J. Cell Sci. 13, 841-861 (1973)
Printed in Great Britain
841
CORRECTION OF GENETIC DEFECTS IN
MAMMALIAN CELLS BY THE INPUT OF
SMALL AMOUNTS OF FOREIGN
GENETIC MATERIAL
YVONNE L. BOYD AND H. HARRIS
Sir William Dunn School of Pathology
South Parks Road, Oxford OXi 3RE, England
SUMMARY
Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking
thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were
cultivated in a selective medium in which possession of these enzymes was essential for cell
survival and growth. Clones of cells able to grow in this medium were isolated and studied. A
detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could
any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising
from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an
inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic
acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes
with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated
in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase
activity.
INTRODUCTION
Small amounts of chick genetic material may be introduced into mouse cells by the
technique of cell fusion, and it has been shown that the interpolated genes are expressed and replicated in the foreign environment (Schwartz, Cook & Harris, 1971).
When chick erythrocytes were fused with A9 cells (Littlefield, 1964), which lack the
enzyme inosinic acid pyrophosphorylase (E.C. 2.4.2.8), clones of hybrid cells could be
selected which contained chick inosinic acid pyrophosphorylase, but which showed
no evidence of any chick chromosomes in standard metaphase preparations. Nor did
these cells containing chick inosinic acid pyrophosphorylase show any chick-specific
surface antigens. It has been suggested that the reactivated chick erythrocyte nucleus
might undergo' premature chromosome condensation' or' chromosome pulverization'
when the heterokaryon enters mitosis (Schwartz et al. 1971; Johnson, Rao & Hughes,
1970), and fragments of the 'pulverized' genetic material might then be incorporated
into the A9 nucleus during its postmitotic reconstitution.
When A9 cells were fused with frog erythrocytes a similar result was obtained:
cells containing frog inosinic acid pyrophosphorylase, but no detectable frog chromosomes, were produced (P. R. Cook, in preparation). This indicates that the pheno-
842
Y. L. Boyd and H. Harris
menon is not restricted to situations in which a chick nucleus acts as the donor. The
present paper reports an extension of this work aimed at determining whether the
carriage of the foreign genetic material is a peculiar feature of the A9 cell and whether
genes other than those determining the expression of inosinic acid pyrophosphorylase
can be carried in this way. The effects of fusing chick erythrocytes with Chinese
hamster cells lacking inosinic acid pyrophosphorylase and with mouse cells lacking
thymidine kinase (E.C. 2.7.1.21) were therefore explored.
MATERIALS AND METHODS
Cell lines. The Wg3-h line was derived from the Chinese hamster Don line in this laboratory
(Handmaker, 1971). It is resistant to 10/tg/ml of 8-azaguanine and has very low levels of inosinic
acid pyrophosphorylase activity (IPP"). The mouse 3T3(tk~) cells used were resistant to
30 /ig/ml of bromodeoxyuridine and had little thymidine kinase activity (Matsuya & Green,
1969). Chick erythrocytes were prepared as described by Bolund, Ringertz & Harris (1969).
Cell culture. All cells were grown as monolayers in Eagle's Minimal Essential Medium
(MEM) containing 10 % foetal calf serum, or in HAT medium which is MEM containing
hypoxanthine, aminopterin, thymidine and glycine at concentrations of 1 x io~* M, 4 x io~' M,
i-6 x 10"' Mand 3 x io~* M respectively (Szybalska & Szybalski, 1962). In this medium only cells
containing both inosinic acid pyrophosphorylase and thymidine kinase can survive.
Cell fusion. 1 x 10' Wg3-h cells or 1 x io° 3T3 (tk~) cells were fused with 10 x 10" chick
erythrocytes by the method of Harris & Watkins (1965). 1000 haemagglutinating units of
Sendai virus inactivated by ultraviolet light were added to mediate fusion. The resultant heterokaryons were maintained in M E M for 24 h and then transferred to H A T medium.
