cytoplasmic inheritance in mammalian tissue culture cells

IN VITRO Volume 12, No. 11, 1976

C Y T O P L A S M I C I N H E R I T A N C E IN M A M M A L I A N T I S S U E C U L T U R E C E L L S 1

DOUGLAS C. WALLACE, Y. POLLACK, C. L. BUNN, AND J. M. EISENSTADT

Department of Human Genetics, Yale School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

SUMMARY

A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol {CAP} resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids {CAP-R human X C AP-S mouse} occurred either at a moderate frequency and were stable or at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspe- cific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA~ of all mouse-human cybrids and most HAT and then CAP- selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.

Key words: mammalian genetics, mitochondrial genetics, mitochondrial DNA, hybrid, cybrid, phylogenetic divergence.

INTRODUCTION

The genetics of mitochondria and chloroplasts has been extensively studied in lower eukaryotes such as Saccharomyces, Paramecium and Chlamydomonas { 1 ~. Mutants resistant to specific mitochondrial protein synthesis inhibitors, such as chloramphenicol {CAPk erythromycin qERY) and spiramycin (SPR}, have been isolated in Saccharomyces (2,3t and Paramecium (4}. Cyto- plasmic inheritance of these mitochondrial markers has been demonstrated by a variety of criteria, such as transfer by microinjection of iso-

1Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Cul- ture Association, Philadelphia, Pennsylvania, June 7-10, 1976.

lated mitochondria, non-Mendelian meiotic inheritance, asexual segregation, drug elimination and recombination (1,4, 5}.

In Saccharomyces a mitochondrial gene "¢o" has been discovered which affects recombination frequency and transmission of the CAP, ERY and SPR genes (6L The gene has two naturally occurring alleles o~ + and o~- and a neutral mutation co" (7}. Crosses between cells having different ¢~ alleles (¢o ÷ X o~-, heterosexual~ cause a polarity in the frequency of marker transmission and reciprocal recombinants, while crosses between cells having the same ¢0 allele (¢o ÷ X ¢o ÷ or ¢o- X c0- homosexual} do not show these effects. A model has been proposed for Saccharomyces which suggests that recombination occurs by gene conversion. In homosexual crosses gene conversion is random and hence reciprocal. In heterosexual crosses gene

758

MAMMALIAN CYTOPLASMIC GENETICS

conversion starts with co- and proceeds through CAP, ERY and SPR in turn, converting co- alleles to co÷ I6,7).

Asymmetric transmission of mitochondrial markers is affected by nuclear genes such as mating type and can be eliminated by treatment of zygotes with inhibitors of cytoplasmic protein synthesis and nuclear transcription, but not by inhibitors of mitochondrial protein synthesis (8-12 ). A variety of models have been proposed to explain biased transmission such as selective replication or destruction of mitochondrial DNA (mtDNA) (Il l .

A marked asymmetric transmission of chloroplast genes in Chlamydomonas has also been correlated with nuclear genes linked to the mating type locus imt) (1,13). A restriction and modification model has been proposed to explain this phenomenon based on the correlation of initial bias toward the mt÷ markers with the loss of the mt- chloroplast DNA (chDNA) and the modification of the mt ÷ chDNA within 6 hr after zygote formation (141.

In Paramecium mitochondrial marker transmission and segregation can be examined by mix- ing the mitochondria during conjugation or by microinjection of purified resistant mitochondria into sensitive cells (4,15). In mixed cytoplasms re- suiting from conjugation of cells from the same syngen tsubspecies or species), both mitochondrial markers are retained for extended periods (151. Formation of mixed cytoplasms by microinjection of mitochondria from cells of the same syngen, followed by selection, results in a high frequency of marker transfer. Microinjection between syugens can give three possible results: a high frequency of transfer, a low frequency of transfer or no transfer. In addition, transfers in one direction may succeed while the reverse transfer may not (16,17).

In Saccharomyces, the molecular nature of cytoplasmic mutations rendering resistance to CAP, ERY and SPR seems most probably to result from an altered ribosomal RNA lrRNA) of the large subunit of the mitochondrial ribosome t3). This is supported by deletion mapping cor- relating the loss or retention of the CAP and ERY resistance loci to the presence or absence of the mtDNA sequences coding for the rRNA ~ 18).

Human HeLa and mouse L cell mutants resistant to CAP have also been isolated. The resistance of these mutants is expressed at the level of mitochondrial protein synthesis in vitro (19-221.

759

This paper summarizes the evidence that the mammalian CAP-R mutations are inherited through the cytoplasm. Using the method of transferring CAP resistance from one cell to another via enucleated ~en) cells (cybrid formation), the genetic interaction and phylogenetic relationship between the nucleus and the cytoplasm have been explored. It has been found that the expression of CAP resistance in cybrids and hybrids decreases rapidly with increased phylogenetic divergence of the two parental cells. Aspects of these studies have been mentioned in previous reports (22-27).

METHODS AND RESULTS

Cell lines and cybrids. Cell lines of mouse, human and hamster origins have been used in this study. The cell lines employed, their genetic origin and the drug resistance markers they carry are listed in Table 1. Mouse cell lines were derived from three separate inbred lines tC3H, BALB/c and DBA/2). The human cell lines were derived from two separate origins ~HeLa and WI-L2t, and a Syrian hamster cell line was also used.

A mouse CAP-R line, 501-1, and a human CAP-R line, 296-1, have been used as donors of CAP resistance (Table 1). Cytoplasmic inheritance of CAP resistance in these cell lines has been demonstrated by fusion of an enucleated fragment of the CAP-R cell to a CAP-S cell tcybrid formation) (22,23). Table 2 summarizes the cybrid crosses previously reported within the same species (22-25). It is clear that cybrids are readily prepared between cells of the same strain [enC3H X C3H, enHeLa X HeLa] and between different strains and differentiated states (enC3H X BALB/c, enC3H X DBA, enHEB7A X WAL2A]. In addition, resistance can be passed from cell to cell in succession. The mitoehondrial resistance of 501-1 was transferred to LM~TK-~ to yield LEA. Subsequently, LEA was enucleated and fused to A9 to yield AEL, a line genetically identical to 501-1. Similarly, 296-1 mitochondrial resistance was transferred to BU25 yielding HEB, and then HEB mitochondrial resistance was transferred to S3AG1 to give BES (Table 2).

Cybrid formation occurred at a high frequency, and the mode of selection, either BrdU or AG/TG, had no affect on this frequency. When the nuclear characteristics of cybrids were examined and compared to those of the two parents, the cybrid was always found to have only the nuclear traits of the CAP-S parent ~22-24). The level

760 WALLACE ET AL.

TABLE 1

THE GENETIC ORIGIN AND MARKERS OF CELL LINES USED 1N CROSSES

Cell Species Line

501-1 LEA2A

M o u s e LM(TK-) A9 RAG GF7

296-1 HEB7A

Human BES7A BU25 S3AG1 WAL2A

Hamster BHK-B1

aAll mouse C3H cells ori

Genetic Origin a

C3H C3H C3H C3H BALB/cd DBA

HeLa HeLa HeLa HeLa HeLa WI-L2

BHK-21/13

inated from them,

BrdU c

S R R S S R

S R S R S S

R

Drug Sensitivity b

TG/AG d Sensitivity e

R R S R S S R S R S S S

S R S R R R S S R S R S

S S

CAP

R Source f

Mutant Cybrid

Mutant Cybrid Cybrid

Re:terence

(19,22) (22) (33) (34) (35) (36) (19,20) (23) (23) (37) (23) (23)

- - (38)

ruse L cell explant 1281; the DBA cells from the leukemic line, L5178Y (29); and the BALB/cd cells from an adenocarcinoma (30L The human cells were derived from two unrelated pa- tients, one having a cervical carcinoma yielding HeLa (3D and the other donating lymphocytes yielding WI-L2 (32). Details of these pedigrees have been reported (27).

bR indicates the line is resistant and can grow in the drug indefinitely. S indicates the line is sensitive and will be killed within 7 to 14 days of exposure.

