Non-Mendelian Segregation in Hybrids between Chinese Hamster Cells MORGAN HARRIS D e p a r t m e n t of Zoology, University o f C a l i f o r n i a , Berkeley, C a l i f o r n i a 94720

ABSTRACT Mechanisms of segregation have been examined in hybrids between Chinese hamster cells, where chromosome loss in comparison to other systems is minimal. Hybrid cells were grown in HAT medium and subjected to back selection with bromodeoxyuridine (BUDR) or azaguanine (AZG). In AZG or BUDR at 30 Pg/ml, segregation began with a random high frequency event that gave rise to cells capable of growth in both HAT and back selection medium, unlike the precursor hybrid or original parental cell types. BUDR-resistant segregants were propagated serially in the presence of BUDR, and were examined by clonal analysis for changes in plating properties during long term culture. Over a period of 300 days the HAT/BUDR plating ratio for segregant cells declined continuously. A parallel decrease was observed in the rate of H3-thymidine incorporation, along with a drop in thymidine kinase activity. These shifts took place only in the presence of BUDR, and could be reversed by altered selection in HAT medium. Clonal studies showed that the evolution of segregant properties occurred in most if not all cells of the population, and did not arise from variation and selection of minority cell types. These properties of the segregating system are not consistent with models based on gene mutation, chromosome rearrangements, or chromosome loss. The evolution of segregants resembles more closely a sorting-out progress, taking place by intracellular selection over many generations. The segregating units may conceivably be cytoplasmic determinants linked functionally to nuclear genes, and which serve to modulate the events of phenotypic expression. Several lines of evidence which bear on this concept are discussed.

Segregation is a classic tool for genetic studies, and in recent years has been applied extensively to the analysis of changes in hybrids constructed between somatic cells. The applications to date have centered mainly on chromosomal segregation, following the observation that chromosome losses are frequent in hybrid cells, particularly in those established between the cells of different species, e.g., mouse cells and human cells. In such combinations, deletion of particular chromosomes can often be associated with changes in marker characteristics at the phenotypic level. These correlations provide a basis for localizing gene loci, and have been used with conspicuous success to construct chromosome maps for human cells and those of other species (Ruddle, '72, '74; Ephrussi, '72). For segregational studies at a more genJ. CELL.PHYSIOL.,86: 413-430.

era1 level, drug resistance markers offer convenient experimental systems. Most hybrids are obtained by HAT selection (Littlefield, '66), in which one cell type resistant to bromodeoxyuridine (BUDR) is fused with another refractory to azaguanine (AZG) or thioguanine (THG). Although normal drug sensitivity is restored to hybrid cells by complementation, resistance of either type can reappear as a segregant property during back selection in the inhibitor concerned. This procedure was used by Marin and his collaborators to obtain segregants to BUDR or THG resistance from hybrids between BHK21 Syrian hamster cells (Marin and Littlefield, '68; Marin, '69, '71). Segregants were also derived from combinations of mouse cells Received Jan. 14, '75.Accepted Mar. 14, '75. 1 Supported by grant CA12130, National Cancer Institute, U . S . Public Health Service.

413

414

MORGAN HARRIS

and Chinese hamster cells (Marin and Pugliatti-Crippa, '72; Marin and Manduca, " 7 2 ) . In each system the resistant cells appeared to represent segregants which had lost non-overlapping groups of chromosomes during the process of back selection. A curious and unexplained phenomenon in these studies was the non-random character of chromosome deletion. The experiments of Marin and Manduca ('72) showed that asymmetry in chromosome loss did not depend on differences in replication patterns for the two sets of parental chromosomes. The direction of chromosome reduction seemed rather to be determined by selective pressure, as demonstrated by Marin and Pugliatti-Crippa ('72) in hybrids between mouse and Chinese hamster cells. In their experiments, selection against an enzymatic activity carried by one' of the parent cells, either thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT), brought about the preferential loss of a group of chromosomes from the same parent. The basis for such group shifts in karyotype was not determined. In the light of these observations it is reasonable to ask whether chromosome changes are the only mechanism leading to the segregation of phenotypic properties in hybrid cells. Other possibilities can be examined by using hybrids which have a relatively stable chromosome pattern, e.g., combinations made between different lines of Chinese hamster cells, where chromosome loss is often minimal (Barski, '70; Sobel et al., '71). Such hybrids have been used for segregational studies by Chasin ('72), who constructed combinations between sublines of CHO cells differing in drug resistance markers. Reappearance of recessive markers under selection pressure was regularly observed. The segregation rate for THG resistance was determined by fluctuation tests, and found to be similar to the value reported by Marin ('69) for Syrian hamster cells. However, karyotypic changes occurred at a lower rate in the Chinese hamster cell hybrids, and thus the mechanism of segregation in the latter was less clear. Chasin advocated an explanation based on chromosome loss, but offered no experimental data to support

the presumed correlation of karyotypic and phenotypic changes. In fact, when 10 segregant Chinese hamster lines resistant to THG were examined, the average modal chromosome number was found to be 39, identical to that of the precursor hybrid cells. In the present study we have used clonal analysis to examine more closely segregational mechanisms in hybrids between Chinese hamster cells. By this means it is possible to monitor early stages in the establishment of segregant phenotypes, and to interpret the events which take place in terms of population dynamics. We have observed a conspicuous further evolution in cellular properties when segregant clones were maintained for long periods under back selection. These transitional changes, thus f a r characterized only for segregants resistant to BUDR, appear to be mediated by mechanisms other than gene mutation or chromosome loss. Our results suggest that gene expression may be modulated in segregants by cytoplasmic determinants which undergo intracellular shifts during back selection; such changes may be of importance even in segregating systems where chromosomal loss predominates. MATERIALS

AND METHODS

Cell lines and culture techniques Most of the experiments to be described were carried out with two hybrid lines of Chinese hamster cells designated 272 and 599 respectively. Both hybrids have a neartetraploid chromosome complement and were isolated originally from single colonies in HAT medium. Hybrid 272 represents a cross between line 129, which is resistant to AZG and deficient in HGPRT, and line BI50, which is deficient in TK and is resistant to BUDR. Hybrid line 599 has a similar origin except that the strain of 129 cells used also carried a marker for resistance to cytosine arabinoside (ara-C). Detailed descriptions of these hybrids and of chromosome markers in parental cells may be found in preceding papers (Harris, '73, '74a). Stock populations were maintained as monolayers in prescription bottles (Brockway Glass Co. ) in nutrients which included

