THE PATTERN OF DNA SYNTHESIS I N THE CHOMOSOMES OF HUMAN CELLS

CONTAINING AN ISOCHROMOSOME FOR THE LONG ARM OF AN X

CHROMOSOME By LEONARD ATKINS’ and BERTA SANTESSON

INSTITUTE FOR MEDICAL GENETICS, UPPSALA, SWEDEN

(Received September 21st, 1963)

INTRODUCTION

UTORADIOGRAPHIC studies of the pattern of DNA synthesis in A a presumptive iso-chromosome for the long arm of the human X chromosome in cultured leukocytes have been reported by MULDAL et al. (1963), GIANNELLI (1963) and GRUMBACH et al. (1963). The iso-X chromosome was found to replicate late during the DNA synthetic pe- riod, and its labelling pattern was similar to the long arm of a morpho- logically normal X ch,romosome which duplicates late. MULDAL and co- workers concluded that the “hot” iso-X was the Barr-body- producing X in their patient and that the symmetrical labelling pattern of the two arms of the “hot” X chromosome confirmed the impression that this chromosome was indeed an iso-chromosome.

The present report concerns the pattern of DNA synthesis in the chro- mosomes of cultured skin cells with an iso-chromosome for the long arm of an X chromosome obtained from a patient with Turner’s syn- drome.

Materials and Methods

Peripheral blood cultures were carried out by a modification of the method of MOORHEAD et al. (1960). The second transplantation of a skin culture was used for karyotypic, sex chromatin and autoradio- graphic studies.

’ Permanent address: Massachusetts General Hospital, Boston 14, Massachusetts, U.S.A.

68 LEONARD ATEINS AND BERTA SANTESSON

The skin culture was pulse-labelled with tritiated thymidine (New England Nuclear, specific activity 2 curies per mM), 1 pc per ml. of medium, for 15 minutes at 37’ C, and the cells were harvested 4 hours later with colchicine added during the final half hour. Culture, labelling and harvesting procedures were identical to those used by ATKINS et al. (1963). 111 adequately spread, analyzable metaphase plates were photo- graphed before application of the autoradiographic stripping film (Ko- dak AR 10). Film exposure time was 5 days. A Feulgen stain of cul- tured skin cells growing on a coverslip was done for examination of the sex chromatin of interphase nuclei.

RESULTS

20 of 40 leukocytes analyzed had 45 chromosomes and an XO sex- chromosome pattern, and 20 cells had 46 chromosomes with an iso-X,X sex-chromosome constitution, The chromosome counts of metaphase plates obtained from skin were consistently 46 with an iso-X,X sex-chro- mosome constitution in all 11 1 cells. The autosomes appeared normal. A sex chromatin body was found in 61.5 ”/. of interphase nuclei. It was not possible to be certain of any difference in size of the sex chromatin body as compared with the normal.

After development of the autoradiographic film 21 metaphase plates were found to be labelled, and these were photographed and analyzed in detail with the aid of photographs of the same cells taken before the stripping film was applied (Figs. 1, 2 and 3). In all of the labelled plates differential labelling of a large metacentric chromosome compatible with an iso-chromosome for the long arm of the X was noted. In 10 cells the entire iso-X chromosome was covered with grains. Because of the very large number of confluent grains it was not possible to obtain ac- curate grain counts over the “hot” X or over the other chromosomes in the complement. In 7 cells there was symmetrical labelling over both arms of the iso-X chromosome with absence of grains over the region of the centromere. In these cells the average grain count per cell, which was much less than in the cells where the entire iso-X was labelled, ranged from 60 to 120, and each “hot” X had a count ranging from 10 to 45. The remaining 4 cells were lightly labelled throughout the chro- mosome Complement with an average grain count per cell ranging from 65 to 80. In these cells 7-15 grains were scattered over each iso-X chro- mosome.

In the cells with heavy labelling over the entire iso-X chromosome a heavy concentration of grains was found over the centromere of both

4

Fig. 1 . l lctal~hasc plnte and karyolypc prepared from a cell photographed before application o f the, aiitoradioRra1,hic stripping film. The sex vliroiiiosonics lic to the right o f the vertical line. Note the relntivctly sinall size o f the i so-S cliroiiiosonic.

Fig. 2. Metaphase plate and and karyotype prepared from the same cell as in Fig. 1 after development of the autoradiographir film. Note the heavy labelling of the entire iso-X chromosome which lies at the periphery of the metaphase plate. Centronieric labelling is present in one member of pair 1 and labelling adjacent to the centromere is seen in its homologue. Heavy labelling over pair 16 and over two members of group 13-15 is present. Several chromosomes of the 6 1 2 group

show considerable labelling. Note absence of grains ovw the members of group 19-20.

f

f Fig. 3. P a i r 3, the iao-S and the G-S group froin 3 metaphnsr platcs in which the entire i s o - S w a s lal~rl led (left 1 and f r o m 3 plates in which the i so-S w a s S ~ I I I I I I ~ I - rically lalwllctl in the arnis (right I . The first two chromosoiiies o n thr l e f t o f r:ich set arc pair 3 . the neat rhromosome is the i so-S . and the threc rcniaining chroiiio- sonirs a r c the 6-S group. Each set consists o f clironiosoiiics I'roiii platc~s photo-

graphed before and after npplication of tlir stripping f i h .

