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Uptake of 3H-thymidine and patterns of DNA replication in nuclei and chromosomes of Vicia faba
Experimental
Cell Research 35, 381-393
UPTAKE
381
(1961)
OF 3H-THYMIDINE
REPLICATION
AND PATTERNS
OF DNA
IN NUCLEI AND CHROMOSOMES OF V1CIA FABA H. J. EVANS
Medical Research Council, Radiobiological Research
Unit, Harwcll, Berks.,
England
Received August 16, 1963
SINCEth e
initial demonstration that thymidine is a highly specific precursor of Dr\TA [lo, 341, considerable use has been made of radioactively labelled thymidine in studying various aspects of DSA synthesis. The compound, especially when labelled with tritium [40], is particularly useful and is widely used in cytological experiments designed to study aspects of Di\lA synthesis, chromosome duplication, and as a marker in studying kinetics of cellular proliferation. Although a considerable number of autoradiographic studies using root-tip tissues exposed to 3H-thymidine have been carried out, there is little available information on the rate of penetration and incorporation of thymidine into the l>Klz of root-tip cells, and no published information on the influence of post-treatment with high concentrations of inactive thymidinc-a procedure which is routinely carried out in a number of laboratories. The present paper reports the results of two experiments carried out on Vicin faba root-tip cells to investigate (i) the rate of uptake of 3H-thymidinc into
DSA
fluence
as measured
from
of post-treatments
grain
count
with inactive
determinations,
thymidinc
and (ii)
the in-
on grain count and on the
proportion of meristem cells which are labelled. Information having a bearing on the relative rates of uptake of 3H-thymidine into DNA at different stages of development
of the synthesis
some. observations and a discussion metaphase chromosomes.
MATERIALS
phase is also presented, on the patterns
AND
together
of labelling
with
found
in
METHODS
EXPERIMENT l.-Fifty primary roots of Vicia faba seedlings, cultured by our usual procedures [7], were exposed to 100 ml of an aerated solution of 3H-thymidine (s.a. 3.0 c/m&f), at a concentration of 2 PC/ml, for various time intervals up to 30 min. The roots were fixed in a modified Flemmings fluid immediately on removal from the Experimental
Cell Research 3.5
H. J. Evans 3H-thymidine solution and hydrolysed for 10 min in N HCl at 60°C. Cytological preparations were made from the terminal 2 mm of root-tip as described previously [5] and autoradiographs were prepared with Kodak AR 10 stripping film, the slides being exposed for 3 weeks at 4°C and developed in Kodak Dl9b. EXPERIMENT 2.-Fifty roots were exposed to 3H-thymidine, as described above, for a period of 30 min. On completion of the 3H-thymidine treatment all roots were quickly rinsed in water and half were transferred to a solution of “cold” thymidine made up to 50 x the concentration of the active solution, i.e. to 8 pg/ml, and the remaining roots were transferred to water. Fixations were made at intervals of up to 1 hr after completion of 3H-thymidine treatment and autoradiographs prepared as described above.
Scoring procedures.-In both experiments grain count determinations were made on 25 prophase nuclei and 50 interphase nuclei scored along a random transverse on each of three slides used per fixation time. The shortest duration of the G, phase in Vicia roots is about 3 hr at 19°C [9], whereas the longest interval between 3Hthymidine exposure and fixation is 90 min. Prophase ments should therefore be unlabelled and thus offered
nuclei in the present experia good control for estimating
background grain count. Furthermore, this estimate of background would be an overestimate in that prophase nuclei expose a much larger area to the film than interphase nuclei. All slides were coded and scored in random order and the grain count data on each slide was analysed separately. For each control set of 25 prophase nuclei the 95 per cent confidence limits of the mean grain count was determined and the upper limit
taken, to the nearest whole number, and used as the background grain count for that slide. This background count was then subtracted from each individual count on the 50 interphase
nuclei
on that
slide so that
50 net grain
counts
on each slide
were obtained. RESULTS
AND
DISCUSSION
Experiment 1.-Roots were fixed immediately on removal from 3Hthymidine and a total of 150 interphase nuclei scored at each fixation time. Since only a proportion of these nuclei showed a significant amount of labelling over the background count, the results are expressed as net labelling per labelled nucleus (Fig. 1). From Fig. 1 it may be seen that the mean net grain count increases with increasing duration of exposure and that the rate of increase could be a constant, the points giving a good fit to a straight line. However, the data are clearly not sufficient to distinguish between relatively small changes in rate and there is some indication of a plateau between 30 and 60 min. The background counts on most of the slides in this experiment were between 1 and 2 grains per 100 ,u2. Despite this low background, when one observer scored these same slides for the presence or absence of labelling in 500 interphase nuclei on each slide, significant differences between long and Experimental
Cell Research
35
3H-fhymidine
and patterns
of DNA
replication
383
short durations of 3H-thymidine treatment were found. The proportions of nuclei scored as labelled at 5, 10 and 20 min were similar with a mean of 25 + 5 per cent, whereas at 30, 40 and 60 min a higher frequency of labelled cells was scored, the mean value for these three treatment times being 41 i 6 per cent. During this scoring the presence of label \vas determined subjectively
Fig. I.-Relation between mean net grain count per labelled nucleus and duration of SH-thymidine treatment.
TlME
IN H%HYMIDINE
(MINI)
and not by actual grain counting, and it seems likely that part of the observed difference was due to subjective scoring error, fewer cells being scored as positive when the differential between background and positive labelling was relatively low. It is also possible, however, that part of the observed difference may be a reflection of different rates of uptake of 3H-thymidine in different zones of the root, although considerably larger cell samples than those used in the present experiment and the use of sectioned material would be required to investigate this possibility. In conclusion we may say that significant incorporation of 3H-thymidine into DNA occurs within 5 min of exposure of roots to the active solution, but that at the concentration of 3H-thymidine used exposures of 30 min or more are required to obtain reasonable labelling and to overcome any possible time lag in the transport of the 3H-thymidine through the root tissues. Experiment 2.-On the basis of the results of the first experiment, roots were exposed to 3H-thymidine for 30 min and then washed and transferred either to water or to the solution of “cold” thpmidine. Grain counts were determined as before and the mean net grain count per labelled nucleus is shown for all fixation times in Fig. 2. The results show a marked difference in grain counts between the two post-treatments in the early fixation times. At 10 and 20 min post-3H-thymiExperimenlal
Cell Research
35
384
H. J. Evans
dine treatment the grain counts in roots transferred to “cold” thymidine similar to the count obtained in roots fixed immediately on completion 3H-thymidine treatment, whereas the counts in roots transferred to were significantly higher. In these latter roots the marked increase in count may be seen to have occurred (Fig. 2) during the first 10 min
Fig. grain time Roots (- o prior TIME
AFTER
REMOLfiL
FROM
are of the water grain post-
2.--Relation between mean net count per labelled nucleus and after removal from 8H-thymidine were maintained either in water. -) or “cold” thymidine (- l - ) to fixation.
