- Email: [email protected]
Fine structures of interphase nuclei
FINESTRUCTURESQFINTERPHASENUCLEI 111.Replication Site Anall*sis of DNA During the S Period of Crepis capillaris T. KUROlWA Department
of Biolo,yy,
Fmwlty
o.f Science, Okayama Unirersity,
Oknqama 700, Japan
SUMMARY The replication sites and morphological steps of chromosomal condensation during S period in root tip cells have been studied with light and electron microscopic the nuclei of Crepis cupilluris autoradiography. From light microscopic autoradiographic observations, the S period can be divided with three portions, early S, mid S, and late S period. Labelled nuclei for each portion of the S period have also been found by using electron microscopic autoradiography. With electron microsconic autoradiography it has been found that in early, mid, and late S period, the replication sites’are distributed in the electron transparent regions, interspersed with dense chromatin masses of variable size which are distributed throughout the nucleus. The time-dependent behavior of the label indicates that when compared with either mid or early replicated DNA, a majority of this chromatin, which contains predominantly late replicated DNA, is the earliest chromatin to be organized into the condensed chromatin. They are organized into the condensed chromatin within 15 min after the termination of replication.
All of the nuclear DNA replicated in the dispersed chromatin in S period ([l-5], see Results) is eventually condensed and organized into rod-like condensed metaphase chromosomes of homogeneous density, regardless of when the DNA is replicated [6]. Therefore, there must be some type of regulatory mechanism which leads to the organization of dispersed chromatin into metaphase chromosomes. In a previous publication, we found evidence of asynchronous condensation of prophase chromosomes in Crepis cupilluris: Late replicated DNA condenses faster into the chromonemata-like dense chromatin and further metaphase-like chromosomal segments than chromatin which contains earlier replicated DNA [7, 81. The asynchronous condensation of chromosomes may be one of the reasons why all of the nuclear DNA re-
plicated in dispersed chromatin is organized into the dense metaphase chromosomes in the same manner, regardless of when the DNA is replicated. It is not clear, however, when this asynchronous condensation of chromosomes begins after DNA replication. In order to understand the details of the DNA replication sites and the occurrence of asynchronous chromosomal condensation following the DNA replication, the morphology of the S period nuclei and the behavior of the DNA replicated during different portions of the S period were studied using light and electron microscopic autoradiography.
MATERIAL
AND METHODS
Seedlings. Seeds of Crepis capillavis
were germinated by the method described previously [9]. Seedlings with approx. 4 mm primary roots were used for each experiment. Esptl Cell Res 83 (1974)
388
T. Kuroiw
Fikation and autoradiography .for light microscopic ohsercation. The morphological identification of each
phase of the nuclei during S period was achieved by observing differences in the number and the distribution of the silver grains appearing over the nuclei after 3H-thymidine (3H-TdR) treatment for 10 min. After 2 ,Ki/ml (spec. act. 5 Ci/mmol) 3H-TdR treatment for IO min, the roots were immediately fixed in ethanol&acetic acid, hydrolysed and stained by the usual Feulgen technique. According to the procedures employed previously [9], autoradiographic preparations were prepared. Labelled nuclei showed various patterns which fit into 3 classes(fig. I ). To determine whether or not each of these 3 typical nuclei correspond to specific portions of the S period, the incorporation rate of 3H-TdR in various portions of the S period was studied. The rate of incorporation of 3H-TdR during various portions of the S period was examined by placing in 3H-TdR 2 @i/ml (spec. act. 5 Ci/mmole) for IO min. The roots were removed, rinsed in distilled water, and placed in a solution containing a 100 excess unlabelled TdR. The roots were harvested from 0 to I2 h later at 30 min intervals. Autoradiographs were prepared by the procedures described above. After a 2 week exposure, the mean grains count over the mitotic chromosomes was determined by examining approx 20 roots. The background was negligible. Fixation and autoradiography Jor electron nricroxopic obxewation. In order to determine the minimum in-
cubation period for pulse labelling with 3H-TdR, the percentage of labelled interphase nuclei fixed 2 to 30 min after the beginning of the exposure to 3H-TdR was determined in the root tip cells of Crepis capillarix. The results showed that it took at least 3 to 4 min for exogenous 3H-TdR to be incorporated into the nuclei at root tip cells, Accordingly, in these experiments an incubation time of 6 min was selected as the pulse labelling time. After a 6 min incubation in 3H-TdR 20/Ki/ml (spec. act. 25.3 Ci/mmole) treatment, the roots were removed from the isotopic solution and rinsed in distilled water. One-half of them were fixed immediately and the others were placed in a non-isotopic solution containing a lOO-fold unlabelled TdR and harvested 15 min later. Autoradiographic preparations for electron microscopy were prepared by the procedures previously described [IO]. Exposure time was 2 months. Analysis of incorporation by electron microscopic autoradiography. The distribution of silver grains was
determined for the dense chromatin region and for the electron transparent region by the technique described previously [6]. The distribution of silver grains over the nuclei is relation to the nuclear membrane was estimated by the following method. The peripheral region was delinated by lines drawn corresponding to 0.1 {ml on either side of the nuclear membrane. The number of silver grains over the entire nucleus and over just the inner 0.1 /Cm shell were scored. Peripheral activity was estimated for each nucleus as the ratio of peripheral grains and silver grains over the entire nuclear region. Exptl Ceil Res 83 (1974)
RESULTS Identification of nuclei from each portion of the S period Light micrographs shown in fig. I a-d are S period nuclei fixed immediately after 3H-TdR treatment for 10 min. They are classified into 3 types by morphological differences and the different numbers and distribution patterns of silver grains. The first type (type I) is characterized by small nuclei, and the small number of silver grains roughly scattered over the entire nucleus, with the exception of the dense chromatin masses in certain parts of the nucleus (arrow in fig. 1a). The second type (type II) is characterized by slightly larger nuclei and the large number of silver grains appearing over the entire nucleus (fig. I h). The third type (type III) is characterized by large nuclei and a relatively small number of silver grains which are localized over specific portions of the nuclei (fig. 1 c, d). These results indicate that S period nuclei do not synthesize DNA at a uniform rate throughout S period, and that it is possible to identify three types of nuclei (type I, type I1 and type 111) which correspond to specific portions of the S period. Fig. 2 shows that the average chromosomal grain counts increase steeply up to a peak at 4 h and decline abruptly until the 6 h and any further decrease is slight. Since it has been reported that the duration of G2 and S period is 2.20 and 5.14 h, respectively, in Crepis cupilluris, these results suggest that the highest rate of DNA synthesis takes place in the midportion of the S period. Therefore it seems that type II with many silver grains corresponds to nuclei in mid S period. On the other hand, the remaining two types (I and 111)cannot be distinguished from each other by the number of grains alone since the numbers of silver grains over the nuclei are similar (fig. 1a, c, d). But type
Asynchonous condensation of ckromosomes during S period
389
Fix. I. Light microscopic autoradiographs of nuclei root tip cells fixed immediately after 3H-TdR treatment for
IO min. The nuclei were labelled lightly over less than half of the nuclear area and none appeared over the dense chromatin mass (arrolz) (a), the nuclei were labelled heavily over the nucleus (O), and nuclei with silver grains localized over certain parts of the nucleus (c, d). (a-d) ~7000.