CBrdU (Bromodeoxyuridine) is added at 30 #g per ml. aAG (8-azaguanine) and TG (6-thioguanine) are added at 10 -s M or more. eCAP is added at 50 gg per ml. fThe origin of cytoplasmic CAP resistance in each resistant line. 501-1 and 296-1 are the original mutants. Cybrids

are produced by cytoplasmic transfer (22,23).

of resistance in cybrids to various concentrations of CAP is always found to be similar to the original donor parent. In addition, mitochondria iso- luted from cybrids show comparable levels of resistance to CAP in protein synthesis in vitro as do the mitochondria of the donor parent. Thus mitochondrial CAP resistance transferred in the cytoplasm results in CAP-R mitochondria in the cybrid (22,23; Molony, unpublished dataL

When hydrids were made between resistant and sensitive ceils of the same species, it was found that the frequency of hybrids was the same in medium containing HAT plus CAP as in HAT medium alone. This is true in crosses between cells of the same strain (C3H X C3H, HeLa X HeLa) as well as between cells of different strains (C3H X BALB/c) (Table 2). Hybrids are selected by using nuclear gene complementation in HAT medium (39) so the inclusion of CAP simply requires that one of the two groups of donated mitochondria be retained. Since equal numbers of resistant and sensitive mitochondrial determinants are present in these cells, this result indicates that CAP resistance is dominant in mixed cytoplasms and that

resistant mitochondria are not deleterious to hybrid formation.

Stability of CAP-R determinants in intraspecific cybrids and hybrids. The demonstration of cytoplasmic inheritance of CAP resistance and of the dominance of resistance in hybrids raised the question of whether the determinants for resistance were stably inherited in cybrids and hybrids in the absence of selection (24-26; Table 3). Con- sistently, fusions between two mouse cells and two human cells of the same genetic origin have been given identical results in relationship to stability of CAP resistance. Fusions between cells and cytoplasts of the same strain generated cybrids (AEL and BES) which were fully and stably CAP-R. This stability of CAP resistance was also observed in hybrids between cells of the same strain (ALM and HAS}, even though these cells were never exposed to CAP. This was true even after 90 doublings in the absence of CAP.

When these intrastrain hybrids were cloned, they consistently generated a colony of smaller size in CAP than in parallel cultures without CAP. This suggests that both sensitive and resis-

MA~VIALIAN CYTOPLASMIC GENETICS

TABLE 2

INTRASPECIFIC CROSSES

761

Genetic Fusion Product Origin Product Cross a Selection b Frequency c Designation d

C3H tCAP-R)

× C3H

iCAP-S)

BrdU + CAP 180 LEA

Cybrid

AG/TG -t- CAP 21 AEL

Hybrid HAT ± CAP 180 ALM

C3H Cybrid AG/TG + CAP 600 LER

(CAP-R) X

BALB/ed [ ~CAP-S) Hybrid HAT + CAP 300 LAR

C3H tCAP-RI X Cybrid BrdU + CAP + GEA

DBA/2 (CAP-SI

en501-1 X

LM(TK-)

enLEA2A X A9

501-1 X

LM(TK-)

enLEA2A X

RAG

LEA2A X

RAG

en501-1 X

GF7

en296-1 X

BU25

enHEB7A ×

S3AG1

HEB7A X

S3AG1A

enHEBTA X

WAL2A

BrdU + CAP 150

Cybrid

HEB HeLa (CAP-R)

X HeLa

(CAP-S) TG + CAP 1200 BES

Hybrid HAT ± CAP 420 HAS

tteLa ICAP-R) X Cybrid TG + CAP + HEW

WI-L2 ICAP-S) "All fusions contained between 3 and 5 ), 106 cells of each )arent with 1,000 HAU of inactive Sendal Virus (22,23).

EnLEA2A X A9 was an inefficient fusion because of poor vrrus. Lymphocytes were preincubated with trypsin ~23), at a concentration of 0,01% for GF7 and 0.001% for WAL-2A. Fused ceils were serially diluted and inoculated into selective media.

ball cybrid and hybrid cultures were selected as previously described ~22-25). The GEA cybrids were selected in 30 ~g per ml BrdU and 50 ~g per ml CAP, first in MEM-E plus 10% fetal bovine sermn and then in RPMI 1640 plus 15% heat-inactivated fetal bovine serum.

CThe mean of the frequency of colonies observed at various dilutions and expressed as colonies per 106 parental cells. Parental cells were treated in the same manner as the fused cells. In all cases the number of parental colonies was negligible or zero.

dThe designation of the hybrid or cybrid resulting from the cross. All cybrid lines have the middle letter "E" for enucleated.

tant mitochondria are retained indefinitely and indicates a complete compatibility of these mito- ehondria with nuclei of the same genetic origin. All hybrids had chromosome numbers equal to the sum of the chromosomes of the two parents (24-26).

Fusions between mouse or human cells of different genetic origin revealed quite different re- suits. CAP resistance was unstable in all such fusions studied (Table 3). The L E R cybrids, formed from cells having a 70% nuclear homology (27,401, were unstable for C A P resistance even af-

762 WALLACE ET AL.

TABLE 3

STABILITY OF C A P RESISTANCE IN INTRASPECIFIC FUSIONS

Species

Mouse

H.m~n

Genetic Origin C3H

CAP-R) ×

C3H (CAP-S)

C3H (CAP-R)

X BALB/cd (CAP-S)

HeLa (CAP-R)

× HeLa

(CAP-S)

HeLa (CAP-Rt

X WI-L2

{CAP-S)

Fusion Product

Cybrid

Hybrid

Cybrid

Hybrid

Cybrid

Hybrid

Cybrid

Cell Line

AEL

ALM

LER

LAR

BES

HAS

HEW

Population Doublings

+ CAP a

22

20 102

17

0-41 [SI >41IF]

- cApb

22

0 11 89

22 44

0 12 90

22

0 14 48

26 30

% CAP-R c

95

104 65 %

0.7 0.4

97 9.8 3.8

93

98 79 93

<2 110

aThe number of cell or population doublings grown m 50 gg per ml CAP immediately following fusion. bTbe number of cell or population doublings grown without CAP after initial selection. CThe percentage of the cells which remained CAP-R following growth without CAP. The percentage of CAP-R

cells was calculated from the cloning efficiency of the line in 50 ~g per ml CAP compared to that without CAP.

ter prolonged growth in CAP, 102 doublings. Therefore these cybrids must maintain both CAP- S and CAP-R determinants for long periods in CAP. During this unstable period, all LER cybrids were observed to clone better in the absence of CAP than in its presence, even when taken directly from medium containing CAP (24,26).

When interstrain hybrids (LAR), formed by using the same mouse lines as the LER cybrids, were examined for the stability of the CAP-R determinants, they were also found to be rapidly lost in the absence of CAP. This loss of CAP resistance does not correlate with chromosome loss (24).

Human HEW cybrids between strains HeLa and WI-L2 were unstable for CAP resistance (Table 3). Two periods of HEW growth were observed (Fig. 1). The first growth phase IS] is char- acterized by a long and variable doubling time, 02 sensitivity, poor cloning in CAP and instability of the CAP-R determinants. The second growth phase [F] has a short and constant doubling time, lack of 02 sensitivity, good cloning in CAP and stability of the CAP-R determinants (25,26L

Since the HEW S phase cultures can revert to sensitivity in the absence of selection, both sensitive and resistant determinants must be present in the cell. Because the CAP-R determinants are lost without selection and the CAP-S determinants are retained in selection, the elimination of the CAP-S determinants by 50/~g per ml CAP must be bal- anced by some other force excluding the CAP-R determinants. This balance was examined by varying the concentration of CAP in the medium and examining an energy dependent process, cell growth rate (Fig. 2).

