In the previous report (@) it was demonstratedthat the synthesis of DNA in synchronized cultures of HeLa cells was a complex, nonlinear process even at the level of individual cells, and thatthe synthesis of at least part of the DNA was dependent on a puromycin-sensitive process, presumably protein synthesis. The possibility waspresented that there are two or more differenttypes of physical association of the DNA within asingle nucleus, one of which under the conditionsof study was competent for DNA synthesis at thetime this process was initiated, while the remaining DNA first had to be altered in some way beforeits synthesis could take place.

In the present study further support for thisconcept has been obtained through an autoradiographic analysis of the chronological pattern ofDNA synthesis in specific chromosomes of theHeLa cell. It has been found that the DNA synthesis during the first hour of this process within agiven nucleus takes place nonrandomly among thevarious chromosomes of the HeLa cell and alsononrandomly along the length of a specific chromosome which could be analyzed. It has been dem

S This work was supported by grant CY-1897 from the

United States Public Health Service and by funds from theAlexander and Margaret Stewart Trust.

t PredoctoralFellowoftheNationalScienceFoundation.Received for publication May 11, 1962.

onstrated that the synthesis of DNA is a focalizedphenomenon in which certain chromosomal segments differ reproducibly from other segmentswith respect to their ratios of early and late labeling DNA. These results are in accord with andprovide an explanation for the observation ofother workers that certain chromosomes containDNA which is synthesized after the completion ofDNA synthesis elsewhere (8—6,and 8). It is suggested that the existence of two different DNAassociations within a chromosome may have significance in the phenomena of genetic expressionand cellular differentiation.

MATERIALS AND METHODS

Culture methods were described in the previousreport (@). Routinely, one volume of a suspensionculture containing approximately S X 10@HeLacells per ml. was diluted with 5 volumes ofBEHM (Eagle's HeLa Medium containing 10 per

cent bovine serum), and aliquots of the resultingcell suspension were placed into replicate culturevessels. After @8hours of incubation at 37°C. themedium was replaced with fresh BEHM containing 10' M amethopterin and 5 X 10' M adenosine. Following an additional 16 hours of incubation, the addition of thymidine (1.0 @g/ml) tothe culture resulted in an immediate initiationof DNA synthesis in more than 90 per cent of theculture, as was reported previously (a).

1091

Molecular Events in the Reproduction of Animal Cells

II. The Focalized Synthesis of DNA in theChromosomes of HeLa Cells*

ELTON STUBBLEFIELDt AND GERALD C. MUELLER

(McArdte MemOrial Laboratory, University of Wisconsin Medical School, Madison, Wi@courmn)

SUMMARY

HeLa cells from synchronized cultures were pulsed with tritiated thymidine duringthe synthesis of specific portions of their DNA. The distribution of this labeled DNAwas determined subsequently in specific chromosomes. Evidence was obtained forthe nonrandom and focalized nature of DNA synthesis among both the variouschromosomes of a single nucleus and the individual segments along a particularchromosome. It is proposed that the reproducible patterns of early and late labelingDNA in chromosomes reflects the existence of two different states of association ofDNA with some other chromosomal constituent. The possible biological significance ofthis finding is discussed.

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109@2 Cancer Research Vol. @,October 196@

The incorporation of thymidine-Q@-C'4 (1.0 mc.per mmole) into the perchloric acid-precipitableresidue of the cell was used as an assay for DNAsynthesis in synchronized cell populations. Tenml. cultures of about one million cells in S-ouncepharmacy bottles were used for such studies. TheC'4 assay procedures were described in the prey

ous report (@).Autoradiographic studies were done with cells

grown on 10 X 50-mm. coverslips in Leightontubes. Total culture volume was 1 ml. The DNAsynthesized during the various intervals studiedwas labeled with tritiated thymidine (0.3 curiesper mmole) . Cultures were labeled in four differenttiming patterns : (A) tritiated thymidine was present in the culture throughout the synthesis periodand was not removed; (B) the isotope was addedat the initiation of the synthesis period, but wasremoved after 1 hour of DNA synthesis, and freshBEHM containing unlabeled thymidine was then

added; (C) the isotope was added at the initiationof the synthesis period but was replaced with unlabeled thymidine after 4 hours of DNA synthesis;and (D) the culture was allowed to synthesizeDNA with unlabeled thymidine for 5 hours, atwhich point the tritiated thymidine was added andleft in the culture until it was harvested. In theseexperiments coichicine (10@ M) was added 6 hoursafter thymidine reversal, so that no cells whichentered anaphase before this time could contributechromosomes for analysis. Colchicine treatmentwas continued for 8 hours.

