J . Cell Sci. 12,809-830 (1973)
Printed in Great Britain
H U M A N CHROMOSOMES I N 18 MAN-MOUSE
SOMATIC H Y B R I D CELL L I N E S A N A L Y S E D
BY QUINACRINE FLUORESCENCE
P. W. A L L D E R D I C E , 0. J. M I L L E R
Department of Human Genetics and Development, and Department of Obstetrics and
Gynecology,College of Physicians and Surgeons, Colt~mbiaUniversity, New Yoriz roogz,
U.S.A.
P. L. P E A R S O N *
M.R.C. Clinical Population Genetics Unit, Headington, Oxford, England
G. K L E I N
Department of Tumor Biology, Karolinska Institutet, S-104 01 Stockholm 60, Szveden
H. H A R R I S
Sir William Dunn School of Pathology, University of Oxford, Oxford
England
AND
0x13RE,
SUMMARY
Chromosome studies were done on 18 somatic hybrid cell lines produced by fusing cells of
the mouse A 9 line with cells of the human Daudi lymphoblastoid line derived from a patient
with Burkitt's lymphoma. The human chromosomes were identified by their quinacrine
fluorescent banding patterns. In one hybrid line the human chromosomes were identified also
by the centromeric heterochromatin staining technique. Every human chromosome was
identified in one or more of the hybrid lines. Some lines were homogeneous in terms of their
human chromosome content, while others were quite heterogeneous. Detailed analysis of the
A 9 chromosomes in one hybrid line showed very few changes in comparison with the chromosome constitution of the average A g cell.
INTRODUCTION
Human chromosomes tend to be eliminated from man-mouse somatic cell hybrids
(Weiss & Green, 1967). Retention of a specific human chromosome can be achieved
by the use of a selective growth medium which requires the retention of a chromosome
bearing a gene that specifies an enzyme essential for growth. By this technique the
human thymidine kinase gene locus, earlier shown to be on a human E-group chromosome (Matsuya, Green & Basilico, 1968; Migeon & Miller, 1968), was assigned to
chromosome 17 (Miller et al. 1971), the human isocitrate dehydrogenase locus to
chromosome 20 (Boone, Chen & Ruddle, 1972), the human lactate dehydrogenase-A
locus to chromosome 11 (Boone et al. 1972) and, by the use of cells bearing an Xautosome translocation, the human phosphoglycerate kinase locus to the long arm of
the X chromosome (Grzeschik et al. 1972).
* Present address: Department of Genetics, University of Leiden, Leiden, T h e Netherlands.
52
G E L 12
810
P. W. Allderdice and others
In most of the earlier studies it was not possible to identify precisely the other
human chromosomes retained in the hybrid cells. However, estimates of their number
were made, based on the increase in the frequency of biarmed (Migeon & Miller,
1968) or acrocentric chromosomes (Kusano, Long & Green, 1971). With the quinacrine fluorescence technique, exact identification of each normal human chromosome
is possible (Caspersson, Lomakka & Zech, 19716), and the identification of several
human chromosomes in hybrid cells has been reported (Caspersson et al. 1971a;
Boone et al. 1972).
We have applied this technique to a series of man-mouse hybrid cells lines and can
now report that each of the 24 different human chromosomes is sometimes retained.
We also present a method for evaluating the heterogeneity (or instability) of such
hybrids with respect to their content of chromosomes of each parental type.
MATERIALS AND METHODS
The two cell lines used in the production of hybrids were the near-diploid human line,
Daudi, and the heteroploid mouse line, A 9. Daudi is a lymphoblastoid line derived from a
patient with Burkitt's lymphoma. It grows as a suspension culture and shows surface IgM
(Klein et al. 1967). The A 9 line, which is deficient in the enzyme inosinic acid pyrophosphorylase (EC 2.4.2.8), was derived from mouse L cells (Littlefield, 1964). Its quinacrine
fluorescence karyotype has been studied in some detail (Allderdice et al. 1973). The Daudi and
A 9 cells were fused by the u.v.-inactivated Sendai virus technique (Harris & Watkins, 1965)
late in 1970 and early in 1971. The mixture of fused and unfused cells was grown in H A T
medium, thus providing a double selection system: Daudi cells do not attach to glass, and A 9
cells, lacking inosinic acid pyrophosphorylase, do not grow in H A T medium. Thus only
A 9/Daudi hybrid cells grew on the surface of the culture vessels. Within 2-3 weeks isolated
hybrid colonies appeared in the cultures, and we shall refer to their descendants as clonal lines.
