Two series of interspecific hybrids have been generated between liver cells (which actively secrete several serum proteins) and fibroblasts (which do not). In each series, one of the parental cells was a normal diploid cell: mouse hepatoma cells were fused with normal diploid rat fibroblasts, and normal rat liver cells were fused with mouse fibroblasts of the permanent line A9. The production of albumin, α-fetoprotein (AFP) transferrin and the third component of complement (C3) was analysed in these hybrids. Most hepatoma cell hybrids exhibit extinction of albumin, AFP and (to a lesser extent) transferrin; they retain the capacity’ to secrete C3. Normal liver cell hybrids are also characterized by the absence of albumin and transferrin production and by retention of C3 secretion. These results, when compared to previous results obtained with hybrids derived exclusively from different differentiated cells of permanent and transformed lines show that the phenotype of such hybrids is not determined by the abnormal character per se of the aneuploid parental cells. Amongst the rat fibroblast-mouse hepatoma cell hybrids, a few clones retain the capacity to actively secrete mouse albumin, AFP and transferrin, without the concomitant production of the rat serum proteins. These hybrids have lost more rat (fibroblast) chromosomes than the other clones and also have an increased number of mouse (hepatoma) chromosomes. Thus, their phenotype must result from either the complete loss of ‘extinguisher’ chromosomes, or gene dosage effects. The significance of the lack of rat serum protein production is also discussed, and it is suggested that retention, without concomitant activation, could be explained in terms of diffusible regulators and heritable differences in chromatin conformation.

Liver functions, like other differentiated traits, generally fail to be expressed in hybrids between hepatoma cells and other cells (for reviews, see Darlington & Ruddle, 1975; Davidson,. 1974; Ringertz & Savage, 1976). The hybrids examined were derived with very few exceptions from the fusion of cells of hepatoma lines with cells of other permanent lines (either fibroblasts, epithelial cells or lymphoma cells…). The investigations have left unanswered the following question: are the phenotypes of such hybrids determined by the heteroploid and/or neoplastic character of the 2 parental cells (Ephrussi, 1972) ?The primary aim of the present work is to analyse this question.

We have previously studied hybrids between mouse hepatoma cells (which actively secrete albumin, α-fetoprotein, transferrin and the third component of complement) and fibroblasts of permanent lines. These hybrids exhibit total extinction of albumin and α-fetoprotein (AFP) production and partial extinction of transferrin, but do secrete the third component of complement (C3). Moreover, in interspecific hybrids, activation of C3 secretion has been demonstrated (Szpirer & Szpirer, 1975a; Szpirer, Szpirer & Wiener, 1976).

In order to provide a definite answer to the above question, one would ideally study hybrids derived from fusions between 2 normal diploid differentiated cells (hepatocyte × fibroblast for instance). However, such hybrids would be difficult to isolate and would very probably be incapable of prolonged proliferation. We chose therefore to study hybrids derived from either normal diploid fibroblasts fused with hepatoma cells or normal hepatocytes fused with fibroblasts of permanent lines and asked the question: will extinction of the serum protein production still be observed ?

Hybrids involving normal diploid cells generally have a less stable karyotype than hybrids derived from fusions involving 2 permanent cell lines. In particular, hybrids derived from the fusion of mouse hepatoma cells with diploid rat fibroblasts can be expected to loose preferentially the chromosomes contributed by the diploid rat fibroblast cell. If some specific chromosomes contributed by the fibroblast are responsible for the extinction of hepatic traits, then the loss of these chromosomes might allow the hybrid cell to escape extinction. Identification of these chromosomes would then be possible in an interspecific cross. Part of this work was aimed at testing this hypothesis by trying to isolate clones secreting albumin and/or AFP.