NON-MENDELIAN SEGREGATION IN HYBRID CELLS

415

Incorporation studies Functional comparisons of hybrid cells and of segregant lines were performed by measuring Ha-thymidine incorporation or C'4-hypoxanthine incorporation into log phase cultures. For this purpose groups of cultures were set up in 1 oz prescription bottles in 1OFCSDB and assayed during the period of rapid growth. C"-hypoxanthine (Schwartz/Mann, 53.7 mC/mM) was added to the culture medium at 1 ,C/ml for a 4-hour incubation period; H3thymidine (Schwartz/Mann, 6.0 C/mM) was administered for 24 hours at 1 &/ml. Counts to determine cell number were made in parallel in cultures without label. Upon removal of labelled media, the cultures were rinsed two times with cold Hank's buffer, fixed in 5 % cold TCA, and rinsed with 100% ethyl alcohol and absolute ethyl ether. After air drying, 0.2 ml Nuclear-Chicago solubilizer (NCS) was added to each culture to dissolve the cell Fluctuation tests sheets and the tightly capped bottles incuRates of segregation to BUDR or AZG bated overnight at 45°C. After cooling the resistance were determined for hybrid contents were transferred to scintillation cells by using a modified Luria-Delbruck vials containing Aquasol (New England fluctuation test, as described previously Nuclear) and the radioactivity determined (Harris, '71). Each experiment was ini- in a Nuclear-Chicago Mark I scintillation tiated from a single colony of hybrid cells system. which after four days of incubation in Determination of thymidine kinase lOFCSDB contained approximately 200 Enzyme assays were carried out on excells. The colony was trypsinized and the cells redistributed into several petri dishes tracts from cell populations in logarithmic in 1OFCSDB. After incubation for an growth or plateau phase, using procedures additional seven days, 15 of the daughter based on the methods of Boyd and Harris colonies which appeared were isolated in- ('73) and Bull et al. ('74). Cells were dividually. The resulting cell suspensions obtained by trypsinizing stock cultures 24 were then subcultured, or used to set up hours after a previous fluid change, and assays for colony formation in petri dish the resulting pellets were washed twice by cultures containing 30 pg/ml AZG or 30 centrifugation in ice-cold 0.154 M KC1 M thymidine and ,g/ml BUDR. The subcultures, made from containing 1.6 x 1 0 - ~ a portion of each cell suspension, were car- 3 mM p-mercaptoethanol. A suspension ried as mass populations in lOFCSDB, and was then prepared at 10 X lo6 cells/ml assayed later for segregant frequency when in cold extraction medium (10 mM-Tris the cell number for each population ex- HCl, pH 8.0, 1.6 x lo-' M thymidine, and ceeded 20 x lo6. Plating data for individ- 0.003 M p-mercaptoethanol), and the cells ual cell lines were based on the average disrupted by three rapid cycles of freezenumber of segregant colonies in four petri thawing, using a mixture of methanol-COz dish cultures. The average values obtained and a water bath at 37°C. After centrifuwere evaluated for significance by a Chi- gation at 30,000 g for 30 minutes at 4°C square test, and segregation rates calcu- in a Sorvall SS34 rotor the supernatant lated by the median method as outlined by was decanted and stored at -85°C in a Revco low temperature freezer. The proLea and Coulson ('49).

10% fetal calf serum, 50 pg/ml Gentamycin (Schering Corp.) and 90% Dulbecco's modification of Eagle's medium (10FCSDB). HAT medium (Szybalski et al., '62) was prepared by supplementing lOFCSDB with hypoxanthine ( 1 x M ) , thymidine (1.6 X M ) , and aminopterin ( 4 X lo-' M ) . Nutrients for back selection were prepared by adding AZG or BUDR to lOFCSDB as specified. All cell lines were checked at intervals by culture methods for the presence of mycoplasma; none were found. Plating experiments were carried out in 60 mm petri dishes (Falcon Plastics) which were incubated at 37°C in a humidified CO, incubator. Fluid changes were made two to three times per week and the cultures terminated when colonies were well developed. For scoring, the cultures were rinsed with 0.85% NaC1, stained with a saturated solution of crystal violet in 0.85% NaC1, rinsed with distilled water and air dried.

416

MORGAN HARRIS

tein content of each extract was determined by the method of Lowry et al. (’51). Assays for thymidine kinase were carried out in a total volume of 100 pl, using disposable polypropylene microtubes as reaction vessels. Each sample contained 5 mM-MgClz, 10 mM-Tris-HC1, pH 7.5, 10 mM-ATP (,previously neutralized to pH 7.5 and stored frozen), 10 ,C-H3-thymidine (Schwartz/Mann, 6.0 C/mM), and 60 pl cell extract. The sample mixtures were prepared in an ice bath and when complete were incubated for 15 minutes at 37°C. The reaction was terminated by transferring the sample tubes to ice water, after which 80 ,l of each sample was spotted on a 2 cm square of Whatman DE81 paper in a 60 mm petri dish (Falcon Plastics). The squares were immediately rinsed with 10 ml 0.1 mM ammonium formate (pH 7.3) for 15 minutes, followed by three additional rinse cycles. After a final rinse in absolute methanol the squares were dried under an infrared lamp and transferred to scintillation vials with Aquasol for counting. This procedure effectively removes from DE81 paper essentially all thymidine not phosphorylated by enzyme action; tests showed that less than 0.1 % of H’-thymidine added to blanks was detectable in the final scintillation counts. RESULTS

Incidence and rates of segregation Table 1 provides in outline form a description of the cell lines used for the present experiments. Line 129 is resistant to AZG as a result of deficiency in HGPRT. These cells incorporate C “-hypoxanthine at a rate which is 0.5 percent that of the other parental cell type. B150 cells are deficient in TK activity. They are resistant to BUDR and are unable to incorporate H’-thymidine except at a low level (0.6

percent of the value for 129 cells). When hybrids, e.g., line 272, are constructed between the two parental cell types, high rates of incorporation for both salvage pathways are restored, along with sensitivity to AZG and BUDR. Thus, hybrid cells proliferate vigorously in HAT medium and show the same plating efficiency in this nutrient as in 10FCSDB. In contrast, both 129 and B150 cells are killed rapidly in HAT, and the rates of reversion to HAT+ growth for the parental lines are quite low. No HAT+ colonies have been recovered so far from 129 stock populations in tests with 10* to lo9 cells. Revertants from BI50 in HAT medium were found earlier with a frequency of about 1 per 106-107 cells, but later tests with larger numbers of cells have been completely negative. The experimental plan followed for segregation experiments can be outlined briefly. Hybrids were isolated in HAT medium, and clonal populations were then subjected to back selection with either AZG or BUDR, usually at 30 pg/ml (referred to hereafter as 30 AZG or 30 BUDR). At these inhibitor levels most of the hybrid population was destroyed but a few cells survived to form segregant colonies which could be isolated and propagated serially in the back-selection medium. Table 2 gives the frequencies of segregants obtained by one-step selection of hybrid cells in 30 AZG or 30 BUDR. The incidence of segregants obtained in this way varied significantly among a series of hybrids derived from the same parental cells, and these differences reappeared in successive tests with the cells in question. The frequency of segregation from particular hybrids remained fairly constant during long term culture, particularly for segregants to BUDR resistance. In some

TABLE 1

Characteristics o f parental a n d hybrid lines used f o r segregation experiments Cell line

129 B150 272 (129 X B150)

Enzyme deficiency

C14-hypoxanthine incorporation, CPM/lOe cells

HJ-thymidine incorporation, CPM/lOG cells

HGPRTTKNone

635 120,000 146,000

184,000 1,100 261,000

Resistance markers

AZGR BUDRR None

Growth in HAT medium -

-

++

NON-MENDELIAN SEGREGATION IN HYBRID CELLS

hybrid lines a fall-off in AZG segregation with time was observed, as Marin ('69) had described earlier. However, in other sublines increases in the incidence of AZG segregants were observed (table 2).

