309
J. gen. Virol. (I979), 45, 3o9-32I
Printed in Great Britain
Somatic Cell Genetics of Human Interferon Production in
H u m a n - R o d e n t Cell Hybrids
ByA. M E A G E R , 1 H . E . G R A V E S , 1 J . R . W A L K E R ,
1D.C.BURKE,
D. M. S W A L L O W 2 A N D A . W E S T E R V E L D 3
1
1 Department of Biological Sciences, University of Warwick, Coventry C V 4 7AL, 2 M R C
Human Biochemical Genetics Unit, The Galton Laboratory, University College London,
Wolfson House, 4 Stephenson Way, London NWr 2HE, England and s Department of
Cell Biology and Genetics, Erasmus Universiteit Rotterdam, Postbus 1738, Rotterdam,
The Netherlands
(Accepted 8 May I979)
SUMMARY
Forty-two primary human-mouse cell hybrids, derived in two separate experiments, were treated with Newcastle disease virus (NDV): eight hybrids were found
to produce human interferon and this was shown in every case to be predominantly
of the fibroblast type. An extensive analysis was made in terms of karyotype and
marker enzymes on all the eight hybrids producing interferon and also on five
hybrids which did not produce interferon, five randomly selected hybrids and
eleven subclones resistant to diphtheria toxin. The results suggest that, contrary
to previous reports, a gene on chromosome 5 is not involved in production of
human interferon. Its production was however correlated with the presence of
chromosome 9 in the hybrids. Analyses of two sets of human-Chinese hamster
hybrid subclones from two different crosses were also consistent with the assignment of a human interferon gene to chromosome 9.
INTRODUCTION
Interspecific somatic cell hybrids, in which one parent cell is human and the other rodent,
generally lose human chromosomes and retain those of the rodent. Assignment of human
genes to particular chromosomes can be made by correlating the presence or absence of
gene products (enzymes or other proteins) with the presence or absence of individual
chromosomes or of proteins coded by genes already assigned to particular chromosomes
(for review, see McKusick & Ruddle, 1977). Analysis of human-rodent hybrids has to date
provided conflicting evidence concerning the number and location of genes involved in the
production of human interferon, but genes on chromosomes 5 (Tan et al. t974; Morgan &
Faik, 1977; Tan, 1977) and 2 (Tan et al. 1974) have been implicated. Tan (1977) has tentatively assigned a gene for interferon production in human cells to the long-arm of chromosome 5, but has suggested that a gene on chromosome 2 is also required for interferon
production in human-mouse hybrids (Tan et al. I974).
Two types of human interferon have been identifed, namely the leucocyte type which
can be obtained from lymphocytes and lymphoblastoid cells and the fibroblast type which
is made by fibroblasts. Leucocyte and fibroblast interferons differ antigenically (Havell
et al. 1975; Paucker et al. 1975) and in the tool. wt. of their polypeptides (Stewart &
Desmyter, I975; Knight, I976 ; T6rma & Paucker, I976; Havell et al. 1977).
The purpose of this study was to identify the type of human interferon produced in
oo22-1317/79/oooo-361o $02.00 ~ 1979 SGM
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3Io
A. M E A G E R A N D O T H E R S
different hybrid cells and to clarify the role of human chromosomes 2 and 5 in this production. To this end, we have examined 42 primary human-mouse hybrids (four with
human lymphocyte parents and 38 with human fibroblast parents). Eight hybrids producing
human interferon (two with human lymphocyte parents) were identified by means of
sensitive bio-assays, and the type of interferon produced was determined with specific
antisera to leucocyte and fibroblast interferons. All the hybrids tested produced interferon
of the fibroblast type.
Since the gene conferring sensitivity to diphtheria toxin has been assigned to chromosome
5 (Creagan et al. I975), we tested I9 of our hybrids for their sensitivity to this toxin in an
attempt to show a correlation with human interferon production. Two series of subclones
resistant to diphtheria toxin, which produced high levels of human interferon, were also
selected from sensitive hybrids.
Analysis of I3 primary hybrids, five randomly selected subclones and II diphtheria
toxin-resistant subclones in terms of their karyotype, isozymes and interferon production
suggests that the structural gene for human fibroblast interferon is not located on chromosome 5; however, the data are compatible with the assignment of this gene to chromosome 9This assignment is supported by our findings in a small series of human-Chinese hamster
hybrids.
METHODS
Materials. 5-aH-uridine (26 Ci/mmol) and L-4,5-3H-leucine (55 Ci/mmol) were purchased
from The Radiochemical Centre, Amersham. The sources of other chemicals were : ouabain,
cycloheximide, hypoxanthine and thymidine from Sigma; aminopterin from Nutritional
Biochemicals (Cleveland, Ohio); polyethylene glycol (mol. wt. 6ooo) from BDH; concentrated ( I o × ) o r powder culture medium and foetal bovine serum (FBS) from Flow
Laboratories; colcemid from Gibco Biocult (Paisley, Scotland). Diphtheria toxin, purified
to 825 Lf/ml, was kindly provided by D. C. Edwards, Wellcome Research Laboratories,
Beckenham, Kent. (An Lf unit is defined as the volume in ml of toxin that gives the most
rapid flocculation with one standard unit of antitoxin. Fo: diphtheria toxin I Lf/ml is
approximately 3OHM.) Purified human leucocyte interferon (P-IF, batch 8991, 9 × I O ~
reference research units/ml) and anti-human leucocyte interferon (serum from sheep liver,
neutralizing titre I :36oooo) were provided by Dr K. Cantell, Central Public Health Laboratory, Helsinki, Finland; anti-human fibroblast interferon (monospecific anti-human FS4
interferon, rabbit globulin pool no. 29-33) by Dr J. K. Dunnick, National Institute of
Allergy and Infectious Diseases, Bethesda, Maryland, U.S.A.
