premature chromosome condensation: a mechanism … · cells tha to attact failedh to the dish withi...

J. Cell Sci. 10, 495-513 (1972) 495Printed in Great Britain

PREMATURE CHROMOSOME CONDENSATION:

A MECHANISM FOR THE ELIMINATION OF

CHROMOSOMES IN VIRUS-FUSED CELLS

P. N. RAO* AND R. T. JOHNSONDepartment of Biophysics and Genetics and the Eleanor Roosevelt Institute forCancer Research, University of Colorado Medical Center, Denver,Colorado, U.S.A., and Department of Zoology, University of Cambridge,Cambridge, England

SUMMARY

The fate of prematurely condensed chromosomes (PCC) that are induced following fusionbetween a mitotic and an interphase cell was studied in the homokaryons of HeLa and2 glycine-requiring mutants of Chinese hamster ovary cells. The data indicate: (1) the prema-turely condensed chromosomes are often incorporated into daughter nuclei of the fused cellsand hence they are functionally retained by the progeny; (2) in general, the only viable productsof fusion between mitotic and interphase cells are those where PCC has been induced; (3) fusionbetween mitotic and 5-phase cells leads to the most rapid loss of chromosomes, while thehomophasic fusions tend to produce more stable hybrids with regard to their chromosomenumber.

INTRODUCTION

Formation of visible, condensed chromosomes can be induced in an interphasenucleus by placing it in a proper environment; that is, within a mitotic cell. Thevisualization of interphase chromosomes achieved by the fusion of mitotic with inter-phase cells is termed premature chromosome condensation or PCC (Johnson & Rao,1970). The factors present in a mitotic HeLa cell are capable of inducing PCC indifferentiated or tissue culture cells from a wide variety of animal species includingmosquito cells (Johnson, Rao & Hughes, 1970). The condensed Gx and G2 chromo-somes so produced are discrete units with one and two chromatids, respectively. Thecondensed S chromosomes on the other hand, present a 'pulverized' appearance. Inthe present study the fate of the prematurely condensed chromosomes from Glt

S and G2 nuclei is examined in heterophasic homokaryons which complete divisionafter fusion. The data presented indicate that the PCC are randomly segregated intothe daughter nuclei, and that in many instances are retained by the progeny of thefused cells without loss of their genetic activity. The significance of these findings inrelation to the chromosome losses that are common among heterokaryons producedby cell fusion is discussed.

* Present address: Department of Developmental Therapeutics, University of Texas M.D.Anderson Hospital and Tumor Institute, Houston, Texas, U.S.A.

496 P. N. Rao and R. T. Johnson

MATERIALS AND METHODS

HeLa cells and auxotrophic mutants of Chinese hamster ovary (CHO) cells were used inthis study. The procedures for the maintenance of HeLa cells in suspension culture weredescribed earlier (Rao & Engleberg, 1965). HeLa cells were synchronized by the use of excessthymidine (2-5 HIM) double block technique (Rao & Engleberg, 1966). Synchronized popula-tions of mitotic HeLa cells were obtained by the application of a nitrous oxide block (Rao,1968). The reversal of the nitrous oxide block results in the rapid restoration of the mitoticspindle and the subsequent completion of the cell division.

Mediumchange

Harvest cells in

Colcemideadded

1

1M*

1—1

1 SG,

1 \1 1

1S

11

IG

- 2 0 2 3 9 13Hours

Fig. 1. Synchronization procedure for CHO cells. A schematic presentation of thetime schedules for obtaining synchronized populations of Gly-A and Gly-B mutantsof Chinese hamster ovary cells. • Mitotic cells collected, colcemide block reversed andcells plated in fresh medium.

The 2 glycine-requiring mutants (Gly-A and Gly-B) of Chinese hamster ovary cells werederived from the parental culture CHO-K1 and were grown as described previously (Kao &Puck, 1967; Puck & Kao, 1967). They belong to different complementation groups so thattheir fusion hybrids will grow in the absence of glycine even though neither parental cell canmultiply without glycine in the medium (Kao, Chasin & Puck, 1969a, b).