1 nuclei in which DNA replication is just initiated and type III nuclei in which DNA replication is nearly complete can be distinguished morphologically. It has been reported previously [6, 111that the localization of large dense chromatin masses at one side of the nucleus, as seen in type I, is a characteristic feature of nuclei in early S or Gl period, whereas the presence of chromonemata-like masses, distributed over the entire nucleus, as seen in type III, is a characteristic feature of
nuclei in late S or G2. Therefore, type I may be defined as an early S period nucleus, and type III as a late S period nucleus. These results (fig. 1) suggest that these 3 types of nuclei are sequential and representative of early S, mid S and late S period nuclei, respectively. The duration of each type of nuclei was studied. In a normal population consisting of asynchronous cells, the number of each labelled type is proportional to the period Exptl Cell Res 83 (1974)
390
T. Kuroiwa
7Or
TdR treatment for 10 min are, nS -7 I 163; nl -65; nil -839; nIlI -259. By substitution, the duration of the three types of nuclei, type I, II and III, was calculated as 0.29, 3.71 and I. 14 h, respectively.
I
40-
Electron microscopic ohsercutions of replication site und time-dependent hehal~ior of DNA replicated in each portion of S period
30
20.
10 /
L,-!!i 0
2
4
6
8
10
12
Fig. 2. Abscissu: time (hours) after 3H-TdR treatment for 10 min; ordinate: mean grain count per mitotic figure.
length for each of these types. Therefore, it is possible to use an equation analogous to that described by Quastler & Sherman [12] and others [9, 13, 141 to calculate the length of these 3 phases;
ns nl nil nlll ts tl :tlr tIIl
(1)
where the total number of cells in the synthesis period is designated as nS, and those for type 1, II, and 111 are designated as nI, nil, and ~111,respectively. Time spent in each phase is expressed as tS for S period, rl for type I, etc. Values of all the numerators can be estimated by microscopic observation, while the value of the denominator, tS, has been reported previously [9]. Values obtained by scoring S period nuclei of five root tips fixed after “H-
Figs 335 show electron micrographs of type I (fig. 3). type II (fig. 4) and type III nuclei (fig. 5), respectively, fixed immediately (figs 3n, h, 40, h, 5~7,h) and fixed following chase treatment with non-isotopic excess TdR for 15 min (figs 3~, d, 4c, d, 5c, d) after “H-TdR treatment for 6 min. In type I nuclei, the dense chromatin masses of various size are distributed in right (fig. 30) or lower (fig. 3~) half of the nucleoplasm of the nucleus: while in the left (fig. 30) or upper (fig. 3c) half, the small dense chromatin and the electrontransparent region can be seen. The distribution of the chromatin in type I nuclei is similar to that seen in late G 1 period nuclei [6, II, 151. Figs 3 n, c, 40, c, 5 a, c are higher magnification micrographs of a portion of figs 3h, cl. 46, d, 5h, d, respectively. The silver grains in fig. 30 are distributed throughout the nucleus rather than localized in the peripheral region of the nucleus. In addition, almost all of the silver grains are distributed over the electrontransparent regions in the nucleus rather than over the dense chromatin masses (fig. 3~). Similar distribution of the chromatin and silver grains was observed in the nuclei fixed
Fig. 3. Electron microscopic autoradiographs of early S period nuclei fixed immediately after 3H-TdR treatment for 6 min (a, b) or fixed after chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treattttent for 6 min (c, d). (a) :- 26 000; (6) :- 8 500; (c) ,L29 000; (d) ?:9 000. Fig. 4. Electron microscopic autoradiographs of mid S period nuclei fixed immediately after 3H-TdR treatment for 6 min (a, b) or fixed following chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treatment for 6 min (c, d). (a) x 26 500; (b) x 6 700; (c) %26 000; (d) :’ 6 700. Fig. 5. Electron microscopic autoradiographs of late S period nuclei fixed immediately after “H-TdR treatment for 6 min (a, b) or fixed following chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treatment for 6 min (c, d). (a) > 24 000; (b) x 6 900; (c) A:24 000; (d) ,’ 7 000. Exptl Cell Res 83 (1974)
Fig. 3. Exptl Cell Res X3 (1974)
Fig. Exptl
4. Cell Res 83 (1974)
Fig. 5.
Exptl Cell Res 83 (1974)
394
T. Kuroiwa
Table I. Distribution of grains owr the peripheral region (P.re) and the entire nuclear region (N.re) of nuclei during early S (ES), mid S (MS), and late S (LS) period fixed immediatei~ af’ter “H-TdR
treatment
,for 6 min Labelled region
Phase
No. of nuclear cross sections analysed
P.re
N.re
Peripheral activity (” I,)
ES MS LS
12 13 9
2.6 10.5Y (31)” 8.211.15 (114) 2.9+ 1.10 (26)
224.4 + 22.3 (2693) 658.6 t60.0 (8432) 274.1 im28.5 (2472)
1.2 1.2 1.1
’ Mean nutnber of grains with S.D. * Total grains appearing over cross sections analysed.