The coverslips with accumulated mitoses wereimmersed in warm tap water for 5 minutes tospread the chromosomes and then fixed in niethanol-acetic acid (3 : 1) for 30 minutes. After rinsingcarefully in a fresh change of fixative, the coverslips were rapidly air-dried to flatten the cells (1).The dried, unstained preparations were taped temporarily to microscope slides for detailed studywith the cells sandwiched between the coverslip andthe slide. Cells with sufficiently spread chromosomes were located and photographed with phasecontrast optics. The coverslip was then removed,inverted, and remounted on a slide with the cellsexposed. Stripping film (Kodak AR-b) was applied, and exposed at 4°C. for 1—7days, dependingon the amount of DNA synthesized during thelabeling period. The autoradiographs were developed in Kodak D-19 developer for 7 minutes(18° C.), rinsed in distilled water, and fixed in

Kodak Acid Fixer. After extensive washing inseveral changes of distilled water, the slides wereag@iinair-dried, and a second coverslip was mounted with Permount. Finally, the autographs corre

sponding to the selected chromosome complementswere located and photographed.

Photographic enlargements of the chromosomes were made with the negative reversed, sincethe coverslip culture was also inverted before theautoradiograph was prepared. Enlargements of thecorresponding autographs were made to exactlythe same size as those of the chromosomes, so thatthe two photographs could be superimposed.

For grain counting the photograph of the autoradiograph was superimposed over the photograph of the corresponding set of chromosomes ona backlighted frosted glass illuminator. The silvergrains over the chromosomes under study werethen counted from the photograph, and in the caseof the number@ chromosome a sketch of the graindistribution was made. Finally, a true count wasdetermined by direct observation of the autoradiograph with phase-contrast microscopy inorder to verify the count from the photomicrographs.

RESULTSThe experiments to be described involve the

pulse labeling with tritiated thymidine of DNAsynthesized during limited portions of the totalsynthesis period and the subsequent localization ofthat DNA in the autoradiographs of the chromosomes. In order for this to be successful, it wasfirst necessary to demonstrate that the pulsingtechnic could be used. The cultures were synchronized with amethopterin treatment for 16 hoursfollowed by the addition of thymidine-@C'4 asshown in Chart 1. Removal of the thyxnidine by achange of medium resulted in immediate cessationof DNA synthesis in the culture. Change of themedium in itself did not affect the rate of synthesisas long as thymidine was supplied at the same concentration. It was therefore concluded that anyintracellular pool of thymidine nucleotides wasnegligible and would not interfere with the labelingof specific portions of the DNA synthesis period.Since the cells have no endogenous source of thymidine while under treatment with amethopterin, it is possible to quantitate the amount of DNAsynthesis in particular chromosomal segments bya statistical analysis of silver grain distribution inautoradiographs. This overcomes one of the pitfalls of previous autoradiographic studies and provides interesting opportunities for future research.

As seen in Figure 1, the chromosomes of thisstrain of HeLa cells are remarkably similar tothose of normal human cells, although the totalnumber of particular pairs per cell is quite vanable; thus the standard nomenclature for humanchromosomes was used (7) . Several chromosomes,

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STUBBLEFIELD AND MUELLER—DNA Synthesis in HeLa Chromosomes 1093

however, have no normal counterparts and mayhave arisen through translocations and deletions;these have been designated with letters instead ofnumbers (the female chromosome X excepted) andinserted in their most likely position in the sequence. The minute chromosome could be thecentromeric region of any of the other chromosomes and was designated Z.

The chromosomes illustrated in Figure@ werefrom a cell in which only the DNA synthesizedduring the initial hour of the total synthesis periodwas labeled with tritiated thymidine (ProcedureB). In comparing a series of such preparations, it

9

8

is readily apparent that the radioactivity incorporated initially was not uniformly distributed alongthe length of chromosomes, since many chromosomal segments appear to have much less radioisotope than others. Even more striking is the frequent symmetrical distribution of the label between the daughter halves of the chromosomes.Furthermore, in many cases, chromosomes of likemorphology have similar silver grain distributionsalong their length.