Cells of each mutant were synchronized by the reversal of a colcemide block. Mitotic cellsof 96-97 % purity were harvested by gentle shaking after the cultures were exposed to colcemide(0-05 /ig/ml) for 2 h. The mitotic cells were centrifuged, resuspended in fresh medium andplated in several plastic dishes. Cells that failed to attach to the dish within 1 h after platingwere removed by changing the medium in order to improve the degree of synchrony. About2 h after the reversal of the colcemide block virtually all the cells had completed mitosis. Thesedishes were trypsinized at appropriate times to obtain Glt S or Gt populations as shown inFig. 1. A 15-min exposure of the S cells to 3H-TdR resulted in the labelling of 9 4 % of thepopulation indicating a high degree of synchrony. However, the degree of synchrony amongthe G, population is rather poor in comparison to the other phased populations.

Hybridizations between random and synchronized populations of Gly-A and Gly-B cellswere carried out, using synchronization in the same or different phases of the life-cycle (Table 1).The procedures for cell fusion are essentially the same as followed by Kao et al. (19696). About5 x 10* cells of each mutant were washed once in cold (4 °C) Hanks's salt solution to removethe serum completely before adding 65 haemagglutinating units (HAU) of ultraviolet-inactivatedSendai virus. The fusion mixture was kept at 4 °C for 15 min and then transferred to a 37 °Cwater bath. After incubating for 45 min at 37 °C the fused cells were diluted and plated in F 12minus glycine medium (Kao et al. 1969a) in a number of plastic dishes. Each dish was inocu-lated with 4000 cells of the fusion mixture. This figure refers to the cell count taken before thecell fusion was initiated and hence does not represent the exact numbers of fused or singlecells plated. The plating efficiencies as measured by the number of colonies which developedin the selective medium were scored after 7 days.

Tab

le I

. P

roto

col f

or t

he c

olle

ctio

n of

G,,

S,

G,

and

M c

ells

from

Gly

-A a

nd G

ly-B

mut

ants

for

fusi

on

Pha

se i

n

whi

ch

cell

s ar

e H

ou

rs

Dis

h

to b

e A

I

,

no.

coll

ecte

d o

2

4 6

7 9

I I

13

15

Col

lect

M

cell

s, r

ever

se

colc

emid

e bl

ock

and

pl

ate*

i T

ryp

sin

ize

and

co

llec

t G,

cell

s -+

--+

Try

psi

niz

e an

d

coll

ect S

cell

s -+

d

-- >

T

ryp

sin

ize

and

co

llec

t G

2 ce

lls

Add

col

cem

ide

Col

lect

M c

ells

an

d re

vers

e co

lcem

ide

bloc

k

%

-+

%

Try

psi

niz

e an

d

m

coll

ect S

cel

ls

3

--+

-+

-

Try

psi

niz

e an

d

E

coll

ect

G2

cell

s c &

Try

psi

niz

e an

d

coll

ect

G,

cell

s ---f

Col

lect

M

( ce

lls,

rev

erse

1 co

lcem

ide

I bl

ock

and

pl

ate

L A

dd c

olce

mid

e C

olle

ct M

cel

ls

and

reve

rse

colc

emid

e bl

ock

-.

+

-+

-+

0

a

Add

col

cem

ide

Col

lect

M c

ells

, T

ryp

sin

ize

and

re

vers

e co

l-

coll

ect G

, ce

lls

%

cem

ide

bloc

k 2

and

pla

te

%

-+

Add

col

cem

ide

Col

lect

M c

ells

R

and

rev

erse

F

0

colc

emid

e bl

ock

-+

-+

a A

dd

col

cem

ide

Col

lect

M c

ells

, T

ryp

sin

ize

and

z

reve

rse

col-

co

llec

t G

, ce

lls

3 ce

mid

e bl

ock

and

pla

te

2 -+

-+

-+

-+

T

ryp

sin

ize

and

coll

ect S

cel

ls

-+

Add

col

cem

ide

Col

lect

M c

ells

, re

vers

e co

l-

cem

ide

bloc

k an

d p

late

--+

4

--

Ad

d c

olce

mid

e C

olle

ct M

cel

ls,

reve

rse

col-

ce

mid

e bl

ock

* 7

x 105 ce

lls

wer

e pl

ated

in

100

-mm

pla

stic

pla

tes

at 44 h

pri

or

to t

he

com

men

cem

ent

of t

he

expe

rim

ent.

t

Fol

low

ing

the

reve

rsal

of

colc

emid

e bl

ock

the

cell

s w

ere

resu

spen

ded

in

2 m

l of

med

ium

an

d p

late

d in

go-

mm

pla

stic

dis

hes.

a,

Mu

tan

t G

ly-A

; b, M

uta

nt

Gly

-B.