Re-expression associated with chromosome loss has already been described: reexpression of kidney esterase 2 (Klebe, Chen & Ruddle, 1970), of liver enzymes (Croce, Litwack & Koprowski, 1973; Bertolotti, 1977; Bertolotti & Weiss, 1974; Weiss & Chaplain, 1971) and more recently, re-expression of DMSO-inducible haemoglobin synthesis, in erythroleukemic × bone marrow cell hybrids, which was found to be associated with the loss of an X chromosome contributed by the bone marrow cell (Benoff & Skoultchi, 1977).

Parental cells and culture media

Three permanent lines have been used: A9, an L-cell variant (Littlefield, 1964a) deficient for the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8); BWTG3, a clonal line of mouse hepatoma cells, deficient for HGPRT (Szpirer & Szpirer, 19756); Fa32, a subclone of Faza 967 (Deschatrette & Weiss, 1974), a clonal line of HGPRT-rat hepatoma. Faza967 was kindly provided to us by Professor B. Ephrussi and Dr M. Weiss. The mouse and rat hepatoma cells secrete albumin, transferrin and C3; BWTG3 also secretes mouse AFP. BWTG3 was grown in Eagle’s Medium, Dulbecco’s modification (DMEM), supplemented with 10 % foetal calf serum; A9 and Fa32 were grown in Eagle’s minimal essential medium supplemented with 10 % foetal calf serum. Freshly isolated adult rat hepatocytes were provided to us by Drs Drochmans and Wanson. The cells were isolated according to their enzymic perfusion technique (Drochmans, Wanson & Mosselmans, 1975). Rat fibroblasts were obtained from the skin of embryos and cultured in DMEM for about 2 weeks before fusion. Concentrated culture media from the fibroblasts, or from A9, never gave a positive reaction when tested for the presence of albumin, AFP, transferrin or C3.

Cell fusion and selection of hybrid clones

The rat hepatocytes were kept in suspension before cell fusion. The other cells were harvested by trypsinization. After rinsing with culture medium without serum, the cells were fused in suspension using u.v.-irradiated Sendai virus (Harris, Watkins, Ford & Schoefl, 1966). Hybrids were selected in DMEM supplemented with 10−4 M hypoxanthine, 4 × 10−6 M aminopterin and 1·6×105M thymidine (HAT medium, Littlefield, 1964b): A9 and BWTG3 which lack HGPRT fail to grow in this medium and the normal diploid cells, which have a limited life span, do not form clones susceptible of propagation. HAT resistant colonies (one per dish) were isolated about 2 weeks after fusion with the aid of stainless steel cylinders.

Chromosome preparations

These were made by the standard procedure, described previously (Szpirer & Szpirer, 1975 a). C-banded metaphases were prepared according to the method of Dev, Miller, Allderdice & Miller (1972) modified by Marshall (1975).

Cell incubation and double immunodiffusion

All the antisera were rabbit antisera. Anti-mouse C3 and anti-rat AFP were purchased from Nordic Pharmaceutics and Diagnostics, Tilburg, Holland. Anti-rat transferrin was purchased from Microbiological Associates. Anti-mouse AFP was a gift from Dr R. Hooghe. Anti-mouse albumin, anti-rat albumin and anti-rat C3 were obtained after repeated injections of albumin or of zymosan-rat C3 complex (Mardiney & Müller-Eberhard, 1965).

The methods used were as previously described (Szpirer & Szpirer, 1975 a). At the end of the log phase, cells were incubated with fresh medium and counted at the end of the incubation period (3 days). To estimate the amount of protein secreted by the hybrid cells, the concentrated media were serially rediluted. Dilutions of the concentrated media from the hybrid cells were compared by immunodiffusion with dilutions of concentrated medium from hepatoma cells. In this way, and taking into account the number of incubated cells, the relative rates of serum protein produced by the hybrid clones and the parental hepatoma cells could reproducibly be estimated. The sensitivity of the immunodiffusion tests was determined in the same way; in the case of the anti-rat antiserum, the sensitivity of the assay was determined using a solution of purified rat AFP.