H3THYMlDlNE(MINS)
treatment and no further increase is observed between 10 and 60 min. The pattern in roots post-treated with “cold” thymidine is quite different, showing a gradual but continued increase in grain count with increasing post-treatment time, until at 40 min the grain count is the same as that found in roots post-treated with water. When scoring, as in experiment 1, was carried out to estimate the proportion of interphase cells which were labelled, no significant differences were observed between treatments, the pooled data giving a mean value of 43 per cent of the cells labelled. However, there appeared to be a tendency for a slightly higher proportion of labelled nuclei in the roots post-treated with water, and in the later “cold” thymidine fixation times, relative to the proportion of cells labelled at the end of 3H-thymidine treatment. We interpret the above data as showing that the reserve pool of 3H-thynudine in the root cells is very small (cf. the experiments of Wimber [41] on Tradescantia roots) and on removal of the cells from the active solution it is used up in a matter of a few minutes. On the other hand, when roots are given a post-treatment with “cold” thymidine the “hot” pool is not immediately used but is slowly depleted, so that labelled thymidine is available for incorporation into DSA for up to 40 min post-treatment. It would seem Experimental
Cell Research 35
3H-thymidine therefore that with root-tip practice of following an “cold” thymidine is to be Grain count distribution tion of 3H-thymidine into
and patterns
of DNA
replication
385
cells of Vicia, and probably Tradescantia, the 3H-thymidine treatment with a long exposure to avoided. and patterns of labeIling.-If the rate of incorporaDNA is constant throughout the DNA synthesis
Fig. S.-Distribution of net grain counts between positively labelled nuclei from roots exposed to 3H-thymidine for 30 min and then fixed, after transfer to water, between IO and 60 min later.
“0 p
phase, and if the time of exposure to 3H-thymidine is short in relation to the total synthesis time, then the distribution of grain counts between labelled cells should accord with the Poisson distribution, the variance of the distribution being equal to the mean grain count. The observed distribution of net grain counts between positively labelled nuclei from roots exposed to 3H-thymidine for 30 min and then fixed, after transfer to water, between 10 and 60 min later is shown in Fig. 3. Statistical analysis of the data in Fig. 3 shows that the grain counts are not distributed as a Poisson, there being considerable over-dispersion, but the data give a good fit to a negative binomial distribution, xi =9.5, p = 0.3. The parameters of the distribution [19] were c = 1.676 X 1O-2 and p = 1.0, the data strongly suggesting a basic exponential rate of uptake of 3H-thymidine by the cell nuclei (cf. [‘Ll]). The finding that the grain count distribution did not conform with a Poisson series was not surprising, in that Howard and Devvey [15] have presented data on uptake of 3H-thymidine into Vicia root cell nuclei which can be interpreted as indicating that the rate of DNA synthesis is high in early and late S and somewhat lower in the mid-synthesis phase. Similarly, our own experiments on the production of X-ray-induced chromatid aberrations in nuclei exposed to 3H-thymidine also indicate a variable rate of DNA synthesis during the S phase [8]. In view of this we have made an attempt to determine whether there is any regular pattern of DNA synthesis in Experimental
Cell Research
35
H. J. Evans these nuclei. Our observations on the distribution of grains in metaphase chromosomes were made on nuclei which were exposed for 30 min or 1 hr to 3H-thymidine and then fixed at hourly intervals from 3 to 16 hr later; these fixations covering a period which spans the whole of the S phase [Y]. Our results are described below. A number of investigations on patterns of labelling in plant chromosomes and nuclei have been carried out, and the early observations on interphase nuclei of Vicia fuba by LaCour and Pelt [20] suggested that heterochromatin and euchromatin underwent replication at different times. Similarly, Woodward, Rasch and Swift [43] also showed that certain chromosome regions in this species were preferentially more heavily labelled with 3H-thymidine at different times during synthesis. Studies on Crepis chromosomes by I’aylor [38] showed that in some cells of this species there was a gradient of labelling from the ends of the chromosomes to the centromere and it was concluded that replication began at the chromosome ends and proceeded regularly towards the centromere. A similar conclusion was arrived at by Pelt and LaCour [33] from their studies, once more on interphase nuclei, in FritiZlrwin lanceolata and Scilla sibirica; they concluded that I>X\;A synthesis began at the ends of the chromosomes and proceeded in sequence along the length of the chromosome in both eu- and heterochromatic regions alike. Furthermore, it was concluded that the last portion of the chromosome to synthesize DNA was the centromere region and that synthesis in the chromosome arms proceeded from either end towards the centromere, but not across it. hlost of these observations were made on Frifillaria nuclei and since the heterochromatin in this species is located proximally to the centromere, replication in the heterochromatin occurred late in the synthesis phase. In Vicia fabn the chromosome complement consists of one pair of metacentric (Izl) and five pairs of acrocentric (S) chromosomes (Fig. 4), and heterochromatic zones, as defined by Heitz [ 131, which show positive heteropycnosis in early mitosis can be recognized. These heterochromatic
Fig. 4.-The metaphase centric S chromosomes Fig. 5.-Two label confined
chromosome complement of Viciu and the single pair of metacentric
interphase nuclei, one showing a general to the chromocentres. x 1600.
faba showing the M chromosomes.
distribution
of label
five pairs x 2000.