Detection of chick-specific surface antigens. This was done by the technique of Watkins &
Grace (1967).
Extraction of inosinic acid pyrophosphorylase. This was done by the method of Harris & Cook
(1969).
Assay of inosinic acid pyrophosphorylase. A modified version of Cook's assay was used (Harris
& Cook, 1969). The reaction mixture contained 1-16 nmol P 4 C]hypoxanthine (specific activity
58 mCi/ml; Radiochemical Centre, Amersham), 4 x i o ! nmol s'-phosphoribosylphosphate,
5 x io 3 nmol Tris buffer at pH 7-4 and 5 x io J nmol MgCl, in a total volume of 50 /tl. 20 /i\ of
enzyme solution were added to the reaction mixture to start the reaction, and the mixture was
then incubated for 30 min at 37 °C. The reaction was stopped by cooling the samples to 4 °C,
and 50 /A aliquots were immediately pipetted onto 2 x 2 cm Whatman DE 81 disks (W. R.
Balston Ltd., Springfield Mill, Maidstone, Kent). The disks were then treated as described by
Harris & Cook (1969) to remove unincorporated ["C]hypoxanthine, dried and assayed for their
radioactive content in a Packard Tri-carb scintillation counter with an efficiency of 60 %.
The scintillation fluid was composed of 0-06 % diphenyloxazole in toluene.
Electrophoresis of inosinic acid pyrophosphorylase. Satisfactory separation of Chinese hamster
and chick inosinic acid pyrophosphorylase was achieved by electrophoresis on cellulose acetate
gels (Cellogel, Serva Biochemicals), in a Tris-EDTA-borate buffer at pH 8 9 . The procedure
used was that of Khan (1971). Instead of visualizing the enzyme activity along the DE 81
paper by autoradiographic procedures, however, the paper was divided into 2 mm sections and
the radioactivity in each section was measured as described above.
Extraction of thymidine kinase. The procedure was similar to that used for inosinic acid
pyrophosphorylase. However, the washing solution was 0154M KC1 and the cells were suspended in Tris buffer at pH 8-o before lysis; both these solutions contained 1 6 x IO~°M
thymidine and 0-003 M /?-mercaptoethanol (Littlefield, 1965).
Assay of thymidine kinase. The DEAE cellulose disk method was used to separate the radioactive reaction products, [ 14 C]thymidine mono-, di- and triphosphate, from the substrate [ 14 C]thymidine (Bollum & Potter, 1958; Breitman, 1963). The reaction mixture for the assay
contained i-i8nmol [ 14 C]thymidine (specific activity 59mCi/ml; Radiochemical Centre,
Amersham), 4 x io* nmol ATP, 5 x i o ' nmol Tris buffer at pH 8-o and 5 x io 1 nmol MgCl,
Correction of genetic defects
843
in a total volume of 50 ji\. The method was otherwise similar to that used in the inosinic acid
pyrophosphorylase assay.
Measurement of the heat sensitivity of thymidine kinase. 20-fil aliquots of the enzyme solution
prepared as above were incubated for o, 5, 10 or 15 min at 65 °C in stoppered tubes. The
samples were then immersed in an ice bath and the residual thymidine kinase activity was
assayed as described above. The residual activity was expressed as a percentage of the activity
at zero time.
Electrophoresis of thymidine kinase. The method was adapted from that given by Migeon,
Smith & Leddy (1969). The starch gel was prepared in a phosphate buffer at pH 70, and the
solution in the vessels was a 002 M phosphate buffer at pH 7-0. The gel was prepared as
described by Smithies (1955). 20 /A of enzyme solution was applied to each slot and the gel
run for 3-4 h at 150 V. DE-81 paper soaked in the reaction mixture was incubated at the cut
surface of the gel for 6-10 h and then washed to remove unincorporated substrate. The paper
was cut into strips and the radioactivity in each strip was measured in a scintillation counter as
described above.