after chase treatment with excess TdR for 15 min (fig. 3 C, d). Since no silver grains were found localized over the nuclear membrane and the labelling pattern of nuclei fixed after chase treatment for 15 min was very similar to that of nuclei fixed immediately after pulse labelling, it seems likely that initiation sites of DNA synthesis are scattered throughout early S period nuclei and that early replicating DNA is synthesized in the dispersed chromatin which appear as electron-transparent regions (though not in the dense chromatin masses). In type II nuclei, the dense chromatin masses are distributed randomly throughout the entire nucleus (fig. 4a-d). Occasionally, large (fig. 4a, b) and small (fig. 4c, c/) vacuoles are observed in the central region of the nucleolus. The distribution of the chromatin masses and the nuclear size are similar to that of “S period nuclei” described previously
1151. The silver grains in fig. 4a, b are distributed homogeneously over the entire nucleus and no localization of silver grains in the peripheral region of nucleus was seen. Nearly all of the silver grains appear over the electrontransparent regions and the peripheral region of the dense chromatin masses, rather than over the dense chromatin masses as a whole (fig. 4~). This distribution pattern is similar to Exptl Cell Res 83 (1974)
that of nuclei fixed after chase treatment with excess TdR for I5 min, except that an appreciable number of silver grains were observed over the dense chromatin masses of the nuclei (arrows in fig. 4~). These results suggest that DNA replication sites are distributed throughout the nuclei and that the replicating DNA of type II nuclei is synthesized in the dispersed chromatin between the dense chromatin masses. Few of the replicated DNA are organized into the peripheral region of the dense chromatin masses or the dense chromatin masses within 15 min. In type 111 nuclei, the dense chromatin masses are distributed throughout the nucleus and there are large (fig. 5 h) and small (fig. 5 d) vacuoles in a large nucleolus. The distribution pattern of the dense chromatin masses, the nuclear size, and the appearance of the nucleolus, are all similar to that of “G2 period nuclei” [15]. As shown with light microscopic autoradiography (fig. I), most of the silver grains are clustered over certain parts in the nucleus (fig. Saul). Almost all of the silver grains in fig. 5a appeared over the electron-transparent region and borders of condensed chromatin and no specific localization of silver grains in the peripheral region of the nucleus was seen. However, after chase treatment with excess TdR for 15 min the silver grains were localized
Asynchronous condensation sf’ chromosomes during S period
395
Table 2. Mean number of silver grains over the electron transparent region (E.T.re) and the dense chomatin region (D.R.re) of nuclei during earljs S (ES), mid S (MS) and late S (LS) fixed either 0 or 15 min gfter “H-TdR for 6 min
Phase ES MS LS
Time (min) after 3HTdR treatment
No. of nuclear cross sections analysed
Labelled regions E.T.re
D.R.re
0 I5 0 I5 0 15
9 IO 8 8 7 9
219.1 i27.9a (1972)” 285.6 k30.0 (2856) 704.4 k72.6 (5635) 739.4 5 140.4 (5915) 353.0 + 35.3 (2469) 60.9 i- I I .7 (548)
20.9i2.7 (188) 26.8 -t4. I (268) 80.5 k 11.2 (644) 109.4? 16.6 (875) 44.0+8.0 (312) 455.0 k 102.4 (4095)
a Mean number of silver grains per nuclear cross section with S.D. ’ Total grains appearing over the E.R. region or the D.R. region in nuclear cross sections analysed.
over a dense chromatin mass adjacent to the nucleolus and the dense chromatin masses (fig. 5 c, d). These results suggest that near the termination of the S period, DNA is synthesized in certain parts of the dispersed chromatin and that the DNA which is replicated in the dispersed chromatin is organized into the dense chromatin masses for 15 min. In order to examine the correlation, if any, between the nuclear membrane and the replication site and between the replication site and the morphological form of chromatin, the distribution of silver grains appearing over the nuclei was studied by the method described previously [6]. Eukaryotic chromosomal DNA replicates at 0.1 to IL2 (16-18) pm/min; therefore, a labelled precursor incorporated into DNA could be displaced approx. 0.1 Urn away from its site of incorporation after 6 sec. This suggests that if newly replicated DNA is preferentially associated with the nuclear membrane, more than 3.3 % of the total grains appearing over the nucleus after 3H-TdR treatment should be contained within the peripheral 0.1 pm shell adjacent to the nuclear membrane. However, the results shown in table 1 indicate that the peripheral
activity is less than 1.3 ‘,L of the total grains in any portion of the S period. Therefore, it is unlikely that the initiation and replication sites of DNA synthesis are located on the nuclear membrane. Table 2 shows the distribution of the silver grains over the electron-transparent region and the condensed chromatin masses of the nuclei in various portions of S period fixed either after 3H-TdR treatment for 6 min or after chase treatment with excess TdR. There are approx. 10 times more grains over the electron-transparent regions than over the condensed chromatin masses of the nuclei fixed immediately after 3H-TdR treatment, regardless of which portion of S period is examined. However, the distribution of silver grains over the nuclei fixed after chase treatment with excess TdR for I5 min shifts from being localized over the electrontransparent region in early S and becomes localized over the dense chromatin region in late S. These results suggest that DNA synthesis occurs in the dispersed chromatin throughout the S period, and only late replicating DNA is organized rapidly into dense chromatin. Exptl Cell Res 83 (1974)
396
T. Kuroiwa DISCUSSION
Studies on the correlation between the nuclear membrane and the initiation and replication sites of DNA synthesis in many mammalian cells indicate these possible relationships: (I ) The initiation and replication sites of DNA synthesis are definitely on the nuclear membrane [19-211 in a manner analogous to the bacterial mesosome-replicator system; (2) DNA synthesis is initiated at the nuclear membrane and the replication site may be on the membrane, but it migrates from the initiation site on the membrane towards the center of the nuclei [22]; (3) the initiation sites of DNA synthesis are distributed throughout the nucleus [23-271 and the replication sites migrate from the initiation sites toward the periphery of the nucleus [4,23-24). The present results (fig. 3, table 1) support the conclusion that the initiation sites are evenly distributed in the nucleus. The replication sites of DNA synthesis in Crepis capillaris do not fit any of the models outlined above. As indicated in figs 335, the direction of advance of DNA replication is not consistent; the replication sites expand uniformly from each initiation site of DNA synthesis, diverging from the inner area of the nucleus throughout the entire nucleus. However, at the termination of DNA replication these sites are located in portions of the dispersed chromatin area associated with the nucleolus. These data and the results in table 1 make it unlikely that DNA replication occurs on the nuclear membrane. These results concerning DNA replication sites conflict with the first model which is supported by many biochemical studies. The basis for this discrepancy is not clear. However, Hanaoka & Yamada 1191have shown by the use of the M-band fractionation technique that DNA treated with lauryl sarcosinate and then sheared, moves from the M-band to Exptl Cell Res 83 (1974)
the supernatant, and pulse-labelled DNA is more resistant to shearing than the bulk of the DNA. These results were taken as evidence that the replication sites of mammalian DNA are on the membrane. Their results certainly indicate that DNA replication sites are on a portion of the DNA which is associated with a segment of the nuclear membrane. Their results do not indicate, however, that the replication sites correspond to the points at which the chromosomal DNA is attached to the nuclear membrane. On the other hand, ultrastructural studies of nuclei obtained by the whole mounting method have shown that a fragment of the nuclear membrane is associated with many long DNA-like fibrils which extend into the inner area of the nucleus [27]. This may suggest that it is difficult to separate only the shorter DNA fibrils associated with nuclear membrane from the nuclear membrane by physical force. Such biochemical investigations do not provide reliable evidence for the suggestion that initiation and replication sites of DNA synthesis are intimately associated with the membrane. If the attachment of DNA replication sites to the nuclear membrane is common to organisms from bacteria to higher eukaryotic organisms, it should be found in primitive organisms, such as Euplotes and Physarum, which lie evolutionarily between bacteria and higher eukaryotic organisms. It is known that DNA synthesis in Euplotes begins synchronously at the tips of the Cshaped macronucleus and progresses toward the center as two narrow waves of synthesis, and these two waves of synthesis are cytologically visible as ‘replication bands’ [28, 291 Electron microscopic autoradiographs shown by Miller & Stevens [30] and others [3l] indicate that incorporation of “H-TdR is restricted to the rear zone of the replication band but no specific incorporation to a por-
