Patterns of chromosome segregation were studied in 2 different intraspecific mouse cell hybrids: (1) A9 × B82, formed by fusing 2 cell lines of heteroploid fibroblasts, and (2) UWE, originating from the fusion of A9 cells with euploid foetal erythrocytes. Detailed analyses of Giemsa (G)-banded chromosomes and chromosome arms of both parental and hybrid cells were made for each hybrid type, in order to determine the specificity of the losses and to assess the influence of ploidy and cell differentiation.

Unlike the A9 × B82 hybrids, which revealed a significant chromosome loss under selective tissue culture pressures only after 9 months, the UWE hybrids showed a sharp reduction in the total chromosome number during the initial 2 months under similar pressures. However, with no additional cloning, UWE remained karyotypically stable after that time. This rapid chromosomal segregation in UWE hybrids may be caused by properties of the parental foetal erythrocytes.

In UWE cells, the majority of the chromosome arms were retained or duplicated. Less than a quarter of the total number of chromosome arms were segregated or lost, and these were all chromosome arms with abnormal mouse G-banding patterns, present only in the heteroploid A9 parental cells. In two of the four A9 × B82 hybrid lines, there was marked segregation of chromosome arms whose banding patterns were identical to those of wild type mouse telocentric chromosomes. For both types of intraspecific cell hybrids, two thirds or more of the chromosome arms had banding patterns which were the same as those of the wild type genome.

Since little is understood about the phenomenon of chromosome segregation in somatic cell hybrids, studies of 2 different intraspecific hybrids were undertaken to examine in detail chromosome loss as a function of specificity, ploidy, and cell differentiation. In intraspecific cell hybrids, usually only moderate losses of chromosomes occur over many generations (Littlefield, 1966; Yoshida & Ephrussi, 1967; Ruddle, Chen, Shows & Silagi, 1970). However, much greater chromosome losses may occur within a year in intraspecific cell hybrids giving rise to segregant cells that resemble the parental cells in total chromosome number, when the hybrid cells are placed under directed tissue culture pressures (Engel, McGee & Harris, 1969b; Engel, Empson & Harris, 1971). Radically segregated hybrids arising several months after the initial cell fusion have also been described in intraspecific hepatoma crosses-subjected to selection in culture (Weiss & Chaplain, 1971).

Chromosome segregation was investigated in a mouse intraspecific cell hybrid (UWE), originating from the fusion of a heteroploid, differentiated fibroblast of the AB cell line with a euploid, mouse foetal erythrocyte. Patterns of chromosome segregation for UWE were obtained by analyses of individual trypsin-Giemsa (G)-banded chromosomes and chromosome arms of both the parental and the hybrid cells. The chromosome changes in UWE cells were compared to those seen in A9 × B82, another intraspecific mouse cell hybrid formed from the fusion of cells from two different lines of heteroploid fibroblasts (Engel, McGee & Harris, 1969a,b; Engel et al. 1971; Russell, Engel, Vaughn & McGee, 1977).

Nucleated erythrocytes were obtained from C2H mouse embryos approximately 12 days old. The embryos were beheaded, and their blood was collected into Petri dishes containing Hanks’ balanced salt solution and 0·1 μg/ml of heparin. To reduce contamination by other embryonic cells, the cell suspension was placed in a Falcon flask for 3 h at 37 °C, which allowed contaminating cells to attach to the plastic substrate.