\

417

Azoguonine Resistonce in 272 Hybrid Cells and Parental Lines

TABLE 2

Ixcidence of segregants from hybrid cells obtained by back selection with AZG or BUDR Frequency of segregants

Hybrid line 1

30AZG

272 599 579 600 196 196C 196E

2

30BUDR

1.6 2.0 4.3 7.5 0.7 3.0 3.1

2.1 0.3 0.2 0.8 0.04 0.8 22.6

~

lHybrid lines derived as independent clones from fusions of 129 x B150 cells i n HAT medium. 196C and 196E are sublines of 196 selected in chloramphenicol and ethidium bromide respectively, as part of another experiment. All other cell lines were maintained in HAT medium. 2 Shown as colonies per 100 cells plated in 30 fig/ml back selection medium. made with a sinele batch of fetal calf serum (Flow' Laboratories)

0 001

I

-

0.001

I

I I8

30

55

I

100

180

300

BUDR, p g / m l

Fig. 1 Frequency of segregation to BUDR resistance i n 272 hybrid cells as a function of inhibitor concentration. Curves for colony formation by the two parental lines are shown for comparison. Assay populations were plated out at graded cell numbers, and each point shown is based o n the average colony count for 6-9 petri dish cultures.

An initial group of studies was performed to measure the frequency of segregation to AZG or BUDR resistance as a function of inhibitor concentration. Fig. 1 and Fig. 2 serve to illustrate experiments of this type, in which the incidence of segregants from hybrid cells was compared to the plating efficiency of the corresponding parent cells under the same conditions. Colony formation in AZG by B150 parent cells was unaffected by subcloning and did not differ greatly from values reported for AZG-sensitive V79 cells (Harris, '71 ). Similarly, the plating efficiency in BUDR of 129 populations or subclones was comparable to that of wild-type cells. The incidence of segregants from hybrid cells in both inhibitors was intermediate between that of the parent cells, and appeared to decrease in linear fashion on a log-log scale. Curves for BUDR segrega-

418

MORGAN HARRIS

tion from 272 hybrids were relatively similar when carried out with different batches of fetal calf serum. However, levels of segregation for AZG resistance differed systematically by as much as 10-fold when cells were assayed in different sera, although the rate of change with inhibitor concentration remained the same (fig. 2). These differences presumably can be traced to varying content of hypoxanthine or other competitive inhibitors of AZG within the sera used (Albertini and De Mars, '70). Actual rates of segregation from hybrid cells, as opposed to mere incidence of segregants, were determined by fluctuation tests. Table 3 provides a summary of the data obtained by back selection in AZG or BUDR at 30 ,g/ml. In all three assays, the numbers of segregant colonies were in good agreement for replicate cultures from a single subline. However, significant variation was observed between independent sublines, as shown by the high variance and Chi square tests. Thus, segregation began as a random and spontaneous event, in agreement with the findings of Marin ('69) and Chasin ('72). The segregation rates obtained in the present study were quite high, and were substantially similar for both markers. This is perhaps surprising since linkage data show that HGPRT activity in Chinese hamster

cells is associated with glucose 6-phosphate dehydrogenase (G6PD) and 3-phosphoglycerate kinase (PGK) activities (Westerveld et al., '72). Thus, the loci for these enzymes may be X-linked, as in man. However, the T K locus, which in man is autosomal (Miller et al., '71), was found by Westerveld and coworkers to be unlinked to HGPRT, GGPD, or PGK markers. A gene dosage effect in Chinese hamster cells would thus be expected in comparing the rates of BUDR and AZG segregation, but none was observed (tables 2, 3). The high rates of segregation would seem to imply frequent chromosome loss, if deletion at this level is the implementing mechanism. In fact, if losses were random in hybrid cells with 44 chromosomes, and if the appearance of segregants for AZG resistance took place by the elimination of the X chromosome carrying the normal HGPRT allele, chromosome deletion at approximately 15% of mitotic divisions would be required ( 1 / 7 x 1/44 = 3.2 X close to the value shown in table 3 ) . For the expression of BUDR resistance, a proportionately higher incidence of chromosome loss would be necessary, if the T K locus occurs in a pair of autosomes and both were present. Actually, it was a simple matter to ascertain by direct observation that chromosome losses of this magnitude did not occur in

TABLE 3 Rates of segregation to BUDR and AZG resistance for 272 hybrid cells under back selection 30 BUDR

30 AZG

Total cells/subline

8176

22.4 X lo6

33.4 x 1 0 6

Cells/assay culture

33-1070

100-1000

100-1000

37.2

61.2

Selection medium

30 BUDR

~~

Segregant colonies/ 1000 cells Variance (corrected for sampling) variance Ratio -______ mean

XZ P Segregation rate

37.4 1998 53.4 762

< 0.001 3.7 x 10-3

1043

1785

29.1

28.0 406

422

< 0.001

< 0.001

x

3.6 x 10-3

2.4

10-3

Aliquots of cells from each of 15 sublines were inoculated into 60 mm petri dishes for test, and were incubated i n lOFCSDB containing 30 &g/ml BUDR or AZG (FLOW serum). Segregation data are based on colony counts for four dishes per subline. Cell numbers in assay cultures for the first BUDR experiment were determined by parallel platings of each subline in lOFCSDB alone. 2 Segregants per hybrid cell per generation. 1

419

NON-MENDELIAN SEGREGATION IN HYBRID CELLS

the hybrid combinations studied. The modal chromosome number in segregant populations remained at or near the initial hybrid value, as Chasin ( ' 7 2 ) had reported earlier. Segregation for BUDR or AZG resistance clearly was not accompanied by the elimination of non-overlapping groups of chromosomes, as found by Marin ('69) in hybrids between BHK21 cells. On the contrary, karyotypic variation was minor, and some of the segregants studied showed remarkable constancy in chromosome pattern during back selection, as illustrated by examples shown in table 4. It is well known, however, that the Chinese hamster karyotype undergoes frequent and extensive rearrangement in the cells of established lines, even though the modal chromosome number remains at a pseudodiploid or pseudotetraploid level (Deaven and Petersen, '73; Rommelaere et al., ' 7 3 ) . Segregation might therefore be mediated by chromosomal alterations other than numerical shifts. Such a concept is difficult to test directly but can be examined in terms of expected segregant properties. According to this model, drug-resistant segregants would appear by chromosomal rearrangements (e.g., somatic crossing over or balanced non-disjunction) which uncovered the recessive parental allele (s) without introducing a change in chromosomal number. Variants obtained should thus correspond in phenotype to the parental drug-resistant cells. However, as the following section will make clear, segregants for BUDR or AZG resistance were in fact quite different phenotypically from the precursor cells. Segregation, therefore, cannot be accounted for in these hybrid

combinations by a simple uncovering of parental alleles.