When S phase cultures of HEW5 were grown in varying concentrations of CAP, the doubling time was seen to increase exponentially from cultures without the drug to cultures containing 100/~g per ml CAP. Since only sensitive mitochondria are affected by CAP, this result implies that both sensitive and resistant mitochondria must be con- tributing to the cell's energy metabolism.

When the same parameter was tested on an F phase culture of HEW5, it became clear that CAP concentration had no effect on doubling time. This is consistent with the stability of resistance in


G PHASE [ F PHASE I

~ 7o ~ 60 . . . . . .

i , 0 3 0 . . . . . . . . . . . . . .~ zo

O IO :?0 30 40 50 60 70 SO 90 I00 NUMBER OF DOUBLINGS

PHYSIOLC~ICAL T R A I T ' ] ~i GROWTH RATE SLOW FAST RATE VARIABILITY VARIABLE C STANT 02 SENSITIVITY S I CLONING t CAP L I CLONING - CAp H RESISTANCE STABILITY U

FIG. 1. The HEW culture was maintained in log phase in deep medium containing 50 gg per ml CAP by regular counting and periodic dilution. A series of sequential growth curves was obtained. The slope of each curve is plotted as a bar spanning the number of doublings of that growth curve. Zero is the time of the first transfer. A slow (S) and fast iF) growth rate (the doubling time vari- ance of S phase is 159 while that of F is 29), O5 sensitivity (S is sensitive, R is resistant), cloning efficiency with and without CAP (L is low and H is high cloning efficiency) and the stability of CAP resistance in the absence of selection (U is unstable and S is stable).

these cells, suggesting that sensitive mitochondria do not contribute to their energy metabolism. The same result has also been obtained for a stably resistant BES cybrid from an intrastrain HeLa cross (Fig. 2).

In summary, both human and mouse interstrain cybrids have been found to be unstable for CAP resistance in contrast to the intrastrain cybrids previously described. Each maintains sensitive and resistant mitochondrial determinants during selection for extended periods and shows an impaired ability to clone in CAP during this time. These results implicate an exclusionary process acting against the resistant determinants in opposition to the CAP selection acting against the sensitive determinants.

It is possible that not all interstrain crosses are unstable. Cybrids of CAP-R C3H and CAP-S DBA cells (GEA cybrids, Table 2) cloned very well in CAP immediately after formation, grew at a normal rate and were not O2 sensitive. These characteristics are more similar to intrastrain cybrids. These lines have a 75 to 80% nuclear homology as compared to 70% for C3H and BALB/c cells (27,40). If these cybrids prove to be stable and this homology difference is significant,

763

then this will suggest a direct correlation between nuclear homology and stability of CAP resistance.

Intrastrain and interstrain hybrids follow the same pattern as found in the cybrids, even though both nuclear genomes are present. Hybrids be- twecn cells of the same species occur at the same high frequency whether selected in HAT or HAT plus CAP. However, HAT-selected hybrids from the same strain are stable for CAP resistance, while those between strains are not. It was of interest to know what the fate of the CAP-R determinants would be in cells having even less genetic homology. To examine this, cybrids and hybrids between cells of different species were prepared and analyzed.

Studies on interspecific hybrids and cybrids. Formation of cybrids between CAP-R mouse cells (Mus musculus) and CAP-S Syrian hamster cells (Mesocricetus auratus) showed two stages of growth tTable 4). Initially, at 25 days, a high frequency of cybrid colonies was observed, about 220 colonies per 106 parental cells. At this time the cybrid clones were quite large and the parental

4 0 0

200

100

80

V- 60

Z

-5 40 ~ , g

2C

BEG5

HEWSA r.- FAST

Io I I I I 0 25 50 75 I00

~9/,=1 CAP

FIG. 2. The effect of CAP concentration on growth rate was examined for an HEW cybrid during the S phase (O) and F phase (A) as well as for a BES cybrid i []). Cells were inoculated into parallel cultures containing various concentrations of CAP. The cell number of these cultures was monitored and the doubling time calculated from the slope of each curve. The HEW S phase culture was grown in deep medium to avoid O2 toxicity.

764 WALLACE ET AL.

TABLE 4

INTERSPECIFIC RODENT CROSSES

Genetic Fusion Product Origin Product Cross Selection a Frequency b Designation

220 C3H

ICAP-R) X

BHK21/13 (CAP-St

Cybrid

Hybrid

en501-1 X

BHK-B1

501-1 X BHK-B1

BrdU + CAP initial

BrdU + CAP final

HAT

HAT -4- CAP

3.4

310

BEL

BAL

BLA

aAll cultures were selected in 2XMEM-E plus 20% fetal bovine serum (38L BrdU was employed at 30/Jg per ml and CAP at 50 ~g per ml. Both were added on day 3 after fusion for cybrid selection. "Initial" represents the colony frequency after 25 days selection and "final" is the frequency of permanent cell lines obtained. HAT and CAP were usecd in hybrid selection as described in Table 2.

~'The mean frequency of colonies per 106 fused parental cells. Inoculating the parents under identical selection conditions resulted in no viable colonies.

cells were virtually eliminated. The growth of the cybrids then stopped and the colonies slowly degenerated. Later, a few colonies began to grow again and gave rise to the BEL clones at a frequency of about 3.4 per 106 parental cells (Table 4L The BEL cybrids grow well but are sensitive to medium depletion at high cell densities.

When hybrids were prepared between the same cell lines, colonies appeared in HAT at a high frequency {310 per 10 ~ parental cells~ in only 10 days. When the same fusion mixture was inoculated into medium containing HAT plus CAP, a 100-fold decrease in colony frequency was observed. Colonies grew slowly, appearing between 10 and 95 days. These HAT-R and CAP-R hybrids have been designated BLA {Table 4). This result is in marked contrast to similar hybrids prepared within the same species in which colony frequency in HAT was also equal to that in HAT plus CAP (Table 2 L These results are comparable to those obtained for cybrid formation and suggest that increased genetic differences between the mitochondria of the CAP-R cell and the nucleus of the CAP-S cell may be inhibiting hybrid and cybrid formation.

HAT-selected hybrids were also removed from HAT medium and challenged with a rich medium containing CAP. In addition to all of the amino acids and vitamins, the medium contains 0.3% glucose, 15 mM HEPES and 1 mg per liter folinic acid to counter the aminopterin blockage of HAT. The medium is called SUDUSO with aminopterin included and SUDUSO-Am with it deleted (27L Four mixed cultures composed of cells from between 66 and 107 colonies and six cultures from individual purified clones were removed from HAT after about 22 doublings and challenged

with CAP using SUDUSO-Am. All cultures were rapidly killed. However, after extended selection, three of the mixed cultures and four of the clones gave rise to CAP-R colonies at frequencies between 3 X 10 -6 and 5 X 10 -4 of the cells challenged. These colonies were designated BAL.

Karyotypic analysis of these cybrids and hybrids was carried out using Hoechst 33258 dye, which brightly stains the pericentric regions of mouse chromosomes but not those of human, Chi- nese hamster {41,42} or Syrian hamster origins {Table 51. The resistant mouse parent, 501-1, is

TABLE 5

CHROMOSOME COMPOSITION OF R EPRESENTATIVE MOUSE-HAMSTER HYBRIDS AND CYBRIDS

Cell Line

501-1 BHK-B1 BEL4A BLA1A

Chromosome Number a

With pericentric

fluorescence b

46.5 0 0

45.2

Without pericentric

fluorescence c

7.4 42.3 41.2 24.9

aChromosome spreads were prepared as previously described (23). The chromosomes were stained using Hoechst 33258 ~411 at between 0.04 and 0.05/~g per ml for 10 min at 37°C. The slides were rinsed in distilled water, mounted in glycerol, and examined with a fluor- escent microscope. Between 20 and 32 individual spreads were counted and the mean number of each type of chromosome reported. BHK-B1 and BEIAA tend to generate tetraploid and octaploid cells. These were ex- cluded from the calculation.

bChromosomes with pericentric fluorescence display a brilliant block of fluorescence around the centromeres of the mouse chromosomes.

cChromosomes without pericentric fluorescence have a uniform fluorescence for the entire length of the chromosome.


seen to contain about 47 chromosomes with and only seven without bright pericentric fluorescence. Except for seven mouse chromosomes, the chromosomes of these lines can be easily distin- guished. The karyotype of a representative BEL cybrid is clearly that of the CAP-S BHK-B1 parent. Yet this and the other BEL cybrids are fully resistant to CAP, and thus meet the criteria for cybrids as established intraspecific studies.