The isolation of hybrid lines in H A T medium was made by one or two methods. Primary
clonal lines P1C1, P1C2, P1C3, Cl 3, Cl 5, Cl 6, Cl 7, Cl 9 and Cl 10 were each derived from
a separate fusion event: individual primary hybrid colonies were isolated and trypsinized in
steel cylinders. Line Pi was a wild type culture derived from the flask from which clonal lines
P I C I , P1C2 and P1C3 had been isolated earlier. Lines Or, P i , P2, P3, P4, PA3, PA4, PA5
and PA6 were wild type lines each derived independently from a single flask containing the
original population of fused cells. Each line was continuously grown in H A T medium except
the Or line, which was transferred to Eagle's MEM after its initial selection in HAT.
Metaphase chromosome preparations were made of the Daudi cells in June 1971 and of the
A 9 cells (grown continuously in mass culture in H.H.'s laboratory since 1967) in July and
August 1971. Samples of the hybrid cell lines were harvested for chromosome study at the
same time. Mitotic cells were collected by shaking growing cultures and removing the detached
cells; these were suspended for 3 min in 10 ml of medium containing 1 drop of 0-04 % colcemid,
transferred to hypotonic KCl solution (007 M) and then fixed in several changes of acetic acidmethanol (1:3). Small drops of the cell suspension were placed on coverslips (thinness no. 1)
and air-dried. For fluorescent banding studies, these preparations were stained with a 5 mg/ml
solution of quinacrine dihydrochloride in ethanol, washed and mounted in distilled water.
Fluorescence microscopy was done by means of a Leitz Ortholux microscope with an HBO
200-W mercury lamp, epi-illumination, BG 12 or Schott 436 excitor filter, 490-nm barrier
filter, and a x 90 oil-immersion objective with a funnel stop. Photographs were taken on Kodak
Panatomic X film with exposures of 25-40 s, developed in Kodak Microdol X and printed on
Ilford paper contrast grade 4. In one case, centromeric heterochromatin was stained by the
method of Dev, Miller, Allderdice & Miller (1972). This involved treating the preparations
with 95 % formamide in SSC (0-15 M sodium chloride plus 0-015 M trisodium citrate) at 65 °C
for 20 min and then staining the cells with Giemsa for 30 min.
Chromosomes in man-mouse hybrid cells
811
RESULTS
Parental lines
These have been described elsewhere, but the most significant karyotypic features
are described here because of their relevance to the observations made on the hybrid
cells.
The A 9 cells contained only 16 of the 21 normal mouse chromosomes (Committee, 1972;. Of the other 5, the Y chromosome was never seen, while chromosomes
15, 16 and 17, and the X were present in various centric fusion translocation
chromosomes. A detailed analysis of the karyotypes of 11 of these A 9 cells has been
reported (Allderdice et al. 1973). The median number of chromosomes per cell was 59
(range 55-63), with up to 45 morphologically distinctive chromosomes per cell. A total
of 58 different chromosomes was seen in the eleven A 9 cells, i.e. 16 normal mouse
chromosomes plus 42 identifiable marker chromosomes.
The Daudi cell line appeared to be slightly heterogeneous, as noted already by
Manolov, Manolova, Levan & Klein (1971). Of the 10 cells karyotyped, 5 showed a
normal human diploid male karyotype (46, XY), 4 were trisomic for chromosome 7
(47, XY, +7) and 1 was monosomic for chromosome 14 (45, XY, — 14). In 1 of the
trisomic cells there was a constriction near the end of the long arm of a chromosome
11. Several chromosomes had polymorphic characteristics which could be used to aid
identification in hybrid cells. Both chromosomes 3 had an intensely fluorescent band
adjacent to the centromere. One chromosome 13 had an intensely fluorescent satellite,
while the other had intense fluorescence of the short arm. The satellite of each
chromosome 22 was more brightly fluorescent than the long arm of the chromosome.