Measurement of protein content. The amount of protein in the sample of enzyme was estimated
by the method of Lowry, Rosebrough, Farr & Randall (1951). Bovine serum albumin was used
as the reference standard.
RESULTS
Chinese hamster-chick erythrocyte hybrid cell lines
When Chinese hamster \Vg3-h cells and chick erythrocytes were fused together
clones of cells able to survive in HAT medium regularly appeared within 2-3 weeks.
Up to 50 HAT-resistant clones appeared for each million \Vg3-h cells plated out after
cell fusion. Spontaneous revertants of \Vg3-r1 cells are very rare. A total of 5 x io 8
\Vg3-h cells were treated with Sendai virus in the absence of chick erythrocytes and
Table 1. Properties of Wgyh Chinese hamster-chick erythrocyte
hybrids and the parental cell lines
Chromosome number
N
Cell
Inosinic acid
pyrophosphorylase
activity*
nmol IMP formed/h/mg
protein
Mode
Range
22
21-23
032
22
20-26
82-28
1
22
2
22
5'5i
539
8-07
325
319
629
602
33°
Chinese hamster
Wg3-h(IPP-)
Chinese hamster (IPP+)
Hybrid cell lines
3
4
5
6
7
Chick red blood cells
22
21-23
21-24
21-24
21-25
21-24
21-25
20-24
78
—
Chick embryo
fibroblasts
78
—
22
23
22
22
•
36-35
• Mean value calculated from at least 3 independent assays, each in triplicate.
844
Y. L, Boyd and H. Harris
were plated out in HAT medium; not a single HAT-resistant clone appeared. Seven
HAT-resistant clones arising from different primary fusions between \Vg3-h cells and
chick erythrocytes were studied in detail. The hybrid cells were similar in morphology
and growth rate to the parent \Vg3-h cells. When tested for the presence of chickspecific surface antigens, these cells showed no evidence of such antigens; chick
fibroblasts tested under the same conditions gave a strongly positive result. A summary
of the properties of these hybrid lines and of the parental cells is given in Table 1.
These lines have been cultivated in vitro for 12-18 months.
Table 2. Chromosomal analysis of Wgyh cells with and without
chick inosinic acid pyrophosphorylase
Mean number of chromosomes in group
Cell line
Wg3-h population before
cloning
\Vg3-h clonal line
A
B
C
D
E
F
G
H
Total
2-14
[•00
[•14
1-04
6-18
5-12
760
1 80
2602
2 0 0
[•00
[•00
100
5-02
5-oo
604
094
2202
c-oo
coo
[ 0 2
1 00
I-O2
C96
5-02
498
602
504
500
6-io
[•OO
i-oo
i-oo
0-96
5-12
533
5-02
5-O4
5-oo
5-oo
498
496
22-04
22-l6
22O8
23-53
23-O4
2I-98
[OO
100
498
5-oo
] •00
I 02
5-24
500
Wg3-h—chick hybrid lines
1
2-OO
2
2OO
3
4
2-00
5
6
7
I 98
Hybrid line 4 after the
loss of chick inosinic
acid pyrophosphorylase
1-98
[•00
[•00
[•02
[•00
[•04
2OO
] •00
2OO
2-OO
[•OO
[•OO
[•02
[•04
600
i-oo
i-oo
i-oo
7-22
0-96
700
602
6-04
i-oo
i-oo
0-96
22-OO
7-40
i-oo
23-66
Karyological analysis
As shown in Table 1 the modal number of 6 out of 7 of these clones is the same as
that of the \Vg3-h parent cell. Clone 4 had a mode of 23 chromosomes instead of the
more usual 22. The karyotype of the \Vg3-h clonal line used is given in Fig. 6.
Although this line has a mode of 22 chromosomes, it is not strictly diploid. The
chromosomes can be divided into 8 distinct groups A-H as shown (Fig. 6). The small
proportion of cells with 23 chromosomes usually had an extra small metacentric
chromosome in group G. A chick metaphase spread is given in Fig. 7; it is composed
of 12 macro- and 66 dot or microchromosomes. Preparations of a mixture of Wg3-h
and chick embryo fibroblast metaphase spreads were studied. Chinese hamster and
chick chromosomes were compared in spreads within the same microscopic field.