Evolution of segregnnt properties A new perspective on the mechanisms of segregation emerged when the characteristics of segregants were examined during the initial stages of back selection, and at further time points after isolation. Initial studies of this kind revealed that segregation for AZG or BUDR resistance is not necessarily accompanied by the loss of ability to grow in HAT medium. Mass populations obtained by one-step selection in 30 pg/ml of either AZG or BUDR were found to retain the potential for growth in HAT medium as well. One possible explanation was the prolonged survival of drug-sensitive cells. Thus, the segregant mass population might initially include cells which were still HAT+ and drugsensitive although able to grow temporarily in inhibitor, as well as true segregants which were HAT- and drug resistant. To test this possibility we sampled early segregant populations by isolating a series of small clones derived from single cells. These were located as individual colonies six days after plating strain 272 hybrid cells into 30 AZG or 30 BUDR. Each colony was trypsinized and the cells redistributed in equal numbers into petri dishes containing HAT and 30 BUDR or 30 AZG respectively. Tables 5 and 6 provide a summary of the data obtained. It can be seen that nearly every clone retained the ability to form colonies in HAT as well as in the back selection medium, and some grew better in HAT. The plating efficiencies for individual populations varied, but

TABLE 4

Chromosome n u m b e r s in hybrid a n d segregant ceEl lines Cell line

272 hybrid AZGR segregant

No. of cells examined

Chromosome made

Numerical range

HAT

50

45

3949

30 AZG 45 days

50

45-46

42-51

HAT

50

44

40-50

50

44

4048

Selective medium

599

hybrid

BUDRR segregant

30 BUDR 188 days

420

MORGAN HARRIS TABLE 5

Clonal analysis o f segregation in hybrid cells a f t e r buck selection in AZG

1

1

2

3

4

5

6

7

8

9

10

HAT 30 AZG

25 22

2 1

15 11

12 13

0 4

19 11

24 18

22 29

18 65

18 14

Total no. of colonies

47

3

26

25

4

30

42

51

83

32

Clones

Segregant colonies isolated six days after plating 272 hybrid cells into 30 ag/ml AZG. Each colony trypsinized and cells divided equally into petri dishes containing 30 AZG or HAT medium. 1

TABLE 6

Clonal analysis o f segregation in hybrid cells a f t e r back selection in B U D R Clones

1

2

3

4

5

6

7

8

9

10

HAT 30BUDR

27 14

8 1

23 37

58 24

12

11

1

2

53 99

46 49

56 0

54 44

Total no. ofcolonies

31

9

60

82

13

13

152

95

56

100

Segregant colonies isolated six days after plating 272 hybrid cells into 30 ag/ml BUDR. Each colony trypsinized and cells divided equally into petri dlshes containlng 30 BUDR or HAT medium. 1

the total number of cells in each of these nearly every clonal colony isolated for test test populations was so small that the gave rise to colonies in HAT and in 30 ability to form colonies in both media BUDR. Over the period of study, there was could not reasonably have arisen second- a conspicuous shift in plating characterisarily by variation and selection. Thus, the tics. The formation of colonies in HAT data argue against the occurrence of two relative to BUDR decreased progressively, contrasting cell types in early segregant and essentially in parallel for the individual clones of a given group. This decline cultures. Segregants from hybrid cells, once es- can be summarized in index form by tablished, may undergo a conspicuous fur- changes in the HAT/BUDR plating ratio. ther evolution of properties when main- Clonal analysis thus suggested that tained under back selection in serial cul- changes in segregant properties during ture. Detailed study of this phenomenon back selection occurred simultaneously in has to date been confined to BUDR-resist- most if not all cells of the population, and ant sublines from hybrid cells, although was not mediated by variation and selecthere are indications of a similar transition tion of a minority cell type. On the basis of this finding we followed in AZG-resistant segregants under appropriate experimental conditions. In one the further evolution of 950 segregant cells experiment, a subline designated 950 was with mass populations in long term culture. isolated from 599 hybrid cells by back Subcultures were made twice a week in selection in 30 BUDR. After 25, 39, and 30 BUDR, and the cells plated out at fre60 days in the presence of inhibitor, quent intervals to determine colony formagroups of small colonies were isolated and tion in 30 BUDR and in HAT. Figure 3 assayed for plating efficiency in HAT and summarizes the results of the complete 30 BUDR. Table 7 gives the results of this experiment, carried out over a period of study. It should be pointed out that the 330 days. The plating efficiency of 950 growth rate of the segregant population in cells in 30 BUDR rose to a level of approximass culture increased progressively dur- mately 70% soon after isolation from the ing the 25-60-day period in BUDR, and original hybrid population and remained thus the total number of cells obtained in high in subsequent subculture. However, test colonies over a 5-6-day period was the potential for colony formation in HAT greater at successive time intervals. At medium decreased throughout the period each of the three time points, however, of study, levelling off after 300 days at a

42 1

NON-MENDELIAN SEGREGATION IN HYBRID CELLS TABLE 7

Clonal analysis of phenotypic s h i f t s in 950 segregant cells during serial culture in 30 B U D R Days of selection in BUDR

1

HATIBUDR plating ratio, average

Clones

1

2

3

4

5

HAT BUDR

20 126

67 84

0 123

2 53

13 31

Clones

1

2

3

4

5

6

HAT BUDR

84 283

72 206

8 266

11 253

11 123

5 234

Clones

1

2

3

4

HAT BUDR

8 1830

3 600

2 1680

8 2460

25 0.245

39 0.140

60 0.003

I Assays performed with cells from individual colonies, isolated 5-6 days after previous subculture of mass population in 30 pg/ml BUDR. Each colony trypsinized and cells divided equally into HAT and 30 BUDR.

value approximately equal to that of the BUDR-resistant parent line, BI50. This trend is depicted by the HAT/BUDR plating ratio (fig. 3 ) , which declined as an unbroken curve over seven orders of magnitude, without discontinuity or stepwise change. 100.0

SEGREGATION IN 599 HYBRID CELLS DURING SERIAL CULTURE I N BUDR I0 0

The evolution of plating properties during serial culture in BUDR is not a unique feature of 950 segregant cells, or of the precursor 599 hybrid population. Table 8 summarizes the results of similar experiments performed with segregants from 272 hybrid cells. Subline 282 was isolated in 30 BUDR in a pilot study and showed the same progressive decline in HAT/ BUDR plating ratio described above. Subsequently, sublines 296, 297, and 298 were isolated from 272 cells at higher levels of BUDR, to see whether the shift in plating characteristics was a function of inhibitor concentration. The HAT/BUDR ratio deTABLE 8

Evolution of plating characteristics in segregants f r o m 272 hybrid cells i n graded levels of BUDR 1 Subline BUDR, pg/ml

282 30

Days of mcubotion

8”

30 BUDR

Fig. 3 Evolution of plating characteristics i n segregants from 599 hybrid cells during serial culture in BUDR. See text for experimental details. Assay populations were plated out at graded cell numbers. Each point shown is based on an average for three petri counts i n 30 BUDR, and 6-9 petri fish cultures i n HAT medium.

