H E R E 5 8 - 3

CHROMOSOME DNA SYNTHESIS I N CULTURED NORMAL

HUMAN FEMALE SKIN CELLS By LEONARD ATKINS' and EERTA SANTESSON

(Received January loth, 1966) INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDEN

INTRODUCTION

ATA concerning the patterns of DNA synthesis of human chronio- D somes have been published by a number of workers (LIMA-DE- FARIA et al., 1961; MORISHIMA et al., 1962; GILBERT et al., 1962; MOOR- HEAD and DEFENDI, 1963; SCHMID, 1963; GERMAN, 1963).

The present study was undertaken to examine the replication pat- terns of normal chromosomes of cultured female skin cells throughout the S period. Furthermore, special attention was given to the late-repli- cating X chromosome and its relationship to the sex chromatin of inter- phase nuclei.

Material and methods

A biopsy specimen of skin was obtained from a normal twenty-two- year-old woman and cultured according to the method of B ~ O K et al. (1961). The third subculture was used for autoradiographic studies. Labelling procedures were similar to those used by ATKINS e f al. (1963). Aliquots of the same culture (5 Petri dishes each) were harvested at in- tervals of 3, 4, 6, 12, 21 and 24 hours after exposure to the isotope. Col- chicine (0.01 pgm per nil of medium) was added 15 minutes before harvesting. Film exposure time was 4 days. After development of the autoradiographic film previously photographed nietaphase plates were relocated on the slides and the percentage of labelled plates for each time interval after exposure to the isotope was determined. The labelled plates were then photographed and analyzed with the aid of karyotypes prepared from photographs of the same cells made before application of the stripping film.

' Present address: Massachusetts General Hospital, Boston, Massachusetts, 02114, U.S.A.

40 LEONAHI) ATKINS AND BERTA SANTESSON

Before the isotope was added several coverslips from the same sub- cultures were stained with the Feulgen reagent and examined for the presence of sex chromatin in interphase nuclei. Only heterochromatic bodies lying against the inner nuclear membrane were considered to be sex chromatin.

ItESULTS

The proportion of labelled metaphase plates and the percentage of labelled interphase nuclei for each time interval after a pulse exposure to the isotope are shown in Table I. No labelled metaphase plates were present after 3 hours, and the first labelled plates appeared after 4 hours. Thus the G, period was between 3 and 4 hours. The peak num- ber of labelled nietaphases occurred after 12 hours, and somewhere be- tween 12 and 21 hours after labelling synthesis was complete since single chromatid labelling, indicative of an additional replication after exposure to the isotope, was seen in the 21- and 24-hour samples. Al- lowing 3-4 hours for the G , period, then the second S period must have ended about 17-18 hours after completion of the first S period. Hence the generation time was 17-18 hours.

A total of 52 labelled diploid nietaphase plates was studied in detail with analysis of karyotypes. In addition, two tetraploid cells, one har- vested 6 hours and the other 4 hours after a pulse exposure to the iso- tope, were studied. Each of these cells contained 2 heavily labelled XS. Thirty-five of the 52 diploid metaphase plates contained a relatively heavily labelled chromosome in the 6-X-12 group which was presumed to be the late-replicating X chromosome. The distribution of these plates by time intervals after a pulse exposure to the isotope is shown in Table 2.

The general labelling pattern in all cells, including those labelled early as well as those labelled late in the S period, showed asynchrony in D N 4 synthesis anlong the different chromosomes as well as con- siderable homologue asynchrony. Late-labelled cells were considered to be those containing a heavily labelled X chromosome and, except where noted, the patterns described are for the late-labelled diploid cells. The late-labelled cells in the various time intervals after exposure to the iso- tope showed similar patterns of DNA synthesis, and the data for these cells are pooled (Figs. 1, 2, 3, 4 and 5).

The patterns seen in Pairs 1, 3 and 16 are summarized in Tables 3 and 4. Pair 2 showed a tendency for labelling to occur in the centro-

Fig. 1 . Karyotype o f cell harvested -I hours after :I pulse exposure to tritiated thyinitlinc. Centroiiirric. l:il)eIling is p r c w i i t in Pairs 1 ant1 3. The presumed sex rhroniozoiiies are pI:irrtl to the right o f the vertical line. The heavily labelled X, wliirh Iny ;it the periph(~ry of the nlot:iphnse plate, appe:irs inurh smaller than its

presuiiictl ~ l 0 I i l O l O g l i e .

1;ig. 2. Iiaryotypc o f rrll harre\tctl 1' hours after n pulse exposurc to tritintetl thymidine. N o t e vom1):iratircly hclavy labelling of llic :inii\ of Pair 1 with alnence

o f label in region of centroincrc.