In 3 days, 106 confluent BWTG3 cells secrete about 25 μg of albumin and of AFP. The secretion rate of such confluent cells is constant during the first 2 days of incubation (± 10 μg/ 106 cells/24 h) and then levels off; this secretion rate is slightly higher than during log phase (D. Cassio, personal communication). The approximate rates of transferrin and C3 secretion are 3 and 1 μg/106 cells/24 h respectively (Szpirer & Szpirer, 1975a).

Cross between mouse hepatoma cells and diploid rat embryo skin fibroblasts (BS clones)

Twenty-four clones were isolated and characterized from this type fusion. These clones could easily be classified into two groups on the basis of their morphology. Nineteen clones showed a fibroblast-like morphology and grew as monolayers (type I clones); their saturation density was low (manuscript in preparation). On the other hand, the cells of three other clones (6841, 50, 130) were more epithelial-like and very refractile; they were inclined to pile up and did not adhere strongly to the surface of the culture dish (type II clones). The two remaining clones (BS30 and BS231) appeared to be a mixture of the two cell types described above; they were thus subcloned and subclones of each type isolated. We have not yet been able to determine whether or not the original BS30 and BS231 clones were true clones, one cell type being derived from the other.

Characterization of type I hybrids

The type I clones possess a complete hepatoma cell genome (62 mouse chromosomes or more) combined with at least 29 rat chromosomes (this represents 70 % of the rat genome; 10 clones have retained 90 % of the rat genome). The data on the karyotypes of the BS hybrids are illustrated in Fig. 1 and summarized in Table 1.

Table 1.

Karyotypes of parental and BS hybrid cells

Karyotypes of parental and BS hybrid cells
Karyotypes of parental and BS hybrid cells
Fig. 1.

C-banded metaphases of the parental mouse hepatoma cells (BWTG3), the rat fibroblasts (RSF), a Type I subclone (BS30-6), and a Type II clone (BS41).

Fig. 1.

C-banded metaphases of the parental mouse hepatoma cells (BWTG3), the rat fibroblasts (RSF), a Type I subclone (BS30-6), and a Type II clone (BS41).

Each clone was analysed for the secretion of albumin, AFP, transferrin and C3. Albumin and AFP could never be detected in any concentrated culture medium from the type I hybrids. Given the sensitivity of the method, and taking into account the final number of incubated cells, we can say that these BS cells, depending upon the clone, secrete less than 2-10% of the amount of albumin or AFP produced by the parental hepatoma cells. It thus appears that albumin and AFP production is, if not totally extinguished, considerably lowered. Several type I clones were found to secrete mouse transferrin in low amounts, as indicated by the fact that, when present, the precipitin band (see Fig. 2c) with the anti-transferrin serum was weak or even barely detectable (the strongest reaction was observed with BS6o for which the secretion rate was estimated to be about 40 % of that of BWTG3). The type I BS hybrids secrete mouse C3 (Fig. 2D), and some lines (5/21) also rat C3. The failure to detect rat C3 in some clones is not significant, as the sensitivity of the assay for C3 detection is not very high (see Fig. 2E and Table 2).

Table 2.

Secretion pattern of the BS hybrids

Secretion pattern of the BS hybrids
Secretion pattern of the BS hybrids
Fig. 2.