Examination of the chromosomes of the HAT- plus CAP-selected BLA hybrids has consistently revealed the pattern shown by BLA1A (Table 51. All have a full mouse complement and a partial hamster complement, in this case about half of the hamster chromosomes. In previous studies comparable hybrids have been observed to segregate the mouse chromosomes (43L This suggests that selection for a subset of the hybrids which retain the full mouse complement and lose the hamster chromosomes. Finally, when the chromosomes of the HAT-selected then CAP-challenged BAL hybrids were examined, every one had a mouse karyotype. Selection for CAP resistance from HAT-selected hybrids removed from HAT seems to have resulted in the isolation of rare ceils which have lost all hamster chromosomes and returned to something similar to the original mouse parent. If it is assumed that the mtDNA is the vehicle of CAP resistance, then these results are consistent with the previous observation that loss of mouse or hamster mtDNA correlates with loss of homologous chromosomes (441.

Studies on interorder cybrids and hybrids. The apparent success of transfers between two rodent genera indicated that comparable transfers of CAP resistance might be attempted between human and mouse cells. The first series of experiments used a human CAP-R parent and a mouse CAP-S parent. These results are reported in Table 6, in which clear strain differences were observed in these experiments. Transfer of CAP resistance from HeLa cells to LMtTK-~ cells of C3H origin generally were more successful than transfers from HeLa cells to RAG cells of BALB/c origin. In crosses between HeLa and LM(TK-) cells ~Table 6}, the first cybrid formation occurred at the moderate rate of 51 colonies per 106 parental cells. Most of the colonies had appeared by 38 days after fusion and were designated EEL. When each parent was inoculated alone under identical selective conditions, the 296-1 parent died but a few LMtTK-~ flasks grew (Table 6~.

765

All of the EEL cultures examined for chromosome composition using Hoechst 33258 dye have been found to contain chromosomes of LM(TK-~ origin only (Table 7}. Most of the chromosomes contained the distinctive mouse pericentric fluorescence. The remaining three to four chromosomes can be readily identified as of LM(TK-t origin t27). As is true of all cybrids, the EEL lines contain only the nuclear information of the CAP- S parent but are now CAP-R. The EEL cybrids differed from the BEL cybrids in not showing an initial growth period followed by arrest. Further- more, they are less sensitive to starvation at high cell densities. The EEL's require supplementation of the glucose level to 0.3% and addition of HEPES buffer in the medium. The increased glucose requirement probably indicates that the cells are more reliant on glycolysis resulting from de- bilitation of their mitochondria.

When hybrids were prepared using CAP-R HeLa cells and CAP-S LMtTK-~ cells, colonies were readily selected in HAT tTable 6~. Selection of hybrids in HAT plus CAP, on the other hand, was always unsuccessful, and no hybrids were obtained from inoculation of 9 )< 106 cells. Hybrid formation in CAP in these crosses is less than the 30 colonies per 107 parents observed in the mouse- hamster hybrids.

Hybrids selected in HAT were called LAB. When two of these hybrids were removed from HAT at 41 doublings after fusion and challenged with CAP in SUDUSO and SUDUSO-Am, CAP- R cultures emerged after an extended lag period. The two resistant cultures which emerged were designated LABIA and LAB2A. When these CAP-R lines were karyotyped, virtually no human chromosomes were detected ~Table 71.

To circumvent the apparent toxicity of aminopterin and CAP to interspecific hybrids, a selection regime was designed to eliminate the use of HAT. Mouse cells are naturally a thousand times more resistant to ouabain than are human cells ~27,45~. By treating mouse-human fusion mixtures with ouabain for 7 to 14 days after fusion, all of the human fusion mixtures were killed while the mouse ceils and the hybrids remained. By also including either HAT or CAP to select against the mouse parent, viable hybrid colonies were obtained.

Hybrids between 291-1 and LMtTK-~ cells arose at very high frequencies when selected in ouabain plus HAT (Table 61. These cultures were called LOA. Selection of the same fusion mixture in ouabain plus CAP gave a colony frequency of

766 WALLACE ET AL.

TABLE 6

INTERORDER CROSSES

Genetic Fusion Product Origin Product Cross Selection a Frequency b Designation

en296-1 BrdU + CAP Cybrid X 51 EEL

LM(TK-) + G + H

HAT 6 LAB HeLa

(CAP-R) X

C3H ~CAP-S)

HeLa {CAP-R)

× BALB/cd {CAP-S)

C3H ~CAP-R)

X HeLa

{CAP-S)

Hybrid

Cybrid

Hybrid

Cybrid

Hybrid

BES7A ×

LM(TK-)

296-1 X

LM~TK-)

en296-1 ×

RAG

HAT + CAP <0.1 - -

HAT + Oua 2600 LOA

296-1 ×

RAG

CAP -4- Oua

TG + CAP initial

TG + CAP final

610

0.6

0.2

LOH

CAP + Oua

HER

HEBTA HAT 38 HAR X

RAG HAT + CAP <0.07 - -

HAT + Oua 40 ROA

6.5 ROH

BrdU -4- CAP

AG/TG + CAP

en501-1 X

BU25

enLEA2A X

S3AG1A

501-1 X

BU25

<0.1

<0.03

<0.1

<0.1

HAT

HAT -4- CAP

aAU cultures were selected in MEM-E ,lus 10% fetal bovine serum. G indicates the medium was supplemented with glucose to 0.3% while H indicates supplementation to 15 mM HEPES at a final pH of 7.3. BrdU, CAP, and HAT were used as in Table 2. "Oua" means ouabain was added to the cultures at 10 -6 n from day 7 to 14 after fusion. TG was added at 10 -4 on day 0 in the fusion en296-1 X RAG where "initial" frequency was reported for day 20, while "final" frequency is the number of viable CAP-R cybrids obtained. In the fusion en501-1 X BU25, BrdU was added on day 3 while CAP was added at either 0 or 3 days after fusion. AG/TG were added to cultures of enLEA2A X S3AG1A between 5 and 8 days after fusion at concentrations of 5 X 10 -s M TG and either 2 X 10 -4 or 2 X 10 -s M AG with or without G and H.

bThe mean frequency of colonies per 10 ~ parental cells calculated from various dilutions of the fusion mixture. Fre- quencies listed as less than a value, i.e. <0.1, indicate that no colonies were obtained. In all experiments, the parents were inoculated alone under the same selective conditions. These controls were uniformly negative except in the case of the fusion en296-1 X LM(TK-) and the ouabain-selected fusions. Three of 12 LM(TK-) control flasks were positive in the en296-1 X LM(TKq fusion. In the ouabain fusions, about 0.03 296-1 colonies per l0 G cells survived the ouabain selection and 0.2 LM{TK-) colonies per 10 ~ cells survived the ouabain and CAP selection.

610 colonies per 10 ~ paren ta l cells. These cultures were called LOH. T he h igh frequency of colony format ion in these fusions is difficult to equate wi th HAT-se lec ted hybr id frequencies. A survey of the chromosome composi t ion of the LOA and

LOH cultures us ing Hoechst 33258 dye revealed t ha t most conta ined only HeLa chromosomes (Tab le 7).