Fig. 3. Jlrtapliase plale ant1 liaryotype of ccll harvested 2 1 hours after a pulse exposurr to tritiatcvl thymitliiir. ‘ IIe heavily la1)cXllrd S is ~ n u c h smaller than ils presuiiird homologuc and shows cliromatid 1nl)clling. Note :ilso criitromeric lalwl- ling in Pairs 1 m t l 3 :tiid labelling in the rentronicre-proxiInal portion of long arin

region in I’air 1G.

Fig. 5 . Karyolypc o f c(,lI harvrstcd 24 hours after a pulsc exposure to tritiatrd thymidiiir. l'lie late-rcplicating S shows c1iroiii:ilid Iabrlling. Note asynchrony in

Pair 1 . Thero is labelling o w r a sccont1:iry constriction in l'air 16.

6 a 6 b

I i g . 0 a-c. Jlctaphasr plate (F a, F 1)) ant1 karyotypc i G c i of cell harvested 6 hours after :I pulse csliosiirc to tritintcd tligmidinc. The 1atc-rq)Iicating S lics at the periphery of the inetaphase platc. In the centrr of the plat(, there is an :issociation 1)rlween tlic satrllited end o f a chroniosonie o f Group 1:1--15 ant1 tlic scrontlnry constriction of :I presnnietl Number 9. The satellite region and thc sccont1:rry constriction Iioth show latc replication. I n Grouli l:i-l5 two pairs sliow l:ilwlling inostly in l l i e sntrllitr-cc~titroiiierr-~,rosi~iinl portion o f the long arni region, and thc tlrirtl pair shows 1nl)clliiig iiiostly in the tliatnl half of the

long ariii .

DNA SYNTHESIS IN HUMAN SKIN CELLS 41

nieric and proximal portions of both arms in some plates but in the ma- jority of plates there was no consistent pattern of DNA synthesis.

In Group 4-5 grains appeared concentrated over the short arm of one chromosome in 9 cells and over the short arm of two chromosomes in 8 cells. There was comparatively heavy labelling throughout this group.

In the 6-X-12 group scattered “hot” foci were seen in all cells but be- cause of the difficulty in classification of the pairs in this group definite patterns could not be ascertained save for the intense labelling over the late-replicating X and late replication of a secondary constriction in a chromosome tentatively identified as Number 9. A prominent secondary constriction in the proximal portion of the long arm of this chromo- some was seen in one member of the pair in 6 cells and in both meni- bers in 1 cell. One of these labelled secondary constrictions showed an association with the heavily labelled satellite region of a member of Group 13-15 (Fig. 6).

In 15 of 35 late-labelled metaphase plates the late-replicating X chro- mosome showed unlabelled gaps in the midportion of one or both arms. Although this type of internal labelling pattern tended to be more ap- parent toward the end of the latter portion of the S period it was not a constant finding. Diffuse labelling of the X was also seen in some cells with comparatively low grain counts, and an internal labelling pattern could be seen in some cells with relatively high grain counts. About one- half of the cells with the lowest grain counts showed a n internal label- ling pattern in the X. The four cells with the highest grain counts showed diffuse labelling of the late-replicating X. The presumptive ho- mologue of the late-replicating X chromosome as well as the other chro- mosomes in the 6-12 group showed no specific labelling pattern. Dif- fuse heavy labelling was seen over both Xs in each of the tetraploid cells labelled late in the S period.

Table 2 shows the proportion of late-replicating X chromosomes found at the periphery of the metaphase plate. The late-replicating X chromosome was considered to be peripheral if it were at the edge of the metaphase plate and its centromere lay equidistant from the center of the nietaphase plate or beyond when compared with the centronieres of the adjacent chromosomes. 54.5 ”/o of late-replicating Xs were pe- ripheral. 299 of 506 (59 %) interphase nuclei from the same subcul- tures contained a sex chromatin body lying against the inner nuclear membrane. Thus there was a reasonably good correlation between the proportion of cells with a sex chromatin body in interphase and the

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DNA SYNTHESIS IN HUMAN SKIN CELLS 43

S u n i l i c ~ of l l i e t a p l l ~ s ~

pl:rtes allalyzetl

TABLE 2. Frequency of late-replicating X chromosomes.

Sumlirr of late- replicating S cliromosonws

found at peripliery of

iiietapliase plate

Nuriiber of niet:i-

Paining late replicating S elirniiiosoiiie

pll:1sc plates con- Ti me i 11 tc r w I after exposure

to tritiatetl thgniidine

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1,alielling in centro-

portion of long arm region of both

honlologues

I ~ l i e l l i n g in centro-

portion of long arm region of oiic

11 o 111 olog ue n ii I y

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21 ,. 24 ,.