and the other
of acroshowing
Fig. 6.-Autoradiograph of a metaphase nucleus which was exposed to 3H-thymidine whilst in the last hour of the S phase. Note that the label is localized at or near the mid zones of the S chromosomes and on either side of the centromere in the M chromosomes. x 2000. Fig. 7.-Autoradiograph ning of the S phase. Experimental
Cell
Note Research
of a metaphase nucleus exposed to 3H-thymidine whilst at the beginthe absence of label in the heterochromatic regions, cf. Fig. 6. x 2000. 35
3H-thymidine
and patterns
of DNA
replication
Experimental
387
Cell Research
35
H. J. Evans zones are difficult to distinguish at metaphase, but appear as relatively more contracted deeper staining regions than the euchromatin in prophase and telophase nuclei, and are clearly observed in interphase as discrete Feulgenpositive chromocentres (Fig. 5). The heterochromatin is distributed throughout the chromosome complement and the largest blocks are localized to and on either side of the centromere in the M chromosomes and just proximal to or at the mid-zone of the S chromosomes [B, 271. Our observations on the first of the labelled cells to arrive at metaphase following a brief 3H-thymidine treatment show that in some of the cells 3H-label is distributed over much of the chromosome lengths. However, in a large proportion of these cells, which were fixed between 5 and 7 hr after treatment and hence were in the last 2 hr of the S phase at the time of exposure to 3H-thymidine, the label is mainly confined to particular chromosome zones. A typical pattern of labelling is shown in Fig. 6, from which it may be seen that the label is almost exclusively confined to the two regions adjacent to and on either side of the centromere in the M chromosomes and to bands in the mid-regions of the S chromosomes. In the S chromosomes, label is completely absent in the centromere regions, short chromosome arms and distal regions of the long arms in all the chromosomes, and also absent in the proximal regions of the long arms in some of the chromosomes. Similarly no label is found in the distal regions of the M chromosomes. This particular pattern of labelling therefore is specifically associated with the distribution of heterochromatin in the chromosome complement. -4 localization of labelling to heterochromatin may be also observed in a proportion of the relatively lightly labelled interphase nuclei, where the silver grains are localised over the chromocentres (Fig. 5). These observations thus suggest that a high rate of DNA synthesis in the heterochromatic chromosome zones of Vicia nuclei occurs at the end of the DNA synthesis period, and that replication in the euchromatin is completed sometime before replication in heterochromatin. Observations on metaphase cells fixed 12 to 16 hr after 3H-thymidine treatment, i.e. on cells which were entering synthesis at the time of treatment, show that in many of these cells the label is present over a number of regions of most of the chromosomes, but is absent over the heterochromatic zones. This observation indicates that replication starts in a number of different places, even in a single chromosome, but commences first in euchromatic regions, synthesis in heterochromatin being delayed. The absence of label in the heterochromatic regions of cells treated whilst in early S is shown in Fig. 7, where it may be noted that in one of the M chromosomes the presence Experimental
Cell Research
35
3H-thymidine
and patterns
of DNA
replication
of label may be seen in the very short euchromatic regions which are interposed between the heterochromatin and the centromere, but label is absent in the regions occupied by the heterochromatin itself. The fact that not all the cells at any given fixation time show exactly the same pattern of labelling is not unexpected, since a considerable variation in the rate of cell development through the mitotic cycle is evident. Because of this the sequence of events in the mid S phase is difficult to resolve. However, in some of the cells which were in the mid-S phase when given a brief exposure to 3H-thymidine the longest pair of S chromosomes is sometimes seen to contain relatively little label, in contrast to the heavy label over the rest of the chromosome complement. It would seem then that in this chromosome pair the replication of euchromatin is completed more rapidly than in the other chromosomes, but all the heterochromatic chromosome zones appear to start their replication at about the same time. This synchronous and heavy incorporation of label into the heterochromatin which occurs during the last few hours of the S phase suggests an explanation for the high rate of DNA synthesis observed in late S nuclei by Howard and Dewey [ 151. Further, this finding also accords with our observation that late S nuclei are sensitive to the production of chromatid aberrations by maleic hydrazide, the aberrations being confined to the heterochromatic chromosome zones [9]. Because of the differences between the distribution of heterochromatin in the Vicia as opposed to the Fritillaria chromosome complements, our results show clearly that the pattern of replication in Vicia does not involve a simple progression from one end of the chromosome to the other, but that the replication of heterochromatin, regardless of its position, occurs after the completion or near completion of replication in euchromatin. If, as in many species, the heterochromatin is localized to the centromere regions, then the observation of late labelling in these regions could be erroneously construed as indicating that replication proceeded from the distal to the proximal ends of the chromosome. It is of interest to note that Wimber [42] has reported the reverse of this situation in Tradescantia, a species with no clearly demonstrable heterochromatin, where the chromosome zones which show a late localized labelling are the distal chromosome regions. Similarly, Taylor [39] has shown that in Chinese hamster cells in tissue culture DNA replication may be initiated simultaneously in different parts of the same chromosome, and similar observations have been made on grasshopper spermatocytes [23] and on human cells [ll, 24, 291. A further observation on a difference between the time of DNA synthesis in euchromatin and heterochromatin, which does not involve sex chromoExperimental
Cell Research
35
H. J. Evans somes, has been reported for the leaf nuclei of Secale [22]; although from this investigation it was not known which type of chromatin underwent late synthesis. Some of our own experiments, carried out in collaboration with Dr. C. E. Ford, suggest that in bone marro\\- and thymus cells of the mouse DNA synthesis in late S is localized to the proximal chromosome regions, in this species much of the heterochromatin is also localized to these regions [31]. The above observations when taken collectively indicate not only that the pattern of chromosome replication differs between different species, but strongly suggests that there is a general rule governing the time of DNA synthesis in the heterochromatic zones of autosomes. It would seem that chromosome regions showing positive heteroppcnosis in interphase and early mitosis always replicate late in the S phase, usually after the completion of replication in the euchromatin. In addition to the observations on the time of replication of autosomal heterochromatin, much information has been acquired on the time of replication of sex chromosomes which show positive heteropycnosis. Lima de Faria [22, 231 showed that DNA synthesis in the heterochromatic block formed by the sex chromosome of grasshopper (Melanoplus spp.) spermatocytes occurred later than in the euchromatic autosomes, and Taylor’s [39] in vitro studies on Chinese hamster cells revealed that in female cells the whole of one X chromosome and the long arm of the other X chromosome underwent DNA4 replication late in S, whereas in male cells the Y chromosome and the long arm of the S chromosome replicated late. More recently there have been numerous reports on the patterns of replication in human chromosomes, usually from peripheral blood cells, and these have revealed that in females there is a differential behaviour between the two S chromosomes: one of the S chromosomes, which is positively heteropycnotic and is now generally held to constitute the heterochromatic Barr body of interphase nuclei [30, 32], undergoes a late replication at the end of the S phase, the other showing no such late labelling [ll, 291. In abnormal human cells having more than two X chromosomes, except in cases of mosaicism following non-disjunction, all but one show late labelling and there is a direct relationship between the number of late labelling S chromosomes and the number of Barr bodies [la, 361. Morphologically the heteropycnosis in sex chromosomes may be indistinguishable from positive heteropycnosis in autosomes-whether this involves parts of the autosomal regions, whole chromosomes or even the whole of a haploid complement [17]-and the relative lateness of DNA replication in these two sorts of heterochromatic Experimental
Cell
Research
35
3H-fhymidine
and pafferns
of DNA
replication
391
conditions indicates their further similarity. However, heterochromatin is quite variable in its cytological expression, and its presence in a given genotype-at certain development stages only or throughout the whole life cycleresulting in hemay be governed by a number of factors. The conditions terochromatinization of sex chromosomes could therefore bc quite different from those influencing autosomal heterochromatin. The nature and function of heterochromatin has been the subject of considerable discussion since Heitz [IJ] first suggested that the heterochromatic condition was the cytological expression of genetical inertness. If the heteropycnotic state in interphase is equivalent to the condensed state of the chromosome at mitosis then, by analogy, we would expect little genetic information to be released from chromosomes or chromosome zones in this condition. Interest in this aspect has recently been renewed following the proposal by Lyon [23, 261 that the heteropycnotic S chromosome in mammals is genetically inactive. It is clear that genetical inactivity is associated with heterochromatin in certain instances, e.g. in the supernumerary 13 chromosomes of higher plants [a], although there are a number of well documented reports of genetic activity in heterochromatin [28, 35, 371. \1Te may well ask whether a reduced genetic activity, or more properly the cause for such a reduced activity, is associated with or is responsible for the late DNA synthesis in heterochromatin? Structurally, heterochromatic zones simply appear as tightly coiled chromosome regions and there is but little indication of specific histochemical differences between hetero- and euchromatic regions. The elegant experiments of Huang and Conner [16] and of Allfrey, Littau and Mirsky [a], using extracted plant chromatin and isolated calf thymus nuclei respectively, have shown that the complexing of histone to DNA, particularly of histone rich in arginine [2], inhibits DNA-mediated RNA synthesis. Similarly, Izawa et al. [18] have shown that treatment of the giant “lampbrush” chromosomes of amphibian oocytes with arginine-rich histone results in the disappearance of the DNA containing lateral loops, the presence of these loops being intimately associated with the capacity for synthesizing RNA. On a more general level it may be noted that in a variety of animal species a transition from a lysine-rich to an arginine-rich histone occurs in the condensing nuclei of spcrmatids [l, 31; it may be presumed that such nuclei are doing little “genetic work”, and it seems likely that this transition may be an important feature of genetically inactive compacted chromosomes or chromosome regions. These observations naturally lead to the suggestion that the variety of different DNA’s which may constitute heterochromatic regions may all Experimental
Cell Research
35
H. J. Evans be tightly complexed with an arginine-rich histone, whose responsible for the late DNA replication in these regions. is open to experimental attack, but at present the reason delay of DNA replication in heterochromatin is unknown.
late removal is This possibility for the specific
SUMMARY
1. A significant incorporation of 3H-thymidine into the DNA of root tip cells of Vicia faba is shown to occur within 5 min of exposure to the labelled precursor. 2. When roots are exposed to water after a 30 min 3H-thymidine treatment, all the labelled compound is used up or washed out within 10 min of removal from the active solution. However, post-treatment of roots with a high concentration of inactive thymidine, following a water wash, results in a slow depletion of the “hot” pool, labelled thymidine being available for incorporation into DNA for up to 40 min after the removal of the roots from the “hot” solution. 3. The distribution of grain counts between nuclei exposed for short periods to 3H-thymidine does not conform to a Poisson distribution, the data suggesting a basic exponential rate of incorporation by the cell nuclei. .+. Observations on interphase nuclei and metaphase chromosomes of cells exposed to 3H-thymidine at various periods during the S phase show (a) that DNA replication is initiated at a large number of sites over the chromosome complement, there being no sequential progression from the distal to proximal chromosome regions, and (b) that replication in the heterochromatic chromosome zones is confined to late S. 5. The available information on the time of DNA replication in eu- and heterochromatin in a variety of species is reviewed, the evidence indicating that positive heteropycnotic regions, whether in sex chromosomes or in autosomes, always undergo a late replication. The possibility that this delayed replication of heterochromatin is associated with genetic inactivity and the presence of arginine-rich histone is discussed. The author is indebted to Miss J. Bodycote and Mr. T. R. L. Bigger for their able technical assistanceand to Mr. D. G. Papworth for statistical advice.
Experimental
Cell
Research
35
3H-thymidine
and patterns
of DNA
replication
