the synthesis of deoxyribonucleic acid and nuclear histone...

J. Cell Sci. 5, 321-332 (1969) 321

Printed in Great Britain

THE SYNTHESIS OF DEOXYRIBONUCLEIC

ACID AND NUCLEAR HISTONE OF THE

X CHROMOSOME OF THE REHNIA

SPINOSUS SPERMATOCYTE

D. P. BLOCH AND CHRISTINA TENGDepartment of Botany and Cell Research Institute, University of Texas,Austin, Texas, U.S.A.

SUMMARY

The X chromosome of the Rehnia spinosus (Orthoptera) spermatocyte exists in a vesicleseparate from the rest of the nucleus during its replication. This chromosome is typicallyheterochromatic, and late replicating. After replication the chromosome vesicle fuses with thenucleus. Cytophotometric determination of DNA and histone during replication of the chromo-some revealed two types of histone. One class increases in amount in proportion to the DNA.The second class remains constant as DNA doubles, and probably increases later. Autoradio-graphic studies of incorporation of amino acids indicates that histone labelling occurs duringchromosome replication. However, a lag in amino acid incorporation suggests that DNAreplication in the X chromosome, while accompanied, or closely followed, by complexing withhistone, is not necessarily coupled with its synthesis.

INTRODUCTION

Chromosome replication entails synthesis of chromosomal constituents and theirassembly into the whole. Microspectrophotometric studies of DNA and histone ofproliferating cells have shown that the staining of these two substances increasesproportionately during interphase (Bloch & Godman, 1955; Alfert, 1955; Gall, 1959;Woodard, Rasch & Swift, 1961) and this has been interpreted as indicating theirsimultaneous synthesis. Later it was pointed out that histone unbound to DNA maybe labile and elude staining (Umana, Updike, Randall & Dounce, 1962) and thathistone synthesized earlier than DNA might not have been detected. A recent auto-radiographic approach used in such a manner as to preclude loss of a labile inter-mediate histone supported the interpretation of simultaneous synthesis (Bloch,MacQuigg, Brack & Wu, 1967). This interpretation has been reinforced further bythe findings that HeLa cells in the S period contain a small polysome fraction thatsynthesizes proteins whose properties are similar to those of histones (Robbins &Borun, 1967; Borun, Scharff & Robbins, 1967) and that DNA replication is dependenton continued protein synthesis (Bloch et al. 1967; Borun et al. 1967; Brega et al. 1968).

In the last analysis, however, simultaneity must be relative. The degree to which atight relationship between the two syntheses is maintained at the chromosome, or atthe gene level, is unknown. The question is a significant one from a theoretical stand-point, for if histone is involved in the control of gene activity, and a specific pattern of

21 Cell Sci. 5

322 D. P. Block and C. Teng

permissible activity is maintained during division of differentiated cells, then a mech-anism should operate to ensure the true replication of the deoxyribonucleoprotein com-plex. If so, the synthesis or at least the presence of histone should be a prerequisite forDNA synthesis, so that assembly of the chromosome might accompany DNA repli-cation. Otherwise information of the nature of the original DNH might not survivethe process of DNA replication.

The present work is the result of an attempt to determine whether DNA and histonesynthesis occur together at the level of a single chromosome. The heterochromaticX chromosome of the Rehnia spinosus spermatocyte was selected for study becausethis chromosome is very large, and is amenable both to microspectrophotometric andautoradiographic analysis. Also of value is the fact that the chromosome is late repli-cating, and is separate from the rest of the nucleus during the 5 period, existing in its•own vesicle, or subnucleus. This is apparently devoid of RNA, suggesting that thevesicle may have to import its histone, if this material is made in the generally acceptedmanner.

MATERIALS AND METHODS

Adult Rehnia spinosus males were collected in the vicinity of Austin, Texas. This animal isA carnivorous katydid, splendidly coloured and of magnificent proportions. Unfortunately itis rare and all the experiments described here were done on specimens obtained during thesummers of 1964 and 1965. It has not been seen since then. The testes were dissected in Shaw's.grasshopper culture medium (Shaw, 1956) and either fixed immediately in 10% neutralbuffered formalin, or incubated in the medium with isotope prior to fixing. The tissues were•embedded in paraffin and sectioned in the standard manner. Sections used for autoradiography,where subsequent microspectrophotometric analysis was not required, were cut, cold, at 1 fin\.Those used for microspectrophotometric analysis were cut at 5 or 10/tm, the former formeasurement of the X chromosomes, the latter for measurement of nuclei.