Double immunodiffusion tests showing the presence of mouse albumin (A), mouse AFP (B), mouse transferrin (c), mouse C3 (D), and rat C3 (E) in the culture media from BS hybrids. The wells contained: (A) aMA, anti-mouse albumin; aRA, antiratalbumin absorbed withmouse albumin; MA, mouse albumin; RA, rat albumin; BS 50, concentrated culture medium (× 10) of BS50 (107 cells), (B) aMF, anti-mouse AFP; aRF, anti-rat AFP absorbed with mouse AFP; RF, rat embryo serum (diluted 1:5); BW and BS50, respectively, concentrated culture media (× 10) of BWTG3 (2 × 107 cells) and of BS50 (107 cells), (c) aT, anti-rat transferrin; BW’, BS41, Fa’, respectively, concentrated culture media (× 20) of BWTG3 (2 × 107 cells), BS41 (107 cells), Fa32 (4 × 107 cells); BS20 and BS60, respectively, concentrated culture media (× 100) of BS20 (3×107 cells) and BS60 (2 × 107 cells), (D) aMC, anti-mouse C3; Fa’, concentrated culture medium (× 20) of Fa32 (4 × 107 cells); BW’, concentrated culture medium (× 50) of BWTG3 (2 × 107 cells); BS60, BS30-6 and BS331, respectively, concentrated culture media (× 100) of BS60 (2 × 10 6 cells), BS30-6 (107 cells) and BS331 (4 × 10 6 cells), (E) aaRC, anti-rat C3 absorbed with mouse serum; MS, mouse serum; RS, rat serum; BS30-6, concentrated culture medium (× 100) of BS30-6 (107 cells). Note: (A) The identity reaction between mouse albumin and BS50, the spur formed by rat albumin in the reaction with anti-rat albumin and the absence of reaction of BS50 with anti-rat albumin absorbed with mouse albumin, (B) The identity reaction between mouse AFP (BWTG3) and BS50 when tested with anti-mouse AFP, and the absence of reaction of BS50 with anti-rat AFP (which does not cross-react with mouse AFP), (c) The identity reaction between the hybrids and BWTG3, and the spur formed by rat transferrin (present in Fa32) indicating the absence of rat transferrin in the hybrids. And (E) the weak but positive reaction of BS30-6 with aaRC.

Fig. 2.

Double immunodiffusion tests showing the presence of mouse albumin (A), mouse AFP (B), mouse transferrin (c), mouse C3 (D), and rat C3 (E) in the culture media from BS hybrids. The wells contained: (A) aMA, anti-mouse albumin; aRA, antiratalbumin absorbed withmouse albumin; MA, mouse albumin; RA, rat albumin; BS 50, concentrated culture medium (× 10) of BS50 (107 cells), (B) aMF, anti-mouse AFP; aRF, anti-rat AFP absorbed with mouse AFP; RF, rat embryo serum (diluted 1:5); BW and BS50, respectively, concentrated culture media (× 10) of BWTG3 (2 × 107 cells) and of BS50 (107 cells), (c) aT, anti-rat transferrin; BW’, BS41, Fa’, respectively, concentrated culture media (× 20) of BWTG3 (2 × 107 cells), BS41 (107 cells), Fa32 (4 × 107 cells); BS20 and BS60, respectively, concentrated culture media (× 100) of BS20 (3×107 cells) and BS60 (2 × 107 cells), (D) aMC, anti-mouse C3; Fa’, concentrated culture medium (× 20) of Fa32 (4 × 107 cells); BW’, concentrated culture medium (× 50) of BWTG3 (2 × 107 cells); BS60, BS30-6 and BS331, respectively, concentrated culture media (× 100) of BS60 (2 × 10 6 cells), BS30-6 (107 cells) and BS331 (4 × 10 6 cells), (E) aaRC, anti-rat C3 absorbed with mouse serum; MS, mouse serum; RS, rat serum; BS30-6, concentrated culture medium (× 100) of BS30-6 (107 cells). Note: (A) The identity reaction between mouse albumin and BS50, the spur formed by rat albumin in the reaction with anti-rat albumin and the absence of reaction of BS50 with anti-rat albumin absorbed with mouse albumin, (B) The identity reaction between mouse AFP (BWTG3) and BS50 when tested with anti-mouse AFP, and the absence of reaction of BS50 with anti-rat AFP (which does not cross-react with mouse AFP), (c) The identity reaction between the hybrids and BWTG3, and the spur formed by rat transferrin (present in Fa32) indicating the absence of rat transferrin in the hybrids. And (E) the weak but positive reaction of BS30-6 with aaRC.