Although the rates of labeling were nonuniform,there were no chromosomes that did not incorporate detectable amounts of thymidine during thefirst hour of synthesis. This was also found inchromosomes from cells labeled for only 10 mmutes at the beginning of DNA synthesis: all chro

mosonws began synthesis together but accumulatednewly synthesized DNA at different initial rates.

The variation in initial synthesis rate betweenchromosomes can be demonstrated by a companison of chromosomes 16 and 17 (Fig. 3). Although16 was slightly larger than 17, 16 accrued only

about half as much isotope in the first hour ofDNA synthesis as did 17 (Table 1). It was alsoshown that, when the cells were allowed to makeall of their DNA in the presence of the tritiatedthymidine (Procedure A), the level of isotope inchromosome 16 was slightly more than that foundin 17, in agreement with their relative lengths.

A tentative explanation for the variation in mi

tial rates of replication between chromosomes wasthat each chromosome contained a characteristicnumber of “initiallabeling sites― and that theabundance of such sites determined the initial rateof DNA synthesis for that chromosome. In such acase it was also to be expected that such “initiallabeling sites― and, correspondingly, the “latelabeling sites― would be specifically localized incertain segments of the various chromosomes. Inorder to test this concept, a detailed statisticalanalysis of the labeling process was undertaken inspecific chromosomes.

Chromosome No. @,the second largest chromosome of normal morphology, was chosen for themost detailed studies since it is easily identified

Control

0

2.5 hrs.

7

8

5

4

3

2

00 1 2 3 4 5 6 7 8 9 10

. . . 14Hours after tiymidine-2-C add*tton

U

0

E

a.@

CHART 1.—The interruption of DNA synthesis in synchronized HeLa cultures by the removal of the exogenous thymidine.

Replicatecultures weremade thymidinelesswith amethopterm for 16hours and then reversedwith thymidine-2-C―.The cultureswere then again fed thymidine-deficient medium after the indicated intervals, and the accumulation of radioisotope was measured.DNA synthesis was immediately blockedby removal of the thymi&ne@indicating the lack of a pool of stored thymidine withinthecells.

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INITIAL HOURLABELINGTOTAL PERIODI.ABELU@GCell

no.Chromosomes

16 17Cell

no.Chromosomes

16171

6

12

19

21

24

Average grains!chromosome8

687548

12111010115510131178

8.422

14141718

18231421

91715241217

1759

61

62

68

649

7

159

91416109

117

12

1115

128876

9.6

1094 Cancer Research Vol. @,October 196@

and there is no possibility of confusing the longand short arms. Chromosome@ averaged approximately 10 microns in total length, and the armratio (long:short) was 1.4g. For statistical studiesof the isotope distribution along its length, thechromosome was arbitrarily divided into five segments; the short arms were bisected, and the longarms were trisected (Fig. 4). This resulted inslightly smaller segments in the long arms than inthe short arms, since the arm ratio was less than1.50.

TABLE 1

LABELINGOF CmtoMoso@tEs 16 AND 17 DURINGTHE IN!.TIAL HouR AND TOTAL PERIOD OF DNA SYNTHESIS

chromosome it was first necessary to establish thedegree of uniformity in the distribution of DNAalong the chromosome. This has been done inChart @,which depicts the mean grain distribution

of a number of chromosomes@ which were labeledthrough the entire synthesis period. Although theobserved differences in grain distribution are notsignificant, they probably reflect suspected minorvariations in the DNA content of the five segments. As stated earlier, the three sites on the longarms are slightly smaller than the other two sitesand the observed distribution of grains is in agreement with this fact.

Chromosomes@ labeled during the initial hourof synthesis (Table 3, Fig. 4, and Chart 3) yieldeda grain distribution that was significantly differentfrom the totally labeled chromosome @.Site “c―wassynthesized initially at a relatively slow rate, whilesite “e―was quite active in comparison. The datafrom Table@ were used as expected values to compute the chi-square values for the total graincounts in Table 3, and the low probabilities forstatistical agreement indicate the significance ofthe data.