Asynchronous
condensation
tion of the rear zone associated with the nuclear membrane. Accordingly, it is unlikely that there is a specific correlation between DNA replication site and the nuclear membrane. In Physarum polycephalum plasmodia nuclear division is naturally synchronous [32]. The dense chromatin in the S period nucleus forms patch-like masses in the nucleoplasm making the discrimination between dense chromatin masses and the dispersed chromatin very easy. Using plasmodial nuclei and electron microscopic autoradiography, it is possible to accurately localize silver grains over the nuclei, while excluding effects due to artificial synchronizing inhibitors [4, 331. The results obtained with electron microscopic autoradiography support the suggestion that DNA initiation and replication sites are evenly distributed in the nucleus [26]. From the discussion above, it seems that in many eukaryotic organisms DNA replication sites are not restricted to the nuclear membrane but rather to a portion of the DNA which is attached to the nuclear membrane and extends into the inner area of the nucleus. Another important problem related to DNA replication sites is whether DNA synthesis occurs in specific morphological forms of chromatin. Two hypotheses exist concerning DNA synthesis in specific forms of chromatin. (I) DNA synthesis occurs only in dispersed chromatin [l-5, 341; (2) DNA synthesis occurs in both dispersed chromatin and dense chromatin [35-371. It was generally agreed that chromatin containing early replicating DNA is synthesized in a dispersed form. However, there is no agreement concerning the replication form for heterochromatin containing late replicating DNA. The data in figs 3-5 and table 2 support the first hypothesis. Some investigators have shown by light microscopic autoradiography that 3H-TdR,
of chromosomes
during S period
397
at the terminal stages of the DNA synthetic period, seems to be incorporated into condensed chromatin, and from these results, they have proposed that DNA synthesis occurs in condensed chromatin. However, figs I, 3-5, indicate that even when silver grains appear to be homogeneously distributed throughout a nucleus in a light microscopic autoradiograph, the grains are actually localized over dispersed chromatin between the dense chromatin masses. This suggests that the resolution of light microscopy is inadequate to distinguish between silver grains over dense chromatin masses and dispersed chromatin. Another explanation of these contradictory results stems from the velocity of the organization of DNA after replication. It has been shown (fig. 5) that late replicating DNA as well as mid- and late-replication DNA is synthesized in dispersed chromatin between the dense chromatin masses. For a few min after replication this DNA is located in the peripheral region of chromonemata-like dense chromatin masses, and within 15 min this DNA is organized into large dense chromatin masses. These steps of condensation of late replicating DNA are much faster than the early and mid S period replication of DNA (table 2). It has been demonstrated in Crepis capillaris [9] as well as in many higher organisms 1381 that late replicating DNA is contained in heterochromatin. These data suggest that decondensation, replication and recondensation within the heterochromatin during the 6 min pulse and heterochromatin mass results from the rapid condensation of DNA synthesized in dispersed chromatin regions. Thus it is possible that silver grains localized over heterochromatin actually represent late replicating DNA synthesized in dispersed chromatin and rapidly organized into dense chromatin masses. Exptl Cell Res 83 (1974)
398 T. Kuroiwa These results and previous work [S] with high resolution electron microscopic autoradiography suggest that all of the DNA in the nucleus is replicated in the dispersed chromatin regardless of when the DNA is replicated. It is then organized into rod-like condensed chromosomes. These results do not conflict with the results indicating that late replicated DNA condenses faster during prophase into the chromonemata-like masses than chromatin which contains earlier replicated DNA, since this asynchronous condensation of the chromosomal segments occurs just after DNA replication and continues until late prophase. These observations may be very important in understanding the organisation of eukaryotic chromosomes. The author thanks .I. E. Sherwin, Ph. D. (Division of Biological and Medical Research, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, III. 60439, USA) for his invaluable suggestions and critical reading of the manuscript.
REFERENCES I. Hay, E D & Revel, J P. J cell biol 16 (1963) 29. 2. Meek, G A & Moses, M J, J roy microscop sot 81 (1963) 187. 3. Mimer, G R & Hayoe, F G, Nature 218 (1968) 785. 4. Blonde], B, Exptl cell res 53 (1968) 348. 5. Mimer, G R. J cell sci 4 (1969) 569. 6. Kuroiwa, T &Tanaka, N, Exptl cell res 65 (1971) 177. 7. - J cell biol 49 (1971) 939. 8. Kuroiwa, T, Exptl cell res 69 (1971) 97. 9. Kuroiwa, T & Tanaka, N, Cytologia (Tokyo) 35 (1970) 239.
Exptl Cell Res 83 (1974)
IO. Kuroiwa, T, Exptl cell res 88 (1973) 240. I I. Kuroiwa, T & Tanaka, N, J electron microscopy I9 (1970) 63. 12. Quastler, H & Sherman, F G, Exptl cell res I7 (1959) 420. 13. Wimber, D E, Am j bot 47 (1960) 828 14. Sparvoli, E, Gay, H & Kaufmann, B P, Caryologia 19 (1966) 65. 15. Kuroiwa, T & Tanaka, N, Cytologia (Tokyo) 36 (1971) 143. 16. Okada. S. Bionhvs i 8 (1968) 598. 17. Cair 1s: J,‘J mbl bidl I5 (1966) 372. 18. Taylor, J H, J mol biol 31 (1968) 579. 19. Hanaoka, F & Yamada, M, Biochem biophys res commun 42 (1971) 647. 20. Mizuno, N S, Stoops, C E & Peiffer, R L, J mol biol 59 (1971) 517. 21. O’Brien, R L, Sanyal, A B & Stanton, R H, Exptl cell res 70 (1972) 106. 22. Comings, D E & Kakefuda, T, J mol biol 33 (I 968) 225. 23. Williams, C A & Ockey, C H, Exptl cell res 63 (1970) 365. 24. Erlandson, R A & Harven, E, J ceil sci 8 (1971) 353. 25. Fakan, S, Turner, G N, Pagano, J S & Hancock. R, Proc natl acad sci US 69 (1972) 2300. 26. Kuroiwa, T, Chromosoma (Berl.). In press 27. DuPraw, E J, Proc natl acad sci US 53 (I 965) 161. 28. Gall, J G, J biophys biochem cytol 5 (1959) 295. 29. Prescott, D M, J histochem cytochem 10 (1962) 145. 30. Miller, D L & Stevens, A R, in Methods in cell physiology (ed D M Prescott) vol. 2, p. 267. Academic Press, New York (1966). 31. Evenson, D P & Prescott, D M, Exptl cell res 63 (1970) 245. 32. Haward, FL,Amj bot lS(l931) 116. 33. Ockey, C H, Exptl cell res 70 (1972) 203. 34. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc natl acad sci US 52 (1963) 93. 35. Comings, D E, Cytogenetics 6 (1967) 20. 36. - Ibid 6 (1967) 120. 37. Klinger, H P, Schwarzacher, H G & Weiss, J, Cytogenetics 6 (1967) I. 38. Lima-de-Faria, A, Nature 217 (1968) 138. Received June 28, 1973 Revised version received August 26, 1973
of Biolo,yy,
Fmwlty
o.f Science, Okayama Unirersity,
Oknqama 700, Japan
SUMMARY The replication sites and morphological steps of chromosomal condensation during S period in root tip cells have been studied with light and electron microscopic the nuclei of Crepis cupilluris autoradiography. From light microscopic autoradiographic observations, the S period can be divided with three portions, early S, mid S, and late S period. Labelled nuclei for each portion of the S period have also been found by using electron microscopic autoradiography. With electron microsconic autoradiography it has been found that in early, mid, and late S period, the replication sites’are distributed in the electron transparent regions, interspersed with dense chromatin masses of variable size which are distributed throughout the nucleus. The time-dependent behavior of the label indicates that when compared with either mid or early replicated DNA, a majority of this chromatin, which contains predominantly late replicated DNA, is the earliest chromatin to be organized into the condensed chromatin. They are organized into the condensed chromatin within 15 min after the termination of replication.