Total

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Total cells patterrl analyzed

So specific lalielling

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52 I 35 1 19 (54.5%)

Number of cells in which late- replicating S

chroIIlosollle \\.as smaller than its

presuined homologtie

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16(45 .7 96) 1

1 Number of 1 cells 1 9 1 9 1 3 5 1

proportion of late-replicating Xs found at the periphery of the cell in metaphase. Of the late-replicating Xs found in the 21- and 24-hour spec- imens 9 of 14 (64.3 %) were at the periphery of the metaphase plate. The late-replicating Xs in the late-labelled tetraploid plates were not peripherally situated.

The late-replicating X was the same size as its presumed homologue in 19 out of 35 cells, and in the remaining 16 (45.7 5 ) it was definitely smaller than its presumptive homologue. Among the relatively small late-replicating Xs it was very common to find a pyknotic bent chromo- some which in some instances was comparable in size to one of the smallest members of the 6-X-12 group.

In Group 13-15 the most frequent finding was the presence of late labelling in the satellite-short arm-centromere region. This type of pat- tern was seen in all 6 chromosomes of this group in 10 cells and in 5 chromosomes in each of 5 cells. In 23 other cells from 1 to 4 chromo- somes per cell showed this type of labelling pattern. Comparatively

__ 44 LEONARD ATKINS A N D HEHTA SANTESSON

heavy labelling in the long arni, particularly in its distal half, was seen in 2 clironiosonies in each of 1 1 cells and in 1 chroinosome in each of 7 cells.

Groups 17-18 aiid 19-20 were, in geiier:il, lightly labelled compared with the remainder of the coinplement. In Group 17-18 two members were relatively more heavily labelled than the other two in 4 cells. Two members of Group 19-20 were more heavily labelled than the other two chromosomes in this group in 4 cells.

In Group 21-22 two members were more heavily labelled than the other two in 7 cells, and in 8 cells one was more heavily labelled than the other three chromosomes. A tendency for labelling to occur in the satellite-short arm-centromere region was noted. The autosomal label- ling patterns of both late-labelled tetraploid cells were similar to those described for the diploid cells.

In the cells labelled earlier in the S period without a heavily labelled X such as was found in all the labelled plates analyzed frdin the 12- hour sample the general pattern of DNA synthesis appeared more dif- fuse than in the late-labelled plates. In Pairs 1 and 3 the centromeric region usually was not labelled and the labelling was throughout both arms. In Groups 17-18 and 19-20 the labelling was diffuse without any consisteritly heavily labelled reb' tions.

DISCUSSION

Asynchronous replication of DNA was a frequent finding in nost chromosonie pairs although certain patterns of DNA synthesis were seen, particularly in the latter portion of the S period. In general these results confirm the observations of others (GRUMBACH et al., 1963; MOORHEAD and DEFENDI, 1963; SCHIMD, 1963; GERMAN, 1963). The generation time and the lengths of the G, aiid S periods were similar to the data obtained by MOORHEAD and DEFENDI from cultured hunian embryonic cells.

Specific autosomal late-labelling pat terns were observed in the cen- tromeric region of Pair 1 and the centromeric-proximal portion of the long arm in Pair 16. These foci of late replication correspond to the sites of secondary constrictions that are frequently observed adjacent to the centromeric region in these chromosomes (SAKSELA and MOORHEAD, 1962). In addition, late-labelling was also observed in the centromeric region of Pair 3, although not as frequently :IS in Pair 1 . Late repli- cation was seen in a secondary constriction in a pair designated as num-

DNA SYNTHESIS IN HUMAN SKIN CELLS 45

ber 9. Other late-replicating foci were present in Group 4-5, frequently in the short arm of one or two chromosomes; the satellite-short arm- centromere regions in Groups 13-15 and 21-22 as well as in the long arm of a variable number of other large and small acrocentrics. The association between a late-replicating satellite region of a member of Group 13-15 and a similarly late-replicating secondary constriction of a Number 9 chromosome is of interest in view of the observations of FERGUSON-SMITH and HANDMAKER (1963). These authors found a ten- dency for the short arms of satellited chromosomes to lie close to sites where secondary constrictions have been demonstrated, and they sug- gested that these associations may indicate the position of active nu- cleolar-organizer sites. The autosomal labelling patterns in the second generation metaphase plates with chromatid labelling were similar to the late-labelling patterns noted in the first generation.