For cell fusion, 1·6 × 107 nucleated erythrocytes were mixed with 5 × 106 A, cells, a derivative of the L cell line (Littlefield, 1964), in the presence of 6000 haemagglutinating units of u.v.-inactivated Sendai virus at 4 °C for 5 min. The cell suspension was then incubated at 37 °C for 30 min. Three flasks, containing Minimum Essential Medium (MEM) with 15 % foetal calf serum, were seeded. The medium was replaced by a selective medium containing hypoxanthine, aminopterin, and thymidine (HAT) (Littlefield, 1964) with 15 % foetal calf serum after 3 days. Hybrid colonies termed UWE appeared in all of the flasks; the first harvest and karyotypic analysis by conventional staining procedures were done 3 weeks later. At that time, chromosome-banding techniques were not available. Six UWE clones were transferred from the primary flasks and harvested 7 weeks after the initial fusion. One clone was maintained for over 3 years in HAT before being frozen in liquid nitrogen. Two years and eight months after the original fusion this UWE clone and the parental A9 cell line, grown continuously in MEM, were studied by the G-banding technique as described in a previous paper (Russell et al. 1977).

Twenty high-quality G-banded metaphases were selected for both the A9 line and the UWE clone. The standardized guide for the Q- and G-bands of normal mouse telocentric chromosomes (Committee on Standardized Genetic Nomenclature for Mice, 1972) and the characterization of the A, line by Allderdice et al. (1973) and Russell et al. (1977) were all used as references for karyotyping the parental and hybrid chromosomes. The A9 cell line used in this fusion was found to differ significantly (P < 0·05) from the AB previously described (Russell et al. 1977) when the mean number of chromosomes of the groups and subgroups in each line were compared by an analysis of variance of the completely randomized design. Although both A9 cell lines had a common origin, cultivation under different laboratory conditions appears to have resulted in different heteroploid genomes.

The hybrid cells

There was marked segregation of chromosomes in the UWE hybrid cells within 3 to 7 weeks after the initial fusion. The sum of the 2 parental chromosome sets contributed to the UWE hybrid cell would be in the range of 90 chromosomes; but 7 weeks after fusion, examination of 6 clones revealed that the number of chromosomes per cell ranged from 63 to 72. The clone having the lowest number of chromosomes per cell, which was approximately 63, was maintained and studied for over 3 years.

The first detailed study of this hybrid clone was done by routine staining 3 months after the initial fusion. In 22 cells examined, the average total number of chromosomes was 63·27 with a range of 44 to 77. Of the 3 major chromosome groups, the hybrid cell line contained a mean number of 19·68 biarmed (B) chromosomes, 4·31 telocentric (T) chromosomes, and 2·14 dot (D) chromosomes. There was also a fourth group, ring (r) chromosomes, which were found in this hybrid cell line only at this stage; these were present at an average of 0·14 chromosomes per cell.

This hybrid clone was further studied by the G-banding technique, 2 years and 8 months after the original fusion (Fig. 1). The average total number of chromosomes for the 20 cells examined was 63·60, with a range of 53 to 67. The mean number of B chromosomes present was 20·15; T chromosomes, 42·05; and D chromosomes, 1·40 (Table 1). The mean numbers of chromosomes of each major group found in the previous conventional study were compared with those found in the G-banding study; an analysis of variance of completely randomized design was used. There was no significant difference in the total number of chromosomes in the B and T groups, but the D groups were significantly different (P < 0·05).

Comparison of the parental and hybrid cells

A G-banded karyotypic analysis of A9, maintained under the same tissue culture conditions as UWE, was made at approximately the same time as the G-banding study of UWE. A9 averaged 52·80 chromosomes for 30 cells, with a range of 50 to 60 chromosomes. These chromosomes were divided into 3 major groups as previously described for the hybrid cell line (Table 1). The other parental cell type, the mouse foetal erythrocyte, had a normal diploid number of 40 chromosomes, which were all T chromosomes.

The mean numbers of chromosomes in the major groups and subgroups of A9 and of the hybrid clone were compared by the analysis of variance. The B and T chromosome groups of A9 and of UWE were significantly different (P < 0·05), while there was no significant difference for the D chromosome group. The metacentric and submetacentric subgroups were significantly different (P< 0·05) for these 2 cell lines. However, there was no significant difference for the isochromosomes.