Although one in te rpre ta t ion of these results is t h a t some form of metabol ic coopera t ion between the 296-1 and L M ( T K - ) cells rescued the HeLa cells f rom ouaba in killing, the absence of colonies in parental controls argue against such a high protect ion rate. An a l ternat ive explana t ion could be t ha t hybr ids did form, passed t h rough ouaba in

selection, and then lost the mouse chromosomes .


TABLE 7

CHROMOSOME COMPOSITION OF REPRESENTATIVE HELA-LM~TK-) CYBRIDS AND HYBRIDS

Cell Line

291-1 BES7A LM(TK-D EEL5 LABIA LOA4 LOH4A

Chromosome Number a

With pericentric

fhtore~ence

0 0

44.4 42.4 43.1 0 0

Without pericentric

fluorescence

63.7 65.4 3.5 3.4 4.3

67.0 67.7

aSee Table 5 for explanation. The data reported for LOA4 and LOH4A are the mean chromosome counts of 10 karyotypes each. The mean chromosome counts of between 20 and 31 karyotypes are reported for the remaining cultures.

The inheritance of CAP resistance in fusions between CAP-R HeLa cells and CAP-S RAG cells was quite different from that in fusions between HeLa and LM(TK-} cells. When cybrids were made between en296-1 and RAG ceils (Table 6}, only 0.6 colonies per 106 parental cells were obtained, a total of 11 colonies in two experiments. These colonies appeared within 18 days but stopped growing. Two-thirds of the original colonies then slowly died and the remaining cultures grew into the HER cybrids. This general course of events is very similar to that seen for the BEL cybrids except that the frequencies were much lower and the appearance of HER colonies did not follow a dilution series. Treatment of the two parental lines under identical selective conditions did not yield any colonies (a total of 1.5 X 107 RAG cells was tested }. Supplementation of the medium with 0.3% glucose and 15 mM HEPES increased the cybrid yield only slightly.

The HER cybrids were observed to be quite sensitive to medium depletion. At a density higher than 104 cells per cm 2, the cells would rapidly shrivel and die. This effect was countered by rais- ing the glucose level and adding HEPES buffer. The initially unstable colony formation and sensitivity to medium depletion were similar for the BEL and HER cybrids but quite different from the EEL cybrids.

Using Hoechst 33258, the chromosome complements of the HER cybrids and their parents were analyzed (Table 8}. Every chromosome of the H E R cybrids was found to be of RAG origin. Thus the HER eybrids, like BEL and EEL cy-

767

brids, contain only the nuclear genetic material of the CAP-S parent but are now CAP-R.

Selection of hybrids between CAP-R HeLa and RAG cells gave a moderate number of hybrids in HAT at a rate of 38 colonies per 106 parental cells ITable 6}. Inoculation of large numbers of fused cells into HAT plus CAP gave no viable hybrids. This result is parallel to the HeLa fusion with LM(TK-j .

Nineteen of the HAT selected hybrids (HAR hybridM were picked, recloned, and then challenged with HAT plus CAP followed by SUDUSO and SUDUSO-Am. Four of these gave rise to continuous CAP-R cultures failing into two classes. Three of the hybrids (represented here by HAR9B} grew very slowly, required high levels of glucose and rich medium, produced excessive acid and were highly vacuolated. Two of these have been karyotyped and found to have similar chromosome complements. Both had one and one-half RAG complements and between 12 and 16 human chromosomes ~Table 8 }.

The hybrid HAR16C was quite different from the other hybrids. For normal growth it required high levels of glucose but much less rich medium. The chromosome complement of this line is composed of a single RAG chromosome complement and about five HeLa chromosomes ~Table 8}.

An attempt was also made with 296-1 X RAG hybrids to select directly for CAP resistance using CAP and ouabain. The selective regime was the same as that used in the 296-1 X LM(TK-I experiments. Selection of the fusion mixture in HAT

TABLE 8

CHROMOSOME COMPOSITION OF REPRESENTATIVE HELA-RAG CYBRIDS AND HYBRIDS

Cell Line

296-1 HEB7A RAG HER1 HAR9B HARI6C ROA5C ROH8A ROH9A

Chromosome Ntmlber a

With pericentric

fluore~ence

0 0

63.9 57.3 91.9 57.2

121.0 41.6 0

Without pericentric

fluorescence

63.7 57.4 0 0

16.4 5.0

27.2 90.6 63.6

aSee Table 5 for explanation. The mean chromosome counts for ROH9A are based on the counts of 10 chromosome spreads; those for ROHSA, 52 spreads; and those for the remaining lines, between 21 and 31 spreads.

768

plus ouabain resulted in colonies with a frequency similar to that found with HAT selection (Table 6L These cultures have been designated ROA. Inoculation of the same fusion mixture into medium containing ouabain plus CAP resulted in a colony frequency of 6.5 colonies per 10 ~ parental cells. These cultures were designated ROH. A clear difference in frequency is seen between colonies formed in the fusions of 296-1 X LM(TK-} and 296-1 X RAG.

The ROA cultures selected in ouabain plus HAT were found to contain three types of cells. Some contained only HeLa chromosomes. Others contained only RAG chromosomes, though presumably the human HPRT gene must be present. The third type, the typical hybrid form, contained both HeLa and RAG chromosomes. One example of this latter class, ROA5C, is reported in Table 8.

The karyotypes of cultures selected in ouabain plus CAP were unique. Though most of the clones examined contained primarily human chromosomes as indicated by clone ROH9A (Table 8}, one clone, ROH8A, was found to contain one and one-half HeLa chromosome complements and only two-thirds of a RAG complement. This un- usual hybrid between permanent mouse and human cell lines segregated the mouse chromosomes. Extreme cases of this phenomenon could explain the appearance of HeLa cells among the fusion products. Previous reports on segregating mouse-human hybrids using permanent lines have observed the selective loss of the human chromosomes ~46-48}. The only other clear cases of reverse segregation occur when permanent human lines are fused to primary explant rodent cells (49L These observations suggest that, as in the BLA hybrids, selection for CAP resistance has selected for cells which contain all of the chromosomes of the resistant parent. Previous studies have shown that there is a correlation between the parental origin of chromosomes and mtDNA retained ~47,48, 50-521. Therefore selection for human CAP resistance might also select for cells which retain all of the human chromosomes.

Efforts to construct cybrids by fusing enucleated CAP-R mouse L cells to HeLa cells were consistently negative regardless of the cell line used or the selective system employed ~Table 6L In the fusion en501-1 X Bu25, 107 cells were inoculated into selective medium without any permanent clones appearing. This experiment was done in parallel with the fusion of en501-1 X BHK-B1 in which numerous colonies were obtained (Table

WALLACE ET AL.

4~, indicating that the conditions used should have resulted in cybrid formation. When enLEA2A was fused to S3AG1A, 3.6 X 107 total cells were inoculated into selective medium. Four separate experiments were carried out using six different variations on the selective regime. No viable colonies were obtained in marked contrast to the successful reciprocal cross which resulted in the EEL cybrids. A similar observation of differences between reciprocal transfers has also been reported in interspecies transfers of Paramecium (17L

On two separate occasions, attempts were made to prepare hybrids between 501-1 and BU25 cells. In each experiment, 5 X 10 ~ fused cells were inoculated into medium containing HAT and medium containing HAT plus CAP. No colonies appeared in any of the HAT selected cultures. One colony appeared in a HAT plus CAP culture but then stopped growing at about 160 ceils and died. These negative results are not the result of variations in experimental conditions, for the fusion 501-1 X BHK-B1 (Table 4} was run concurrently with one of the 501-1 X BU25 hybrid fusions. The mouse-hamster fusion was clearly positive while the mouse-human fusion was negative.

