraut, C L, Marmur, J & Doty, P, J mol biol 4 (1962) 795. 25. Bennett, M D, Proc roy sot London B 178 (1971) 277. 26. Whitehouse, H L K, Nature 199 (1963) 1034. 27. Holliday, R, Genet res 5 (1964) 282. 28. Howell, J & Stern, H, J mol biol 55 (1971) 357. Received June 26, 1972 Revised version received August 30, 1972