Three sets of chick chromosomes cannot be distinguished from those of the Chinese
hamster: the largest pair of metacentric chick chromosomes resemble those of group
E of the Wg3-h cell, the largest telocentric chromosomes those of group F and the sex
chromosomes those of group G.
The karyotype of each of the hybrid clones was examined in detail. All of the 6
Correction of genetic defects
845
clones with a mode of 22 chromosomes had idiograms identical to that of the \Vg3-h
cell (Fig. 8). Clone 4 had an extra small metacentric chromosome in group E (Fig. 9).
The mean number of chromosomes in each of the groups A-H was calculated for each
of the hybrid cell lines and for the \Vg3-h line. The data were collected from 50 metaphase preparations of each cell line and are shown in Table 2. Hybrid clones 4 and 5
tended to have a more variable chromosome number than the clonal Chinese hamster
line and showed a higher mean value for the chromosomes in group G. However, the
wild type \Vg3-h population also had a higher mean number of chromosomes in group
G than the clonal line. It therefore seemed possible that the higher mean number of G
group chromosomes in hybrid clones 4 and 5 was due not to the presence of a chick
chromosome but to the karyotypic variation of the \Vg3-h line. This idea was tested
by subjecting hybrid clone 4 to back selection in 8-thioguanine. A clonal population
was isolated that was resistant to io/tg/ml of 8-thioguanine and lacked inosinic acid
pyrophosphorylase activity. The chromosome constitution of this population was then
studied. These cells, which had lost the chick enzyme, nonetheless still had a mode of
23 chromosomes and an extra chromosome in group E. The loss of inosinic acid
pyrophosphorylase activity was thus not accompanied by the loss of a chromosome
(Table 2; Fig. 10).
Analysis of the enzyme
All the hybrid cell lines showed a measurable level of inosinic acid pyrophosphorylase activity. There was variation in the level of enzyme in the different hybrid cell
T
12
16
20
24
28
Strip number from origin to anode
32
36
40
Fig. 1. Electrophoretic mobilities of inosinic acid pyrophosphorylase isolated from
different sources. —O-O-, Chinese hamster inosinic acid pyrophosphorylase;
—•-•—, chick erythrocyte inosinic acid pyrophosphorylase; - A - A - , inosinic acid
pyrophosphorylase isolated from \Vg3-h-chick erythrocyte hybrid clone 1.
54
CEL
13
846
Y. L. Boyd and H. Harris
lines (Table 1). This variation was greater than that produced in any one line by
changes in cultural conditions.
Electrophoretic examination of the enzyme in the hybrid cells showed it to possess
the same mobility as the enzyme isolated from chick erythrocytes, from primary
cultures of chick embryo skin fibroblasts and from homogenates of whole chick
embryos. The mobility of the enzyme extracted from 2 Chinese hamster cell lines
containing normal levels of inosinic acid pyrophosphorylase was quite different (Fig. 1).
Table 3. Properties of mouse 2T2(tk~)-chick hybrids and parental lines
Chromosome number
Mode
Range
3T 3 (tk-)
66
3T3(tk+)
66
61-69
129-133
60-71
126-134
Cell
Thymidine kinase activity*
nmol TMP
formed/h/mg protein
048
17-27
Hybrid cell lines
1
66
2
66
3
65
4
66, 132
5
66
6
67
Chick red blood cells
Chick embryo
fibroblasts
—
61-69
128-133
62-70
127-132
63-70
128-133
55-70
113-137:254
60-69
130-133
64-70
127-133
1-09
I 05
1-40
I-07
098
I-2I
OO4
113
Mean value calculated from at least three independent assays, each in triplicate.