These results are surprising since the hybrid cell contains the nuclei and cytoplasms of both parents. All genes necessary for a successful interaction between the nucleus and the mitochondria should be present. In addition, hybrids have been prepared in HAT between human HeLa BES7A cells and mouse C3H LM(TK-D ceils, which have identical genetic origins to BU25 and 501-1, re- spectively. The only difference in the crosses is the reversal of nuclear and cytoplasmic drug resistant markers. Assuming that fusion took place, the reversal of the CAP-R marker from human to mouse cells resulted in a severe depression of hybridization frequency.

The negative hybrid results may be due to the same factors as the negative cybrid results or they may be the product of additional factors specific to this fusion. In all previous experiments, the inheritance of CAP resistance has been similar in cybrids and hybrids having the same parents. This observation suggests that the factors responsible for the failure to transfer CAP resistance in the en501-1 X BU25 cybrids may also be responsible for the failure to retain CAP resistance in the 501-1 X BU25 hybrids. Alternatively, the reasons for the lack of success in forming CAP-R hybrids may be different from those responsible for the negative cybrid results. Models for the cybrid in-


compatibility and lack of reciprocity will be considered in the discussion.

The failure of hybrid colonies in the fusion of CAP-R mouse and CAP-S human cells in medium containing HAT plus CAP is consistent with comparable lack of success in forming viable hybrids in fusions of CAP-R human and CAP-S mouse cells selected in HAT plus CAP. Pre- viously, it was concluded that the combination of aminopterin, CAP-R mitochondria, and a mixed mouse-human nucleus was inhibitory. This was partially supported by the fact that CAP-R colonies can be obtained by stepwise selection in HAT and then CAP or by direct selection in ouabain and CAP.

The lack of viable hybrids in fusions of CAP-R mouse and CAP-S human cells in HAT can be explained based on previous work. Reports have shown that, in segregating mouse-human hybrids, the mouse mtDNA is retained and human mtDNA is lost (47,48). Assuming that CAP resistance resides in the mtDNA, this predicts that in the 501-1 X BU25 hybrids the genetic configura- tion of the cell would be a mixed nucleus plus the mouse CAP-R mtDNA, whether or not CAP was included in the medium. Consequently, hybrid formation would be suppressed.

Though it is unknown why this combination of factors is inhibitory, the effect of aminopterin could be attributed to the inhibition of formyla- tion of mitochondrial methionyl-tRNA. High levels of aminopterin have been shown to suppress mitochondrial protein synthesis (53,54L

Preliminary analysis o/ mtDNA in mouse- human hybrids and cybrids. As mentioned above, the human chromosomes and mtDNA are lost in mouse-human hybrids between permanent cell lines (47,48L Human mtDNA is also lost in human-Syrian hamster hybrids (55}. In all of ,oc these studies, differentially radioactive labeled • 8¢ mtI )NA was purified from the parents and hybrids and mixed in CsCI density gradients. Since = the densities of human and rodent mtDNA differ, : : 4 o

the origin of the hybrid's mtDNA can be "~ 201

identified. Hybrids which are prepared using permanent

IO0 human cells and primary rodent cells can segregate either human or rodent chromosomes (49). The mtDNA of such hybrids has been analyzed by hybridization of differentially labeled mitochondrial complementary RNA (cRNA) from the two parental species to whole cell DNA or purified mtDNA. A correlation has been made between the origins of the nuclear DNA and

769

mtDNA retained. In addition, mtDNA of both species has been found to be maintained up to 150 doublings in certain cell lines (50-52}. Similar re- suits have been reported in mouse-Syrian hamster hybrids. However, one hybrid having predominantly mouse chromosomes was found to have hamster mtDNA (44).

Finally, the structure of the mtDNA from hybrids between permanent human and primary rodent cells has been analyzed using CsC1 gradients and cRNA hybridization. Several hybrids were found to contain human-rodent recombinant mtDNA (51,52,56L

These techniques were applied to the mtDNA's of the human-mouse hybrids and cybrids produced in the present study (Table 9L The HER cybrids, made by the fusion of en296-1 )< RAG (Table 6L were analyzed by comparing the densities of differentially labeled mtDNA from the parents and the cybrids. The cybrid mtDNA was found to be equivalent to RAG mtDNA (Table 9, columns 2 and 3L

The mtDNA of various cybrids and hybrids was also analyzed by hybridizing differentially labeled human and mouse mitochondrial cRNA to whole cell DNA extracted from cybrids and hybrids. By calculating the ratio of 32P-human cRNA and 3H-mouse cRNA counts hybridized to the cybrid and hybrid DNA and comparing these ratios to ones obtained from known mixtures of human and mouse whole DNA cell and mtDNA (Fig. 3), the percentage of human and mouse mtDNA sequences can be determined. (Table 9, columns 4 and 5 L

I0 1.0 32p CPMI3H CPM

at

so

BO

0 I O0

FIG. 3. Known mixtures of human and mouse whole cell DNA (/X) and mtDNA (©) were bound to cellulose nitrate filters and hybridized to 32P-labeled human mitochondrial cRNA and 3H-labeled mouse mitochondrial cRNA. The counts were corrected for nonspecific binding to E. coli DNA and the 32p to 3H ratios plotted against the percentage of the DNA from each species.

770 WALLACE ET AL.

TABLE 9

ANALYSIS OF THE MITOCHONDR1AL DNA IN HUMAN-MOUSE CYBRIDS AND HYBRIDS

Labeled mtDNA [ Disc Hybridization Cell Gradientsa t Hybridizati°nb to Gradients c

Line % % % % % I % Mouse Human Mouse Human Mou~ I Human

HER1 100 0 100 0 NT NT

HER2 100 0 100 0 NT NT

EEL5 NT NT 96 4 NT NT

HAR9B NT NT 91 9 + +

HAR16C NT NT 95 5 -4- - -

LAB1A NT NT 97 3 + - -

LAB2A NT NT 1 99 1 + - -

aRadioactiveb labeled mtDNA from the parents, 296-1 and RAG, and the HER cybrids was ~urified, mLxed in pairs and centrifuged to equilibrium in a CsCI gradient (47}. Parental mtDNA was labeled with l~C-thymidine and cybrid mtDNA with 3H-thymidine. Coincidence in density of RAG and the HER mtDNA's is represented here as 100% mouse sequences.

bWhole cell DNA was purified from each cybrid and hybrid and between 15 and 50 ~g were bound to cellulose- nitrate discs. Known mixtures of mouse and human whole cell DNA and mtDNA were also bound to discs as was E. coli DNA. All discs were hybridized to 32P-labeled human mitochondrial cRNA and 3H-labeled mouse mitochondial cRNA in 50% formamide and 4XSSC at 40°C for 18 hr. The filters were washed, treated with RNase A, rewashed, dried and counted. All counts were corrected for nonspecific binding to E. coli DNA. The 32p to ~H ratios for the cybrids and hybrids were then compared to the standard curve (Fig. 3), permitting determination of the percentage of mouse and human mtDNA in the cell (50).

eMtDNA was purified from each cybrid and hybrid, centrifuged to equilibrium in CsC1 density gradients, and the fractions of the gradients were collected and bound to discs. Discs for a standard curve and an E. coil background were also prepared and hybridized to 3~P-human and 3H-mouse cRNA as described above. E. coli background was subtracted from all fractions. Peaks of mouse mtDNA hybridizing to the 3H cRNA and mtDNA hybridizing to the 32p cRNA were then identified. The purity of each peak was assessed by comparing its 32P/3H ratio to a standard curve i56}. " + " means a peak was found; " - - " means no peak was observed; and NT means not tested.

The mouse origin of the H E R m t D N A is confirmed by the lack of any detectable human sequences in these cybrids. The percentage of human m t D N A in the E E L cybrids and HAR16C, L A B I A and LAB2A hybrids is too close to the resolution of the technique to be considered significant. H A R 9 B may contain human m t D N A .