All of the nuclear DNA replicated in the dispersed chromatin in S period ([l-5], see Results) is eventually condensed and organized into rod-like condensed metaphase chromosomes of homogeneous density, regardless of when the DNA is replicated [6]. Therefore, there must be some type of regulatory mechanism which leads to the organization of dispersed chromatin into metaphase chromosomes. In a previous publication, we found evidence of asynchronous condensation of prophase chromosomes in Crepis cupilluris: Late replicated DNA condenses faster into the chromonemata-like dense chromatin and further metaphase-like chromosomal segments than chromatin which contains earlier replicated DNA [7, 81. The asynchronous condensation of chromosomes may be one of the reasons why all of the nuclear DNA re-
plicated in dispersed chromatin is organized into the dense metaphase chromosomes in the same manner, regardless of when the DNA is replicated. It is not clear, however, when this asynchronous condensation of chromosomes begins after DNA replication. In order to understand the details of the DNA replication sites and the occurrence of asynchronous chromosomal condensation following the DNA replication, the morphology of the S period nuclei and the behavior of the DNA replicated during different portions of the S period were studied using light and electron microscopic autoradiography.
MATERIAL
AND METHODS
Seedlings. Seeds of Crepis capillavis
were germinated by the method described previously [9]. Seedlings with approx. 4 mm primary roots were used for each experiment. Esptl Cell Res 83 (1974)
388
T. Kuroiw
Fikation and autoradiography .for light microscopic ohsercation. The morphological identification of each
phase of the nuclei during S period was achieved by observing differences in the number and the distribution of the silver grains appearing over the nuclei after 3H-thymidine (3H-TdR) treatment for 10 min. After 2 ,Ki/ml (spec. act. 5 Ci/mmol) 3H-TdR treatment for IO min, the roots were immediately fixed in ethanol&acetic acid, hydrolysed and stained by the usual Feulgen technique. According to the procedures employed previously [9], autoradiographic preparations were prepared. Labelled nuclei showed various patterns which fit into 3 classes(fig. I ). To determine whether or not each of these 3 typical nuclei correspond to specific portions of the S period, the incorporation rate of 3H-TdR in various portions of the S period was studied. The rate of incorporation of 3H-TdR during various portions of the S period was examined by placing in 3H-TdR 2 @i/ml (spec. act. 5 Ci/mmole) for IO min. The roots were removed, rinsed in distilled water, and placed in a solution containing a 100 excess unlabelled TdR. The roots were harvested from 0 to I2 h later at 30 min intervals. Autoradiographs were prepared by the procedures described above. After a 2 week exposure, the mean grains count over the mitotic chromosomes was determined by examining approx 20 roots. The background was negligible. Fixation and autoradiography Jor electron nricroxopic obxewation. In order to determine the minimum in-
cubation period for pulse labelling with 3H-TdR, the percentage of labelled interphase nuclei fixed 2 to 30 min after the beginning of the exposure to 3H-TdR was determined in the root tip cells of Crepis capillarix. The results showed that it took at least 3 to 4 min for exogenous 3H-TdR to be incorporated into the nuclei at root tip cells, Accordingly, in these experiments an incubation time of 6 min was selected as the pulse labelling time. After a 6 min incubation in 3H-TdR 20/Ki/ml (spec. act. 25.3 Ci/mmole) treatment, the roots were removed from the isotopic solution and rinsed in distilled water. One-half of them were fixed immediately and the others were placed in a non-isotopic solution containing a lOO-fold unlabelled TdR and harvested 15 min later. Autoradiographic preparations for electron microscopy were prepared by the procedures previously described [IO]. Exposure time was 2 months. Analysis of incorporation by electron microscopic autoradiography. The distribution of silver grains was
determined for the dense chromatin region and for the electron transparent region by the technique described previously [6]. The distribution of silver grains over the nuclei is relation to the nuclear membrane was estimated by the following method. The peripheral region was delinated by lines drawn corresponding to 0.1 {ml on either side of the nuclear membrane. The number of silver grains over the entire nucleus and over just the inner 0.1 /Cm shell were scored. Peripheral activity was estimated for each nucleus as the ratio of peripheral grains and silver grains over the entire nuclear region. Exptl Ceil Res 83 (1974)
RESULTS Identification of nuclei from each portion of the S period Light micrographs shown in fig. I a-d are S period nuclei fixed immediately after 3H-TdR treatment for 10 min. They are classified into 3 types by morphological differences and the different numbers and distribution patterns of silver grains. The first type (type I) is characterized by small nuclei, and the small number of silver grains roughly scattered over the entire nucleus, with the exception of the dense chromatin masses in certain parts of the nucleus (arrow in fig. 1a). The second type (type II) is characterized by slightly larger nuclei and the large number of silver grains appearing over the entire nucleus (fig. I h). The third type (type III) is characterized by large nuclei and a relatively small number of silver grains which are localized over specific portions of the nuclei (fig. 1 c, d). These results indicate that S period nuclei do not synthesize DNA at a uniform rate throughout S period, and that it is possible to identify three types of nuclei (type I, type I1 and type 111) which correspond to specific portions of the S period. Fig. 2 shows that the average chromosomal grain counts increase steeply up to a peak at 4 h and decline abruptly until the 6 h and any further decrease is slight. Since it has been reported that the duration of G2 and S period is 2.20 and 5.14 h, respectively, in Crepis cupilluris, these results suggest that the highest rate of DNA synthesis takes place in the midportion of the S period. Therefore it seems that type II with many silver grains corresponds to nuclei in mid S period. On the other hand, the remaining two types (I and 111)cannot be distinguished from each other by the number of grains alone since the numbers of silver grains over the nuclei are similar (fig. 1a, c, d). But type
Asynchonous condensation of ckromosomes during S period
389
Fix. I. Light microscopic autoradiographs of nuclei root tip cells fixed immediately after 3H-TdR treatment for
IO min. The nuclei were labelled lightly over less than half of the nuclear area and none appeared over the dense chromatin mass (arrolz) (a), the nuclei were labelled heavily over the nucleus (O), and nuclei with silver grains localized over certain parts of the nucleus (c, d). (a-d) ~7000.