The late-replicating X chromosome tended to show an internal la- belling pattern as the end of the S period was approached. However, this was not a constant finding. Variation in the size of the late-repli- cating X has previously been noted (GILBERT et al., 1964; ATKINS et al., 1963; BISHOP et al., 1965). Correlation between the percentage of late- replicating Xs found at the periphery of the metaphase plate and the percentage of chromatin-positive interphase nuclei in cells derived from the same culture was good. Of particular interest in the present study was the equally good correlation obtained for the chromatid-labelled late-replicating Xs of the second generation. This is compatible with the Lyon hypothesis (LYON, 1962) which states that the decision as to which X chromosome will be the “inactive” one and form the Rarr body in a particular cell is made early in embryogenesis and that in all the de- scendants of that cell the same X will be “inactive”.

Acknorvledgements. - This investigation was supported by Public Health Service fellowship No. 18,666 awarded to L.A. from the Division of General Medical Sciences, U. S. Public Health Service and by the U. S. National Institutes of Health Grant NB 04662. This work was part of a research program supported by grants to Professor JAN A. B 6 6 ~ from the Swedish Medical Research Council, the Foundations Fund for Research in Psychiatry (New Haven, Connecticut, 1J.S.A.) and the U. S. National In- stitutes of Heallh (Research Grant No. B-4320).

SUMMARY

The pattern of chromosomal DNA synthesis and the generation time and G , period of cultured normal human female skin cells were studied

48 L E O N A R D ATKINS A N D B E R T A SANTESSON

with tritiated thymidine labelling. Toward the end of the S period spe- cific patterns of DNA replication were noted. Among the autosomes the most consistent findings included late replication of the centronieric re- gions of Pairs 1 and 3, the proximal portion of the long arm in Pair 16 and the satellite-shortarm-centromeric regions of the large and small acrocentric chromosomes.

One of the X chromosomes showed heavy labelling late in the S pe- riod. This late-replicating X showed variation in size, frequently being smaller than its presumed homologue. Good correlation between the pe- ripheral localization of the late-replicating X in the metaphase plate and the sex chromatin of interphase nuclei was observed in both the first and second divisions after exposure to the isotope.

Literature cited

ATKINS, L., GUSTAVSON, K.-H. and HANSSON, 0. 1963. A case of XXXXY sex chronio- some anomaly with autoradiographic studies. - Cytogenetics 2: 308-322.

BISHOP, A., LEESE, M. and BLAND, C. E. 1965. The relative length and arm ratio of the human late-replicating X chromosome. - J. Med. Genet. 2: 107-111.

BMK, J. A., SANTESSON, B. and ZETTERQVIST, P. 1961. Association between congenital heart malformation and chromosomal variation. - Acta Paediat. Sthlni. 50:

FERGUSON-SMITH, hf. A. and HANDMAKER, S. D. 1963. The association of satellited chromosomes with specific chromosomal regions in cultured human somatic cells. - Ann. Hum. Genet. 27: 143-156.

GERMAN, J. 1963. The pattern of DNA synthesis in the chromosomes of human blood cells. - J. Cell Biol. 20: 37-55.

human chromosome duplication. - Nature 195: 869-873. GRUMBACH, hl. hl., MORISHIMA, A. and TAYLOR, J. 11. 1963. Human sex chromosome

abnormalities in relation to DNA replication and heterochromatinization. - Proc. Nat. Acad. Sci. U. S. 49: 581-589.

LIMA-DE-FARIA, A., REITALU, J. and BERGMAN, S. 1%1. The pattern of DNA synthesis in the chromosomes in man. - Hereditas 47: 695-704.

LYON, MARY F. 1962. Sex chromatin and gene action in the mammalian X chromo- some. - Amer. J. Human Genet. 14: 135-148.

hfOORHEAD, P. S. and DEFENDI, V. 1963. Asynchrony of DNA synthesis in chromo- somes of human diploid cells. - J. Cell Biol. 16: 202-209.

MORISHIMA, A,, GRUMBACH, M. M. and TAYLOR, J. H. 1962. Asynchronous duplication of human chromosomes and the origin of sex chromatin. - Proc. Nat. Acad. Sci. U. S. 48: 756-763.

SAKSELA, E. and MOORHEAD, P. S. 1962. Enhancement of secondary constrictions and the heterochromatic X in human cells. - Cytogenetics 1: 225-244.

SCHMID, W. 1963. DNA replication patterns of human chromosomes. - Ibid. 2 : 175- 193.

2 17-227.

GILBERT, C. W., MULDAL, S., LAJTHA, L. G. and ROWLEY, J. 1962. Time-sequence Of