REFERENCES 1. ALFERT, M., J. Biophys. Biochem. Cyfol. 2. 109 (1956). 2. ALLFREY, V. G., LITTAU, V. C. and MIRSKY, A. E., Proc. Nat1 Acad. Sci. 49, 414 (1963). 3. BLOCH, D. P. and HEW, H. Y., J. Biophys. Biochem. Cyfol. 7, 515 (1960). 4. DARLINGTON, C. D., Chromosome Botany. Allen and Unwin Ltd., London 1956. 5. EVANS, H. J., Genefics 46, 257 (1961). 6. EVANS, H. J. and BIGGER, T. R. L., Genetics 46, 277 (1961). 7. EVANS, H. J., NEARY, G. J. and TONKINSON, S. M., J. Genef. 55, 487 (1957). 8. EVANS, H. J. and SAVAGE, J. R. K., J. Cell Biol. 18, 525 (1963). 9. EVANS, H. J. and SCOTT, D., Genetics 49, 17 (1964). 10. FRIEDKIN, M., TILSON, D. and ROBERTS, D., J. Biol. Chem. 220, 627 (1956). 11. GILBERT, C. W., MULDAL, S., LAJTHA, L. G. and ROWLEY, .J., iVa/rzre 195, 869 (1962). 12. GRUMBACH, M. M., MORISHIMA, A. and TAYLOR, J. H., Proe. Nafl Acad. Sci. 49, 581 (1963). 13. HEITZ, E., Jahrb.wiss. Bot. 69, 762 (1928). 14. __ Ber Dezzf. Bof. ties. 47, 274 (1929). 15. HOWARD, A. and DEWEY, D. L., Expfl Cell Res. 24, 623 (1961). 16. HUANG, R. C. and BONSER, J., Proc. Nafl Acad. Sci. 48, 1216 (1962). 17. HUGHES-SCHRADER, S., Aduan. Genef. 2, 127 (1948). 18. IZAWA, M., ALLFREY, V. G. and MIRSKY, A. E., Proc. Nat1 Acad. Sci. 49, 544 (1963). 19. KENDALL, M. G. and STUART, A. Advanced Theory of Statistics. Vol. 1, p. 129. Griffin and Co. London, 1958. 20. LACOUR, I,. F. and PELC, S. R., Xafure 182, 506 (1958. 21. LAJTHA, L. G., OLIVER, R., BERRY, R. J. and HELL, E., Nature 187, 919 (1960). 22. LIMA-DE-FARIA. A.. J. Bioohus. Biochem. Cufol. 6. 457 (1959). 23. -Heredifas 47, k74 (19ilc 24. LIntA-DE-limu. A.. REITALU. J. and BERGMAN. S., Heredifas 47, 695 (1961). 2% LYON, hf. I’., safzzre 190, 37i (1961). 26. __ Am. .I. Human Genef. 14, 135 (1962). 27. MCLEISH, J., Heredify (Suppl.) 6, 125 (1953). 28. MATHER, K., Proc. Roy. Sot. (London), B 132, 308 (1945). 29. MORISHIMA, A., GRUMBACH, M. M. and TAYLOR, J. H., Proc. Nat1 Acad. Sci. 48, 756 (1962). 30. OHNO, S. and HAUSCHKA, T. S., Cancer Res. 20, 541 (1960). 31. OHNO, S., KAPLAN, \V. D. and KINOSITA, R., Expfl Cell Res. 13, 358 (1957). 32. OHNO, S. and MAKIKO, S., Lancef 78, 000 (1961). 33. PELC, S. R. and LACOUR, L. F., in The Cell Nucleus, p. 232, J. S. MITCHELL (ed.) Butterworth, London, 1959. 34. REICHAHD, P. and ESTBORN, B., J. Biol. Chem. 188, 839 (1951). 35. RHOaDES, M. M., in Heterosis p. 66, J. W. GOWEN (ed.) Iowa State College Press, 1952. 36. ROWLEY, J., MULDAL, S., GILBERT, C. W., LAJTHA, L. G., LINDSTEN, J., FRACCARO, M. and KAIJSER, K., Nature 197, 251 (1963). 37. SCHULTZ, J., Cold Spring Harbor Symp. Qzzanf. Biol. 12, 179 (1947). 38. TAYLOR, J. H., Expfl Cell Res. 15, 350 (1958). 39. __ J. Biophzfs. Biochem. Cyfol. 7, 455 (1960). 40. TAYLOR, J. H.; WOODS, P. S.-and HUGH&, W. L., Proc. Nat1 Acad. Sci. 43, 122 (1957). 41. WIMBER, D. E., Am. J. Bof. 47, 828 (1960). 42. -Expfl Cell Res. 23, 402 (1961). 43. WOOOARD, .J., RASCH, E. and SWIFT, H., J. Biophys. Biochem. Cyfol. 9, 445 (1961).
Experinzerzfal
Cell Research
35
Cell Research 35, 381-393
UPTAKE
381
(1961)
OF 3H-THYMIDINE
REPLICATION
AND PATTERNS
OF DNA
IN NUCLEI AND CHROMOSOMES OF V1CIA FABA H. J. EVANS
Medical Research Council, Radiobiological Research
Unit, Harwcll, Berks.,
England
Received August 16, 1963
SINCEth e
initial demonstration that thymidine is a highly specific precursor of Dr\TA [lo, 341, considerable use has been made of radioactively labelled thymidine in studying various aspects of DSA synthesis. The compound, especially when labelled with tritium [40], is particularly useful and is widely used in cytological experiments designed to study aspects of Di\lA synthesis, chromosome duplication, and as a marker in studying kinetics of cellular proliferation. Although a considerable number of autoradiographic studies using root-tip tissues exposed to 3H-thymidine have been carried out, there is little available information on the rate of penetration and incorporation of thymidine into the l>Klz of root-tip cells, and no published information on the influence of post-treatment with high concentrations of inactive thymidinc-a procedure which is routinely carried out in a number of laboratories. The present paper reports the results of two experiments carried out on Vicin faba root-tip cells to investigate (i) the rate of uptake of 3H-thymidinc into
DSA
fluence
as measured
from
of post-treatments
grain
count
with inactive
determinations,
thymidinc
and (ii)
the in-
on grain count and on the
proportion of meristem cells which are labelled. Information having a bearing on the relative rates of uptake of 3H-thymidine into DNA at different stages of development
of the synthesis
some. observations and a discussion metaphase chromosomes.
MATERIALS
phase is also presented, on the patterns
AND
together
of labelling
with
found
in
METHODS
EXPERIMENT l.-Fifty primary roots of Vicia faba seedlings, cultured by our usual procedures [7], were exposed to 100 ml of an aerated solution of 3H-thymidine (s.a. 3.0 c/m&f), at a concentration of 2 PC/ml, for various time intervals up to 30 min. The roots were fixed in a modified Flemmings fluid immediately on removal from the Experimental
Cell Research 3.5
H. J. Evans 3H-thymidine solution and hydrolysed for 10 min in N HCl at 60°C. Cytological preparations were made from the terminal 2 mm of root-tip as described previously [5] and autoradiographs were prepared with Kodak AR 10 stripping film, the slides being exposed for 3 weeks at 4°C and developed in Kodak Dl9b. EXPERIMENT 2.-Fifty roots were exposed to 3H-thymidine, as described above, for a period of 30 min. On completion of the 3H-thymidine treatment all roots were quickly rinsed in water and half were transferred to a solution of “cold” thymidine made up to 50 x the concentration of the active solution, i.e. to 8 pg/ml, and the remaining roots were transferred to water. Fixations were made at intervals of up to 1 hr after completion of 3H-thymidine treatment and autoradiographs prepared as described above.
Scoring procedures.-In both experiments grain count determinations were made on 25 prophase nuclei and 50 interphase nuclei scored along a random transverse on each of three slides used per fixation time. The shortest duration of the G, phase in Vicia roots is about 3 hr at 19°C [9], whereas the longest interval between 3Hthymidine exposure and fixation is 90 min. Prophase ments should therefore be unlabelled and thus offered
nuclei in the present experia good control for estimating
background grain count. Furthermore, this estimate of background would be an overestimate in that prophase nuclei expose a much larger area to the film than interphase nuclei. All slides were coded and scored in random order and the grain count data on each slide was analysed separately. For each control set of 25 prophase nuclei the 95 per cent confidence limits of the mean grain count was determined and the upper limit
taken, to the nearest whole number, and used as the background grain count for that slide. This background count was then subtracted from each individual count on the 50 interphase
nuclei
on that
slide so that
50 net grain
counts
on each slide
were obtained. RESULTS
AND
DISCUSSION
Experiment 1.-Roots were fixed immediately on removal from 3Hthymidine and a total of 150 interphase nuclei scored at each fixation time. Since only a proportion of these nuclei showed a significant amount of labelling over the background count, the results are expressed as net labelling per labelled nucleus (Fig. 1). From Fig. 1 it may be seen that the mean net grain count increases with increasing duration of exposure and that the rate of increase could be a constant, the points giving a good fit to a straight line. However, the data are clearly not sufficient to distinguish between relatively small changes in rate and there is some indication of a plateau between 30 and 60 min. The background counts on most of the slides in this experiment were between 1 and 2 grains per 100 ,u2. Despite this low background, when one observer scored these same slides for the presence or absence of labelling in 500 interphase nuclei on each slide, significant differences between long and Experimental
Cell Research
35
3H-fhymidine
and patterns
of DNA
replication
383
short durations of 3H-thymidine treatment were found. The proportions of nuclei scored as labelled at 5, 10 and 20 min were similar with a mean of 25 + 5 per cent, whereas at 30, 40 and 60 min a higher frequency of labelled cells was scored, the mean value for these three treatment times being 41 i 6 per cent. During this scoring the presence of label \vas determined subjectively
Fig. I.-Relation between mean net grain count per labelled nucleus and duration of SH-thymidine treatment.