1 nuclei in which DNA replication is just initiated and type III nuclei in which DNA replication is nearly complete can be distinguished morphologically. It has been reported previously [6, 111that the localization of large dense chromatin masses at one side of the nucleus, as seen in type I, is a characteristic feature of nuclei in early S or Gl period, whereas the presence of chromonemata-like masses, distributed over the entire nucleus, as seen in type III, is a characteristic feature of
nuclei in late S or G2. Therefore, type I may be defined as an early S period nucleus, and type III as a late S period nucleus. These results (fig. 1) suggest that these 3 types of nuclei are sequential and representative of early S, mid S and late S period nuclei, respectively. The duration of each type of nuclei was studied. In a normal population consisting of asynchronous cells, the number of each labelled type is proportional to the period Exptl Cell Res 83 (1974)
390
T. Kuroiwa
7Or
TdR treatment for 10 min are, nS -7 I 163; nl -65; nil -839; nIlI -259. By substitution, the duration of the three types of nuclei, type I, II and III, was calculated as 0.29, 3.71 and I. 14 h, respectively.
I
40-
Electron microscopic ohsercutions of replication site und time-dependent hehal~ior of DNA replicated in each portion of S period
30
20.
10 /
L,-!!i 0
2
4
6
8
10
12
Fig. 2. Abscissu: time (hours) after 3H-TdR treatment for 10 min; ordinate: mean grain count per mitotic figure.
length for each of these types. Therefore, it is possible to use an equation analogous to that described by Quastler & Sherman [12] and others [9, 13, 141 to calculate the length of these 3 phases;
ns nl nil nlll ts tl :tlr tIIl
(1)
where the total number of cells in the synthesis period is designated as nS, and those for type 1, II, and 111 are designated as nI, nil, and ~111,respectively. Time spent in each phase is expressed as tS for S period, rl for type I, etc. Values of all the numerators can be estimated by microscopic observation, while the value of the denominator, tS, has been reported previously [9]. Values obtained by scoring S period nuclei of five root tips fixed after “H-
Figs 335 show electron micrographs of type I (fig. 3). type II (fig. 4) and type III nuclei (fig. 5), respectively, fixed immediately (figs 3n, h, 40, h, 5~7,h) and fixed following chase treatment with non-isotopic excess TdR for 15 min (figs 3~, d, 4c, d, 5c, d) after “H-TdR treatment for 6 min. In type I nuclei, the dense chromatin masses of various size are distributed in right (fig. 30) or lower (fig. 3~) half of the nucleoplasm of the nucleus: while in the left (fig. 30) or upper (fig. 3c) half, the small dense chromatin and the electrontransparent region can be seen. The distribution of the chromatin in type I nuclei is similar to that seen in late G 1 period nuclei [6, II, 151. Figs 3 n, c, 40, c, 5 a, c are higher magnification micrographs of a portion of figs 3h, cl. 46, d, 5h, d, respectively. The silver grains in fig. 30 are distributed throughout the nucleus rather than localized in the peripheral region of the nucleus. In addition, almost all of the silver grains are distributed over the electrontransparent regions in the nucleus rather than over the dense chromatin masses (fig. 3~). Similar distribution of the chromatin and silver grains was observed in the nuclei fixed
Fig. 3. Electron microscopic autoradiographs of early S period nuclei fixed immediately after 3H-TdR treatment for 6 min (a, b) or fixed after chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treattttent for 6 min (c, d). (a) :- 26 000; (6) :- 8 500; (c) ,L29 000; (d) ?:9 000. Fig. 4. Electron microscopic autoradiographs of mid S period nuclei fixed immediately after 3H-TdR treatment for 6 min (a, b) or fixed following chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treatment for 6 min (c, d). (a) x 26 500; (b) x 6 700; (c) %26 000; (d) :’ 6 700. Fig. 5. Electron microscopic autoradiographs of late S period nuclei fixed immediately after “H-TdR treatment for 6 min (a, b) or fixed following chase treatment with excess non-isotopic TdR for 15 min after 3H-TdR treatment for 6 min (c, d). (a) > 24 000; (b) x 6 900; (c) A:24 000; (d) ,’ 7 000. Exptl Cell Res 83 (1974)
Fig. 3. Exptl Cell Res X3 (1974)
Fig. Exptl
4. Cell Res 83 (1974)
Fig. 5.
Exptl Cell Res 83 (1974)
394
T. Kuroiwa
Table I. Distribution of grains owr the peripheral region (P.re) and the entire nuclear region (N.re) of nuclei during early S (ES), mid S (MS), and late S (LS) period fixed immediatei~ af’ter “H-TdR
treatment
,for 6 min Labelled region
Phase
No. of nuclear cross sections analysed
P.re
N.re
Peripheral activity (” I,)
ES MS LS
12 13 9
2.6 10.5Y (31)” 8.211.15 (114) 2.9+ 1.10 (26)
224.4 + 22.3 (2693) 658.6 t60.0 (8432) 274.1 im28.5 (2472)
1.2 1.2 1.1
’ Mean nutnber of grains with S.D. * Total grains appearing over cross sections analysed.