A smaller group of chromosomes@ labeled for

4 hours (Procedure C) yielded the data in Table

4. These data do not differ appreciably from those

of Table @.This indicates that by the time theDNA in chromosome@ was more than half completed, the earlier deviations in synthesis rates hadbeen obliterated.

Although the data obtained from any singlechromosome are not in themselves decisive, someindication of the agreement of the data from singlechromosome data with the aggregate data can beobtained by a relatively simple analysis. For example, the data from chromosomes@ labeled duning the initial hour of DNA synthesis indicatedthat site “c―contained less isotope than the othersites. It might therefore be asked : How many individual chromosomes@ conform to this pattern?

For each chromosome@ the site having the leastnumber of silver grains was scored ; in case of ties,the score was split evenly. If the grains were randomly distributed among the sites, one would expect each site to have the least number of grainsin about one-fifth of the cases. The same reasoningapplies to the site yielding the highest silver graincount. Accordingly, the results of these two assaysshould be complementary: a site which has a highfrequency of highest grain counts should have alow frequency of least grain counts if the data arenonrandom.

The highest and least grain count scores are indicated in Tables @,8, and 4. The results demonstrate the nonrandom nature of the labeling of in

Cells were treated with tritiated thymidine according to labeling procedures A, B, and C (see“Methods―). Before autoradiography the cellshaving No.@ chromosomes favorably positionedfor analysis were selected and photographed.Chromosome@ was identified by its arm ratio andthe ratio of its total length to that of chromosome1. The autoradiographs were exposed for 16.7, 16@,

and 34 hours, respectively. The only chromosomesdiscarded from the analysis were those in cells contaming little or no isotope; only five cells were thuseliminated. The distribution of silver grains in theautographs of cells labeled with tnitiated thymidine under procedures A, B, and C are recorded inTables @,3, and 4, respectively.

In order to attach any significance to the distribution of pulse-labeled DNA along a given

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CELL NO.Ga&zx

COUNTTOTALChromosomal

siteabcde

TABLE 2

DISTRIBUTION OF THYMIDINE H3 OVER CHROMOSOMES 2LABELED THROUGHOUT DNA SYNTHESIS PERIOD

58 2# 5 7 3 9* 265 8* 5 5 2# 25

59 4 6@ 3 4 1# 185 (7*) 5 (7*) S# 27

(4#) (4#) 6 8@ 5 278 2# 4 4 10@ 28

60 6 7* 6 (S#) (8#) 25(5#) 6 6 8@ (5#) 30

61 6 (9*)@ (9*) 4# 339* 6 (4#) (4#) (4#) 27

(9*) 5 4# 5 (9*) 32

Total 63 65 55 60 55 298Mean 5.7 5.9 5.0 5.5 5.0 27.1Leastnumber 2.0 1.5 1.3 0.8 5.3 10.9Highestnumber 1.5 4.0 0.0 3.0 2.5 11.0

62 5 5 (6*) (6k) 4# 2663 8 4 3# 4 10@ 2964 6* 3 5 2# 5 2175 5 7* 3 6 1# 22

4# 7 5 8* 5 2976 8* 4 4 5 2# 23

7 6 4 1# 8@ 269 10@ 8 5 4# 36

77 3# 6 4 10* 4 274 (1#) 3 (1#) 9* 18

78 4 (3#) 4 (3#) 5@ 19

Total 63 56 49 51 57 276Mean 5.7 5.1 4.5 4.6 5.2 25.1Leastnumber 2.0 1.0 1.0 3.0 4.0 11.0Highestnumber 2.0 2.0 0.5 2.5 4.0 11.0

78 10@ 2 4 0# 4 2079 8* (3#) 6 6 (3#) 26

(6k) 5 (3#) (6k) (3#) 2380 3# 4 6 5 7* 2581 3 13* (2#) (2#) 3 2382 (6*) (4#) (4#) (6@) (4#) 24

(7*) (7*) 3# 5 6 286 4 7 9* 2# 28

83 (4#) 6 (4#) 7* (4#) 256@ 3 2# 5 3 194# 6 6 6 8* 30

Total 63 57 47 57 47 271Mean 5.7 5.2 4.3 5.2 4.3 24.7Leastnumber 2.3 0.8 3.7 1.5 2.7 11.0Highestnumber 4.5 1.5 0.0 3.0 2.0 11.0