[3H]Thymidine (19 Ci/mM, Schwarz Bio Research) was injected into the abdomen, or ad-ministered to the medium containing the testes in short-term cultures. The doses varied andare given in the text. The animals, or tissues, were taken at various times after administrationof the label, and prepared for microscopy as described above. [3H]Lysine (105 Ci/mM, NichemInc.) and [3H]arginine (0-2 Ci/mM, Nichem Inc.) were given in a similar manner. Sections were•coated with Kodak AR-10 autoradiographic stripping film, exposed for various lengths of time,developed, using Kodak D-19 developer (see Taylor & McMaster, 1954, for general details),and stained with toluidine blue O (Prescott & Bender, 1962).

Microspectrophotometric analysis was done using the 2-wavelength method of Patau (1952)and of Ornstein (1952). The following schedule was used for the determination of DNA andhistone of the X chromosomes. Autoradiographed preparations were examined and areas foundin which the X chromosomes were labelled, but the nuclei were unlabelled. These werephotographed, and the photographs marked for relocation of X chromosomes on the basis oflabelling or non-labelling. Both the photographs and the serial sections were examined carefullyto ensure that only whole X chromosomes were selected for measurement. Chromosomes wereeliminated where there was any indication of sectioned fragments on sections adjacent to thatcontaining the chromosome. Following selection of the X chromosomes the emulsion wasTemoved with trypsin and the silver grains removed with potassium ferricyanide (Bianchi,Lima-de-Faria & Jawarska, 1964). The sections were then Feulgen-stained. They were hydro-lysed for 25 min at 60 °C in saturated picric acid containing 5 % trichloroacetic acid, then.stained for 15 h with the conventional Schiff's reagent, this reinforced by adding 1 part of10% potassium metabisulphite solution to 5 parts of the Schiff's reagent. Sulphite rinsing ofthe slides was done in the usual manner, except for the substitution of picric acid for HC1.T h e picric acid modification was used for the purpose of ensuring retention of the histones,

DNA and histone synthesis of the X chromosome 323

some of which are quite labile (Bloch, 1966). (The picric acid Feulgen staining had been stan-dardized and subsequently improved (R. Dwivedi & D. Bloch, personal communication) and willbe described in another publication.) The selected X chromosomes were measured with themicrospectrophotometer, then restained for histone as follows. The slides were hydrolysed insaturated picric acid at 60 °C for 6 h, then stained overnight in a o-i % solution of eosin Yrbuffered at pH 8-2 with 007 M tris HC1. The staining was carried out in an ice bath, anddifferentiation in the cold with buffer, 5 min, 70 % ethanol, 5 min, and 95 % ethanol, 5 min.The slides were then dehydrated and mounted in refractive-index oil for measurement.

Measurement of the eosin-stained material proved to be more difficult because theabsorption spectrum changed with time. It became apparent in the course of trying to deter-mine the shape of the absorption spectrum that exposure to light had a profound effect onthe absorption. The slides were therefore exposed to illumination from a fluorescent tablelamp for almost a day, after which the spectral response seemed to be stabilized. The spectralabsorption was again determined, and the two wavelengths for maximal and half-maximalabsorption were found. These were seen to be constant for a number of different nuclei, andwere applied for the determination of histone in those X chromosomes whose DNA values hadbeen determined previously.

After measurement, the slides were subjected to the action of 1 M urea for 1 h, for reasonsgiven below. They were restained as above, and the X chromosomes remeasured.

Electron microscopy was done on material fixed in OsO4 and glutaraldehyde and embedded,in Araldite in the manner of Mollenhauer (1964).