TlME
IN H%HYMIDINE
(MINI)
and not by actual grain counting, and it seems likely that part of the observed difference was due to subjective scoring error, fewer cells being scored as positive when the differential between background and positive labelling was relatively low. It is also possible, however, that part of the observed difference may be a reflection of different rates of uptake of 3H-thymidine in different zones of the root, although considerably larger cell samples than those used in the present experiment and the use of sectioned material would be required to investigate this possibility. In conclusion we may say that significant incorporation of 3H-thymidine into DNA occurs within 5 min of exposure of roots to the active solution, but that at the concentration of 3H-thymidine used exposures of 30 min or more are required to obtain reasonable labelling and to overcome any possible time lag in the transport of the 3H-thymidine through the root tissues. Experiment 2.-On the basis of the results of the first experiment, roots were exposed to 3H-thymidine for 30 min and then washed and transferred either to water or to the solution of “cold” thpmidine. Grain counts were determined as before and the mean net grain count per labelled nucleus is shown for all fixation times in Fig. 2. The results show a marked difference in grain counts between the two post-treatments in the early fixation times. At 10 and 20 min post-3H-thymiExperimenlal
Cell Research
35
384
H. J. Evans
dine treatment the grain counts in roots transferred to “cold” thymidine similar to the count obtained in roots fixed immediately on completion 3H-thymidine treatment, whereas the counts in roots transferred to were significantly higher. In these latter roots the marked increase in count may be seen to have occurred (Fig. 2) during the first 10 min
Fig. grain time Roots (- o prior TIME
AFTER
REMOLfiL
FROM
are of the water grain post-
2.--Relation between mean net count per labelled nucleus and after removal from 8H-thymidine were maintained either in water. -) or “cold” thymidine (- l - ) to fixation.
H3THYMlDlNE(MINS)
treatment and no further increase is observed between 10 and 60 min. The pattern in roots post-treated with “cold” thymidine is quite different, showing a gradual but continued increase in grain count with increasing post-treatment time, until at 40 min the grain count is the same as that found in roots post-treated with water. When scoring, as in experiment 1, was carried out to estimate the proportion of interphase cells which were labelled, no significant differences were observed between treatments, the pooled data giving a mean value of 43 per cent of the cells labelled. However, there appeared to be a tendency for a slightly higher proportion of labelled nuclei in the roots post-treated with water, and in the later “cold” thymidine fixation times, relative to the proportion of cells labelled at the end of 3H-thymidine treatment. We interpret the above data as showing that the reserve pool of 3H-thynudine in the root cells is very small (cf. the experiments of Wimber [41] on Tradescantia roots) and on removal of the cells from the active solution it is used up in a matter of a few minutes. On the other hand, when roots are given a post-treatment with “cold” thymidine the “hot” pool is not immediately used but is slowly depleted, so that labelled thymidine is available for incorporation into DSA for up to 40 min post-treatment. It would seem Experimental
Cell Research 35
3H-thymidine therefore that with root-tip practice of following an “cold” thymidine is to be Grain count distribution tion of 3H-thymidine into
and patterns
of DNA
replication
385
cells of Vicia, and probably Tradescantia, the 3H-thymidine treatment with a long exposure to avoided. and patterns of labeIling.-If the rate of incorporaDNA is constant throughout the DNA synthesis
Fig. S.-Distribution of net grain counts between positively labelled nuclei from roots exposed to 3H-thymidine for 30 min and then fixed, after transfer to water, between IO and 60 min later.
“0 p
phase, and if the time of exposure to 3H-thymidine is short in relation to the total synthesis time, then the distribution of grain counts between labelled cells should accord with the Poisson distribution, the variance of the distribution being equal to the mean grain count. The observed distribution of net grain counts between positively labelled nuclei from roots exposed to 3H-thymidine for 30 min and then fixed, after transfer to water, between 10 and 60 min later is shown in Fig. 3. Statistical analysis of the data in Fig. 3 shows that the grain counts are not distributed as a Poisson, there being considerable over-dispersion, but the data give a good fit to a negative binomial distribution, xi =9.5, p = 0.3. The parameters of the distribution [19] were c = 1.676 X 1O-2 and p = 1.0, the data strongly suggesting a basic exponential rate of uptake of 3H-thymidine by the cell nuclei (cf. [‘Ll]). The finding that the grain count distribution did not conform with a Poisson series was not surprising, in that Howard and Devvey [15] have presented data on uptake of 3H-thymidine into Vicia root cell nuclei which can be interpreted as indicating that the rate of DNA synthesis is high in early and late S and somewhat lower in the mid-synthesis phase. Similarly, our own experiments on the production of X-ray-induced chromatid aberrations in nuclei exposed to 3H-thymidine also indicate a variable rate of DNA synthesis during the S phase [8]. In view of this we have made an attempt to determine whether there is any regular pattern of DNA synthesis in Experimental
Cell Research
35
H. J. Evans these nuclei. Our observations on the distribution of grains in metaphase chromosomes were made on nuclei which were exposed for 30 min or 1 hr to 3H-thymidine and then fixed at hourly intervals from 3 to 16 hr later; these fixations covering a period which spans the whole of the S phase [Y]. Our results are described below. A number of investigations on patterns of labelling in plant chromosomes and nuclei have been carried out, and the early observations on interphase nuclei of Vicia fuba by LaCour and Pelt [20] suggested that heterochromatin and euchromatin underwent replication at different times. Similarly, Woodward, Rasch and Swift [43] also showed that certain chromosome regions in this species were preferentially more heavily labelled with 3H-thymidine at different times during synthesis. Studies on Crepis chromosomes by I’aylor [38] showed that in some cells of this species there was a gradient of labelling from the ends of the chromosomes to the centromere and it was concluded that replication began at the chromosome ends and proceeded regularly towards the centromere. A similar conclusion was arrived at by Pelt and LaCour [33] from their studies, once more on interphase nuclei, in FritiZlrwin lanceolata and Scilla sibirica; they concluded that I>X\;A synthesis began at the ends of the chromosomes and proceeded in sequence along the length of the chromosome in both eu- and heterochromatic regions alike. Furthermore, it was concluded that the last portion of the chromosome to synthesize DNA was the centromere region and that synthesis in the chromosome arms proceeded from either end towards the centromere, but not across it. hlost of these observations were made on Frifillaria nuclei and since the heterochromatin in this species is located proximally to the centromere, replication in the heterochromatin occurred late in the synthesis phase. In Vicia fabn the chromosome complement consists of one pair of metacentric (Izl) and five pairs of acrocentric (S) chromosomes (Fig. 4), and heterochromatic zones, as defined by Heitz [ 131, which show positive heteropycnosis in early mitosis can be recognized. These heterochromatic
Fig. 4.-The metaphase centric S chromosomes Fig. 5.-Two label confined
chromosome complement of Viciu and the single pair of metacentric
interphase nuclei, one showing a general to the chromocentres. x 1600.