As a further confirmation of these results, m t D N A ' s from the four hybrids were purified, centrifuged to equilibrium in CsC1, and the fractions were hybridized to human and mouse cRNA. All of the lines revealed clear peaks at the mouse m t D N A density which hybridized to mouse mitochondrial cRNA. H A R 9 B also had a smaller peak at the density of human m t D N A which hybridized to human mitochondrial c R N A . Although these results are not definitive, the corroboration of the two hybridization procedures lends support to the possible presence of human m t D N A in the HAR9B hybrid. Human m t D N A peaks were not observed in other lines.

Most of the human-mouse hybrids and cybrids

were found to contain little or no human m t D N A . However, if the existence of both mouse and human m t D N A and H A R 9 B can be confirmed, then analysis of the 16 human chromosomes retained by the line may prove valuable in understanding the interaction of the mitochondrial genes of the nuclear D N A and the m t D N A .

DISCUSSION

A series of somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for CAP resistance. These studies have shown that as genetic homology between the two parents decreases, retention of the C A P - R determinants decreases. These results are summarized in Table 10.

CAP resistance was found to be transferred at high frequencies from cytoplasts to cells of the same genetic origin in both mouse and human systems. This transferred resistance was stable in the


TABLE 10

RELATIONSHIP OF GENETIC HOMOLOGY TO INHERITANCE OF CAP RESISTANCE IN CYBRIDS AND HYBRIDS

771

Genetic Homolog:,/

Genetic Origin a

Frequency of Colony Formation b

Hybrids Cybrids R X S HAT HAT--CAP d HAT + CAP

[ntrastrain C3H X C3H + + + + + + + + H + + + + S HeLa X HeLa + + + + + + + + H + + + + S

C3H X DBA + + + + c NT NT NT S [nterstrain C3H X BALB/c + + + + + + + + MS + + + + U

HeLa X WI-L2 + + + + c 0 - - 0 U

[ntergenus C3H X BHK + + + + ~ + + + + + + ML + + US

HeLa X C3H + + + + + + ML 0 US [nterorder HeLa X BALB/c + + - * + ++-4- L 0 UU

C3H X HeLa 0 0 - - 0 - -

Fate oI

CAP-R e

aThe origin of the ~arents of the cross arranged so that the line on the right side is CAP-S, while that on the left is CAP-R.

bThe frequency of colony formation per l0 s parental cells in various experiments. "+ + + +" is greater than 100 colonies; "+ + +" is between 50 and 6 colonies; "-4- + " is between 3 and 0.6 colonies; a n d " + " is 0.2 colonies. "0" means that no colonies were observed, and "NT" means the fusion was not tested in that medium. The lack of hybrids in the fusion HeLa X WI-L2 may be unrelated to CAP resistance.

CThe frequency of cybrid formation in crosses using lymphocytes tC3H X DBA and HeLa X WI-L21 were selected in suspension and could not be measured. However, both fusions grew up readily, suggesting an H rating to be appropriate.

OThis column rates the ease with which HAT-selected hybrids can yield resistant cultures when removed from HAT and selected in CAP. Two factors were considered: the percentage of cultures challenged which yield resistant lines; and the length of incubation in CAP before resistant cultures grew out. High IHI indicates that 100% of the HAT-selected hybrids were immediately resistant to CAP. Medium short IMSt indicates that in interstrain crosses between 3 to 5% of the cells were resistant in 10 days. In interorder crosses, medium long (MLI indicates that 80 to 100% of the challenged cultures yielded resistant lines but required a moderately long period of selection before resistance was expressed. Low (L} indicates that all cultures showed initial toxicity and cell death on CAP challenge but up to 30% yielded resistant cultures with prolonged selection.

e"S" indicates that CAP resistance is stable in hybrids and cybrids in the absence of selection. This rating is postu- lated for C3H X DBA based on secondary criteria. "U" means that initial CAP resistance will be lost in these hybrids and cybrids in the absence of selection. US indicates that HAT-selected clones were sensitive when challenged with CAP but 80 to 100% of those tested still retained the potential to give rise to CAP-R cultures. UU indicates that when challenged all clones were sensitive and less than 30% could give rise to resistant cultures.

absence of CAP. Resistance is also stable in intrastrain hybrids where the frequency of colonies was the same whether selected in H A T or H A T plus CAP.

When cybrids were prepared between cells of the same species but of different strains, two re- suits were observed. In all crosses, cybrids occurred readily and at high frequencies when quan- tifiable. Cybrids between the mouse strains C3H and D B A were found to behave similarly to the intrastrain cybrids having stable CAP resistance. These cells were not O5 sensitive, grew at the same rate as the parental ceils and cloned equally well with and without CAP.

Cybrids prepared between C3H cells and B A L B / c cells were found to contain unstable CAP resistance in the absence of selection. These cybrids also showed a marked depression of cloning

efficiency by CAP. The H E W cybrids, formed by the fusion of HeLa and WI-L2 cells, were also found to have unstable CAP resistance in the absence of selection, grew much more slowly than the parents, were sensitive to O2 and cloned very poorly in the presence of CAP. All interstrain hybrids between resistant C3H and sensitive B A L B / c cells were initially resistant to CAP, but, like the cybrids, their resistance was unstable.

The two different results obtained for the stability of CAP resistance in interstrain cybrids could be due to an increased genetic difference between the C3H and D B A parents, among other possibilities. Isozyme analysis of 16 enzymes has revealed slight differences between the pairs of inbred lines 140~. C3H and D B A have 75 to 80% of their isozymes in common, while C3H and B A L B / c have 70% in common (271. If these dif-

772

ferences are significant, they suggest that genetic divergence could account for the difference in stability between the two cybrids. No comparable data is currently available for the parental lines of the HEW cybrids.

When cybrids and hybrids were prepared between cells of the same order but of different genera, the expression of CAP resistance became more difficult. Intergeneric Imouse-hamster) cybrids had difficulty in retaining CAP resistance even in selection. Though cybrid colonies formed initially at a high frequency, they soon stopped growing and slowly degenerated, thus suggesting that their resistance was unstable. Intergeneric hybrids occurred at a high frequency when selected in HAT, but at one-hundredth the frequency in HAT plus CAP. When HAT-selected intergeneric hybrids were removed from HAT and challenged in CAP, most of the ceils were killed. However, with prolonged selection, some clones developed. The resulting resistant lines were composed of cells having only the chromosomes of the mouse parent. A severe incompatibility seems to exist between the CAP-R mouse determinants and the hamster nucleus. No numeri- cal data on the nuclear homology of these lines are currently available.

The fusion of cells of two different orders, which have less than 5% nuclear DNA homology (50}, revealed two phenomena which influence the inheritance of CAP resistance. Obvious strain differences were found, and reversal of the CAP-R marker from one parent to the other produced different results.

Colonies were obtained at a reasonably high frequency when cybrids were prepared between enucleated CAP-R HeLa cells and CAP-S C3H cells. This frequency was lower than that observed by intraspecific and intergeneric transfer but, unlike the mouse-hamster cybrids, these colonies grew directly into fully resistant lines. So long as selection was applied, these cybrids retained their CAP resistance.

Hybrids between resistant HeLa and sensitive C3H cells ~LAB hybrids} could be readily selected in HAT, but not in HAT plus CAP. Challenging the HAT-selected hybrids with CAP resulted in initial toxicity, but all lines tested ultimately grew. The LAB hybrids, therefore, do not seem to lose their CAP-R determinants. The resistant LAB hybrids contain a full complement of mouse chromosomes and at most one human chromosome. The uniformity of cybrid development dur-

WALLACE ET AL.

ing selection and the prolonged retention of the CAP-R determinants in these hybrids suggest moderate compatibility between the human CAP- R determinants and the mouse nucleus (Table

The pattern of inheritance of CAP resistance in cybrids and hybrids between CAP-R HeLa cells and CAP-S mouse BALB/c cells was much different from that in HeLa X C3H cybrids and hybrids. I t was more analogous to that in the mouse- hamster cybrids and hybrids. Cybrids between en296-1 cells and RAG cells occurred at a low frequency and the colonies which developed were unstable.