All of the normal mouse T chromosomes of the diploid karyotype, except chromosome number 4, were found in the hybrid cells as normal T chromosomes. Over 73 % of the total number of T chromosomes in UWE were normal mouse chromosomes; this figure was 100% for foetal mouse erythrocytes and 60% for A9. These percentages were similar for chromosome arms derived from normal T chromosomes.

To detect overall patterns of duplication, retention, segregation, or complete loss of redundant chromosome material in UWE hybrid cells, the sums of the parental chromosome arms, representing the state of affairs immediately after cell fusion, were compared with the corresponding number of chromosome arms in the hybrids. This procedure has been previously described for A9 × B82 cell hybrids (Russell et al. 1977).

When the identifiable chromosome arms in the UWE hybrid cells were compared with the sum of the identifiable arms in A9 and foetal erythrocytes, 60% were found to be duplicated and 40% retained. Segregation occurred in 14% of the unidentifiable chromosome arms. Also, 14% of the unidentifiable arms had new banding patterns not previously recognized in A9. Over one fifth of the unidentifiable arms were lost, one quarter retained, and an additional quarter duplicated. Replication of the identifiable arms was much greater than that of the unidentifiable arms.

Over 75% of the total number of chromosome arms, including both the identifiable and unidentifiable, in the hybrid cells were retained or duplicated. However, more than 20% of the total chromosome arms that were segregated or lost were chromosomes of unidentifiable origin present in the heteroploid A9 parent cells.

Marked segregation of chromosomes appeared during the early phase of growth of intraspecific hybrid cells formed between heteroploid cells of a permanent mouse cell line and euploid, nucleated foetal mouse blood cells. Approximately 20 to 30 chromosomes were lost from these cells 7 weeks after the initial fusion. From the analyses of the major chromosome groups, it appeared that the hybrids remained karyotypically stable for the majority of the B and T chromosomes for a period of over two and a half years thereafter. When the hybrid chromosomes for the major groups and subgroups were compared to what would be expected for the sum of the parental chromosome groups, there was a loss of chromosomes in the B groups, including the metacentric and submetacentric subgroups, and in the T group, so that the total number of chromosomes in the hybrid cell approached that of one of the parent cells.

In the UWE cells, duplication and retention of chromosome arms occurred in over three quarters of the total number of identifiable and unidentifiable arms: less than a quarter of the total number of chromosome arms were segregated or lost. It is important to note that only unidentifiable chromosome arms were segregated or lost, and this loss was not balanced by the appearance of new chromosome material in the hybrid cells. The data thus suggest that the losses mainly involved abnormal chromosomes derived from the A9 parent cell.

Hybrid cells of A9 · B82 showed little chromosome loss during the early phase of growth in vitro (Engel et al. 1969a), but under selective pressure in vitro over a prolonged period of time, highly significant losses occurred (Engel et al. 1969b, 1971).

This slow but substantial, chromosome segregation in the heteroploid × heteroploid hybrid cells is in marked contrast to the rapid loss (within a 2-month period) of approximately one third of the chromosomes in the heteroploid × euploid cells of UWE.

Over two thirds of the chromosome arms in both the parental A9 and B82 lines and in the four A9 × B82 hybrid lines were shown by G-banding analysis to be identical in banding patterns to the normal mouse T chromosomes. Similarly, two thirds of the total chromosome arms in UWE cells were of normal mouse origin.

G-banding studies of four A9 × B82 hybrid lines revealed that the majority of the redundant chromosome arms were of identifiable, normal mouse origin; and these tended to be segregated in two of the hybrid cells lines, but retained in the other two (Russell et al. 1977). This analysis of A9 x B82 demonstrated that segregation and chromosome recombination accounted for the evolution of the karyotype in the hybrid cells. This could result in a reduction of the chromosome modes to those of the parental cells, and in an increased heterogeneity in the distribution of chromosome classes.