RESULTS

PCC and the cellular factors that influence its induction in HeLa cells

When mitotic cells obtained by treating an exponentially growing culture of HeLacells with colcemide (6*4 x io~7 M) for 22 h were fused with a random population,PCC was induced in 50% of the mitotic-interphase fused cells. The frequency ofinduction could either be increased or decreased by adding suitable chemical com-pounds to the fusion mixtures (Rao & Johnson, 1971). Presently, we are concernedonly with the possible causes for the failure of PCC induction in 50 % of the jnitotic-interphase fused cells. Earlier it was suggested that the rate of induction of PCC varieddepending upon the location of the interphase cell in the cell cycle (Johnson & Rao,1970). Thus the nuclei of Gx phase were more readily induced than those of S or G2

phase. Subsequently it occurred to us that the length of the period for which a cell isheld in mitosis by colcemide might have some effect on its ability to induce PCC in aninterphase nucleus following fusion. When colcemide is added to an exponentiallygrowing culture and mitotic cells collected after 20 h it is clear that some cells areheld in mitosis for a longer period than others. How does this factor affect the rate ofPCC induction? HeLa cells were plated in plastic culture dishes and incubated over-night. The medium, along with the floating cells, was then removed and replacedby fresh medium containing colcemide (6 -4XIO~ 7 M) ; 4h later mitotic cells werecollected by the selective detachment technique. Half of these mitotic cells wasimmediately fused with interphase cells while the other half was used for similarfusion after another 20 h of colcemide treatment. The incidence of PCC was 88 %when freshly collected mitotic cells were used in contrast to 10% when the cells wereheld in mitosis for 20 h or longer, indicating the loss of activity of the inducing factorsas a function of time. Consequently in complementation studies with the glycine-requiring mutants of CHO cells only those mitotic cells which had arrived at mitosiswithin the preceding 2 h were used.

The fate of PCC during cell division immediately following fusion

HeLa cells, reversibly blocked in mitosis by nitrous oxide treatment (Rao, 1968),were separately fused with Glt S and G2 HeLa cells each of which was prelabelledwith pHJthymidine. After the completion of cell fusion the cells were resuspended inculture medium and plated in plastic dishes. One of the dishes was trypsinized everyhour until 7 h after fusion, and cells were prepared for microscopic examinationusing the cytocentrifuge as described earlier (Rao & Johnson, 1970). In most casesthe first mitosis following fusion was completed by 7 h. At this time, colcemide wasadded to the remaining dishes to see whether the PCC, following incorporation intothe daughter nuclei of the dividing cell, would enter into mitosis during the secondcycle after fusion.

Distribution of PCC into daughter cells. Immediately following fusion betweenmitotic and interphase cells the PCC of Glt S or G2 types formed discrete entitieswhich were not interspersed with the metaphase plate of the mitotic component. Theyoften lay alongside the metaphase plate (Fig. 3). We have no evidence to suggest that

Fate of prematurely condensed chromosomes 499

the PCC of any type are integrated into the mitotic spindle. As metaphase progressedthe discrete bodies of PCC became scattered and this was most commonly seen inthe PCC of the S type (Fig. 4A). At this stage some of the PCC become intermingledwith the mitotic chromosomes. During anaphase the PCC were distributed at random.The PCC were incorporated into one or both of the groups of anaphase chromosomesor neither of them (Fig. 4B, c). The random distribution and incorporation of PCCinto daughter cells became evident by the end of telophase. Often only one of thedaughter cells received the PCC (Fig. 5 A, B). In these interphase nuclei silver grainswere localized in certain areas indicating the incorporation of labelled PCC (Fig. 5 c).

Mitosis in PCC during the second cycle. Among the cells of MjGx or MjG2 fusionsto which colcemide was added during the second mitotic cycle, chromosome spreadswere observed where some chromosomes were labelled while others were not (Fig. 6 A).This suggests that the chromatin derived from the PCC and incorporated into daughternuclei would produce normal chromosomes during the subsequent mitosis.

Formation of micronuclei in mitoticjinterphase fused cells. When there was no induc-tion of PCC in mitotic-interphase fused cells, the mitotic chromosomes, in a majorityof the cases, did not complete division but formed micronuclei (Fig. 6B). Thisphenomenon occurred even when the mitotic: interphase ratio was 1:1. These micro-nuclei derived from the mitotic chromosomes did not synthesize either DNA or RNAduring the next cycle and were probably genetically inactive (Fig. 6B). However, insome fused cells, particularly in the M\GX fusion where there was no induction of PCC,the mitotic chromosomes underwent division (Fig. 6c).