Group totals 189 178 151 168 159 845Groupmean 5.7 54 . 4.6 5.1 4.8 25.6Leastnumber 6.3 3.3 6.0 5.3 12.0 82.9Highestnumber 8.0 7.5 0.5 8.5 8.5 33.0

The distribution of silver grains was determined in autoradiographs of chromosomes2 whichhadbeen labeled throughout the total period of DNA synthesis with tritiated thymidine. Each chromesome 2 was divided into 5 nearly equal segments as described under “Results.―The sites of least #and highest@ grain counts for each chromosomehave been tabulated; ties are indicated by bracketsin which case the score is split. For statistical purposes the chromosomeswere divided into threegroups at random before analysis.

1095

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CJnL NO.Gasas

COUNTTOTALChromosomal

siteabcde

1 8 6 5 6 10 359 6 4 8 7 34

2 5 5 5 4 3 22S 5 9 2 1 20

S 7 4 5 3 6 254 10 S 4 5 4 26

10 4 0 6 7 271 6 3 S 11 24

6 3 5 4 6 10 281 6 5 5 10 27

7 4 7 1 7 5 249 7 3 1 8 6 25

5 3 7 6 9 30

Total 73 63 53 69 89 347Mean 5.6 4.8 4.1 5.3 6.8 26.7Leastnumber 3.0 2.0 5.0 1.0 2.0 13.0Highestnumber 4.3 0.8 1.3 1.5 5.0 12.9x2 0.21 1.37 1.31 0.00 8.85 11.74

(F=0 .020)

10 4 6 4 9 5 283 4 6 2 5 20

11 3 2 4 5 2511 3 3 S 1 8 18

7 4 6 5 4 2612 7 7 5 5 9 8814 6 4 8 5 1 1915 11 3 4 2 6 26

4 8 3 9 10 294 5 5 3 6 23

16 5 8 4 4 7 2810 6 1 3 5 258 6 4 4 5 27

Total 88 62 50 56 76 327Mean 6.4 4.8 3.8 4.3 5.8 25.1Leastnumber 0.5 1.0 4.5 5.5 1.5 13.0Highestnumber 6.0 1.0 1.0 1.0 4.0 13.0x2 1.68 0.71 1.37 1.25 3.68 8.69

(P=0 .067)

16 5 S S 6 5 2217 8 4 1 5 7 25

S S S 5 8 243 2 1 4 2 12

18 6 8 S 6 12 354 6 1 8 7 26

19 6 5 2 4 4 215 5 3 7 5 256 4 5 7 7 295 10 4 4 6 299 5 5 . 6 10 35

20 4 6 2 8 7 278 6 8 6 9 32

Total@ 74 67 36 76 89 342Mean 5.7 5.2 2.8 5.8 6.8 26.3Leastnumber 0.0 2.5 10.0 0.5 0.0 13.0Highestnumber 2.0 1.0 0.0 5.5 4.5 13.0x2 0.06 0.35 10.88 0.94 9.77 22.00

(P<0 .0005)

1096

TABLE S

DISTRIBUTIONOFTHYMIDINE H@OVERCHROMOSOMES2 LABELEDDURING THE INimu. HouR OF DNA SYNTHESIS

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CELL NO.Ga&ne

COUNTTOTALChromosomal

siteabcde21

22

23

24

TotalMeanLeastnumberHighestnumberx213

57634710688811

967.40.53.80.73S

932665395778

685.25.02.83.404

2453S2942385

544.25.50.34.506

3106656896864

886.41.01.30.207

511952121595584

977.51.04.77.1538

243528232032458726813727

89880.613.012.915.98

(P=O .003)

CELL NO.Ga.uot

COUNTTOTALChromosomal

siteabcde58

54

55

57

TotalMeanLeastnuniberHighest numberx28

75866467610

16

806.21.04 .50.056

786886545664

695.83.02 .00.345

261666677543

644.93.53 .50.023

4571253574744

705.43.01 .00.0010

4425444

135643

685.22.52 .00.0632

242824372428268827341920

35127.013.013.00.47

(P=0.94)

TABLE 8—Continued

The distribution of silver grains was determined in autoradiographs of chromosomes2 which werelabeled with tritiated thymidine during initial hour of DNA synthesis. The data have been analyzed asdescribed under Table 2. The chromosomes were divided into four groups at random prior to analysis.