These results, summarized in Table 2A, show that the type I BS hybrids have a pattern of serum protein secretion identical to that of hybrids derived from permanent heteroploid fibroblasts: they exhibit extinction of albumin and AFP production, partial extinction of transferrin production and retention of C3 secretion (Szpirer & Szpirer, 1975 a).

Characterization of type II hybrids

In contrast to the type I hybrids, all type II hybrids retained the capacity to secrete albumin and AFP. Using the appropriate antisera, we determined that only mouse albumin and mouse AFP are produced. This is illustrated in Fig. 2 A, B. Given the sensitivity of the assay, we would have detected a production of rat albumin or AFP equal to + 2 % of that of mouse albumin or AFP produced by BWTG3 cells. An estimation of the relative rates of mouse albumin and mouse AFP secretion was made by testing serial dilutions of the concentrated media (see Materials and methods). Table 2 shows that, with the exception of the BS30 subclones, type II hybrids actively secrete these 2 proteins: up to 400% of the amount produced by BWTG3 cells. Similarly, the type II clones actively secrete mouse transferrin as shown in Fig. 2C. Rat transferrin could not be detected in the culture medium of any of the hybrid clones. As expected, the type II hybrids actively produce mouse C3 (with the exception of the BS30 subclones); 3 of them (BS41, 50 and 130) also secrete rat C3, indicating that despite the loss of rat chromosomes, some type II clones retained at least one chromosome carrying the C3 structural gene(s). These results are summarized in Table 2B.

The karyotypes of the type II hybrids (see Fig. 1 and Table 1) are strikingly different from those of the type I hybrids. Firstly they all have more chromosomes than expected: about twice the expected number of mouse chromosomes (or even more, as is the case for BS231 subclones). Secondly, they have undergone a more extensive loss of rat chromosomes than any of the type I clones: they retained at the most 27 rat chromosomes (whereas the type I hybrids retained at least 29 rat chromosomes). The ratio between the mouse and rat chromosome numbers is equal to, or higher than 5. (The high ploidy of BS231 subclones provides an explanation for the fact that they produce more albumin and AFP than BWTG3 cells.)

Cross between rat liver cells and mouse heteroploid fibroblasts (LA clones)

Rat liver suspensions, obtained by the enzymic perfusion technique are essentially made up of well preserved and functionally active hepatocytes (the proportion of cells other than parenchymal cells is less than 4%; Drochmans et al. 1975). These cells continue to synthesize and secrete serum proteins in vitro for several hours. Albumin, transferrin and C3 were identified in the culture medium and the approximative rate of albumin secretion was found to be 2 μg/h/106 cells; AFP, made only in trace amounts by the adult rat, could not be detected in the culture medium by immunodiffusion tests (May, Wanson, Szpirer and Szpirer, unpublished observations).

Such a cell preparation, freshly isolated from an adult rat liver, was fused with A9 mouse fibroblasts, and numerous hybrid clones were isolated. Eleven independent clones were characterized. These hybrids have retained at least 90 and 80% of the mouse and rat chromosome complements respectively.

None of these hybrids were found to secrete albumin or transferrin in detectable amounts (i.e. less than 0 ·01 μg albumin/h/106 cells). On the other hand, rat and mouse C3 could be detected in 4 out of the 6 clones we tested for C3 production.

These results suggest that extinction of albumin and transferrin occurred in LA hybrids, under the infiuenceof thefibroblastgenome. However, we cannot rule out the possibility that the hybrids we tested were derived from cells other than hepatocytes, but this possibility appearsveryunlikely: hepatocytes represent at least 96 %of the cells present in the rat liver preparations isolated by the enzymic perfusion technique. In addition, a control fusion was made between such liver cells and mouse hepatoma cells (BWTG3). We isolated 8 hybrid clones (LB clones). Most of them, contrary to the LA hybrids retained only few rat chromosomes (5–20); nevertheless, one LB clone secreted rat albumin and 3 other LB clones secreted transferrin. This indicates that at least half of the LB hybrids were derived from the fusion of hepatocytes with hepatoma cells (a detailed report on these hepatocytes × hepatoma cell hybrids will be published separately).