RESULTS

Figure 1 shows, schematically, the relationship between the nucleus and the Xchromosome, during the early part of the premeiotic interphase. Separation of the Xchromosome from the remainder of the chromosomes is thought to result from in-complete reconstitution of the nucleus following the last premeiotic mitosis, although

jm jliii

Ci Fusion ofX-vesicleand nucleus

Fig. 1. Relationship between the X chromosome and nucleus during the early premeioticstages. The black dots represent silver grains and are used to depict the pattern ofDNA replication.

it is not known whether this is so. In any event, early premeiotic interphase cells areseen in which the X chromosome is elongated and appears to be separated from therest of the nucleus. This chromosome tends to round up and is almost sphericalduring the S period. The nuclei incorporate thymidine early, the X chromosomeslater. Some sections can be seen in which all the silver grains overlie the nucleus,others in which all the grains overlie the X chromosome. The sections shown inFigs. 3 and 4 are of stages in which labelling can be seen both in the X chromosome andin some of the later replicating chromatin within the nucleus. Some time after DNA


synthesis is completed, the X chromosome elongates slightly, and the vesicle con-taining it fuses with the nucleus.

Figures 5 and 6 show sections through both the nucleus and the vesicle. In Fig. 6a bridge can be seen linking the two. Note that the membrane of the vesicle is similarto that of the nucleus, and is continuous in the regions of the bridge. It is not knownwhether the vesicle and nucleus are ever completely separated, that is, whether thebridge is at any time completely absent during the stages where the two bodies appearto be separate in any one section.

Fig. 2. Relationship between DNA amounts and histone amounts in the X chromo-some during DNA replication. Each point represents data from a single nucleus. Ex-cept for one of the nuclei that fell off the slide during urea extraction, every nucleus isrepresented by a pair of points, the open circles relating DNA content to total histone,and the closed circles relating DNA content to histone that is not extracted by urea.The lines are calculated regression lines relating histone staining to DNA staining.

Sections similar to and including the one shown in Fig. 3 were mapped and stainedas indicated, for comparison of the contents of DNA and histone within the X chro-mosomes during replication. The initial results, shown in the upper line in Fig. 2,indicate a parallel but disproportionate increase in both substances, the DNA doubling,but the histone increasing only by a factor of about 50%. An approximately straight-line relationship is maintained, which when extrapolated does not go through theorigin. It was known from previous studies that condensed chromatin, such as occursin the metaphase chromosome, and also in the heterochromatic X chromosome of theChortophaga viridifasciata (grasshopper) spermatocyte, contains an extra charge ofhistone that binds eosin when this stain and fast green are used together (Bloch, 1966).The protein is usually quite labile and may be easily removed with urea treatment.As it is possible that a different relationship may exist between the syntheses of these


two histones and DNA, the slides were treated with urea and restained, and the re-maining histone, refractory to urea treatment, determined. The result is shown on thelower line of Fig. 2. It is seen that the residual histone, thought to be the typical his-tone, increases proportionately to the DNA during chromosome replication. Here theregression line, calculated by least mean squares, goes very near the origin. It isconcluded that there are two fractions of histone within the X vesicle; a typical, or'ground level' histone, which is constantly associated with the chromatin, and a secondfraction, perhaps the previously described 'eosinophilic' histone (Bloch, 1966), whichis thought to function in the condensation of chromatin and control of nuclear activity.The typical histone doubles along with the DNA. The other histones apparently donot.

Incorporation of amino acids into the X chromosome

An attempt was made to determine whether a pattern of labelling of the X chromo-some with amino acids similar to that obtained with thymidine could be discerned.Needless to say, any such pattern would be at least partly obscured by the proteinsynthesis that occurs generally within the cells. Of primary interest was the comparisonof labelling of the S period and non-5 period X chromosomes (S period with regardto the nucleus and to the X chromosome will henceforth be designated Sn and Sx

periods respectively). The main problem lay in the identification of the Sx period Xchromosomes. An initial approach sought to relate incorporation of [3H]thymidineinto the X chromosome with size of its associated nucleus, using the latter as a para-meter by which incorporation of amino acid into the X chromosome could also berelated.