faba showing the M chromosomes.
distribution
of label
five pairs x 2000.
and the other
of acroshowing
Fig. 6.-Autoradiograph of a metaphase nucleus which was exposed to 3H-thymidine whilst in the last hour of the S phase. Note that the label is localized at or near the mid zones of the S chromosomes and on either side of the centromere in the M chromosomes. x 2000. Fig. 7.-Autoradiograph ning of the S phase. Experimental
Cell
Note Research
of a metaphase nucleus exposed to 3H-thymidine whilst at the beginthe absence of label in the heterochromatic regions, cf. Fig. 6. x 2000. 35
3H-thymidine
and patterns
of DNA
replication
Experimental
387
Cell Research
35
H. J. Evans zones are difficult to distinguish at metaphase, but appear as relatively more contracted deeper staining regions than the euchromatin in prophase and telophase nuclei, and are clearly observed in interphase as discrete Feulgenpositive chromocentres (Fig. 5). The heterochromatin is distributed throughout the chromosome complement and the largest blocks are localized to and on either side of the centromere in the M chromosomes and just proximal to or at the mid-zone of the S chromosomes [B, 271. Our observations on the first of the labelled cells to arrive at metaphase following a brief 3H-thymidine treatment show that in some of the cells 3H-label is distributed over much of the chromosome lengths. However, in a large proportion of these cells, which were fixed between 5 and 7 hr after treatment and hence were in the last 2 hr of the S phase at the time of exposure to 3H-thymidine, the label is mainly confined to particular chromosome zones. A typical pattern of labelling is shown in Fig. 6, from which it may be seen that the label is almost exclusively confined to the two regions adjacent to and on either side of the centromere in the M chromosomes and to bands in the mid-regions of the S chromosomes. In the S chromosomes, label is completely absent in the centromere regions, short chromosome arms and distal regions of the long arms in all the chromosomes, and also absent in the proximal regions of the long arms in some of the chromosomes. Similarly no label is found in the distal regions of the M chromosomes. This particular pattern of labelling therefore is specifically associated with the distribution of heterochromatin in the chromosome complement. -4 localization of labelling to heterochromatin may be also observed in a proportion of the relatively lightly labelled interphase nuclei, where the silver grains are localised over the chromocentres (Fig. 5). These observations thus suggest that a high rate of DNA synthesis in the heterochromatic chromosome zones of Vicia nuclei occurs at the end of the DNA synthesis period, and that replication in the euchromatin is completed sometime before replication in heterochromatin. Observations on metaphase cells fixed 12 to 16 hr after 3H-thymidine treatment, i.e. on cells which were entering synthesis at the time of treatment, show that in many of these cells the label is present over a number of regions of most of the chromosomes, but is absent over the heterochromatic zones. This observation indicates that replication starts in a number of different places, even in a single chromosome, but commences first in euchromatic regions, synthesis in heterochromatin being delayed. The absence of label in the heterochromatic regions of cells treated whilst in early S is shown in Fig. 7, where it may be noted that in one of the M chromosomes the presence Experimental
Cell Research
35
3H-thymidine
and patterns
of DNA
replication
of label may be seen in the very short euchromatic regions which are interposed between the heterochromatin and the centromere, but label is absent in the regions occupied by the heterochromatin itself. The fact that not all the cells at any given fixation time show exactly the same pattern of labelling is not unexpected, since a considerable variation in the rate of cell development through the mitotic cycle is evident. Because of this the sequence of events in the mid S phase is difficult to resolve. However, in some of the cells which were in the mid-S phase when given a brief exposure to 3H-thymidine the longest pair of S chromosomes is sometimes seen to contain relatively little label, in contrast to the heavy label over the rest of the chromosome complement. It would seem then that in this chromosome pair the replication of euchromatin is completed more rapidly than in the other chromosomes, but all the heterochromatic chromosome zones appear to start their replication at about the same time. This synchronous and heavy incorporation of label into the heterochromatin which occurs during the last few hours of the S phase suggests an explanation for the high rate of DNA synthesis observed in late S nuclei by Howard and Dewey [ 151. Further, this finding also accords with our observation that late S nuclei are sensitive to the production of chromatid aberrations by maleic hydrazide, the aberrations being confined to the heterochromatic chromosome zones [9]. Because of the differences between the distribution of heterochromatin in the Vicia as opposed to the Fritillaria chromosome complements, our results show clearly that the pattern of replication in Vicia does not involve a simple progression from one end of the chromosome to the other, but that the replication of heterochromatin, regardless of its position, occurs after the completion or near completion of replication in euchromatin. If, as in many species, the heterochromatin is localized to the centromere regions, then the observation of late labelling in these regions could be erroneously construed as indicating that replication proceeded from the distal to the proximal ends of the chromosome. It is of interest to note that Wimber [42] has reported the reverse of this situation in Tradescantia, a species with no clearly demonstrable heterochromatin, where the chromosome zones which show a late localized labelling are the distal chromosome regions. Similarly, Taylor [39] has shown that in Chinese hamster cells in tissue culture DNA replication may be initiated simultaneously in different parts of the same chromosome, and similar observations have been made on grasshopper spermatocytes [23] and on human cells [ll, 24, 291. A further observation on a difference between the time of DNA synthesis in euchromatin and heterochromatin, which does not involve sex chromoExperimental
Cell Research
35