Our results show that, in mouse hepatoma cell hybrids, extinction of albumin and AFP production and, to a lesser extent of transferrin production can be achieved by the genome of a fibroblast whether the latter is diploid and normal, or aneuploid and transformed. The data on the normal liver hybrids suggest that extinction of albumin and transferrin production takes place in hepatocyte hybrids as well as in hepatoma cell hybrids. These data argue in favour of the idea that the pattern of expression of differentiated functions in cell hybrids is not determined by the abnormal character per se of the aneuploid parental cells. Our results are in agreement with those of Riddle & Harris (1976) who showed that a liver enzyme, tyrosine aminotransferase, is produced at a reduced level in rat hepatoma cell × normal diploid fibroblast hybrids as well as in hepatoma cell × lymphoma cell hybrids.

The fusion of BWTG3 hepatoma cells with normal diploid fibroblasts allowed us to isolate hybrids producing albumin and AFP (BS hybrids of type II), whereas several fusions involving heteroploid fibroblasts of permanent lines never gave rise to this type of hybrid. As expected, the producer BS hybrids have lost a significant number of rat (fibroblast) chromosomes; however, they have also acquired a considerable number of mouse chromosomes. (These hybrids could have arisen from a triparental fusion, from a fusion involving a 2S BWTG3 cell, from a dikaryon in which the mouse chromosomes replicated twice before the first cell division whereas the rat chromosomes replicated only once, or from a type I BS hybrid in which doubling of the whole chromosomal set was followed by segregation of rat chromosomes.)

How can the phenotype of the type II BS hybrid be explained ? One possibility is that the putative ‘extinguisher’ rat chromosomes (which are supposed to bear gene(s) directing the production of repressor) have all been lost in the type II BS hybrids, whereas at least one copy of these chromosomes have been retained in the type I BS hybrids. However, if the loss of rat chromosomes is random, this hypothesis appears statistically unlikely.* Another possibility is that the mouse chromosomes, being largely supernumerary in the type II BS hybrids, overcome the extinction effect directed by the rat fibroblast chromosomes. In other words, the lack of extinction of albumin Gene dosage effects could reflect an overtitration or a dilution of repressors produced by the rat fibroblast genome, or alternatively an increased concentration of activators involved in the maintenance of the activity of the mouse albumin, AFP and transferrin genes.

None of the type II BS hybrids were found to secrete rat albumin, rat AFP or rat transferrin, even though most of them actively produced the mouse serum proteins and some of them secreted rat C3 (in agreement with our previous results; Szpirer et al. 1976). It is statistically very unlikely that all these type II BS hybrids, some of which retained at least one copy of the chromosome(s) bearing the C3 structural gene(s), have lost all the rat chromosomes carrying the structural genes for albumin, AFP and transferrin (the genetic linkage of these structural genes is unknown). Therefore, the lack of production of rat albumin, rat AFP and rat transferrin in these hybrids should not be considered as resulting from the loss of structural genes but rather as the lack of activation of these genes. Retention of differentiated functions without concomitant activation has previously been described (Riddle & Harris, 1976; Bernstine, Koyama & Ephrussi, 1977). Riddle & Harris concluded that their results could not be easily explained by the mere intervention of diffusible repressors or activors, even if these regulators were species specific. A model in which the superstructure of a gene is an important factor in the hierarchy of the mechanisms which govern the expression of the gene, as discussed by Cook (1973, 1974; Colman & Cook, 1977) would appear to be more interesting. This proposal is supported by recent studies indicating that in differentiated cells, active genes are maintained in a conformation that is distinguishable from that of the majority of the transcriptionally inactive DNA in the chromatin (Gottesfeld & Partington, 1977; Wallace, Dub.e & Bonner, 1977; Weintraub & Groudine, 1976). Moreover the maintenance of the active conformation does not seem to be a consequence of the presence of RNA polymerase molecules along the genes (Weintraub & Groudine, 1976; Garel, Zolan & Axel, 1977). The conformation of the genes, whether active or inactive, might be imposed during development and be stably conserved during mitosis (Cook, 1973, 1974). DNA modifications, such as methylation of bases (Scarano, 1969; Holliday & Pugh, 1975) might play a role in the establishment and (or) the maintenance of these superstructures.