PATTERN OF DNA SYNTHESIS IN HUMAN CELLS 69

members of pair 1 in five cells. One cell showed heavy centromeric la- belling in one member of pair 1, and its homologue was heavily labelled in the area adjacent to the centromere. In an additional two cells heavy centromeric labelling was present in one member of pair 1. In one cell there was heavy labelling immediately adjacent to the centromere in one arm of both members of pair 1, and in another cell one member of pair 1 showed heavy labelling on either side of the centromere over the inner third of each arm. Occasional scattered grains were noted through- out the length of both arms of pair 1 in many cells in addition to the above findings. Heavy labelling in the region of the centromere was also present in pair 3 in two cells. In two cells comparatively heavy labelling was found throughout the long arms of 2 members of group 4-5, and the other two members of this group were heavily labelled over the short arms. In three other cells there was comparatively heavy labelling of the short arm of one member of group 4-5. One to three chromo- somes of group 6-12 showed heavy labelling of centromeric and adja- cent regions in each cell except for one cell which showed considerable labelling throughout the complement. In the latter cell eight chromo- somes in the 6-12 group showed heavy labelling in centromeric re- gions. Heavy labelling of the short or long arms was also seen in several chromosomes of the 6-12 group in some cells. The identity of the la- belled chromosomes in this group varied considerably so that 12 mem- bers of the group were at one time or other tentatively identified as being differentially labelled. In three cells two members of group 13-15 showed heavy labelling which involved mostly the long arms. Both members of pair 16 were found to be heavily labelled in 5 cells. Heavy labelling of two members of group 21-22 was seen in one cell, and one member of this group was heavily labelled in two cells.

In the cells with symmetrical labelling of the arms of the iso-X chro- mosome without centromeric labelling both members of pair 1 showed heavy centromeric labelling in four cells. In two additional cells one member of pair 1 showed centromeric labelling. In one of the latter cells the homologue was not labelled, and in the other cell heavy labelling was noted immediately adjacent to the centromere. In two cells there was heavy labelling of the short arm of a member of group 4-5, and in a third cell the short arm and centromeric region of a member of group 4-5 was heavily labelled. Three members of group 6-12 showed heavy centromeric labelling in two cells, and in a third cell the centro- meric region and the medial portion of the long arm of one chromo- some was heavily labelled. The heavily labelled chromosomes were ten-

70 LEONARD ATEINS AND BERTA SANTESSON

tatively identified as numbers 8, 9, 10 and 11. Two cells showed con- siderable labelling of both members of pair 16, and in two other cells one member of pair 16 showed comparatively heavy labelling. It was noteworthy that groups 17-18, 19-20 and 21-22 showed either no labelling at all or at most a few scattered grains.

In the four cells with light labelling of the iso-X centromeric labelling of both members of pair 1 was seen in one cell; in another cell one member of pair one showed centromeric labelling; a third cell showed an area of labelling in one arm adjacent to the centromere; and a fourth cell showed labelling of one member of pair 1 over the medial third of each arm. In these cells only a few scattered grains were noted else- where over the rest of the chromosome complement.

DISCUSSION

MOORHEAD and DEFENDI (1963) found the DNA synthetic period for growing human cells to last between 7 and 7 ' / t hours, and the G , period (interval between completion of DNA synthesis and the onset of mitosis) from 3 to 6 hours. In the present case a 4-hour interval between pulse- labelling with the isotope and harvesting of the cells was used to obtain metaphase plates that had their chromosomes labelled during the latter portion of the synthetic period. One of the X chromosomes in the nor- mal female has been shown to replicate late during the synthetic period at a time when most DNA synthesis has been completed in the rest of the chromosomes, and this late-replicating X chromosome probably forms the sex chromatin of interphase nuclei (GERMAN, 1962; GILBERT et al., 1962; GRUMBACH et al., 1963; ATKINS et al., 2963). Previous authors have concluded that it is the iso-X chromosome which forms the sex chromatin in interphase nuclei of iso-X,X cells on the basis of a direct correlation in the size of the Barr body with the size of abnormal X chromosomes.

The plates in which the iso-X chromosome was symmetrically label- led in both arms without labelling of the centromeric region were pre- sumably labelled later than the cells with the entire iso-X labelled since DNA synthesis seemed to have been nearly completed throughout the rest of the complement as evidenced by the grain counts. The centro- meric region of the iso-X apparently completes its synthesis of DNA be- fore the ends of the arms. This finding was also noted by GIANNELLI.