after chase treatment with excess TdR for 15 min (fig. 3 C, d). Since no silver grains were found localized over the nuclear membrane and the labelling pattern of nuclei fixed after chase treatment for 15 min was very similar to that of nuclei fixed immediately after pulse labelling, it seems likely that initiation sites of DNA synthesis are scattered throughout early S period nuclei and that early replicating DNA is synthesized in the dispersed chromatin which appear as electron-transparent regions (though not in the dense chromatin masses). In type II nuclei, the dense chromatin masses are distributed randomly throughout the entire nucleus (fig. 4a-d). Occasionally, large (fig. 4a, b) and small (fig. 4c, c/) vacuoles are observed in the central region of the nucleolus. The distribution of the chromatin masses and the nuclear size are similar to that of “S period nuclei” described previously
1151. The silver grains in fig. 4a, b are distributed homogeneously over the entire nucleus and no localization of silver grains in the peripheral region of nucleus was seen. Nearly all of the silver grains appear over the electrontransparent regions and the peripheral region of the dense chromatin masses, rather than over the dense chromatin masses as a whole (fig. 4~). This distribution pattern is similar to Exptl Cell Res 83 (1974)
that of nuclei fixed after chase treatment with excess TdR for I5 min, except that an appreciable number of silver grains were observed over the dense chromatin masses of the nuclei (arrows in fig. 4~). These results suggest that DNA replication sites are distributed throughout the nuclei and that the replicating DNA of type II nuclei is synthesized in the dispersed chromatin between the dense chromatin masses. Few of the replicated DNA are organized into the peripheral region of the dense chromatin masses or the dense chromatin masses within 15 min. In type 111 nuclei, the dense chromatin masses are distributed throughout the nucleus and there are large (fig. 5 h) and small (fig. 5 d) vacuoles in a large nucleolus. The distribution pattern of the dense chromatin masses, the nuclear size, and the appearance of the nucleolus, are all similar to that of “G2 period nuclei” [15]. As shown with light microscopic autoradiography (fig. I), most of the silver grains are clustered over certain parts in the nucleus (fig. Saul). Almost all of the silver grains in fig. 5a appeared over the electron-transparent region and borders of condensed chromatin and no specific localization of silver grains in the peripheral region of the nucleus was seen. However, after chase treatment with excess TdR for 15 min the silver grains were localized
Asynchronous condensation sf’ chromosomes during S period
395
Table 2. Mean number of silver grains over the electron transparent region (E.T.re) and the dense chomatin region (D.R.re) of nuclei during earljs S (ES), mid S (MS) and late S (LS) fixed either 0 or 15 min gfter “H-TdR for 6 min
Phase ES MS LS
Time (min) after 3HTdR treatment
No. of nuclear cross sections analysed
Labelled regions E.T.re
D.R.re
0 I5 0 I5 0 15
9 IO 8 8 7 9
219.1 i27.9a (1972)” 285.6 k30.0 (2856) 704.4 k72.6 (5635) 739.4 5 140.4 (5915) 353.0 + 35.3 (2469) 60.9 i- I I .7 (548)
20.9i2.7 (188) 26.8 -t4. I (268) 80.5 k 11.2 (644) 109.4? 16.6 (875) 44.0+8.0 (312) 455.0 k 102.4 (4095)
a Mean number of silver grains per nuclear cross section with S.D. ’ Total grains appearing over the E.R. region or the D.R. region in nuclear cross sections analysed.
over a dense chromatin mass adjacent to the nucleolus and the dense chromatin masses (fig. 5 c, d). These results suggest that near the termination of the S period, DNA is synthesized in certain parts of the dispersed chromatin and that the DNA which is replicated in the dispersed chromatin is organized into the dense chromatin masses for 15 min. In order to examine the correlation, if any, between the nuclear membrane and the replication site and between the replication site and the morphological form of chromatin, the distribution of silver grains appearing over the nuclei was studied by the method described previously [6]. Eukaryotic chromosomal DNA replicates at 0.1 to IL2 (16-18) pm/min; therefore, a labelled precursor incorporated into DNA could be displaced approx. 0.1 Urn away from its site of incorporation after 6 sec. This suggests that if newly replicated DNA is preferentially associated with the nuclear membrane, more than 3.3 % of the total grains appearing over the nucleus after 3H-TdR treatment should be contained within the peripheral 0.1 pm shell adjacent to the nuclear membrane. However, the results shown in table 1 indicate that the peripheral
activity is less than 1.3 ‘,L of the total grains in any portion of the S period. Therefore, it is unlikely that the initiation and replication sites of DNA synthesis are located on the nuclear membrane. Table 2 shows the distribution of the silver grains over the electron-transparent region and the condensed chromatin masses of the nuclei in various portions of S period fixed either after 3H-TdR treatment for 6 min or after chase treatment with excess TdR. There are approx. 10 times more grains over the electron-transparent regions than over the condensed chromatin masses of the nuclei fixed immediately after 3H-TdR treatment, regardless of which portion of S period is examined. However, the distribution of silver grains over the nuclei fixed after chase treatment with excess TdR for I5 min shifts from being localized over the electrontransparent region in early S and becomes localized over the dense chromatin region in late S. These results suggest that DNA synthesis occurs in the dispersed chromatin throughout the S period, and only late replicating DNA is organized rapidly into dense chromatin. Exptl Cell Res 83 (1974)
396
T. Kuroiwa DISCUSSION
Studies on the correlation between the nuclear membrane and the initiation and replication sites of DNA synthesis in many mammalian cells indicate these possible relationships: (I ) The initiation and replication sites of DNA synthesis are definitely on the nuclear membrane [19-211 in a manner analogous to the bacterial mesosome-replicator system; (2) DNA synthesis is initiated at the nuclear membrane and the replication site may be on the membrane, but it migrates from the initiation site on the membrane towards the center of the nuclei [22]; (3) the initiation sites of DNA synthesis are distributed throughout the nucleus [23-271 and the replication sites migrate from the initiation sites toward the periphery of the nucleus [4,23-24). The present results (fig. 3, table 1) support the conclusion that the initiation sites are evenly distributed in the nucleus. The replication sites of DNA synthesis in Crepis capillaris do not fit any of the models outlined above. As indicated in figs 335, the direction of advance of DNA replication is not consistent; the replication sites expand uniformly from each initiation site of DNA synthesis, diverging from the inner area of the nucleus throughout the entire nucleus. However, at the termination of DNA replication these sites are located in portions of the dispersed chromatin area associated with the nucleolus. These data and the results in table 1 make it unlikely that DNA replication occurs on the nuclear membrane. These results concerning DNA replication sites conflict with the first model which is supported by many biochemical studies. The basis for this discrepancy is not clear. However, Hanaoka & Yamada 1191have shown by the use of the M-band fractionation technique that DNA treated with lauryl sarcosinate and then sheared, moves from the M-band to Exptl Cell Res 83 (1974)