The results were equivocal for a number of reasons. No population of X chromo-somes with an appreciably higher amino acid incorporation was at first apparent.The number of grains overlying the cells increased with increasing times of exposureof the tissue to isotope. This increase appeared to occur fairly evenly both in thenucleus and the cytoplasm, precluding the positive-negative scoring that facilitatesthe study of thymidine incorporation. Moreover, the presence of X chromosomes inthe Sx period among the population could not be established indirectly with anydegree of certainty, for nuclear size proved to be a very unreliable index for stagingthe cells within the period in which accurate assessment of stage was crucial. Itbecame clear that comparison of amino acid incorporation into X chromosomes in theSx and non-S'x periods would require a direct and independent identification of theX chromosomes with regard to stage. So, tissues were incubated in a medium con-taining a mixture of [3H]thymidine and pHJlysine, 20 /iCi of the first, and 40 /«Ci ofthe second, per ml. Samples were taken at various times, fixed, sectioned and auto-radiographed. Slides containing adjacent sections were set aside for later use in deter-mining radioactivity of protein. All nuclei and X chromosomes were radioactive,because of the labelled lysine. However nuclei in the Sn and X chromosomes in theSx period could be identified easily because of the high density of grains attributableto incorporation of the thymidine. All that remained was to remove label due to


thymidine after identification of the X chromosomes with regard to their stage, andthen determine the labelling attributable to incorporation of the lysine.

The following preliminary experiment was done to assess the lability of DNA label,and stability of protein label, to hydrolysis with trichloroacetic acid. Adjacent sectionsof testes, one testis labelled with [3H]arginine, another with pHJthymidine, were puton slides. One slide of each was subjected to hydrolysis with 5 % trichloroacetic acidat 90 °C for 15 min. The other was used as a control. Counts were made of the grainsoverlying sections of the same cyst, present on both the control and the hydrolysedslide. Three cysts were scored in the case of the arginine-labelled material. Theresults were, for the control slide, 2239 grains, and for the hydrolysed slide, 2460grains. The increase is not significant. Such an increase might be expected because oflower self-absorption after removal of the nucleic acids by hydrolysis.

Table 1. Incorporation of labelled lysine into the X chromosomesduring spermatogenesis

(The numbers refer to the grains overlying and touching the edges of the X chromosomes. Thestandard errors are also given.)

Stage

Gonial or^-spermatocyte

Gonial or more probablyGL spermatocyte

Gi spermatocyteSn spermatocyteSj spermatocyteG2 spermatocyte, Xstill outside nucleus

Gt spermatocyte, Xfused with nucleus

Spermatids bearingX chromosome

• This is a gross overestimate because

No. of chromo-somes counted

14

6

15162611

27

34

Average number ofgrains ±S.E.

I-2I ± O-27

4-33±O'42

4-80 ±0-59•ic-31 ± 1-32

n-oo± 0-90636 ±076

1'92 ±0-07

i-oo ±0-06

of high cytoplasmic labelling during this stage.

In the case of the thymidine experiment, the control cyst showed 302 grains; thehydrolysed, 26 grains (a loss of about 92% of the DNA). The 92% figure is probablyan underestimate, because of the stability of background grains to hydrolysis, and theremoval of thymidine by trichloroacetic acid may well be quantitative. In any event,such hydrolysis can be relied upon to remove [3H]labelled nucleic acids selectively, sothat pHJthymidine labelling may be used for identification of X chromosomes inthe Sx period in doubly labelled material. Adjacent sections on other slides, con-taining the same cysts (hence the same stages), can be used after hydrolysis for assayof amino acid incorporation.

Several tubules were found that contained cysts in which the stages were readilyidentified. The results of the grain counts are given in Table 1. This preparation was


one in which label had been administered in vitro for 8 h before fixing the tissue. Allthe cells were labelled with amino acid. The cytoplasm of the cells in the Sn periodwas very highly labelled, much more so than in cells during other stages. For thisreason, counts made of grains overlying X chromosomes in the Sn period are un-reliable. Many, if not most, of these grains lie at the periphery of the chromosome,and doubtlessly belong to the surrounding cytoplasm. The counts are given here onlybecause they show an upper limit. The actual incorporation by the X chromosomeduring the Sn period must be somewhat lower than that during the 5X period.