The pattern of autosomal replication in the cells of the case under discussion appeared to be similar in many respects to that previously

PATTERN OF DNA SYNTHESIS IN HUMAN CELLS 71

reported for normal cells. The most consistent findings were late DNA synthesis in pair 16 and in the centromeric region of pair 1. MOORHEAD and DEFENDI also found late replication in the centromeric region of pair number 1 in embryonic cells. In addition they noted late replica- tion of the centromeric region of pair 3. Centromeric labelling was found in pair 3 in two cells of the present study. Homologue asynchrony was noted in pair 1 and in pair 16 in some cells of the present case. GERMAN also noted homologue asynchrony in pair 1 as well as else- where in the chromosome complement of normal leukocytes. In the pre- sent case most of the DNA synthesis appeared to have occurred earlier in the S period in groups 17-18 and 19-20 since there was compara- tively little labelling of these groups in the late-labelled cells. MORISHIMA et al. (1962) noted late replication of a portion of a chromosome of the 4-5 group and earlier replication of one pair of group 19-20, and MOORHEAD and DEFENDI suggested that pairs 19 and 20 are early label- ling.

In 14 out of 21 labelled metaphase plates the iso-X chromosome was either the same size or smaller than the largest member of the 6-X group. The relatively smaller size of the allocyclic X in these cells, which may possibly be related to differential coiling of heterochroma- tin, has been noted by ATKINS and co-workers in a study of XXXXY cells.

In 8 of 21 labelled metaphase plates from the present case the “hot” X was located at the edge of the plate-i.e. the centromere of the iso-X chromosome lay either at the same distance from the center of the plate or more distally than any of the adjacent chromosomes. In 4 other cells the iso-X lay near the edge of the plate. The proportion (57.1 %) of peripherally and near-peripherally located iso-X’s corresponded closely to the percentage of peripherally located sex chromatin in interphase nuclei (61.5 %). GRUMBACH and co-workers found 21 of 36 late-labelled X’s from a normal female to lie in the peripheral portion of the meta- phase plate. Among 10 cells from an XXX individual they found 3 with both late-labelled X’s at the periphery, and in 5 other cells one of these X’s was at the edge of the plate. ATKINS and co-workers found that in 15 of 29 metaphase plates from an XXXXY boy the “hot” X’s were at or near the periphery of the plate; 8 plates showed 2 “hot” X’s at or near the periphery; and in the remaining 6 plates 1 “hot” X was found at or near the periphery of the plate. GRUMBACH and co-workers state that in flattened groups of metaphase chromosomes the late-labelled X chromosome is situated at the periphery more often than is attributable

72 LEONARD ATKINS AND BERTA SANTESSON

to chance, and that this finding is consistent with the peripheral loca- tion of sex chromatin in most somatic cells

SAKSELA and MOORHEAD (1962), using a modification of the usual methods of fixation, were able to cause the allocyclic X chromosome of a normal female to appear negatively pyknotic. This procedure also produced a strong enhancement of secondary constrictions in autosomes 1 , 9 , 16, a member of group 4-5 and the Y chromosome. These changes were presumed to have occurred in heterochromatic portions of meta- phase chromosomes. The areas of late DNA synthesis in some of the autosomes as well as the allocyclic X of the present case appear to cor- respond to the changes described by SAKSELA and MOORHEAD.

Acknowledgements. - 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 04662-01. We are grateful to Dr. OVE STALKLINT, Centrallasarettet, Lidkoping, for re- ferring the patient. This work was part of a research program supported by grants to Professor JAN A. BUUK from the Swedish Medical Research Council, the Founda- tions Fund for Research in Psychiatry (New Haven, Conn., U.S.A.) and the U S . Na- tional Institutes of Health (Research Grant No. B-4320).

SUMMARY

Autoradiographic studies of DNA synthesis in the chromosomes of human skin cells containing an iso-chromosome for the long arm of an X chromosome showed an asynchronous labelling pattern with late rep- lication of the iso-X chromosome in all labelled metaphase plates. The centromeric region of the iso-X completed its DNA synthesis before the arms. Among the autosomes late labelling was found to occur in the centromeric region of pair 1, and pair 16 showed late replication. Late- labelled foci were also found in pair 3 and groups 4-5, 6-12, 13-15 and 21-22. Some cells showed homologue asynchrony. Most of the smaller chromosomes appeared to complete DNA synthesis compara- tively early.

The iso-X chromosome appeared smaller than its expected size in two-thirds of the metaphase plates examined. This finding may be re- lated to differential coiling of heterochromatin.

The late-labelled iso-X chromosome was found at or near the periph- ery of the metaphase plate almost as frequently as the sex chromatin was found in the peripheral portion of the nucleus in interphase. This finding was interpreted as evidence for the derivation of the sex chro- matin body from the iso-X chromosome in these cells.

PATTERN OF DNA SYNTHESIS IN HUMAN CELLS 73

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