On the basis of the above evidence, we assume that the genes coding for albumin, AFP and transferrin are in different conformations in the mouse hepatoma cell on the one hand (active conformation), and in the rat fibroblast on the other hand (inactive conformation). After fusion, the pre-existing conformations would be maintained, but diffusible repressors actively produced by the fibroblast genome would shut off the expression of the structural genes coding for albumin and AFP and to a lesser extent, the expression of the transferrin gene. This would not necessarily alter the conformation of these genes, so that re-expression might subsequently take place, as is the case for liver enzymes (Weiss & Chaplain, 1971; Bertolotti & Weiss, 1972, 1974; Croce et al. 1973; Bertolotti, 1977) after the loss of the chromosomes carrying the repressor gene(s).

Hybrids were described in which activation of a gene was shown to accompany lack of extinction (Peterson & Weiss, 1972; Malawista & Weiss, 1974; Brown & Weiss, 1975; Darlington, Bernhard & Ruddle, 1974). Such an effect would need a modification in the conformation of this normally inactive gene. Conformational changes might or might not occur, depending on the stability of the inactive conformation and on the efficiency of the implicated regulators.

Our working hypothesis reconciles the different results yielded by various hybridization experiments. For instance, the study of albumin production in hepatoma cell hybrids has revealed the following situations: total extinction (Szpirer & Szpirer, 1975; Deschatrette & Weiss, 1975), partial extinction (Peterson & Weiss, 1972; see also Conscience, Ruddle, Skoultchi & Darlington, 1977), retention without activation (this paper) and finally retention with concomitant activation (Malawista & Weiss, 1974; Darlington et al. 1974).

This hypothesis could be tested by studying the conformation of genes (as detected by their sensitivity to nucleases) in hybrids displaying these different phenotypes.

We are grateful to Professor J. Brachet, Professor R. Thomas and Dr A. Kinsella for thoughtful reading of the manuscript. We thank Dr J. C. Wanson and Dr C. May for kindly providing us with suspensions of isolated rat hepatocytes, Dr M. Van de Winckel for giving us purified rat AFP and Dr C. Wuillemaert for his help in statistical calculations.

This work was carried out under a Euratom-ULB contract, an agreement between the Belgian Government and the Université Libre de Bruxelles (‘Actions de Recherches concertées’) and with support from the FNRS.

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*

If only a single pair of rat chromosomes control the extinction of both albumin and AFP production, the probability of randomly losing the 2 copies of this pair, when r chromosomes are lost (out of 42), is equal to being the number of combinations of 42 chromosomes taken r at a time). For BS50, BS41, BS231-12, BS130 and BS30-11 respectively, these probabilities are 0·12, 0·16, 0·41, 0·50 and 0·58. The probability that all these 5 type II clones randomly lost the 2 putative extinguisher chromosomes is equal to 0·0023. and AFP production would be due to a gene dosage effect, as is the case for the rat hepatoma cell hybrids studied by Weiss and coworkers (Peterson & Weiss, 1972; Malawista & Weiss, 1974). However, such gene dosage effects would be observed in mouse hepatoma cell hybrids only when they have retained hepatoma and fibroblast chromosomes in a ratio of at least 5 whereas a ratio of about 2 appears sufficient in Fu 5 (Peterson & Weiss, 1972) and Faza (Malawista & Weiss, 1974) hepatoma cell hybrids.