Incorporation of amino acids into the X chromosome during the Sx period issignificantly higher than that during subsequent and earlier stages.

DISCUSSION

During replication of the X chromosome in the Rehnia spermatocyte there is aprecise and parallel doubling of DNA and of one fraction of its associated histonecomplement, designated here the 'ground level' histone. This fraction is thought torepresent the histone common to the chromatin of most cells. The second fraction,believed to be the 'eosinophilic' histone (Bloch, 1966) found in inert nuclei (Claypool& Bloch, 1967) and in the condensed chromatin of mitotic and meiotic cells, remainsessentially constant during the X chromosome replication period. This latter fractionprobably increases sometime during pre-meiotic interphase. The heteropycnotic con-dition of the X chromosome is maintained during a number of cycles of gonial divi-sions, and it seems likely that the special histone would keep pace with the rest of thechromosomal constituents during the divisions. Some casual observations on Pedio-dectes (unpublished) and recent work of Bogdanov, Liapunova, Sherudilo & Antropova(1968) on the cricket suggest the post-51 period as the time of its increase. The finitevalue of the intercept of the regression of total histone on DNA, on the ordinate, issignificantly different from zero, indicating that the total histone is not proportionalto DNA. The departure from proportionality is attributable almost entirely to thespecial histone, since the proportionality between DNA and 'ground level' histone isvery exact.

The exceptional relationship between the X chromosome and autosomes in this celldeserves comment. The separation of the X in other related species had been notedbefore (Davis, 1908). It is not of very common occurrence however, and was not seenin another genus, Pediodectes, that is in many respects similar to Rehnia. The Xchromosome in many of the Orthoptera is heteropycnotic and late replicating (Lima-de-Faria, 1959) so the separation is not a prerequisite for the precocious or continuouscondensation of this chromosome. Neither is separation essential for the differentialhistone content of this chromosome, as revealed by its staining with the fast green-eosin method. The eosinophilic reaction of the X chromosome was first seen inChortophaga viridifasdata in which the X chromosome remains within the nucleusproper.

The separation in Rehnia during replication is of significance insofar as the methodof analysis is concerned, for it permits determination of DNA and histone of the X


chromosome free from error due to interference by other chromosomes. Autoradio-graphic analysis is similarly facilitated. Of importance also is the implied separationof function, for the vesicle containing the chromosome contains little if any RNA,probably is inert, except for chromosome replication, and may depend upon the parentnucleus for functions that are properly nuclear. There are conflicting ideas concerningthe ability of nuclei to carry out protein synthesis. Allfrey, Mirsky and their collabora-tors (Allfrey, 1963), have indicated repeatedly that the nucleus is capable of carryingout protein synthesis utilizing its contained ribosomal machinery. Perry (1967),on the other hand, holds that nuclei do not contain ribosomes. The question remainsunresolved, perhaps because of the difficulty in generalizing from the observations madeon a few disparate cell types. The likelihood that the X vesicle in Rehnia performs itsown protein synthesis is even less than that for the nucleus. Thus the X affords aunique opportunity for investigating the relationship between histone synthesis andits assembly with DNA.

The synthesis of the histone that is incorporated into the X vesicle during the Sx

period probably occurs during and somewhat earlier than the Sx period itself. Thelack of extensive labelling of the Sx period X chromosome with amino acids afterperiods of incubation of tissue for as long as 8 h suggests that some of the protein ismade earlier. Shorter incubations gave little incorporation. However one set of slidesfrom tissue given an 0-5 h-pulse and exposed for 6 months showed excellent thymidinelabelling, no labelling of the SK X chromosome, and fair labelling of the Sn nucleus.It is unlikely that the low rate of incorporation is due to the presence of large endo-genous amino acid pools, for protein synthesis can easily be detected after less than anhour of incubation of the tissue under the conditions of culture.