Short-term survival studies of heterophasic HeLa cells. In order to demonstrate thatheterophasic homokaryons could survive and multiply, the following experiment wasdone. Labelled and unlabelled synchronized populations were fused and the propor-tions of labelled to unlabelled mono- and multinucleate cells were scored immediatelyafter fusion. The fusion mixture was plated in plastic dishes containing coverslips.After 2 days the coverslips were fixed and processed for radioautography. Coloniescontaining 2 or more cells were scored for the presence or absence of label. The pro-portion of labelled to unlabelled colonies after 2 days was very similar to the initialratio (Table 2). This suggests that most of the heterophasic cells which constitutedbetween 13 and 20% of the fusion mixtures completed one or two divisions.

Hybrids of Gly-A and Gly-B mutants of Chinese hamster ovary cells. The long-rangeeffects of PCC induction with regard to the survival of hybrids and the elimination ofchromosomes in their progenies were studied by making various types of homophasicand heterophasic fusions between the synchronized populations of Gly-A and Gly-Bmutants of the CHO cells (Table 1, p. 497). Use of the these mutants has an advantagein that cell growth is impossible unless genetic components from each parent are activein the hybrid cell.

Effect of colcemide on plating efficiency. When random populations of Gly-A andGly-B were fused the rate of recovery of colonies of hybrids was i'56%. However,the exposure of these populations to colcemide at a concentration of 0-05 /ig/ml for2 h reduced the plating efficiency to a third of the control (Table 3). This suggeststhat not only the cells temporarily blocked in mitosis but also nearly 60% of the

5oo P. N. Rao and R. T. Johnson

interphase cells suffered damage even though colcemide was removed from themedium after a 2 h treatment. This is indicative of the fact that even a brief exposureof interphase cells to colcemide has some residual effect on these cells causing adecrease in plating efficiency.

Rate of survival of hybrids among homophasic and heterophasic fusions of Gly-A andGly-B. The average rate of survival of hybrids among the various homophasicfusions (i.e fusion between cells in the same phase of the cell cycle) was 0-46% andcomparable to the 0-5% survival of the control (Table 3). The control in this casewas the fusion (no. 2 of Table 3) involving random populations of Gly-A and Gly-B

Table 2. Short-term survival studies of heterophasicfused HeLa cells

% labelled and unlabelled cells % labelled and unlabelledType of fusion immediately after fusion colonies after 2 days

M/Gj*

M/S*

MIGt*

SIGX*

SIG2*

GJG,*

i

Unlabelled

357

396

41-6

30-8

38-5

34-6

* Prelabelled with [3H]thymidine.f The figures

mixture.in parentheses indicate

Labelled

643(i6-3)t60-4

(19-2)58-4

69-2(20-0)61-5

(i5-5)654

(18-7)

the percentage

Unlabelled

35

44

32

37

46

45

of heterophasic

Labelled

65

55

68

63

54

55

cells in the fusion

which were exposed to colcemide for a period of 2 h. The extremely low rate ofsurvival was due to the low fusion index of about 15%. Of this 15% only half wereproducts of fusion between Gly-A and Gly-B which can grow in the selective medium.About 50% of these hybrids were multinucleate cells with more than 2 nuclei andwould presumably fail to develop into colonies due to anomalies in mitosis. Thesefactors brought the plating efficiency of the fused cells down to 3 %. A brief exposureto colcemide during the synchronization procedures further reduced the plating effi-ciency to 0-5-1 % and this was close to the value we actually obtained.

The average plating efficiency of the heterophasic fusions, i.e. 5a/G1b, <Sa/G2

b andG1

a/G2b, was 0-28% (Table 3). The plating efficiencies were variable in fusions

involving mitotic/interphase cells. The rate of survival in the M.*\Sb fusion was only0-062 % and was significantly lower than that of any other fusion, including MajG^and Ma/G2

b fusions (Table 3). The possibility of PCC induction among the hetero-phasic fusions such as SjGx, S/G2 and GJG2 is relatively greater than among the


homophasic fusions. The highest incidence of PCC, ranging from 80 to 90%, wasobserved among the binucleate cells of the MjGx, MjS and MjG2 fusions. It thusappears that the high incidence of PCC among mitotic/interphase fused cells is acontributing factor to the low rate of hybrid survival.