Mouse 2T2{tk~)-chick erythrocyte hybrid cell lines
When chick erythrocytes were fused with mouse 3T3(tk~) cells and the fused cell
populations grown in HAT medium the bulk of the cell population died off, but clones
appeared within 2-3 weeks. Up to 50 HAT-resistant clones were produced for each
million 3T3(tk~) cells plated out, whereas control cultures containing 2 x io 8 3T3(tk~) cells treated with the Sendai virus in the absence of chick erythrocytes produced
no HAT-resistant clones. Six HAT-resistant clones derived from separate primary
fusions between the 3T3(tk~) cells and chick erythrocytes were isolated and studied
in detail. The cells in these clones were similar in morphology and growth properties
to the parent 3T3(tk~) cells. No chick-specific surface antigens were found in any of
the populations. A summary of the main properties of these clones is given in Table 3.
847
Correction of genetic defects
40
20
3T3 tk~
l
Clone 1
20
tj.iiin.
Clone 2
20
I
»
o
<-
I
I
I
Clone 3
20
0
i
i
i
i
i^
Clone 4
20
•ibun
i
i
r
Clone 5
20
^liML^
i
i
i
i
r
Clone 6
20
li.li
i
20
u
I
60
I
70
i
i
i
i
Clone 2 without thymidine
kinase
I
I
I
I
80
90
100
110
Number of chromosomes
I
120
130
Fig. 2. Histograms depicting the distribution of chromosome numbers in 3T3(tk~)
cells, hybrid clones i-6, and hybrid clone 2 after the loss of thymidine kinase.
34-2
848
Y. L. Boyd and H. Harris
Karyological analysis
The parent 3T3(tk~) line has a mode of 66 chromosomes all telo- or acrocentric
(Fig. 11). Histograms depicting the distribution of chromosome numbers for the 3T3(tk~) line and for the 3T3(tk~)-chick erythrocyte hybrid clones are given in Fig. 2.
Four of the 6 clones had a mode of 66 chromosomes; clones 3 and 6 had modes of 65
and 67 respectively. All clones except 1 and 2 showed a slightly higher percentage of
zn cells than the original 3T3(tk~) line. No chick metacentric chromosomes or small
10
Min at 65 °C
15
Fig. 3. Heat inactivation kinetics of thymidine kinase. - • - • - , thymidine kinase from
chick embryo fibroblasts; - A - A - , thymidine kinase from 3T3(tk+) cells; - O - O - ,
thymidine kinase from 3T3(tk~)—chick erythrocyte hybrid clone 2; - A - A - , thymidine kinase from 3T3(tk~)—chick erythrocyte hybrid clone 6.
microchromosomes were present in any of the metaphase spreads of the hybrid clones
(Figs. 12, 13). It was possible, however, that one of the larger chick acrocentric
chromosomes might have been present. Hybrid clone 2 was therefore subjected to
back selection in bromodeoxyuridine. One clone resistant to 30 /ig/ml of bromodeoxyuridine and lacking thymidine kinase activity was isolated and examined. This subclone was found to have the same chromosome constitution as the clone 2 cell from
which it was derived (Fig. 2).
849
Correction of genetic defects
Analysis of the enzyme
The results obtained with enzyme extracts heated for various periods of time at
65 °C are presented in Fig. 3. The enzyme from Ehrlich ascites tumour cells, L cells
or 3T3(tk+) cells was almost completely inactivated after 5 min incubation at 65 °C,
while the chick enzyme was much more resistant to heating. The thymidine kinase
extract from primary cultures of chick embryo fibroblasts and that from whole chick
1 0 -
- 8 - 6 - 4 - 2
0
2
4
Strip number from origin
6
Fig. 4. Electrophoretic mobilities of thymidine kinase isolated from different sources.