Hybrid cell lines
In one hybrid line, Cl 7, metaphase spreads were examined first by the quinacrine
fluorescence technique (Fig. 7) and then by a centromeric heterochromatin staining
(C-banding) method (Fig. 8). All the chromosomes identified as mouse on the basis
of their fluorescent banding patterns, except mouse chromosome 14 and its derivative,
the A 9 marker M 5, had large blocks of centromeric heterochromatin, as expected in
the mouse. The chromosomes identified as human on the basis of their fluorescent
banding patterns had much smaller heterochromatic blocks, as expected of human
chromosomes. In addition, in each hybrid cell the size of human chromosomes relative
to mouse marker chromosomes could be used for purposes of identification, particularly for checking the identity of the shorter human chromosomes (Fig. 9). No exceptional chromosomes, of the type one might see following an interspecific translocation,
were seen. Since the accuracy of the identification of the species of origin of each
chromosome by its fluorescent banding pattern was confirmed by the C-banding
technique, we used only the fluorescent banding technique for analysing most of the
hybrid lines.
The results obtained from analysis of 20 cells of line Cl 6 are shown in Table 1.
There were 1-7 different human chromosomes per cell. Chromosome 13 was present
in every cell, usually in 2 copies, one with an intensely fluorescent satellite, the other
52-2
P. W. Allderdtce and others
8l2
Table i. Cell by cell analysis of human chromosomes in line 67 6
Number of copies of each chromosome
poll
no.
2
10
11
12
13
17
19
20
22
X
Y 3P-
I
2
3
4
5
6
7
8
9
10
11
12
13
'4
15
16
17
18
'9
20
with an intensely fluorescent short arm (Fig. 10), like the 2 chromosomes 13 in the
Daudi parent. The X chromosome, which was selectively retained, was present in 14
of the 20 cells. The cell line as a whole contained considerably more kinds of human
chromosomes than any single cell. Eleven different human chromosomes, plus 1
marker of presumptive human origin, were present in at least 1 of the 20 cells, although
only 3 were seen in a majority of the cells.
As Table 1 indicates, there was considerable variation from cell to cell in the content
of human chromosomes, and one might ask whether all 24 human chromosomes would
be found in this hybrid cell line if enough cells were analysed. In order to answer this
question, and the more general one of how to characterize the degree of heterogeneity
of any cell line, we have plotted the cumulative number of different human chromosomes against the number of cells examined (Fig. 1). A rough curve was produced by
taking the 20 cells in the order shown in Table 1. The smooth curve was produced by
considering the 20 cells in 20 different orders, and plotting the mean values. This
minimized the variation from cell to cell and the effect of the 3 cells that contained
one or two chromosomes not present in any of the other 19 cells. From the slope of
the resultant smooth heterogeneity curve (o-2 in the region from 18 to 20 cells) it is
clear that examination of additional cells would probably disclose the presence of
additional human chromosomes, but no more than 1 for every 5 cells analysed. Thus,
if 20 more cells had been examined, some, but probably not more than 4, additional
human chromosomes might have been found, making a total of 16. Thus, it is unlikely
that this line contains every human chromosome.
Chromosomes in man-mouse hybrid cells
8"3
12
S „ 10
•8 I
8
u O
c
E
U
10
Number of cells
15
20
Fig. i. Heterogeneity curve showing the cumulative number of different human
chromosomes in Ao,/Daudi hybrid line Cl 6. +, the cells considered in the order
shown in Table i; O, the cells considered in 20 different orders and mean values
plotted.