the supernatant, and pulse-labelled DNA is more resistant to shearing than the bulk of the DNA. These results were taken as evidence that the replication sites of mammalian DNA are on the membrane. Their results certainly indicate that DNA replication sites are on a portion of the DNA which is associated with a segment of the nuclear membrane. Their results do not indicate, however, that the replication sites correspond to the points at which the chromosomal DNA is attached to the nuclear membrane. On the other hand, ultrastructural studies of nuclei obtained by the whole mounting method have shown that a fragment of the nuclear membrane is associated with many long DNA-like fibrils which extend into the inner area of the nucleus [27]. This may suggest that it is difficult to separate only the shorter DNA fibrils associated with nuclear membrane from the nuclear membrane by physical force. Such biochemical investigations do not provide reliable evidence for the suggestion that initiation and replication sites of DNA synthesis are intimately associated with the membrane. If the attachment of DNA replication sites to the nuclear membrane is common to organisms from bacteria to higher eukaryotic organisms, it should be found in primitive organisms, such as Euplotes and Physarum, which lie evolutionarily between bacteria and higher eukaryotic organisms. It is known that DNA synthesis in Euplotes begins synchronously at the tips of the Cshaped macronucleus and progresses toward the center as two narrow waves of synthesis, and these two waves of synthesis are cytologically visible as ‘replication bands’ [28, 291 Electron microscopic autoradiographs shown by Miller & Stevens [30] and others [3l] indicate that incorporation of “H-TdR is restricted to the rear zone of the replication band but no specific incorporation to a por-
Asynchronous
condensation
tion of the rear zone associated with the nuclear membrane. Accordingly, it is unlikely that there is a specific correlation between DNA replication site and the nuclear membrane. In Physarum polycephalum plasmodia nuclear division is naturally synchronous [32]. The dense chromatin in the S period nucleus forms patch-like masses in the nucleoplasm making the discrimination between dense chromatin masses and the dispersed chromatin very easy. Using plasmodial nuclei and electron microscopic autoradiography, it is possible to accurately localize silver grains over the nuclei, while excluding effects due to artificial synchronizing inhibitors [4, 331. The results obtained with electron microscopic autoradiography support the suggestion that DNA initiation and replication sites are evenly distributed in the nucleus [26]. From the discussion above, it seems that in many eukaryotic organisms DNA replication sites are not restricted to the nuclear membrane but rather to a portion of the DNA which is attached to the nuclear membrane and extends into the inner area of the nucleus. Another important problem related to DNA replication sites is whether DNA synthesis occurs in specific morphological forms of chromatin. Two hypotheses exist concerning DNA synthesis in specific forms of chromatin. (I) DNA synthesis occurs only in dispersed chromatin [l-5, 341; (2) DNA synthesis occurs in both dispersed chromatin and dense chromatin [35-371. It was generally agreed that chromatin containing early replicating DNA is synthesized in a dispersed form. However, there is no agreement concerning the replication form for heterochromatin containing late replicating DNA. The data in figs 3-5 and table 2 support the first hypothesis. Some investigators have shown by light microscopic autoradiography that 3H-TdR,
of chromosomes
during S period
397
at the terminal stages of the DNA synthetic period, seems to be incorporated into condensed chromatin, and from these results, they have proposed that DNA synthesis occurs in condensed chromatin. However, figs I, 3-5, indicate that even when silver grains appear to be homogeneously distributed throughout a nucleus in a light microscopic autoradiograph, the grains are actually localized over dispersed chromatin between the dense chromatin masses. This suggests that the resolution of light microscopy is inadequate to distinguish between silver grains over dense chromatin masses and dispersed chromatin. Another explanation of these contradictory results stems from the velocity of the organization of DNA after replication. It has been shown (fig. 5) that late replicating DNA as well as mid- and late-replication DNA is synthesized in dispersed chromatin between the dense chromatin masses. For a few min after replication this DNA is located in the peripheral region of chromonemata-like dense chromatin masses, and within 15 min this DNA is organized into large dense chromatin masses. These steps of condensation of late replicating DNA are much faster than the early and mid S period replication of DNA (table 2). It has been demonstrated in Crepis capillaris [9] as well as in many higher organisms 1381 that late replicating DNA is contained in heterochromatin. These data suggest that decondensation, replication and recondensation within the heterochromatin during the 6 min pulse and heterochromatin mass results from the rapid condensation of DNA synthesized in dispersed chromatin regions. Thus it is possible that silver grains localized over heterochromatin actually represent late replicating DNA synthesized in dispersed chromatin and rapidly organized into dense chromatin masses. Exptl Cell Res 83 (1974)
398 T. Kuroiwa These results and previous work [S] with high resolution electron microscopic autoradiography suggest that all of the DNA in the nucleus is replicated in the dispersed chromatin regardless of when the DNA is replicated. It is then organized into rod-like condensed chromosomes. These results do not conflict with the results indicating that late replicated DNA condenses faster during prophase into the chromonemata-like masses than chromatin which contains earlier replicated DNA, since this asynchronous condensation of the chromosomal segments occurs just after DNA replication and continues until late prophase. These observations may be very important in understanding the organisation of eukaryotic chromosomes. The author thanks .I. E. Sherwin, Ph. D. (Division of Biological and Medical Research, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, III. 60439, USA) for his invaluable suggestions and critical reading of the manuscript.
REFERENCES I. Hay, E D & Revel, J P. J cell biol 16 (1963) 29. 2. Meek, G A & Moses, M J, J roy microscop sot 81 (1963) 187. 3. Mimer, G R & Hayoe, F G, Nature 218 (1968) 785. 4. Blonde], B, Exptl cell res 53 (1968) 348. 5. Mimer, G R. J cell sci 4 (1969) 569. 6. Kuroiwa, T &Tanaka, N, Exptl cell res 65 (1971) 177. 7. - J cell biol 49 (1971) 939. 8. Kuroiwa, T, Exptl cell res 69 (1971) 97. 9. Kuroiwa, T & Tanaka, N, Cytologia (Tokyo) 35 (1970) 239.
Exptl Cell Res 83 (1974)
IO. Kuroiwa, T, Exptl cell res 88 (1973) 240. I I. Kuroiwa, T & Tanaka, N, J electron microscopy I9 (1970) 63. 12. Quastler, H & Sherman, F G, Exptl cell res I7 (1959) 420. 13. Wimber, D E, Am j bot 47 (1960) 828 14. Sparvoli, E, Gay, H & Kaufmann, B P, Caryologia 19 (1966) 65. 15. Kuroiwa, T & Tanaka, N, Cytologia (Tokyo) 36 (1971) 143. 16. Okada. S. Bionhvs i 8 (1968) 598. 17. Cair 1s: J,‘J mbl bidl I5 (1966) 372. 18. Taylor, J H, J mol biol 31 (1968) 579. 19. Hanaoka, F & Yamada, M, Biochem biophys res commun 42 (1971) 647. 20. Mizuno, N S, Stoops, C E & Peiffer, R L, J mol biol 59 (1971) 517. 21. O’Brien, R L, Sanyal, A B & Stanton, R H, Exptl cell res 70 (1972) 106. 22. Comings, D E & Kakefuda, T, J mol biol 33 (I 968) 225. 23. Williams, C A & Ockey, C H, Exptl cell res 63 (1970) 365. 24. Erlandson, R A & Harven, E, J ceil sci 8 (1971) 353. 25. Fakan, S, Turner, G N, Pagano, J S & Hancock. R, Proc natl acad sci US 69 (1972) 2300. 26. Kuroiwa, T, Chromosoma (Berl.). In press 27. DuPraw, E J, Proc natl acad sci US 53 (I 965) 161. 28. Gall, J G, J biophys biochem cytol 5 (1959) 295. 29. Prescott, D M, J histochem cytochem 10 (1962) 145. 30. Miller, D L & Stevens, A R, in Methods in cell physiology (ed D M Prescott) vol. 2, p. 267. Academic Press, New York (1966). 31. Evenson, D P & Prescott, D M, Exptl cell res 63 (1970) 245. 32. Haward, FL,Amj bot lS(l931) 116. 33. Ockey, C H, Exptl cell res 70 (1972) 203. 34. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc natl acad sci US 52 (1963) 93. 35. Comings, D E, Cytogenetics 6 (1967) 20. 36. - Ibid 6 (1967) 120. 37. Klinger, H P, Schwarzacher, H G & Weiss, J, Cytogenetics 6 (1967) I. 38. Lima-de-Faria, A, Nature 217 (1968) 138. Received June 28, 1973 Revised version received August 26, 1973