Ploidy and cell differentiation both appear to be important factors in determining chromosome loss in these intraspecific hybrids. According to Handmaker (1973), in intraspecific hybrids an unknown property that confers karyotypic stability upon a euploid cell at mitosis is either lost or suppressed when that cell is fused with a heteroploid one. However, fusion of euploid, differentiated cells with heteroploid, differentiated cells of the same species can result in hybrids that appear karyotypically stable (Hashmi & Miller, 1976; Szpirer, Szpirer & Wiener, 1976; le Borgne de Kaoüel, Billard & Macieira-Coelho, 1978). Generally, chromosome loss is small in intraspecific, differentiated euploid × euploid cell hybrids (Handmaker, 1973; Migeon, Norum & Corsaro, 1974; Hoehn et al. 1975); although, recently, recombinant euploid hybrids have been recovered from the fusion of two euploid Indian muntjac cell lines (Yoshida & Sasaki, 1978).

It has been observed in interspecific cells hybrids formed from the fusion of chick erythrocytes with both heteroploid mouse cells and Chinese hamster cells that the reactivated erythrocyte nucleus undergoes premature chromosome condensation, which may result in the loss of almost all the chick chromosome material in the newly formed hybrid cell (Schwartz, Cook & Harris, 1971; Boyd & Harris, 1973). Similar results were obtained when frog erythrocytes were fused with heteroploid mouse cells (P. R. Cook, personal communication). Premature chromosome condensation of the foetal erythrocyte may also occur in intraspecific cell hybrids like UWE and give rise to increased chromosome loss in the early phase of growth. Chromosome loss, although not as great as that seen in UWE, has been reported in an intraspecific hybrid formed between an aneuploid murine foetal liver cell line and a mutant L cell line, 5 months after initial fusion (Rintoul, Colofiore & Morrow, 1973).

Karyotypic analyses need to be done on intraspecific hybrids between diploid, undifferentiated cells and heteroploid, differentiated cells, and between diploid, undifferentiated cells crossed with themselves. Such studies might help to elucidate further the influence of ploidy and cell differentiation on chromosome segregation in intraspecific hybrid cells. It is clear, however, that some intraspecific hybrid combinations may be quite suitable for the purposes of segregation analysis and gene mapping.

This work was initiated by E. E. while on sabbatical leave at the department of Dr. Henry Harris at the Sir William Dunn School of Pathology, Oxford, England. The study was supported by a grant from the United States Public Health Services (M. C. T.-000423-15). M. H. R. is a Fellow of the National Institute of Child Health and Human Development, N. I. H. Training Grant HD07043.