The hybrid lines are listed in Table 2 in decreasing order of the number of different
human chromosomes they contain. This ranges from 21 in the Or line to zero in the
C3P1 line. A total of 274 hybrid cells in the 18 different lines were examined for the
presence of human chromosomes. The human chromosomes had banding patterns
indistinguishable from those of the chromosomes in normal cultured leukocytes. This
is illustrated by a composite karyotype (Fig. 11) in which human chromosomes from
various hybrid cells are paired with a haploid set of human chromosomes from a
cultured leukocyte. Every human chromosome was observed at least once. In addition
to the normal chromosomes, several structurally abnormal marker chromosomes of
probable human origin were seen (Fig. 12). In 9 lines there were marker chromosomes
containing an area of brilliant fluorescence and other attributes of the banding pattern
characteristic of human chromosome 3 (Table 2). In 1 line a marker chromosome had
a segment of brilliant fluorescence similar to that of the Y chromosome of the Daudi
line. Another marker appeared to have arisen from chromosome 5, with a segment of
brighter fluorescence translocated to its distal end.
Although every human chromosome, including the Y, was represented in these
hybrid cells, chromosome 8 was identified in only one of the 274 cells. We feel that the
less distinctive fluorescent banding pattern of chromosome 8 may have been responsible, at least in part, for the rarity of its identification, but preferential elimination of
this chromosome cannot be ruled out.
Curves showing the cumulative number of different human chromosomes for 5
of the hybrid lines are seen in Fig. 2. Of these, the Or line contains the most human
chromosomes, 22. The curve for this line has flattened out after 20 cells, but not
completely. Simple projection along the slope defined by the last 3 points suggests
that one more human chromosome might be discovered in the line if another 20 or so
cells were examined. This hybrid line may, in fact, contain every human chromosome.
P. W. Allderdice and others
Chromosomes in man-mouse hybrid cells
8i5
22
| 20
o
§18
o
"5 16
3 14
.C
§ 12
Z 10
E
y
1._ + — ) . _ . | . _ 4 - . |
+
)._+
i
i i
10
y—(-_ + _ +—y — + y—y
i
15
20
N u m b e r of cells
Fig. 2. Heterogeneity curves for human chromosomes in 5 other A<}/Daudi
hybrid cell lines.
The chromosome content of line P4 is in sharp contrast to that of line Cl 6 (Figs.
1, 10) or line Or. The curve is totally flat, reflecting the fact that 19 out of the 20 cells
contained 1 of the 2 human chromosomes seen. It is unlikely that additional human
chromosomes would be discovered, no matter how many additional cells were
examined. Line PA3 had a similar flat curve, and contained a single chromosome 19
in 12 of the 14 cells examined.
The behaviour of cell line P3 falls between that of the Or and the P4 lines. The total
number of different human chromosomes seen in 20 cells was 14, and the slope of the
curve at that point, about o-i, is such that one might expect to find about one additional human chromosome if 10 additional cells were examined.
The hybrid line P2 has a more steeply rising curve (slope about 0-2 after 20 cells)
than P3. The implication is that more human chromosomes have been missed in the
sample of 20 cells examined. As in line Cl 6 (Fig. 1), we expect that up to one more
human chromosome would be identified for every 5 cells analysed. Even more human
chromosomes are probably present in the line Pi (Fig. 2); 13 different chromosomes
were seen in the first 20 cells, and up to 3 more might be identified if 10 more cells
were examined.
Similar curves of the cumulative number of human chromosomes have been constructed for the remainder of the hybrid lines, and from these heterogeneity curves,
an estimate has been obtained for the maximum number of different human chromosomes that would be detected in each line if 30 cells were examined. This is included
P. W. Allderdice and others
8i6
« r
30
2 20
10
2
1
4
3
6
5
8
7
10
9
12 14 16 18 20 22 Y
11 13 15 17 19 21 X
Chromosome
Fig. 3. Frequency of each human chromosome in the pooled sample of 246 cells from
16 hybrid lines. Open areas, 1 copy; shaded areas, 2 copies.
in the last column of Table 2, in which the percentage of cells containing each normal
or marker human chromosome is given. The presence of more than one copy of a
chromosome, being fairly uncommon, has been disregarded in constructing this table.