The selection of hybrids between HeLa and RAG cells occurred at a moderately high frequency in HAT. No hybrids could be obtained in HAT plus CAP. The challenge of HAT-formed hybrids with CAP usually resulted in death. Fewer than 30% of the hybrids were capable of forming CAP-R cultures and then only after prolonged selection. This suggests that the CAP-R determinants in most of these lines are lost ~Table 10}.

A total lack of reciprocity was observed in cybrids and hybrids between CAP-R HeLa and CAP-S C3H cells and CAP-R C3H and CAP-S HeLa cells. No viable cybrids or hybrids were obtained between CAP-R C3H cells and CAP-S HeLa cells. When compared to the relatively high frequency of cybrids and HAT-selected hybrids obtained with ceils in which the human parent was resistant, it is clear that the reversal of this marker has drastically reduced the frequency of CAP-R fusion products. The same phenomenon has been reported in interspecific mitochondrial transfers in Paramecium (17~.

Three generalizations can be made. First, with increasing phylogenetic divergence between the two parents, there is decreased expression of CAP resistance and decreased compatibility between the cytoplasm of the CAP-R cell and nucleus of the CAP-S cell. This incompatibility does not seem to be entirely the result of inadequate action between the resistant mitochondria and sensitive nucleus, because it is also observed in intrastrain hybrids in which both nuclei are contributed. Sec- ond, there are clear strain differences in interspecific crosses. The similarity in the behavior of mouse-hamster and human-BALB/c crosses was striking when compared to the human-C3H fusions. Finally, there was the lack of reciprocity in interorder fusions. Such evidence should be consi-


dered in any hypothesis proposing a mechanism for these results.

Three possible mechanisms have been described in the literature, which could be applied to these data. The first mechanism has been proposed to explain comparable interspecific transfer data in Paramecium ~16,17). The mitochondrial membrane is hypothesized to assemble in a cry- stalline array. Transfer of mitochondria from one set of nuclear genes to another requires an altera- tion of the lattice resulting in incompatibility. In this model, all three generalizations would be explained by the same mechanism.

A second mechanism could be based on the di- rectional gene conversion system proposed for yeast mtDNA isee Introduction). If in mammalian cells, as in Saccharomyces, the locus of CAP resistance mapped close to an co equivalent, then a CAP-R allele linked to an co allele could be gene- converted to CAP-S in any cross in which the sensitive parent was co ÷. Lack of reciprocity and strain differences are then thought to be a reflec- tion of which co allele is associated with the CAP- R allele and whether the cross in homosexual or heterosexual.

The third mechanism is a restriction and modification system similar to that proposed in Chlamydomonas. As described in the Introduc- tion, following fusion of the two gametes, mt ÷ and mt-, the chDNA of the mt- gamete is lost and that of the mt ÷ gamete is modified. Such a model could be applied to these results. Cybrids would donate CAP-R determinants, presumably mtDNA, to a sensitive cell having a restriction enzyme. When the parental cells are of the same strain, the resistant mtDNA would be properly modified and stable. Improperly modified mtDNA of different strains would be hydrolyzed. In hybrids, the restriction activity from the sensitive parent would still cleave the resistant mtDNA. The lack of reciprocity could then be viewed as differences in the efficiency of the restriction systems of the two parents. One attractive aspect of this model is that such a system has also been proposed to explain chromosome segregation in interspecific hybrids (57).

The remaining aspects of interspecific instability of CAP resistance in both the restriction- modification hypothesis and the gene conversion hypothesis would be attributed to poor mitochondrial and nuclear gene interaction resulting from increased phylogenetic differences.

As mentioned above, the loss of the mtDNA from one of the parents in interspecific hybrids is

773

correlated with the loss of the chromosomes of that species (44,47,48,50), an observation consistent with a restriction system acting on both the nuclear and mtDNA from the same species. As- suming that the mtDNA is the vehicle for CAP resistance, then selection for CAP resistance should also select for interspecific hybrids retaining the chromosomes of the resistant parent. This phenomenon was observed in interspecific hybrids selected directly in CAP. Mouse-hamster hybrids selected in HAT plus CAP contained all of the mouse chromosomes and lost those of the hamster. HeLa-mouse hybrids selected in ouabain plus CAP retained the human chromosomes and lost the mouse chromosomes. The CAP-R HAR9B hybrids contain both mtDNA's and 16 human chromosomes. These cells grow poorly. This may be caused by the retention of chromosomes necessary for the retention of the mtDNA but not those essential for the mitochondria to function efficiently. The CAP-R HAR16C and LAB hybrids and the HER and EEL cybrids all contain predominantly mouse chromosomes and mouse mtDNA. These results are consistent with the parallel loss and retention of homologous chromosomes and mtDNA, but are inconsistent with CAP resistance being coded in the mtDNA.

The lack of an absolute correlation between inheritance of CAP resistance and the appropriate mtDNA requires consideration of three alternative explanations of the results. First, the mtDNA may not be the vehicle of CAP resistance. A large number of cytoplasmic but nonmitochondrial DNA's exist in mammalian cells (22,23L In addition, Saccharomyces mutants resistant to mitochondrial inhibitors have been obtained in which the markers are inherited in DNA which is sug- gested to he both extrachromosomal and extra- mitochondrial (58, 59 j.

A second alternative is that permanent CAP resistance is obtained in interspecific cybrids and hybrids by mutation of the sensitive mtDNA to resistance. Cybrids and hybrids when first prepared would rely on the foreign CAP-R mtDNA. In this hypothesis, the homology between nuclear and mitochondrial genes would be assumed to be inadequate for permanent retention of the mtDNA. A metastable state is created in which both resistant and sensitive mtDNA are maintained until a mutation occurs in the sensitive mtDNA making it resistant. At this point, the new mutant mtDNA would repopulate the cell and the foreign mtDNA would be lost. Strain- specific differences are explained by differences in

774

endogenous mutat ion rates of the various strains to CAP resistance. In this study R A G has very rarely been seen to mutate to CAP resistance (a frequency of less than 3 X 10-8). On the other hand, LM(TK-~ has been observed to generate C A P - R clones in control flasks, and a high endogenous mutat ion rate has been reported by other researchers t60). The metastable state resulting from cybrid formation simply facilitates the expression of the strain-specific differences, differences which could also explain the lack of reciprocity between strains.

The final alternative is that the above metastable state is formed but that it is alleviated by recombination rather than mutation. Interspecific recombinant D N A has been reported for mammalian cells 156). The observed differences between strains simply reflect differences in the rapidity with which recombination occurs. Tha t recombinant D N A is not detected by hybridization could be accounted for by the mutat ion for CAP resistance occurring in a portion of the m t D N A which is highly conserved, such as the r R N A CAP resistance is probably the result of an altered mitochondrial r R N A in yeast ~see Introduction}. There is at least a 3% cross hybridization between mouse and human m t D N A , which could mask such recombinants. The low levels of hybridization seen in many of the cybrids and hybrids may reflect such recombinants. Fur- ther efforts are underway to obtain data to distin- guish between these alternatives.

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We wish to thank Ms. A. Eisenstadt, K. Santoro, M. Freimanis, C. Peter- sen, and K. Vane for their excellent technical assistance. This work was supported by U.S.P.H,S. research grants GM-18186, GM-1948 and GM-21024 (to J. M. E.}, and N.I .H. postdoctoral fellowship No. 1 F22 GM-02655 (to D. C. W.).

cytoplasmic inheritance in mammalian tissue culture cells

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