- A - A - , mouse 3T3(tk+) cells; - O - O - , chick embryo fibroblasts; - # - • - ,
3T3(tk~)-chick erythrocyte hybrid clone 2.
embryo homogenates gave the same pattern of heat inactivation at 65 °C. Hybrid
clones 1 to 6 all showed the same kinetics of inactivation as the enzymes isolated from
murine sources. A mixture of mouse and chick enzymes showed a heat-sensitivity
intermediate between that of mouse and that of chick enzyme, thus showing that the
chick enzyme does not confer resistance to heat inactivation on the mouse enzyme. It
thus appeared that the thymidine kinase present in the hybrid cells was mouse, and
not chick, enzyme.
This was confirmed by the behaviour of the enzymes on starch gel electrophoresis.
At neutral pH, chick thymidine kinase migrated very slowly to the anode and mouse
thymidine kinase to the cathode (Fig. 4). The hybrid clones all possessed a thymidine
kinase which had the same mobility as the murine enzyme. The enzyme extract from
850
Y. L. Boyd and H. Harris
primary cultures of chick fibroblasts had the same electrophoretic mobility as the
enzyme extracted from whole chick embryo homogenate. Large numbers of 3T3(tk~)
cells were harvested and the thymidine kinase extracted. When this extract was run
on the starch gel a small peak of enzyme activity could be found; this appeared in
exactly the same position as the thymidine kinase from the 3 other murine sources
tested (Fig. 5).
- 8 - 6 — 4 - 2
0
2
4
Strip number from origin
6
8
Fig. 5. Electrophoresis of thymidine kinase from different sources. - A - A - , mouse
3T3(tk+) cells; —O-O-, chick embryo homogenate; - • - • - , thymidine kinase
extracted from large numbers of 3T3(tk~) cells.
DISCUSSION
The present studies demonstrate that different kinds of genetic defect in animal
cells from different species can be corrected by the appropriate application of the cell
fusion technique. It therefore seems possible that most genetic defects could be
corrected in this way provided selective procedures could be devised for isolating the
corrected cells. In all cases studied so far, the clones of corrected cells were indistinguishable from the defective parent cells in their gross chromosome constitution; and
none of the corrected cells showed species-specific surface antigens characteristic of
the donor nucleus. These corrected clones could be regularly produced with a
frequency of 1 in ioB fused cells. The frequency of spontaneous revertants in the
Correction of genetic defects
851
population of the defective parental cells was very much lower; not one revertant has
been isolated from 5 x io 8 Wg3-h Chinese hamster cells or 2 x 10s mouse 3T3(tk~)
cells treated with Sendai virus and plated out under conditions identical to those used
in selecting the hybrid clones. It is thus clear that the chick erythrocyte nucleus is the
effective agent in the correction of both the genetic defects studied.
In the case of the hamster cell lacking inosinic acid pyrophosphorylase the restored
enzyme has the properties of chick enzyme. This agrees with the previous work in
which similar experiments were done on mouse cells lacking inosinic acid pyrophosphorylase. When these cells were fused with chick erythrocytes, the corrected cells
contained chick inosinic acid pyrophosphorylase; when they were fused with frog
erythrocytes, the corrected cells contained frog enzyme (Schwartz et al. 1971; P. R.
Cook, in preparation). Schwartz et al. (1971) have suggested that a fragment of chick
chromosome bearing the gene for inosinic acid pyrophosphorylase might be incorporated into the defective mammalian nucleus when the heterokaryon enters mitosis;
for it has been shown that the reactivated chick erythrocyte nucleus usually undergoes
'premature chromosome condensation' and 'pulverization' when the mammalian
nucleus in the heterokaryon enters mitosis. This suggestion is supported by the
observations made in the present series of experiments. 'Pulverization' of the chick
chromosomes was commonly seen 24-48 h after cell fusion; but metaphase plates
containing a full set of hamster chromosomes together with some intact chick chromosomes were not seen. The corrected cells have carried the chick enzyme for 18 months
of continuous culture in selective medium. There is thus no doubt that the interpolated genetic material is expressed and replicated in the foreign environment.