Table 3. Rate of survival of hybrids in homo- and heterophasic fusionsbetween synchronized populations of Gly-A and Gly-B mutants

Random1

2

3Homophasic

4567

Heterophasic(i) Mitosis/interphase

89

1 0

Type offusion

R*/Rb

R* + Rb(v//o virus;

M*/Mb

GS/GSSI'Sb

G,'/Gib

Average

M'/GfM"/Sb

M»/G,b

(ii) Interphase/interphase11

1 2

13

R, Random population; n.ml) for 2 h before fusion.

S*/Gb

S*/Gtb

G?i»/G,b

Average

No. of coloniesper 1000 cells

of eachparental type

1 5 6

) o-o

4-0

5-54-64'4

18-5/4 = 4'6

2-O

O-621-56

3-2

2-7

2'5

8-4/3 = 2-8

Relativeplating

efficiency, %

109-0

ioo-o

43-SI3'S34'O

6i-o

, parent Gly-A; b, parent Gly-B; °, exposed to colcemide (0-05 fig/

Chromosome foss in the progenies of homophasic v. heterophasic fusions. On day 7 afterfusion, cells from a number of hybrid colonies of each fusion were separately pooledinto dishes, incubated for another 5 days and chromosome numbers determined forthe bulk populations on day 12. A total of 100-150 chromosome spreads werecounted for each type of fusion. The frequencies of cells with various chromosomenumbers expressed as a percentage of the total chromosome spreads counted areshown as a histogram (Fig. 2).

The patterns of distribution of the various chromosome frequencies are similar toboth homophasic and heterophasic fusions with the exception of one; that is, theMjS fusion. The modal value for the MjS fusion is 37 while it is 39 or 40 chromosomesfor the rest of them. It is necessary to emphasize here that the lowest plating efficiencywas observed in the MjS fusion indicating a possible relationship between chromosomeloss and hybrid survival. The modal chromosome numbers obtained from Fig. 2


are in good agreement with the mean chromosome numbers for each sample of thepopulation examined (Table 4).

Chromosome anomalies in the progenies of the different hybrids. The procedure ofsynchrony using a colcemide block has contributed to certain chromosomal anomaliesamong the progenies of all the types of fusions between Gly-A and Gly-B. The mostcommon of them all are the chromosome breaks at the kinetochore region which

30

20

10

0

20

10

n

20

10

0

20

10

o

20

10

rv

20

10

0

20

10

0

I iA

- B

" C

- D

-

- E

-

" F

-

- G

-

1 1

20

1 1 1 1 1 1

...llllli||

.Jillill

la • • • • ! 1 I I m

1... llll.lh.

.1.1

• I.I

ill. l.lll

IL . ..l lII...

|,II.. .1 1 I I I

30 40 SO

Chromosome no.

i iR/R ~

-

MjM -

-

MIC, -

-

MjS -

-

G./G, -

cjs :

-

sis -

-

1 160

Fig. 2. Chromosome frequency distributions for various types of fusions involvingmutants of CHO cells. The frequency of each class is expressed as % of the totalchromosome spreads counted. In all these fusions one of the parents is Gly-A andthe other Gly-B. The chromosome preparations were made on the 12th day afterfusion which is equal to about 20 generations.

appeared in approximately 2% of the cases. Dicentric chromosomes were obtained in5 out of 1000 chromosome spreads examined. In the progeny of the MjS fusionnumerous chromosome fragments were observed in one of the cells. A ring chromo-some was found in only one of the spreads from MjGx fusion. No such anomaliescould be seen in 116 chromosome spreads of the control, RjR fusion, where thecells were not exposed to colcemide.


DISCUSSION

The data presented in this paper indicate that in mitotic/interphase fusions whereinduction of PCC occurs the Glt S or G2 chromosomes are wholly or partly incor-porated into the progeny of the hybrid cells. The PCC are either directly incorporatedinto the daughter nuclei and/or remain as fragments of chromatin in the cytoplasm.

Table 4. Variability of chromosome numbers among the progenies ofthe various types of fusions between Gly-A and Gly-B mutants

Type offusion

R/RM/MM\GX

M/S

GJCGJSs/s

1

Lowestrecorded

3127

19262 0

332 2

No. of chromosomes per cell

Highestrecorded

1 2 0

7 i8 0

74737796

Mean valuefor the total

sample counted

4i-339-84 0 63 7 6

38-53 9 64°-S

Mode(from Fig. 2)

4 14 04 0

3739394 0

NOTE. Because of the difficulties in obtaining even a reasonable degree of synchrony amongthe Gt population, the MjGt fusion for chromosome studies was not attempted.