297 180

298 300

H A T I B U D R plating ratio 0.341 0.295 0.218

days in BUDR

13 14 20 27 29 34 48 50

296 100

0.237 0.131 0.033

0.082 0.032

0.119 0.026

0.010 0.004

0.012 0.005

0.017 0.009

0.039 0.010

1 Each subline isolated from segregant colonies in petri dish cultures at 13-14 days, and maintained at low density in prescription bottles with subculture twice per week in BUDR as specified. Plating assays for each value shown are based on counts from three petri dish cultures at 100 cells i n BUDR and nine petri dish cultures at graded cell numbers in HAT medium.

422

MORGAN HARRIS

clined continuously in each of these populations during serial culture, but there was no evidence that the rate of change differed significantly between individual series. These findings are subject to alternative interpretations. Is the progressive decay in HAT, BUDR plating ratio an intrinsic property of segregant cells during serial multiplication, or is the plating shift induced by the back selection agent? To answer this question three sublines were established from a population of 950 segregant cells after propagation for 87 days in 30 BUDR. One subline (low density) was subcultured twice a week in 30 BUDR from an inoculum of 5,000 cells; another subline (high density) was subcultured similarly in 30 BUDR but from a constant inoculum of 100,000 cells. The third subline was carried in lOFCSDB, without inhibitor, from 100,000 cells. All three sublines were assayed at intervals over a period of 52 days by plating in HAT and in 30 BUDR. The data obtained are shown in figure 4. The HAT/BUDR plating ratio continued to decline for both sublines carried in BUDR, but the rate of decrease was somewhat greater in low density cultures. Thus, the falloff in HAT/BUDR ratio appeared to be a function of the number of cell generations rather than time as such, since the total number of doublings between subcultures was demonstrably greater for low density populations. In contrast, the HAT/BUDR ratio was unchanged for the subline cultivated in lOFCSDB, remaining level during the entire test period. Progressive loss of the ability to form colonies in HAT was therefore associated with growth in the presence of BUDR, and this decline did not occur in the absence of the selective agent. On the other hand, segregants grown in lOFCSDB alone maintained a stable plating pattern, and in the absence of BUDR did not revert to the higher HAT/BUDR ratios typical of earlier segregants, or of the original hybrid cells. Although plating shifts are convenient for monitoring the segregation process, other indices can be used as well. Since the level of H3-thymidine incorporation differs greately in hybrid and BUDR-resistant parent cells (table I ) , labelling experi-

CHANGE IN PLATING RATIOS FOR BbDRR SELREGAN'S FROM HYBRID CELLS DURING S E R I A L CULTURE

1c-q

8

IOFCSDB IHD!

i

I0 5

1

0

\

lo

20

30

40

50

60

D a y s of tnCubo+iOn

Fig. 4 Stability of plating characteristics within sublines of 272 segregant cells maintained i n different media. See text for experimental details. Assay populations were plated out a t graded cell numbers. Each point shown is based on a n average for three petri counts i n 30 BUDR, and 6-9 petri dish cultures i n HAT medium.

ments were performed with a number of segregants to see whether incorporation varies at different time points in serial culture. These measurements are presented in figure 5 as a function of the HAT/BUDR plating ratio, which was determined for each population separately. It is clear that the level of incorporation of H3-thymidine parallels closely the plating index for growth in HAT medium. In other experiments cell extracts were prepared and the specific activity of thymidine kinase determined for segregant cells as well as for hybrid and parental cell types. The data obtained are shown in table 9, along with the HAT/BUDR plating ratio for each line. B150 parental cells were low in thymidine kinase, with only 0.5 percent of the activity found in 129 parental cells. This residual activity is probably mitochondrial, since BUDR-resistant lines in several instances have been found to retain a mitochondrial thymidine kinase that is distinct from the cytoplasmic

423

NON-MENDELIAN SEGREGATION IN HYBRID CELLS 10-

Correlotion of Plating Ratios with 'H- Thymidine Incorporation in BUDR Segregant Lines

HAT

6"DR plating

a// /

'LltlO

//

Q

/ 01-

0

/ Q

/

/ /Q ,

/

I 103

105

104

'H-thymidine

mcorporotion. cpm /

106

lo6 cells

Fig. 5 Comparison of plating characteristics with H3-thymidine incorporation in BUDR-resista n t segregants from hybrid cells. The d a t a shown represent pooled experiments with segregants from 272 a n d 599 hybrids at different time points in serial culture. Data for H1-incorporation represent averages for 3 4 assays cultures; HAT/ BUDR ratios were calculated from petri counts o n three assay cultures in 30 BUDR, plus 6-9 assay cultures i n HAT medium.

enzyme which is missing in these cells, but present in normal cells (Clayton and Teplitz, '72; Attardi and Attardi, '72; Berk and Clayton, '73). Hybrid cells showed relatively high levels of thymidine kinase, but activity fell off sharply and progressively during back selection in 30 BUDR. Partial activity was regained in revertants obtained by plating segregant cells in HAT medium (see next section), although the level of thymidine kinase remained below that of the original hybrid in the cell lines studied. Other factors, however, are known to affect the level of thymidine kinase activity. One of these is the well-known periodicity in expression of thymidine kinase during cell and population growth cycles. Thus, thymidine kinase is high during S phase in rapidly dividing cells but declines to low values in stationary populations (Brent et al., '65; Eker, '65; Kit et al., '65). In addition, the specific activity of thymidine kinase drops when growth and protein synthesis are inhibited (Kit et al., '65). This observation may be particularly pertinent for enzyme determinations made with segregants of revert-

TABLE 9

T h y m i d i n e kinase activity in extracts f r o m parental lines and hybrid cells, in segregants f r o m hybrids in 30 B U D R , and in revertants f r o m segregants transferred again t o H A T m e d i u m Cells

Description

Growth phase

TK activity, CPM/fig protein

HAT/BUDR

plating ratio

129

Parent TK+

6200 7177

-

B150

Parent TK-

36 35 4

-

272

Hybrid

812

BUDR segregant, 39 days

7

2636 2960 445 121

57.6

0.57

100

950/22

BUDR segregant, 131 days

10

0.0003

12

944 H

Revertant from segregant, 31 days in BUDR

349 406

22.7

950 H32

Revertant from segregant, 127 days in BUDR

518 597 133

3.2

1

Values shown represent averages for 3-4 determinations per sample.