It is interesting that the greatest grain density is seen over cells in the Sn period, thetime when the DNA of the nucleus is undergoing replication, and that the X vesicletends to cast a shadow in the heavy radioactivity profile of the cells during this period.The extent to which this incorporation in the cytoplasm and the nucleus representshistone synthesis is not known. De (1961) and Zweidler (1965) have also found anincrease in amino acid incorporation by nuclei during the 5 periods of other systems.The parallel increase in the DNA and 'ground level' histone of the replicating Xchromosome can best be described as a DNA replication coupled with assembly withpreviously synthesized histone.

The extent to which asynchronous synthesis of DNA and histone is exceptionalremains to be determined. Perhaps meiosis is atypical in this respect. Bogdanov et al.(1968) found that synthesis of histone lags behind that of DNA during the spermato-genic prophase in the cricket. The X was not analysed, apart from the nucleus in theirstudies, and it was not considered feasible to follow the analysis during later stages inours, because of the fusion of the nucleus and the X vesicle. It is possible that theRehnia X, and the Grillus nucleus might behave similarly, some histone being acquiredduring DNA replication, and another portion later. Recent work dealing with thesynthesis of histone during premitotic interphase puts the time of histone synthesisduring the DNA replication period (Bloch et al. i967;'Robbins & Borun, 1967;Borun et al. 1967). The later works, however, and an earlier paper on synthesis of


spermiogenic histone (Bloch & Brack, 1964) put the site of histone synthesis in thecytoplasm. Spatial separation of the synthesis of DNA and histone might seem topreclude a precise synchrony of synthesis, and rule out synchrony as the result of anobligatory coupling of the two processes. On the other hand, the recent finding ofinitiation of DNA synthesis at the nuclear membrane (Comings & Kakefuda, 1968)leaves open the possibility of coupling in spite of the fact that the two events may occuron opposite sides of the membrane.

The authors are indebted to Mrs Margie Bryant for providing the electron micrographs ofthe Rehnia nuclei, and to Dr H. V. Rao for his contributions to the technique of short-termculture of Rehnia tissue.

This work was supported in part by grants from the Damon Runyon Memorial Fund forCancer Research (DRG 905), the National Science Foundation (GB 6051), and United StatesPublic Health Service (GM 09654).

REFERENCES

ALFERT, M. (1955). Quantitative cytochemical studies on patterns of nuclear growth. In FineStructure of Cells, pp. 137-163. New York: Interscience Publishers.

ALLFREY, V. G. (1963). Nuclear ribosomes, messenger RNA, and protein synthesis. Expl CellRes. (Suppl.) 9, 183-212.

BIANCHI, N., LIMA-DE-FARIA, A. & JAWARSKA, H. (1964). A technique for removing silver grainsand gelatin from tritium autoradiographs of human chromosomes. Hereditas 51, 207-211.

BLOCH, D. P. (1966). Histone differentiation and nuclear activity. Chromosoma 19, 317-339.BLOCH, D. P. & BRACK, S. D. (1964). Evidence for the cytoplasmic synthesis of nuclear histone

during spermiogenesis in the grasshopper Chortophaga viridifasciata (de Geer). J. Cell Biol.22, 327-34°-

BLOCH, D. P. & GODMAN, G. C. (1955). A microspectrophotometric study of the syntheses ofdesoxyribonucleic acid and nuclear histone. J. biophys. biochem. Cytol. 1, 17-28.

BLOCH, D. P., MACQUIGC, R. A., BRACK, S. D. & Wu, J. R. (1967). The syntheses of deoxy-ribonucleic acid and histone in the onion root meristem. J. Cell Biol. 33, 451-467.

BOGDANOV, Y. F., LIAPUNOVA, N. A., SHERUDILO, A. I. & ANTROPOVA, E. N. (1968). Uncoup-ling of DNA and histone synthesis prior to prophase I of meiosis in the cricket Grillus(Acheta) domesticus L. Expl Cell Res. 52, 59-70.

BORUN, T. W., SCHARFF, M. D. & ROBBINS, E. (1967). Rapidly labelled polyribosome associatedRNA having the properties of histone messenger. Proc. natn. Acad. Sci. U.S.A. 58, 1977-1983.

BREGA, A., FALASCHI, A., DECARLI, L. & PAVAN, M. (1968). Studies on the mechanism of actionof pederine. J. Cell Biol. 36, 485-496.