Allderdice
,
P. W.
,
Miller
,
O. J.
,
Miller
,
D. A.
,
Warburton
,
D.
,
Pearson
,
P
, L.,
Klein
,
G.
&
Harris
,
H.
(
1973
).
Chromosome analysis of two related heteroploid mouse cell lines by quinacrine fluorescence
.
J. Cell Sei
.
12
,
263
274
.
Boyd
,
Y.
&
Harris
,
H.
(
1973
).
Correction of genetic defects in. mammalian cells by the input of small amounts of foreign genetic material
.
J. Cell Sci
.
13
,
841
861
.
Committee On Standardized Genetic Nomenclature For Mice
(
1972
).
Standard karyotype of the mouse, Mus musculus
.
J. Hered
.
63
,
69
72
.
Engel
,
E.
,
Empson
,
J.
&
Harris
,
H.
(
1971
).
Isolation and karyotypic characterization of segregants of intraspecific hybrid somatic cells
.
Expl Cell Res
.
68
,
231
234
.
Engel
,
E.
,
Mcgee
,
B. J.
&
Harris
,
H.
(
1969a
).
Cytogenetic and nuclear studies on A9 and B82 fused together by Sendai virus: the early phase
.
J. Cell Sci
.
5
,
93
100
.
Engel
,
E.
,
Mcgee
,
B. J.
&
Harris
,
H.
(
1969b
).
Recombination and segregation in somatic cell hybrids
.
Nature, Lond
.
223
,
152
155
.
Handmaker
,
S. D.
(
1973
).
Hybridization of eukaryotic cells
.
A. Rev. Microbiol
.
27
,
189
204
.
Hashmi
,
S.
&
Miller
,
O. J.
(
1976
).
Further evidence of X-linkage of hypoxanthine phosphoribosyl transferase in the mouse
.
Cytogenet. Cell Genet
.
17
,
35
41
.
Hoehn
,
H.
,
Bryant
,
E. M.
,
Johnston
,
P.
,
Norwood
,
T. H.
&
Martin
,
G. M.
(
1975
).
Non-selective isolation, stability and longevity of hybrids between normal human somatic cells
.
Nature, Lond
.
258
,
608
610
.
De Kaoüel Le Borgne
,
C.
,
Billard
,
C.
&
Macieira-Coelho
,
A.
(
1978
).
Growth characteristics in vitro of hybrids between normal and transformed cell lines
.
Int. J. Cancer
21
,
338
347
.
Littlefield
,
J. W.
(
1964
).
Selection of hybrids from mating of fibroblasts in vitro and their presumed recombinants
.
Science, N. Y
.
145
,
709
710
.
Littlefield
,
J. W.
(
1966
).
The use of drug-resistant markers to study the hybridization of mouse fibroblasts
.
Expl Cell Res
.
41
,
190
196
.
Migeon
,
B. R.
,
Norum
,
R. A.
&
Corsaro
,
C. M.
(
1974
).
Isolation and analysis of somatic hybrids derived from two human diploid cells
.
Proc. natn. Acad. Sri. U.S.A
.
71
,
937
941
.
Rintoul
,
D.
,
Colofiore
,
J.
&
Morrow
,
J.
(
1973
).
Expression of differentiated properties in fetal liver cells and their somatic cell hybrids
.
Expl Cell Res
.
78
,
414
422
.
Ruddle
,
F. H.
,
Chen
,
T.
,
Shows
,
T. B.
&
Silagi
,
S.
(
1970
).
Interstrain somatic cell hybrids in the mouse. Chromosome and enzyme analyses
.
Expl Cell Res
.
60
,
139
147
.
Russell
,
M. H.
,
Engel
,
E.
,
Vaughn
,
W. K.
&
Mcgee
,
B. J.
(
1977
).
Karyotypic analyses of parental and hybrid intraspecific mouse cells, A#/Bet, by Giemsa- and centromeric-banding
.
J. Cell Sri
.
25
,
59
71
.
Schwartz
,
A. G.
,
Cook
,
P. R.
&
Harris
,
H.
(
1971
).
Correction of a genetic defect in a mammalian cell
.
Nature, Neto Biol
.
230
,
5
7
.
Szpirer
,
C.
,
Szpirer
,
J.
&
Wiener
,
F.
(
1976
).
The expression of differentiated functions in somatic cell hybrids: retention and activation of Q, production
.
Differentiation
5
,
139
149
.
Weiss
,
M. C.
&
Chaplain
,
M.
(
1971
).
Expression of differentiated functions in hepatoma cell hrbrids: reappearance of tyrosine aminotransferase inducibility after the loss of chromosomes
.
Proc. natn. Acad. Sri. U.S.A
.
68
,
3026
3030
.
Yoshida
,
M. C.
&
Ephrussi
,
B.
(
1967
).
Isolation and karyological characteristics of seven hybrids between somatic mouse cells in vitro
.
J. cell. Physiol
.
69
,
33
34
.
Yoshida
,
M. C.
&
Sasaki
,
M.
(
1978
).
Euploid somatic recombinants with two active X or XY1 Y2 chromosomes isolated from cultured male Indian muntjac cells after HVJ virus fusion, and their use for gene assignment
.
Somat. Cell Genet
.
4
,
437
450
.