The values obtained are usually only slightly higher than those observed with fewer
cells, but line Cl 10 appears to be an exception. In 10 cells, 17 different chromosomes
were observed, but only 4-11 in any one cell. If the next 20 cells were as variable as
the first 10, then 31 different chromosomes would be expected, 7 more than possible,
unless structural variants were involved; but none was seen in this line.
In order to examine the possibility that differential elimination of specific human
chromosomes may have occurred in Ao./Daudi hybrids, we pooled the data from the
16 lines that had been grown continuously in HAT medium and had retained one or
more human chromosomes. Such pooling may not be valid, but appears reasonable
when approximately equal numbers of cells from a large number of lines are used.
Chromosomes in man-mouse hybrid cells
817
Table 3. Median number of chromosomes
Mouse and human
Mouse
Human
Total
(including
dote)
Biarmed Acrocentric Biarmed Acrocentric Biarmed Acrocentric
Line
Ao
Or
61
23
no
44
33
Cl 10
75
P3
CI9
74
73
P2
72
70
Cl7
Cl5
69
69
69
Pi
CIPI
C 2 Pl
68-69
66
C16
Cl3
P4
65
32
34
29
29
32
26
27
26
26
36
58
38
39
38
38
37
33
38
38
23
37
28
26
26
25-26
26
25
24-25
3°
24
23
23
40
36
56
37
34-35
35
35
35
33
38
38
39-4°
37
25-26
40
22
21
22
37
36
36
35
22
35
34-35
39
36-37
36
36
35-36
28
29
24
29
64-65
27
23
PA6
63
22
PA4
PA 3
23
C3P1
62
62
62
PA5
59
21
22
20
7
5
6
8
2
1
5
3
3
4-5
3-4
2
7-8
0
2
0-1
3
3
3-4
0
2-3
3
1
2
1
1
1
i
1
0
1
0
0
0
4
0
130
u 120
o
0
I 110
u
JZ
u
v
I 100
-
A9
s
Median
E
'o
o 90
7f]
The 18 hybrid lines
Fig. 4. Median number of A9 chromosome arms (excluding dots) in each hybrid and
in a sample of A 9 cells.
P . W. Allderdice and others
8i8
Table 4. Identification and number of copies of each human
or mouse chromosome in hybrid line PA 6
Cell number
Chromosome
Type
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
1
1
1
1
1
1
1
14
HUMAN
2
10
21
1
1
1
1
1
1
1
2
2
2
2
1
2
2
1
2
2
2
3
2
2
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
5
1
1
1
1
1
1
1
1
1
1
2
6
4
3
3
2
2
2
7
8
2
1
2
1
2
3
3
MOUSE
Normal
j
4
9 or 13
1
1
1
2
3
2
1
1
1
1
3
3
3
3
4
3
2
2
2
2
1
1
2
1
2
2
1
2
2
2
1
2
1
2
1
3
3
3
3
3
3
2
3
3
3
3
3
3
4
4
4
4
4
4
4
2
2
2
1
2
5
3
1
1
1
1
1
2
10
2
11
12
2
2
3
2
2
2
2
1
14
19
1
1
1
1
1
1
1
1
Biarmed
1
17/1
18/1
X/3
5/3
15/3
5/4
1
1
1
1
I
15/4
n/5
15/5
18/5
15/10
16/10
17/10
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9 or 13/9 or 13
1
1
1
1
1
1
1
1
19/15
19/17
4/4
5/5
1
1
or 11
17/14
19/14
15/15
18/18
1
1
17/9 or 13
18/0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
2
1
2
1
1
1
1
1
2
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
Chromosomes in man-mouse hybrid cells
819
Table 4 (cont.)
Chromosome
No.
Type
Cell number
1
2
I
I
2
I
I
I
I
2
I
3
5
4
6
7
8
I
I
I
2
.
I
I
I
2
I
9
11
10
12
13
H
MOUSE (cont.)