H. J. Evans somes, has been reported for the leaf nuclei of Secale [22]; although from this investigation it was not known which type of chromatin underwent late synthesis. Some of our own experiments, carried out in collaboration with Dr. C. E. Ford, suggest that in bone marro\\- and thymus cells of the mouse DNA synthesis in late S is localized to the proximal chromosome regions, in this species much of the heterochromatin is also localized to these regions [31]. The above observations when taken collectively indicate not only that the pattern of chromosome replication differs between different species, but strongly suggests that there is a general rule governing the time of DNA synthesis in the heterochromatic zones of autosomes. It would seem that chromosome regions showing positive heteroppcnosis in interphase and early mitosis always replicate late in the S phase, usually after the completion of replication in the euchromatin. In addition to the observations on the time of replication of autosomal heterochromatin, much information has been acquired on the time of replication of sex chromosomes which show positive heteropycnosis. Lima de Faria [22, 231 showed that DNA synthesis in the heterochromatic block formed by the sex chromosome of grasshopper (Melanoplus spp.) spermatocytes occurred later than in the euchromatic autosomes, and Taylor’s [39] in vitro studies on Chinese hamster cells revealed that in female cells the whole of one X chromosome and the long arm of the other X chromosome underwent DNA4 replication late in S, whereas in male cells the Y chromosome and the long arm of the S chromosome replicated late. More recently there have been numerous reports on the patterns of replication in human chromosomes, usually from peripheral blood cells, and these have revealed that in females there is a differential behaviour between the two S chromosomes: one of the S chromosomes, which is positively heteropycnotic and is now generally held to constitute the heterochromatic Barr body of interphase nuclei [30, 32], undergoes a late replication at the end of the S phase, the other showing no such late labelling [ll, 291. In abnormal human cells having more than two X chromosomes, except in cases of mosaicism following non-disjunction, all but one show late labelling and there is a direct relationship between the number of late labelling S chromosomes and the number of Barr bodies [la, 361. Morphologically the heteropycnosis in sex chromosomes may be indistinguishable from positive heteropycnosis in autosomes-whether this involves parts of the autosomal regions, whole chromosomes or even the whole of a haploid complement [17]-and the relative lateness of DNA replication in these two sorts of heterochromatic Experimental
Cell
Research
35
3H-fhymidine
and pafferns
of DNA
replication
391
conditions indicates their further similarity. However, heterochromatin is quite variable in its cytological expression, and its presence in a given genotype-at certain development stages only or throughout the whole life cycleresulting in hemay be governed by a number of factors. The conditions terochromatinization of sex chromosomes could therefore bc quite different from those influencing autosomal heterochromatin. The nature and function of heterochromatin has been the subject of considerable discussion since Heitz [IJ] first suggested that the heterochromatic condition was the cytological expression of genetical inertness. If the heteropycnotic state in interphase is equivalent to the condensed state of the chromosome at mitosis then, by analogy, we would expect little genetic information to be released from chromosomes or chromosome zones in this condition. Interest in this aspect has recently been renewed following the proposal by Lyon [23, 261 that the heteropycnotic S chromosome in mammals is genetically inactive. It is clear that genetical inactivity is associated with heterochromatin in certain instances, e.g. in the supernumerary 13 chromosomes of higher plants [a], although there are a number of well documented reports of genetic activity in heterochromatin [28, 35, 371. \1Te may well ask whether a reduced genetic activity, or more properly the cause for such a reduced activity, is associated with or is responsible for the late DNA synthesis in heterochromatin? Structurally, heterochromatic zones simply appear as tightly coiled chromosome regions and there is but little indication of specific histochemical differences between hetero- and euchromatic regions. The elegant experiments of Huang and Conner [16] and of Allfrey, Littau and Mirsky [a], using extracted plant chromatin and isolated calf thymus nuclei respectively, have shown that the complexing of histone to DNA, particularly of histone rich in arginine [2], inhibits DNA-mediated RNA synthesis. Similarly, Izawa et al. [18] have shown that treatment of the giant “lampbrush” chromosomes of amphibian oocytes with arginine-rich histone results in the disappearance of the DNA containing lateral loops, the presence of these loops being intimately associated with the capacity for synthesizing RNA. On a more general level it may be noted that in a variety of animal species a transition from a lysine-rich to an arginine-rich histone occurs in the condensing nuclei of spcrmatids [l, 31; it may be presumed that such nuclei are doing little “genetic work”, and it seems likely that this transition may be an important feature of genetically inactive compacted chromosomes or chromosome regions. These observations naturally lead to the suggestion that the variety of different DNA’s which may constitute heterochromatic regions may all Experimental
Cell Research
35
H. J. Evans be tightly complexed with an arginine-rich histone, whose responsible for the late DNA replication in these regions. is open to experimental attack, but at present the reason delay of DNA replication in heterochromatin is unknown.
late removal is This possibility for the specific
SUMMARY
1. A significant incorporation of 3H-thymidine into the DNA of root tip cells of Vicia faba is shown to occur within 5 min of exposure to the labelled precursor. 2. When roots are exposed to water after a 30 min 3H-thymidine treatment, all the labelled compound is used up or washed out within 10 min of removal from the active solution. However, post-treatment of roots with a high concentration of inactive thymidine, following a water wash, results in a slow depletion of the “hot” pool, labelled thymidine being available for incorporation into DNA for up to 40 min after the removal of the roots from the “hot” solution. 3. The distribution of grain counts between nuclei exposed for short periods to 3H-thymidine does not conform to a Poisson distribution, the data suggesting a basic exponential rate of incorporation by the cell nuclei. .+. Observations on interphase nuclei and metaphase chromosomes of cells exposed to 3H-thymidine at various periods during the S phase show (a) that DNA replication is initiated at a large number of sites over the chromosome complement, there being no sequential progression from the distal to proximal chromosome regions, and (b) that replication in the heterochromatic chromosome zones is confined to late S. 5. The available information on the time of DNA replication in eu- and heterochromatin in a variety of species is reviewed, the evidence indicating that positive heteropycnotic regions, whether in sex chromosomes or in autosomes, always undergo a late replication. The possibility that this delayed replication of heterochromatin is associated with genetic inactivity and the presence of arginine-rich histone is discussed. The author is indebted to Miss J. Bodycote and Mr. T. R. L. Bigger for their able technical assistanceand to Mr. D. G. Papworth for statistical advice.
Experimental
Cell
Research
35
3H-thymidine
and patterns
of DNA
replication
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