424

MORGAN HARRIS

ant cells which may be under growth inhibition i n selective media. The level of thymidine kinase is therefore likely to be a more variable correlate of plating behavior in segregants and revertants than H'thymidine incorporation. However, table 9 shows the two indices to be in reasonable agreement for the cell lines studied except for 950122 cells, where the persisting TK activity is probably at the mitochondria1 level. The enzyme studies described above are of interest in another context, since it is known that animal cells deficient in thymidine kinase can be transformed to the TK+ state by infection with inactivated herpes simplex virus (Munyon et al., '71; Munyon et al., '72). Of particular interest are the experiments of Davidson et al., ('73), who studied the properties of TK+ transf ormants obtained with inactivated herpes virus from LM(TK-) mouse cells. Several transformed lines were selected by growth in HAT medium and were found to contain viral thymidine kinase with activities 20-50-fold greater than that of the precursor cells. The viral enzyme could be suppressed by a single passage in BUDR, or reactivated fully by a single return passage in HAT, although at a low frequency. While these observations parallel in some respects the segregation data reported in the present investigation, the kinetics are quite different in the two systems. Viral and cellular forms of thymidine kinase can also be distinguished by contrasting patterns of activity during the population growth cycle (Lin and Munyon, '74). As noted previously, TK activity in uninfected cells rises and falls in coordinate fashion with cellular DNA synthesis, and is at a minimum when the stationary phase is reached. However, in cells transformed by herpes virus, viral TK activity remains low during rapid DNA synthesis and rises to a maximum in plateau phase cultures. In the present experiments, thymidine kinase activity was much higher in log phase cells than in plateau phase populations (table 9 ) . Here again the data do not fit the pattern for a herpes-type enzyme, although certain parallels i n the selective properties of viral and cellular enzyme systems remain to be explained.

Reversion in segregant cells Do the progressive changes which take place in BUDR segregants during back selection represent a one-way process, or can the segregation trend be reversed by altering the selection pressure? Pilot experiments to investigate this point showed that if early segregants in BUDR were transferred to HAT medium as mass populations, they grew progressively with a rising HAT/BUDR plating ratio. Studies were therefore initiated to determine whether reversal was occurring by variation and selection at the population level, or by changes taking place in most or all cells individually. Figure 6 illustrates the experimental plan for this work. The initial clone of segregant cells, derived from line 272, was plated into IOFCSDB after 31 days of back selection in 30 BUDR. After four days, 12 small colonies were isolated and the cells from each divided equally into HAT and 30 BUDR to establish clonal sublines. HAT and BUDR sublines were then continued by subculture from individual colonies at 5-day intervals; plating ratios €or the two media were simultaneously determined at each point. In this way, segregant cells of common origin were propagated separately in the two media, and the size of the proliferating populations was held at a low level to prevent the accumulation of minority cell types. After 25 days the clonal sublines of each group were continued as mass cultures for an additional 24-day period, and assayed once more for plating efficiency in the two media. CLONAL SUBCULTUHE

OF SEGREGANT CELLS

i')

4 ~ d o ycultures in IOFCS-Dulbecco t r o m clone of BUDRR segregont cells

every 5 doys. HAT/BUDR plating rofios determined

HAT

BUDR

HAT

BUDR

Fig. 6 Schematic diaeram of clonal culture for siblines of segregants-from 272 cells in HAT and in 30 BUDR.

425

NON-MENDELIAN SEGREGATION IN HYBRID CELLS

Table 10 gives the results of this study. a minor additional increase i n HAT/BUDR Clonal sublines maintained serially in 30 plating ratio (fig. 7 ) but did not reach BUDR continued to shift downward in the higher levels characteristic of normal the HAT/BUDR plating ratio, but the hybrid cells. A similar differential was trend was reversed for clonal populations observed in studies of thymidine kinase carried serially in HAT. There was a clear activity (see table 9 ) ; the specific activity divergence of sublines in the two selective of 950H32 cells clearly exceeded that of media over the 49-day period, a trend segregant lines in BUDR, yet was below which emerged whether the cells propa- the measured values for hybrids and wildgated as small clones or as mass popula- type parental cells. Taken as a whole, the tions. Thus, segregation in terms of plat- unique properties of late passage reverting properties was reversible under these ants suggest that such variants arise by conditions, and proceeded at approximately random and spontaneous change, although the same rate as the forward process. B.oth events appeared to take place by gradual and continuous shifts within the populalootion as a whole, rather than by the appearance and selective multiplication of variant @ @ _ _ _ - ---B *------a cells. @ ,/ Quite different kinetics have been ob- HAT/8LIDR plating 0 served in a few reversion experiments / conducted with late passage segregants, $ carried for extended periods in 30 BUDR. f 10: I In these populations the plating efficiency - I in HAT was reduced to a low level, and - I - 1 the HAT+ colonies that did appear were - 1 often markedly different at the outset - 1 from the immediate precursor cells. In I H A T l E U D R ratio 1 O f precursor one such experiment, a revertant colony pOpulOtlon 00003 was isolated in HAT medium from a popu0.1 L 0 10 20 30 40 50 60 70 lation of 950 segregant cells that had been Doys in HAT medium subcultured for 127 days in 30 BUDR. Fig. 7 Reversion of late passage 950 segreThe revertant clone, designated 950 H32, gant cells after transfer to HAT medium. Each grew well at the first test in both HAT and point shown is based on counts from 3-6 petri BUDR. Over a period of 70 days in HAT dish cultures in HAT medium, and 6-9 cultures medium, cells of the 950 H32 line showed in 30 BUDR. @

~

rDllC

t'

TABLE 10

Reversibility of plating shifts in 81 2 segregant cells during clonal subculture Selective medium

30 BUDR

HAT

HAT/BUDR

Days of selection

No. of colonies tested

No. of cells per colony, average

plating ratio, average

0

12

18.8

0.559

10

10

15 20 25 49

6 6 6 6

54.8 95.0 25.0 21.0

0.286 0.202 0.144 0.077 0.0087

10 15 20 25 49

7 6 5 5 6

mass cultures 40.4 58.0 17.4 36.0

mass cultures

1.62 3.30 4.44 3.09 15.47

1 Line 812 obtained as a single colony by back selection of 272 hybrid cells in 30 BUDR. Reversal experiment initiated after 31 days i n back selection medium. Sublines transferred by clonal subculture for the first 25 days, and by mass subculture thereafter (see text).