Telocentric
markers
Mi
12 —
12 +
M2
M3
M4
M5
M6
M9
M12
M13
M14
MiS
M16
Biarmed
markers
M41
M42
M43
M44
M45
M46
M50
M52
MI/MI
I
I
I
2
I
I
I
I
2
I
I
I
I
2
I
I
I
I
2
I
I
I
I
2
I
I
I
I
1
I
I
I
I
1
I
I
I
I
1
I
I
I
I
1
I
I
I
I
2
I
1
1
1
I
.
1
I
I
I
I
I
I
I
I
.
I
I
I
The' results are shown graphically in Fig. 3. The frequency of occurrence of the
human chromosomes in these 274 cells tends to have a bimodal distribution, with
length as the major determinant of frequency. Most of the longer chromosomes (1-11)
were seen in less than 14% of the cells, while most of the shorter chromosomes
(12-22 + ) were seen in more than 20% of the cells. There were several exceptions,
e.g. chromosome 3 was present in 25 % of the cells. We do not believe this is simply
a reflexion of the easier identification of chromosome 3, with its centromeric zone of
brilliant fluorescence, because chromosome 7, which also has a very distinctive banding pattern, was seen in only half as many cells. Of the shorter chromosomes, numbers
14 and :6 were seen less frequently than would be predicted on the basis of their
length. In spite of these exceptions, length appears to be the major factor determining
chromosome retention or elimination in Ao,/Daudi hybrids.
Despite the fact that human X-linked inosinic acid pyrophosphorylase was essential
for survival of these cell lines, a normal human X chromosome was observed in only
31% of these hybrid cells, and the presence of a translocated segment of the X
chromosome was not evident in any of the other cells. In the Or line, which had been
P. W. Allderdice and others
820
6
—
5
vt
0)
Q.
r
0 4 -
n
*o
-
1
-L
E
3
T
41
i
i
c 2
1
j
L
—J
J.
L._J
1
i
L -
n
i
_.
1
1 1
3 4
5
6
7 8 9 + 10 11 12 14 15 16 17 18 19 X Y
13
Chromosome number
1 2
3 4
5 6
7 8 9 10 11 12 13 40 41 42 43 44 45 46
Marker (M) number
3
o
c
£o
Fig. 5. Comparison of the mean number of copies of each mouse chromosome found
) and PA 6 (
) lines.
in the A 9 (
maintained in a non-selective medium so that human inosinic acid pyrophosphorylase
was no longer essential for its survival, a normal X chromosome was observed in only
20 % of the cells. The other chromosomes showed the same frequencies as those seen
in the lines maintained in HAT medium, even though the Or line retained the largest
number of human chromosomes (Fig. 2). It also contained the largest number of A9
chromosomes and chromosome arms, and may have originated from the fusion of
two A9 cells with one Daudi cell.
Although the mouse chromosomes of A9 origin were not individually identified in
most of the hybrid lines, they were identified by exclusion and characterized as
biarmed, telocentric, or dot. The median number of A9 and human chromosomes in
each of the 18 hybrid lines is shown in Table 3. The mean number of mouse chromosomes in each cell line except the Or line was rather similar to the number in the A9
line. This is shown graphically in Fig. 4, in which the median number of chromosome
arms (excluding dots) in the A9 line is compared with the corresponding number of
chromosome arms of A9 origin in each of the hybrid lines. In 15 of the 18 lines, the
median number of arms deviated by 5 or less from the value of 82 seen in A9. In the
other 3 hybrid lines there were 7 to 51 more chromosome arms than in A9. The
largest excess, 51, was observed in the Or line. This excess is so extreme that it must
Chromosomes in man-mouse hybrid cells
0
821
2
4
6
8
10 12
No. of human chromosomes
Fig. 6. The independence of the median total number of mouse chromosomes and the
median total number of human chromosomes in the 18 hybrid lines.
represent the fusion of two A9 cells or one polyploid A9 cell with one human cell.
With this exception, the data fit well with the idea that fairly 'average' A9 cells enter
into the formation of the hybrids and that A9 chromosomes do not tend to be lost
from the hybrid cells.