TABLE 4

DIsTRIBuTIoN OF THYMIDINE fl3 OVER CHROMOSOMES 2 LABELED DURINGTHE FIRST 4 HoURs OF THE DNA SYNTHESIS PERIOD

Grain counts over chromosomes2 were tabulated as in Tables 2 and 3. Chromosomesare from cellslabeled with tritiated thymidine during the first 4 hours of DNA synthesis (i.e., approximatelytwo.thirdsof the DNA synthesis period).

1097

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Least Number Distribution

@I1@@1Lr@nHighest Number Distribution

1098 Cancer Research Vol.@ October 196@2

dividual segments of chromosome@ labeled duringthe initial hour of DNA synthesis (Procedure B):the highest and least grain-count scores are complementary and agree with the aggregate dataexactly (Chart 4).

Cells labeled with tritiated thymidine accordingto Procedure D give the pattern of chromosomesites which label late in the DNA synthesis period.

CHART 3.—Mean grain distribution of 52 chromosomes 2

labeled during the initial hour of DNA synthesis only (Procedure B). The fraction of the label in site “c―was consistentlylow, whereas site “e―was consistently labeled heavily.

Although an exact quantitation of the late labelingdata is difficult in this procedure, there was anobvious tendency for those sites which were labeled initially to be unlabeled and vice versa. Chromosome 16, which was labeled lightly in the earlystages of DNA synthesis, now appeared to containmore isotope than chromosome 17, which waslabeled initially quite heavily. In fact, chromosome 17 appears to contain very little late-labelingDNA.

During this same period No.@ chromosome accumulated more isotope in the “c―site than elsewhere, in contrast with its early labeling pattern.

The synthesis of a full complement of the DNAalso appears to be prerequisite for mitosis. In synchronized populations, the removal of the thymidine before the completion of DNA synthesisprevents the population increase usually observed(Chart 5). Omission of thymidine after@ hours and

4 hours resulted in a slight increase in cell counts;probably this reflected the asynchronous portionof the culture. Direct observation of the cell monolayer revealed very few mitotic cells. However,after 6 hours the thymidine was no longer neededfor a majority of the cells to divide. It may be thatthe DNA which is synthesized last controls the onset of mitosis through the concurrent release ofspecific information as it duplicates; this possibilityappears highly probable, since other studies havedemonstrated that the synthesis of protein is required also at this point for cell duplication.

Chromosomal Site

CHART 4.—Least and highest grain-count distributions in

thefive sites of chromosomes 2 labeled during the initial hour ofDNA synthesis (Procedure B). The two patterns are complementary. Out of 52 chromosomes assayed, 25 had grain-countdistributions in which site “c―had the least grain count.

DISCUSSIONThe evidence presented in this and the previous

paper of this series (@)supports the conclusion thatthe DNA of the HeLa cell exists in at least two different physiological associations. Under the conditions of these experiments the molecules of DNAin one type of association can be readily duplicated on addition of thymidine to the deficient cultunes, whereas the other requires some additionalevent in its conversion to a competent state. Aspresented, this conversion takes place largely duning the first@ hours of the DNA synthesis periodand depends on a puromycin-sensitive process,presumably protein synthesis (@).

The observation that segments of a particularchromosome, even when limited by the resolutionof the light microscope and the tnitium-autoradiographic technic, have reproducibly differentratios of early and late labeling DNA permits certamadditionalconclusions.First,DNAsynthesiswithin a given cell nucleus is a nonrandom processand must therefore be limited by conditions at a

1@L

Cb d Ca

6

55U)$.. 4

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0 -@@ -@- @- -Chromosornat Site

CHART 2.—Mean grain distribution of 83 chromosomes 2

labeled throughout DNA synthesis (Procedure A). The variations in the grain counts per site are not significant.

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13 14 @S 16 17 18 19 20 21 22 Z

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FIG. 1.—Karyotype of a typical HeLa cell. Chromosomes

“A,―“I.,―and “Z―are not found in normal human cells. Theother chromosomes closely resemble normal human componeiits as indicated, but they may be extensively altered in agenetic sense.