426

MORGAN HARRIS

this possibility remains to be verified by fluctuation tests. It is perhaps no coincidence that similar processes, operating in the reverse direction, serve to initiate segregation from the original hybrid cells. Between these limits, as preceding sections have made clear, segregant or revertant properties seem to evolve along a more graded time course, and by events which take place in most if not all cells, rather than in a small variant fraction. DISCUSSION

In the present experiments we have observed a pattern of segregation which is random and spontaneous in origin, yet non-Mendelian in subsequent phases of development. These features can be seen clearly when back selection of hybrids in BUDR is used as a model system. Thus, segregants appear by stepwise change but undergo a progressive alteration in properties when continued serially in the presence of BUDR Shifts in plating characteristics are a direct expression of this process, but segregants also show a continuous decline in ability to incorporate H3-thymidine, and decreasing levels of thymidine kinase activity. Clonal analysis suggests that evolution of segregant properties takes place in most if not all cells of the population, and is not mediated by variation and selection of minority cell types. Finally, the progression in segregant properties can be reversed, within limits. by altering the selection pressure. ‘These are characteristics of the segregation process that are difficult if not impossible to explain on the basis of gene mutation, chromosome rearrangement, or chromosome loss. The broad occurrence of segregational shifts, for example, even within small clones, would seem to rule out gene mutation as an underlying mechanism, Similarly, the reversibility of such shifts cannot be accounted for under the hypothesis of chromosome loss. The evolution of segregant properties by gradual, uninterrupted change poses a particular problem for any of the above models. Discrete, stepwise alterations at the phenotypic level would be expected if gene or chromosome alteration took place; no such discontinuities in the evolution of segregants were observed.

A more promising explanation might be built on the assumption that gene expression shifts progressively during back selection and is responsible for the observed continuity of change (Harris, ’74b). However, one difficulty with this epigenetic concept in its simplest form is the long drawn-out time course for the events concerned. For segregants in BUDR, over 300 days and more than 500 cell generations were required for the process to go to completion. Thus, if shifts in gene action alone are responsible, the time frame is unique. By contrast, induction or repression is known to occur rapidly in enzyme biosynthesis, and in other models of gene expression that have been examined to date. Actually, the evolution of segregants resembles more closely a sorting-out process of some kind; one that occurs progressively within individual cells over a series of generations. The observed kinetics are formally analogous to a model in which two kinds of determinants for phenotypic expression are contributed to hybrids by the parental cell types. Evolution of segregant properties would then correspond to a change in the relative numbers of these determinants, induced by differential replication in the presence of a selective agents. Such a concept draws supports from the non-Mendelian behavior of mitochondria1 drug resistance markers in Paramecium (Adoutte and Beisson, ’72; Beale et al., ’72; Beale, ’73). Resistance to erythromycin and chloramphenicol in Paramecium, for example, are inherited through the cytoplasm and resistance to either inhibitor can be conferred specifically by injecting mitochondria from resistant to sensitive cells. Of particular interest are the results obtained from conjugation of erythromycin-resistant and chloramphenicol-resistant paramecia. Cytoplasmic exchange between the conjugants can be induced to occur, resulting in the formation of “mixed” cells that are able to grow in either antibiotic separately, but not in a combination of both. If such cells are propagated serially in the presence of one drug alone, the potential for resistance to the second drug is found to decrease over a series of generations, and is finally lost. Thus, i t ap-

NON-MENDELIAN SEGREGATION IN HYBRID CELLS

pears that drug-resistant mitochondria multiply progressively in the presence of a selective agent, while sensitive mitochondria within these same cells are inhibited, and are gradually eliminated. Such experiments show that intracellular shifts in cytoplasmic determinants can lead to continuous, graded changes in cellular phenotypes. On the other hand, the generality of this mechanism is an open question. Specifically, are such phenomena confined to a few characteristics such as chloramphenicol and erythromycin resistance, which appear to be coded for directly by the mitochondria1 genome? At first sight, segregation of BUDR or AZG resistance in mammalian cells would seem to have little more than a formal similarity to the Paramecium model, since these are nuclear markers, the genetic loci for which can be assigned to particular chromosomes. However, the more general case may be a functional link in phenotypic expression between nuclear genes and a population of cytoplasmic determinants which serve to modulate the appearance of specific markers. If these determinants were subject to selective changes, intracellular remodeling could lead to new patterns of phenotypic expression, e.g., reduction or loss of the ability to synthesize certain enzymes. Variants arising in this way would be analogous to phenocopies, i.e., cells showing a modified phenotype without any necessary alteration of nuclear genes or chromosomes. The concept of intracellular selection offers an explanation for certain properties of hybrids and segregants that are difficult to account for in more conventional terms. One of these is the varied spectrum of segregation frequencies that is observed among hybrid clones of similar origin (table 2 ; also Marin ('71) and Marin and Manduca ('72)). This disparity could reflect a variable mix in two types of cytoplasmic determinants received from the original parental cells, leading to differences in the probability of segregation within individual hybrid clones. The random high frequency event that initiates segregation (table 3 ) might then be a differential loss or irregular distribution of determinants within a few hybrid cells,

427

leading to altered phenotypic expression and survival in back selection medium. Further shifts in the ratio of determinants under selection pressure could account for the observed gradual rise in growth rate of early segregants in BUDR, the continuous decline over long periods in the HAT/ BUDR plating ratio, and the reversal of this trend on return to HAT medium. These suggestions are provisional in nature, but may be useful as a basis for experimental tests. More direct evidence may conceivably be obtained from fusion studies with specific cytoplasmic organelles (Radsak et al., ' 7 2 ) . The present discussion has been concerned primarily with intracellular selection as a mechanism which may lead to hybrid segregation in the absence of chromosome changes. On the other hand, it is worth emphasizing that there is no necessary conflict between intracellular selection and chromosome loss as segregational mechanisms. Deletion of chromosomes occurs in varying degree in nearly all hybrid systems, and is particularly prominent within interspecific hybrids. Segregation in these cells is clearly associated with chromosome loss, and seems to take place as an abrupt, stepwise alteration. However, it is difficult to distinguish cause and effect in this relationship. I n terms of mechanisms, the underlying sequence could be a random loss of chromosomes, leading to disappearance of functionally associated determinants at the cytoplasmic level. On the other hand, little or nothing is known as yet of the factors that regulate stability or change in hybrid karyotypes. Conceivably, cause and effect in this case may be reversed; i.e., chromosome loss could be a secondary event, triggered by spontaneous or selective remodelling of cytoplasmic determinants. These possibilities can be evaluated with appropriate experimental systems, and deserve careful study. LITERATURE CITED Adoutte, A,, and J. Beisson 1972 Evolution of mixed populations of genetically different mitochondria in Paramecium aurelia. Nature, 235: 393-396. Albertini, R. J., and R. De Mars 1970 Diploid azaguanine-resistant mutants of cultured hum a n fibroblasts. Science, 169: 482-485.