CLAYPOOL, C. J. & BLOCH, D. P. (1967). Synthesis of ribonucleic acid and histone change during8permatogenesis in the grasshopper Chortophaga viridifasciata. Nature, Lond. 215, 966—969.

COMINGS, D. & KAKEFUDA, T. (1968). Initiation of DNA replication at the nuclear membranein human cells. J. molec. Biol. 33, 225-229.

DAVIS, H. S. (1908). Spermatogenesis in Acrididae and Locustidae. Bull. Mus. comp. Zool. Harv.50, 58-158.

DE, D. (1961). Autoradiographic studies of nucleoprotein metabolism during the division cycle.Nucleus 4, 1-24.

GALL, J. G. (1959)- Macronuclear duplication in the ciliated protozoan, Euplotes. J. biophys.biochem. Cytol. 5, 295-308.

LIMA-DE-FARIA, A. (1959). Differential uptake of tritiated thymidine into hetero- and euchro-matin in Melanoplus and Secale. J. biophys. biochem. Cytol. 6, 457-466.

MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy.Stain Technol. 39, 111-114.

ORNSTEIN, L. (1952). Distributional error in microspectrophotometry. Lab. Invest. 1, 250-262.


PATAU, K. (1952). Absorption microphotometry of irregular-shaped objects. Chromosoma 5,341-362.

PERRY, R. P. (1967). The nucleolus and the synthesis of ribosomes. Prog. Nucleic Acid Res.molec. Biol. 6, 219-257.

PRESCOTT, D. M. & BENDER, D. A. (1962). Synthesis of RNA and protein during mitosis inmammalian tissue culture cells. Expl Cell Res. 26, 260—268.

ROBBINS, E. & BORUN, T. W. (1967). The cytoplasmic synthesis of histories in HeLa cells andits temporal relationship to DNA replication. Proc. natn. Acad. Set. U.S.A. 57, 409-416.

SHAW, E. I. (1956). A glutamic acid-glycine medium for prolonged maintenance of high mitoticactivity in grasshopper neuroblasts. Expl Cell Res. n , 580-586.

TAYLOR, J. H. & MCMASTER, R. D. (1954). Autoradiographic and microphotometric studies ofdesoxyribonucleic acid during microgametogenesis in Lilitim longiflorum. Chromosoma 6,489-521.

UMANA, R., UPDIKE, S., RANDALL, J. & DOUNCE, A. L. (1962). Histone metabolism. In TheNucleohistones (ed. J. Bonner & P. O. P. Ts'o), pp. 200-229. San Francisco: Holden Day.

WOODARD, J., RASCH, E. & SWIFT, H. (1961). Nucleic acid and protein metabolism during themitotic cycle in Viciafaba.J. biophys. biochem. Cytol. 9, 445-462.

ZWEIDLER, A. (1965). Strukturund Replikation der Chromosomen. Autoradiographische Unter-suchung mit H'-Thymidin, H'-Arginin und H3-Lysin an Wurzelspiten von Allium ccpa.Zurich: Truninger.

(Received 14 February 1969)

Fig. 3. Toluidine blue-stained autoradiographed section of Rehnia testis after ad-ministration of PH]thymidine. Comparison with Fig. 4 shows that the cyst at thebottom of the photograph contains numerous cells whose X chromosomes exhibitincorporation. Grains overlying some of the late replicating regions in the nucleusproper can also be seen, x 550.Fig. 4. The same section as in Fig. 3, after Feulgen staining following removal ofsilver grains, x 550.

DNA and histone synthesis of the X chromosome


Fig. 5. Early primary spermatocyte or spermatogonial cell showing separation of thenucleus and X vesicle, x 15000; scale line represents 1 /tm.Fig. 6. Part of a cell similar to that shown in Fig. 5. This cell is thought to represent alater stage because of evidence of fusion of the X vesicle and the nucleus. Note con-tinuity of membranes surrounding the nucleus and vesicle, x 30000 approx.; scaleline represents 1 /tm.

the synthesis of deoxyribonucleic acid and nuclear histone...

Documents