Table 5. The genetic consequences of fusion between mitotic and interphase cells

Type offusion PCC induction

Probabilityof genecomple-

mentation No PCC induction

Probabilityof genecomple-

mentation

M/GAM/S \M/G2j

PCC are wholly orpartly incorporated intodaughter nuclei ofhybrid cells (Figs. 4, 5)

or

PCC not incorporatedinto daughter nuclei

+ The uninduced G1 nucleusbecomes part of one of thedaughter cells followingcompletion of mitosis bythe mitotic component(Fig. 6 c)

or— The chromosomes of the

mitotic component formpycnotic micronuclei whichare genetically inactive(Fig. 6 B)

Such fragments are likely to be eliminated during subsequent mitoses. When there isno induction of PCC in mitotic/interphase fused cells the mitotic component com-monly becomes pycnotic and will presumably be eliminated. Only rarely does themitotic component undergo normal division in these cells. Immediately followingfusion the mitotic/interphase cell will probably follow one of the variety of courseslisted in Table 5. The question remains whether the prematurely condensed chromo-


somes of the fused cell are retained in the progeny. From the experiments dealingwith short-term survival of mitotic/interphase HeLa cells it is evident that boththe mitotic and the PCC components are retained in the progeny for at least 2 days(Table 2, p. 500).

To examine whether the PCC become genetically integrated into the progeny ofthe fused cells experiments were performed using glycine-requiring mutants of CHOcells. The rate of recovery of hybrids between Gly-A and Gly-B mutants is dependentupon the type of fusion. Homophasic fusions yielded almost twice the number ofhybrids of heterophasic {SjG^ 5/G2 and G^G^j fusions. The lowest rate of survivalof hybrids (0-062 %) occurred in the M/S fusion closely followed by MjG2 and MjGx

(Table 3). The differences in the rates of hybrid survival between homophasic andheterophasic fusions can be explained partly on the basis of the frequency of PCCinduction. The incidence of PCC which is nearly 90% in the mitotic/interphasefusions probably accounts largely for the low rate of survival of these hybrids. Thepycnosis and micronucleation of the mitotic component in cells where there is noinduction of PCC also contributes to the low rate of survival of these cells since themicronuclei, which synthesize neither RNA nor DNA, are probably geneticallyinactive. PCC is completely absent among homophasic fusions where the rate ofsurvival is markedly greater than in the mitotic/interphase fusions. The rare occurrenceof PCC in the heterophasic interphase/interphase fusions (Johnson & Rao, 1970)suggests that there are other factors involved in reducing the rate of survival to about50% of the homophasic fusions.

It is pertinent to ask whether all the hybrid cells with PCC fail to develop intocolonies. From the experiments with HeLa cells there is conclusive evidence that inmany cases the prematurely condensed chromosomes become part of the daughternuclei of the fused cell and are thereby retained (Fig. 5). When no PCC occurs themitotic component becomes genetically inactive by the process of micronucleationand under these conditions no genetic complementation seems possible. This observa-tion lends further support to the conclusion that most of the surviving colonies in themitotic/interphase fusions were derived from homokaryons in which the interphasenuclei had undergone PCC. Since the mitotic populations of CHO cells used in theseexperiments contained 4% of interphase cells it is possible that a small proportion(approximately 4% of 0-28% = o-oi%; refer to lines 11, 12 and 13 of Table 3) ofthe surviving colonies was derived by fusion between interphase cells of the twomutants.

The pulverization of chromosomes observed in the MfS fusion was not present inthe M/G1 or M/G2 fusions. The high degree of scattering of the prematurely con-densed chromosomes of the S phase and the subsequent failure of substantialincorporation into the hybrid nucleus may contribute to the loss of chromosomesand to a lower rate of survival of the MjS hybrids. On the other hand the PCC ofthe G± or Ga phases usually remain as intact groups and are incorporated into one ormore of the daughter nuclei. The modal chromosome numbers of 40 for the M/Gfusion and 37 for the MjS fusion support this point of view (Table 4). This leads usto the conclusion that hybrid cells with PCC of G± or G2 phases have a better chance


of survival than those with PCC of S phase. A modal chromosome number of 40might be expected for all the fusions involving interphase cells. However, in the lightof Table 5 one would expect a significant loss of chromosomes within a short timein the progenies of mitotic/interphase fusions. The fact that we did not find a greatdeal of chromosome loss in the progenies of the mitotic/interphase fusions suggeststhat for complementation to occur between these 2 mutants retention of a majorityof the prematurely condensed chromosomes is necessary.