A more rigorous test of the idea that each hybrid contains an 'average' A9 chromosome set is possible if each mouse chromosome has been identified. The mouse
chromosomes present in 14 cells of the PA 6 line (Table 4) have been compared with
the 11 A9 cells studied previously (Allderdice et al. 1973). The mean number of
copies per cell of each chromosome in the 2 lines is shown graphically in Fig. 5.
Despite minor differences, the similarity of these two graphs is obvious.
Since there is such a slight change in the average number of A9 chromosome arms
in any of the hybrid cell lines, it might be possible to estimate the number of human
chromosomes present in the A9/human hybrids from the total number of chromosomes in each hybrid line, as others have done. As seen in Fig. 6, this measure could
be used, since the number of mouse chromosomes, like the number of mouse chromosome arms, is fairly constant. Nevertheless, the method is now unnecessarily crude,
since more exact identification of the human chromosomes is possible with the banding
technique.
822
P. W. Allderdice and others
DISCUSSION
Identification of human chromosomes
The most striking finding in this study is that every human chromosome in the
hybrid cells can be identified by its quinacrine fluorescent banding pattern against a
heteroploid background of mouse chromosomes. The identification of the species of
origin of each chromosome can be confirmed by the centromeric heterochromatin
(C-banding) technique, as reported by Boone et al. (1972). In our experience, the
combined use of both techniques has served to confirm the reliability of the fluorescent
banding technique and to identify a translocation chromosome with a human centromere (Fig. 12). However, interspecific translocation chromosomes, whose occurrence
has been suggested by Migeon & Miller (1968), might be more easily demonstrated
by a combination of the 2 techniques than by either one alone (Boone et al. 1972),
especially if both were applied to the same cells.
An interesting finding in this study is that man-mouse hybrid lines can be either
quite homogeneous or quite heterogeneous from cell to cell with respect to their
content of human chromosomes. The degree of heterogeneity can now be accurately
estimated by a simple graphical method. Many of the hybrid lines we studied were
highly variable despite their presumptive clonal origin. In some lines, no two cells
had the same complement of human chromosomes. Assuming these are true clones,
the existence of so much heterogeneity in a line suggests that human chromosomes
are still being lost. Miggiano, Nabholz & Bodmer (1969), using a different method of
analysis, described similar variability in several man-mouse hybrid clones. One of
their lines, 2W1, showed almost no karyotypic change over a 4-month period of
cultivation, while another, 4W10, lost nearly 50% of its chromosomes in the same
period of time. These authors were unable to distinguish clearly between loss of
human chromosomes and loss of mouse chromosomes, whereas the use of a banding
technique makes this distinction easy and reliable. The rate of loss of human chromosomes can now be estimated fairly accurately in a cloned line; it is proportional to the
slope of the curve for the cumulative mean number of different human chromosomes
in the hybrid line after about 10 cells have been examined.
Implications for mapping
The assignment of a human gene to a specific chromosome by the use of interspecific somatic cell hybrids is based on a high correlation between the presence of a
given human gene product and the presence of a specific chromosome. Our results
indicate that even clonal hybrid lines can be quite heterogeneous with respect to the
human chromosomes they contain. This is both a handicap and an opportunity: a
handicap in that the reliability of gene assignment is reduced because a human
chromosome which may not be observed in a small sample of cells examined at
metaphase may be present in a sufficiently large proportion of the hybrid cells to
allow phenotypic expression of one of its genes, especially if the cytological and genetical studies are done at different times; and an opportunity in that further cloning may
Chromosomes in man-mouse hybrid cells
823
permit the isolation of hybrid cell lines containing different human chromosomes,
which would facilitate gene assignment.
The discovery that all 24 human chromosomes are retained in one or more of the
lines in this relatively small series of 19 man-mouse hybrid lines, the variation in the
human chromosome complement among individual lines, and the evidence that
human chromosomes are probably still being lost from some of the lines, indicate that
the raw material is available for assigning genes to each human chromosome.
Interspecific hybrid cells may also be useful in determining the linear order of
genes. For this, translocation chromosomes may be quite helpful. Grzeschik et al.