FIG. 2.—HeLa chromosomes and autoradiograph. This

cell was labeled with tritiated thymidine during the initialhour of DNA synthesis (Procedure B). Although the hackground exposure was unfortunately high in this autoradiograph, it is readily apparent that the label distribution is nonuniform along the chromosomes.

FIG. 3.—Variation in initial labeling rates in chromosomes16 and 17 of HeLa. The chromosomes shown in (A) were froma cell labeled during the initial hour of I)NA synthesis. In the

autoradiograph of this cell (B), note the relatively heavierlabeling of chromosome 17 (circles) compared with chromosome 16 (arrows).

FIG. 4.—The early labeling pattern of HeLa chromosome 2.

‘fliechromosome was arbitrarily divided into the five sites“a—c―as shown. Seven autoradiograms from different chromosomes 2 show the typical early labeling pattern. Note the lowerlabeling potential of site “c.―

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STUBBLEFIELD AND MUELLER—DNA Synthesis in HeLa Chromosomes 1099

particular focus. Second, since each segment of achromosome which was studied obviously contamed many molecules of DNA, it would appear

: also that the DNA of a particular associationisfrequently aggregated along the length of a chromosome. The significance of this latter finding,which has been noted also in the earlier studies ofTaylor (4—6),is not yet clear, but may reflect thelocation of a particular enzyme involved in theconversion and/or more interestingly relate tosome common genetic interaction. Since these chro

2.0

1.5

ACKNOWLEDGMENTSThe authors wish to acknowledge the excellent assistance

of Mrs. Eleanor Erikson, Mrs. Kathleen Deighton, and Miss

Patricia Larson in the execution of these experiments. We arealso grateful to Dr. R. L Jennrich for evaluation and suggestions for the statisticaistudies involved and to Dr. K. Kajiwarafor the availability of HeLa cell stocks.

REFERENCES1. Moounnar@, P. S.; NeWELL,P. C.; MEu@ii, W. J.; BAT

TIPS, D. M. ; and Hunozuroiw, D. A. Chromosome Preparations of Leukocytes Cultured from Human PeripheralBlood. Exper. Cell Res., 20:618—16, 1960.

2. MUELLER, G. C. ; KAJIwARA, K. ; STUBBLEFIELD, E. ; andmosomal labeling patterns have been derived from

2.5

a,U0

I0 0.5

Medium changed

I 1@I,

4th

Thymidine, 5 iig per culture

0 4 8 12 16 20 24 28

Hours after thymidine addition

CHART 5.—The effect of limited exposure to thymidine on the ensuing mitosis of synchronized HeLa cells. Replicate cultures

were treated with amethopterin for 16 hours and then supplied with 5 gig. of thymidine. The medium was replaced with thymidinedeficient medium after 2, 4, and 6 hours. The majority of the cells did not acquire mitotic competency until after 4 hours' exposure to thymidine.

the evaluation of a population of cells, they mustat least reflect some genetic effect with respect tothe organization of a particular chromosome.Third, the existence of early and late labeling sitessuggests that a single DNA molecule, while possibly linearly arranged within the chromosome, extends only over a relatively short segment. In thiscase the chromosome structure itself would haveto provide for the linear arrangement of geneticfactors.

In accord with these suggestions, experimentsare in progress to ascertain the relative genetic importance of the early and late labeling DNA to thesurvival of the cell. Also, the possibility that enviromnental factors and cellular differentiation mayplay a determinant role in the chromosomal labeling pattern is under study.

RUECKERT, R. R. Molecular Events in the Reproductionof Animal Cells. I. The Effect of Puromycin on the Duplication of DNA. Cancer Research, 22: 1084—90, 1962.

8. PAINTER,R. B. Asynchronous Replication of HeLa 53Chromosomal Deoxyribonucleic Acid. J. Biophys. & Biochem. Cytology, 11:485—88, 1961.

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1962;22:1091-1099. Cancer Res   Elton Stubblefield and Gerald C. Mueller  Focalized Synthesis of DNA in the Chromosomes of HeLa CellsMolecular Events in the Reproduction of Animal Cells: II. The

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