Although the loss of chromosomes among the progeny of the MjS fusion is notvery extensive, it is significant. Kao et al. (19696) produced hybrids by fusing randompopulations of 2 CHO mutants. Among the progenies of these hybrids it required116 generations for the modal chromosome number to drop from 40 to 37. In the MjShybrids of the present study a similar state is reached within the duration of about20 generations (Fig. 2).

The relatively small loss of chromosomes observed in this study and that of Kaoet al. (19696), in which the modal value decreased from 40 to 37 chromosomes andremained stable even after 144 generations, may be related to the common origin ofthe cells involved. The degree of chromosomal loss in the hybrid progenies appearsto depend largely upon the species differences between the 2 parental types. Theseauxotrophic mutants of CHO cells are identical except for the specific genes regulatingtheir nutritional requirements and hence their hybrids are relatively more stable.The extensive loss of chromosomes in the hybrids from different species is illustratedby the mouse-human and human-Chinese hamster heterokaryons (Migeon &Miller, 1968; Matsuya & Green, 1969; Kusano, Long & Green, 1971; Kao & Puck,1970). The loss of chromosomes among hamster-hamster or human-human cellhybrids is not as great as in the interspecific hybrids (Kao et al. 19696; Siniscalco et al.1969). In the hybrids of A9 cells and chick erythrocytes the genetic material of thelatter cell type was virtually eliminated except for a small fragment carrying the genethat is necessary for the survival of the hybrids under the selective environment(Schwartz, Cook & Harris, 1971). In this case the authors suggested that the loss ofchick genetic material was probably due to premature condensation of the chicknucleus. When cells of the same strain or species are fused the nuclei in thehomokaryons become rapidly synchronized either at the time of the initiation ofDNA synthesis or mitosis during the first mitotic cycle (Rao & Johnson, 1970). Theachievement of synchrony and subsequent fusion of nuclei largely eliminate thechance of induction of PCC leading to the formation of stable hybrids with twocomplete genomes.

This investigation is a contribution from the Department of Biophysics and Genetics(No. 488) and the Eleanor Roosevelt Institute for Cancer Research, University of ColoradoMedical Center, Denver, Colorado, and the Department of Zoology, University of Cambridge,Cambridge, England. We thank Dr Theodore T. Puck for critical reading of the manuscriptand Meribel Halcrow, George Barela and David Peakman for technical assistance. Thisinvestigation was aided by research grant no. DRG-mo from the Damon Runyon MemorialFund for Cancer Research, by U.S. Public Health Service Grant no. 5 Poi HD02080 from theNational Institute of Child Health and Human Development, and by a grant from the MedicalResearch Council, England.

506 P.N. Rao and R. T. Johnson

REFERENCES

JOHNSON, R. T. & RAO, P. N. (1970). Mammalian cell fusion: Induction of premature chromo-some condensation in interphase nuclei. Nature, Lond. 226, 717-722.

JOHNSON, R. T., RAO, P. N. & HUGHES, S. D. (1970). Mammalian cell fusion. III . A HeLa cellinducer of premature chromosome condensation active in cells from a variety of animalspecies. J. cell. Physiol. 77, 151-158.

KAO, F. T., CHASIN, L. & PUCK, T. T. (1969a). Genetics of somatic mammalian cells. X.Complementation analysis of glycine-requiring mutants. Proc. natn. Acad. Sci. U.S.A. 64,1284-1291.

KAO, F. T., JOHNSON, R. T. & PUCK, T. T. (19696). Complementation analysis on virus-fusedChinese hamster cells with nutritional markers. Science, N. Y. 164, 312-314.

KAO, F. T. & PUCK, T. T. (1967). Genetics of somatic mammalian cells. IV. Properties ofChinese hamster cell mutants with respect to the requirement for proline. Genetics 55,513-524.

KAO, F. T. & PUCK, T. T. (1970). Genetics of somatic mammalian cells: Linkage studies withhuman-Chinese hamster cell hybrids. Nature, Lond. 228, 329-332.

KUSANO, T., LONG, C. & GREEN, H. (1971). A new reduced human-mouse somatic cell hybridcontaining the human gene for adenine phosphoribosyltransferase. Proc. natn. Acad. Sci.U.S.A. 68, 82-86.

MATSUYA, Y. & GREEN, H. (1969). Somatic cell hybrid between the established human line D98(Presumptive HeLa) and 3T3. Science, N.Y. 163, 697-698.