(1972) have used a naturally recurring human X-autosome translocation to show that
the phosphoglycerate kinase locus is carried by the long arm of the X chromosome,
while the glucose-6-phosphate dehydrogenase and inosinic acid pyrophosphorylase
loci are probably on the short arm. In the same way, one might use translocations
which arise in man-mouse hybrid cells. Migeon & Miller (1968) proposed the
occurrence of an interspecific chromosomal rearrangement to account for the fact that
hybrid cells, derived from the fusion of human cells with mouse cells lacking thymidine kinase, could still survive in HAT medium despite the disappearance of the
human E-group chromosome carrying the structural locus for thymidine kinase.
Boone et al. (1972) have presented cytogenetic evidence of the presence of such an
interspecific translocation chromosome.
We have been able to identify a series of human marker chromosomes derived from
part of a normal chromosome. Markers derived from chromosomes 3, 5 or the Y were
observed (Fig. 12), frequently in a minority of cells, indicating either a recent origin
or loss of the marker. In 5 lines, a specific human marker chromosome was present in
90% or more of the cells, indicating either an origin closer to the formation of the
hybrid cell line or selective retention of the marker, perhaps because of the incorporation of a segment of A9 chromosome essential to the survival of the hybrid cell. It is
surprising that such a large proportion of the structural alterations we have observed
involved human chromosome 3 or the Y. Either these chromosomes are more likely
than others to undergo rearrangement in these hybrid cells or else rearrangements of
these chromosomes are easier to recognize, possibly because they usually have a region
of intense quinacrine fluorescence. The idea that structural changes can occur but that
the quinacrine banding technique is just not sensitive enough to pick them up is
supported by the absence of a recognizable human X chromosome or segment of an
X in a high proportion of cells grown under conditions in which a product of an
X-linked gene is essential for survival. The existence of a high level of structural
chromosome instability in hybrid cells increases the difficulty of making gene assignments to specific chromosomes by the now standard method of correlating the
presence of a specific human gene product with the presence of a specific chromosome.
However, this difficulty could be overcome by analysing larger numbers of hybrid
lines.
This work was supported by grants from the National Cancer Institute, the National
Foundation-March of Dimes, the Cancer Research Campaign and the Swedish Cancer Society.
824
P. W. Allderdice and others
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MIGEON, B. R. & MILLER, C. S. (1968). Human-mouse somatic cell hybrids with single human
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(Received 11 August 1972)
Chromosomes in man-mouse hybrid cells
825
See Figs. 7-12 on pp. 826-830.
53
C E L 12
826
P. W. Allderdice and others
',0
M40
M41
M42
M43
M44
I
I *
M45
M46
11/
Fig. 7. Quinacrine fluorescent karyotype of a cell from A 9/Daudi
line Cl 7, cell 21.
Chromosomes in man-mouse hybrid cells
827
>l(Chlll«
nuuiiiii
Mil
Hi
t
MM
1
I ( lit*
It III I I " *
'
•
Fig. 8. C-banding patterns of the chromosomes in the same cell as in Fig. 7,
arranged in the same order.
53-2
828
P. W. Allderdice and others
Fig. 9. Relative sizes of selected A 9 and human chromosomes
in three A 9/Daudi cells.
829
Chromosomes in man-mouse hybrid cells
< III
I Mil
II ft *
I I
M40
Ml
M41
M2
M42
M3
M43 M44
M45 M46
M51
I ft*
Fig. 10. Quinacrine fluorescent karyotype of an A 9/Daudi cell from line Cl 6.
§3°
P. W. Allderdice and others
Fig. i i . Composite karyotype. The chromosome on the left in each pair is from a
man-mouse hybrid cell; the chromosomes on the right are from a single human
leukocyte.
Fig. 12. Human marker chromosomes from the hybrid cells. All have intensely
fluorescent material which, with the banding patterns, is consistent with the following
derivations: a, b, c, d, from chromosome 3; e, from the Y chromosome; and f, from
chromosome 5.