428

MORGAN HARRIS

Attardi, B., and G. Attardi 1972 Persistence of thymidine kinase activity i n mitochondria of a thymidine kinase-deficient derivative of mouse L cells. Proc. Nat. Acad. Sci., 69: 2874-2878. Barski, G. 1970 Cell association and somatic cell hybridization. Int. Rev. Exp. Path., 9: 151-1 90. Beale, G. H. 1973 Genetic studies on mitochondrially inherited mikamycin-resistance in Paramecium aurelia. Molec. Gen. Genetics, 127: 241-248. Beale, G. H., J. K. C. Knowles and A. Tait 1972 Mitochondria1 genetics in Paramecium. Nature, 235: 396-397. Berk, A. J., and D. A. Clayton 1973 A genetically distinct thymidine kinase in mammalian mitochondria, J. Biol. Chem., 248: 2722-2729. Boyd, Y. L., and H. Harris 1973 Correction of genetic defects in mammalian cells by the input of small amounts of foreign genetic material. J. Cell Sci., 13: 841-861. Brent, T. P., J. A. V. Butler and A. R. Crathorn 1965 Variations i n phosphokinase activities during the cell cycle i n synchronous populations of HeLa cells. Nature, 207: 176-177. Bull, D. L., A. T. Taylor, D. M. Austin and 0. W. Jones 1974 Stimulation of fetal thymidine kinase i n cultured human fibroblasts transformed by SV40 virus. Virology, 57: 279-284. Chasin, L. A. 1972 Non-linkage of induced mutations i n Chinese hamster cells. Nature New Biology, 240: 50-52. Clayton, D. A,, and R. L. Teplitz, R. L. 1972 Intracellular mosaicism (nuclear - /mitochondrial+) for thymidine kinase i n mouse L cells. J. Cell Sci., 10: 487-493. Davidson, R. L., S . J. Adelstein and M. N. Oxman 1973 Herpes simplex virus as a source of thymidine kinase for thymidine-kinase deficient mouse cells: suppression and reactivation of the viral enzyme. Proc. Nat. Acad. Sci., 70: 1912-1916. Deaven, L. L., and D. F .Petersen 1973 The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-band, C-band, and autoradiographic analyses. Chromosoma, 41 : 129144. Eker, P. 1965 Activities of thymidine kinase and thymidine deoxyribonucleotide phosphatase during growth of cells i n tissue culture. J. Biol. Chem., 240: 2607-2611. Ephrussi, B. 1972 Hybridization of somatic cells. Princeton University Press, Princeton, New Jersey. Harris, M. 1971 Mutation rates i n cells at different ploidy levels. J. Cell Physiol., 78: 177184. __ 1973 Phenotypic expression of drug resistance in hybrid cells. J. Nat. Cancer Inst., 50: 423-429. __ 1974a Comparative frequency of dominant and recessive markers for drug resistance in Chinese hamster cells. J. Nat. Cancer Inst., 52: 1811-1816. 1974b Mechanisms of de nouo variation in mammalian cell cultures. In: Somatic Cell Hybridization. R. L. Davidson and F. de la Cruz, eds. Raven Press, New York, pp. 221-227.

Kit, S., D. R. Dubbs and P. M. Frearson 1965 Decline of thymidine kinase activity in stationary phase mouse fibroblast cells. J. Biol. Chem., 240: 2565-2573. Lea, D. E., and C. A. Coulson 1949 The distribution of the numbers of mutants i n bacterial populations. J. Genetics, 49: 264-265. Lin, S., and W. Munyon 1974 Expression of viral thymidine kinase gene i n herpes simplex virus-transformed L cells. J. Virol., 14: 11991208. Littlefield, J. W. 1966 The use of drug resistance markers to study the hybridization of mouse fibroblasts. Exp. Cell Res., 41: 190-196. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J . Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Marin, G. 1969 Selection of chromosomal segregants i n a “hybrid” line of Syrian hamster fibroblasts. Exp. Cell Res., 57: 29-36. 1971 Segregation of morphological revertants i n polyoma-transformed hybrid clones of hamster fibroblasts. J. Cell Sci., 9: 61-69. Marin, G., and J. W. Littlefield 1968 Selection of morphologically normal cell lines from polyoma-transformed BHK2l/ 13 hamster fibroblasts. J. Virol., 2: 69-77. Marin, G., and P. Manduca 1972 Synchronous replication of the parental chromosomes i n a Chinese hamster-mouse somatic hybrid. Exp. Cell Res., 75: 290-293. Marin, G., and L. Pugliatti-Crippa 1972 Preferential segregation of homo-specific groups of chromosomes in heterospecific somatic cell hybrids. Exp. Cell Res., 70: 253-256. Miller, 0. J., P. W. Allerdice, D. A. Miller, W. R. Breg and B. R. Migeon 1971 Human thymidine kinase gene locus: assignment to chromosome 17 i n a hybrid of man and mouse cells. Science, 173: 244-245. Munyon, W., R. Buchsbaum, E. Paoletti, J. Mann, E. Kraiselburd and D. Davis 1972 Electrophoresis of thymidine kinase activity synthesized by cells transformed by herpes simplex virus. Virol., 49: 683-689. Munyon, W., E. Kraiselburd, D. Davis and J. Mann 1971 Transfer of thymidine kinase to thymidine kinaseless L cells by infection with ultraviolet irradiated herpes simplex virus. J. Virol., 7:813-820. Radsak, K., W. Sawicki and H. Koprowski 1972 Fusion of isolated mitochondria with tissue culture cells. Zeitschr. f. Naturforsch., 27b: 4 19-423. Rommelaere, J., M. Susskind and M. Errera 1973 Chromosome and chromatid exchanges in Chinese hamster cells. Chromosoma, 41: 243-257. Ruddle, F. 1972 Linkage analysis using somatic cell hybrids. Adv. Human Genetics, 3: 173-235. 1974 Human genetic linkage and gene mapping by somatic cell genetics. In: Somatic Cell Hybridization. R. L. Davidson and F. de la Cruz, eds. Raven Press, New York City, pp. 1-25. Sobel, J. A., A. M. Albrecht, H. Riehm and J. L.

NON-MENDELIAN SEGREGATION IN HYBRID CELLS Biedler 1971 Hybridization of actinomycin D and amethopterin-resistant Chinese hamster cells in nitro. Cancer Res., 31: 297-307. Szybalski, W., E. H. Szybalska and G. Ragni 1962 Genetic studies with h u m a n cell lines. Nat. Cancer Inst. Monogr., 7:75-88. Westerveld, A., R. P. L. S. Visser, M. A Freeke

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and D. Bootsma 1972 Evidence for linkage of 3-phosphoglycerate kinase, hypoxanthineguanine-phosphoribosyl transferase, and glucose 6-phosphate dehydrogenase loci in Chinese hamster cells studied by using a relationship between gene multiplicity and enzyme activity. Bioch. Genetics, 7:33-40.