MIGEON, B. R. & MILLER, C. S. (1968). Human-mouse somatic cell hybrids with single humanchromosome (group E): Linked with thymidine kinase activity. Science, N. Y. 162, 1005-1006.

PUCK, T. T. & KAO, F. T. (1967). Genetics of somatic mammalian cells. V. Treatment with5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc.natn. Acad. Sci. U.S.A. 58, 1227-1234.

RAO, P. N. (1968). Mitotic synchrony in mammalian cells treated with nitrous oxide at highpressure. Science, N.Y. 160, 774-776.

RAO, P. N. & ENGELBERG, J. (1965). HeLa cells: Effects of temperature on the life cycle.Science, N.Y. 148, 1092-1094.

RAO, P. N. & ENGELBERG, J. (1966). Effects of temperature on the mitotic cycle of normal andsynchronized mammalian cells. In Cell Synchrony: Studies in Biosynthetic Regulation (ed.I. L. Cameron & G. M. Padilla), pp. 332-352. New York: Academic Press.

RAO, P. N. & JOHNSON, R. T. (1970). Mammalian cell fusion: Studies on the regulation ofDNA synthesis and mitosis. Nature, Lond. 225, 159-164.

RAO, P. N. & JOHNSON, R. T. (1971). Mammalian cell fusion. IV. Regulation of chromosomeformation from interphase nuclei by various chemical compounds. J. cell. Physiol. 78,217-223.

SCHWARTZ, A. G., COOK, P. R. & HARRIS, H. (1971). Correction of a genetic defect in a mam-malian cell. Nature New Biol., Lond. 230, 5-8.

SINISCALCO, M., KLINGER, H. P., EAGLE, H., KOPROWSKI, H., FUJIMOTO, W. Y. & SEEGMILLER,

J. E. (1969). Evidence for intergenic complementation in hybrid cell derived from twohuman diploid strains each carrying an x-linked mutation. Proc. natn. Acad. Sci. U.S.A. 62,793-799-

(Received 27 September 1971)

Fig. 3. PCC in M/Glt M/S and M/G, fusions of HeLa cells at 30 min after fusion.The mitotic cells were obtained by the application of NSO block. The interphase cellswere prelabelled with [3H]thymidine prior to cell fusion.

A, M\GX. PCC of the Gj type which is lightly stained lying alongside the metaphaseplate (darkly stained) of the mitotic cell.

B, M/S. A radioautograph showing labelled 5 type PCC (indicated by arrows)lying around the metaphase plate.

c, M/G,. A radioautograph showing labelled PCC of the G, type (indicated by thearrow) lying adjacent to the metaphase chromosomes of the mitotic cell.

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Fig. 4. Scattering of PCC in M/S fused HeLa cells at 4 h after fusion. S-phase cellsprelabelled with 3H-TdR were fused with mitotic cells reversibly blocked with N,O.

A, the mitotic chromosomes are completing anaphase while the PCC are scatteredaround them. PCC will probably be distributed randomly between the daughternuclei.

B, radioautograph of an M/S fused cell during anaphase where the labelled PCCbecome localized to one side of the diving cell.

C, in this M/S fused cell the PCC form a ring around one of the anaphase chromosomegroups and in all probability will become part of that daughter cell.

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Fig. 5. Distribution of PCC during anaphase and telophase.A, MjS fused cell. The 5 type of PCC is going to be incorporated exclusively into

one of the daughter cells.B, M\GX fused cell in telophase. The labelled PCC are incorporated into the larger

of the 2 telophase chromosome groups.c, M/G1 fused cell at the end of the first mitosis after fusion. The Gj-PCC are in-

corporated into 2 of the 3 daughter nuclei as indicated by the presence of label.

Fate of prematurely condensed chromosomes

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512 P.N. Rao andR.T. Johnson

Fig. 6. A, M/Gt hybrid cell during the second mitosis after fusion showing labelledand unlabelled chromosomes on the same metaphase plate. The labelled chromo-somes were derived by the incorporation of labelled PCC of the G2 type.

B, M/S fused cell in which there was no induction of PCC of the S nuclei. Themitotic chromosomes were transformed into micronuclei under the influence of theinterphase nuclei. The presence of 3H-TdR in the growth medium followingfusion resulted in an increase in the number of grains on the S nuclei.

c, M/G1 cell in which the Gx nucleus failed to undergo PCC induction. Themitotic component (shown by the arrow) has completed anaphase. The Gx nucleusmay be incorporated into one of the daughter cells.

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