In the case of the mouse cells lacking thymidine kinase, the role of the chick erythrocyte nucleus in correcting the genetic defect is more indirect, for the corrected cells
show mouse, not chick, enzyme. This indicates that the defect in the tk~ cells is
clearly not a deletion of the structural gene, a conclusion in any case implicit in the
finding that these cells contain low levels of a thymidine kinase having the electrophoretic properties of the normal mouse enzyme. There is evidence that the thymidine
kinase deficiency produced by exposure of cells to bromodeoxyuridine is not a onestep mutational process of the conventional kind (Freed & Sooy, 1972; Mezger-Freed,
1972). There appears first to be some change resulting in defective transport of thymidine across the cell membrane, and this change may not be mutational in character.
The low level of thymidine kinase activity in the tk~ cells may be explained by the
fact that, in the absence of thymidine, thymidine kinase is extremely unstable (Littlefield, 1965). The heritable chick factor that corrects the thymidine kinase deficiency
might thus act by restoring normal transport of thymidine across the cell membrane.
The question remains why at least some of the corrected tk~ cells do not contain
both chick and mouse enzyme. One possible explanation for this might be provided
by the observation of Schwartz et al. (1971) that the interpolated chick genes are
rapidly lost if the selection pressure for their retention is removed. Since the restoration of the mouse enzyme permits the corrected cells to survive in HAT medium, the
selective pressure to retain the structural genes for chick thymidine kinase would be
removed; and if the findings of Schwartz et a/. (1971) for inosinic acid pyrophosphory-
852
Y. L. Boyd and H. Harris
lase are applicable to thymidine kinase, one would expect the superfluous genetic
determinants to be eliminated. The absence of chick thymidine kinase might, on the
other hand, be explained by simple statistical considerations. If the frequency of
production of HAT-resistant clones (1 in io5) reflects the frequency with which a
piece of chick genetic material is stably incorporated into the mouse nucleus, then the
frequency with which two unlinked markers would be incorporated would be 1 in io10.
If the genetic factor that restores mouse thymidine kinase activity and the structural
gene for chick thymidine kinase are not linked, then cells having both mouse and
chick enzymes would be exceedingly rare.
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(Received 29 March 1973)
Correction of genetic defects
853
%
t! I X I m i x
A
B
C
D
A * 1 ft *
F
«
X 1 •
G
1
H
Fig. 6. Karyotype of the Chinese hamster \Vg3-h cell line.
Y
- L- B°yd
8.i;4
and H
- Harris
M \
Chinese Hamster
*• ^
Chick
Mm
B
C
D
it
it
I •
I ft 0 • •
« X a t a«
H
Fig. 7. Karyotypes of a cell of the Chinese hamster \Vg3-h line and of a chick embryo fibroblast
taken from metaphase spreads lying in the same microscopic field.
Correction of genetic defects
< * •
IX ft A A
B
A
A
n
C
D
A A
* x * «
H
Fig. 8. Karyotype of \Vg3-h-chick erythrocyte hybrid line 1.
856
Y. L. Boyd and H. Harris
Fig. 9. Karyotype of \Vg3-h-chick erythrocyte hybrid line 4. Note the extra small
metacentric chromosome in group G.
Correction of genetic defects
W
A
857
BC
i IIU
I
F
Jt I I I B I
_G_
H
858
Y. L. Boyd and H. Harris
*r
uin
B
C
D
X Xx* * «*
G
H
Fig. 10. Karoytype of clone 4 after the loss of chick inosinic acid pyrophosphorylase.
Correction of genetic defects
Jiillttauiifl
Fig. I I . Karyotype of 3T3(tk-) cells.
859
$6o
Y. L. Boyd and H. Harris
I IIMIIII II Illll
l i l t I l l l l l l 1MH
Fig. 12. Karyotype of 3T3(tk~)-chick erythrocyte hybrid clone 2.
861
Correction of genetic defects
IIIMIAIRMIIIII
!*••
*••
• » » * * t * # »ftA * * * A *
ftft*0A
AA
Fig. 13. Karyotype of 3T3(tk~)-chick erythrocyte hybrid clone 4.
M
CEL I J