Viruses. Newcastle disease virus (NDV), strains Texas and F, and Sendai virus were
grown in embryonated eggs (Lomniczi, I97O; Lomniczi et al. I971); stocks of clarified
virus-containing allantoic fluids were stored frozen at --7o °C. The stock of NDV strain
Texas had an infectivity of l × Io 9 p.f.u./ml in chick embryo cell monolayers (Waiters et al.
1967). The stocks of NDV strain F and Sendai virus (which do not form plaques in chick
cells) had haemagglutinating titres of z × lo 3 haemagglutinating units (HAU)/ml and
5 × toa HAU/ml, respectively. Semliki Forest virus, the challenge virus used in interferon
assays, was grown in chick cell suspensions (Kennedy & Burke, I972); stocks containing
about I × Io 9 p.f.u./ml were stored frozen at --70 °C.
Cell cultures. The human diploid foreskin cell line, FS4, at passage 20, was obtained from
Dr J. Vilcek, Department of Microbiology, New York University School of Medicine, New
York, U.S.A. Cat lung cells, originally supplied by Flow Laboratories, were obtained from
Dr J. Desmyter, Rega Institute, Leuven, Belgium. These two cell lines were cultured in
Glasgow modified Eagle's minimal essential medium (GMEM) with 1o% FBS, penicillin
(200 units/ml) and streptomycin (Ioo #g/ml).
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Genetics o f human interferon
311
Table I. Parentage of the human-mouse and human-hamster hybrids used in this study
Parental lines
Hybrid
designation
CTP34
CTP4 i
DUR4
HORP9
l
HORPI4
HL6-4z
HL43-64
46.6. i
Ch3
C123
A
c"
Human
Mouse* Hamster*
PG19
T lymphocytes
IR
DUV fibroblast (X; 15 translocation)
IR
Lymphocytes
L-A9
Embryo fibroblast
(HEF)
Fibroblast (X; I translocation)
Fibroblast
-
-
Reference
Jonasson et al. 1977
Solomon et al. I976
van Heyningen et aL 1973
This paper
E36 This paper
Wg3h This paper
* All the mouse and Chinese hamster cells were hypoxanthine-guanine phosphoribosyltransfe-ase (HPRT)
deficient and grew as adherent fibroblasts.
The mouse cell line P G I 9 and hybrid cell lines D U R 4, H O R P 9, H O R P I 4 , CTP34 and
CTP41 were obtained from Dr W. Bodmer's group at Oxford University, courtesy of Dr E.
Jones and Dr E. Solomon. These five hybrid lines were cultured in R P M I I64o medium
containing H A T (hypoxanthine, 13-6/zg/ml ; aminopterin, o'19#g/ml; and thymidine,
3"9 #g/ml) with 1 o ~ FBS.
A further 37 hybrid clones, HL6 to 42 and HL43 to 64, were derived in two experiments
from fusion between a normal diploid embryo fibroblast (HEF) cell line, derived from skin
and muscle of an aborted female embryo, and the mouse line L-A9, which is deficient in
both adenine phosphoribosyltransferase (APRT) and H P R T (Cox et al. I972; Tischfield &
Ruddle, I974). Hybrid clones were selected after fusion of io 5 H E F and Io 6 L-A9, mixed in
a monolayer culture, with 5 o ~ (w/w) polyethylene glycol, mol. wt. 6ooo (Davidson &
Gerald, I976) by culture in H a m ' s F I o medium containing H A T and ouabain (Io -5 M);
these kill respectively, the L-A 9 and H E F parents, whilst hybrids between these cells are
able to grow (Mankovitz et al. I974). Hybrid cell colonies were ring-cloned after 14 to 2I
days at 37 °C, separately transferred to 3o mm plastic dishes and grown out to large
numbers ( > ~on) in selection medium to establish hybrid cell lines (Table I). These were
then routinely subcultured in B H K medium ( G M E M plus tryptose phosphate broth)
containing I O ~ FBS with or without HAT. The human-Chinese hamster hybrids used
were: 46.6. i, C h 3 and C123 (Table t). The latter two hybrids (obtained from Dr R.
Buckland, M R C Clinical and Population Cytogenetics Unit, Western General Hospital,
Edinburgh) were subclones of a parental hybrid containing human chromosomes 3, 5, 6, 8
9, ~o, i2, I4, 17, 2o, 2i, (22) X. All hybrids and Chinese hamster parental cell lines, E36 and
Wg3h, were grown in H a m ' s F12 medium (Flow Laboratories) with added IO~o FBS,
penicillin (2oo units/ml) and streptomycin (Too/zg/ml). Medium for growing hybrids was
additionally supplemented with HAT.
Random subclones of CTP34, HLI5, HL53 and 46.6. t were obtained by plating these
hybrids at low cell density (I × IOa to 5 × IOa cells/too mm plastic dish) in R P M I I64O, or
H a m ' s F I o or FI2 medium, with or without HAT, and ring-cloning colonies after Io to 2o
days at 37 °C. Subclones are suffixed sc, for example, H L I 5 sc9. Diphtheria toxin resistant
subclones of CTP34, H L I 5 and H L I 5 sc9 were selected by plating to 4 to IOn cells in appropriate growth medium containing H A T and diphtheria toxin (o-t Lf/ml). After ringcloning colonies of resistant cells, the resulting cultures were grown for several generations
in the presence of the same concentration of diphtheria toxin and eventually subcultured in
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312
A. MEAGER AND OTHERS
Table 2. Isozyme analyses*
Enzyme
Enolase- 1 (ENO1)
Peptidase C (PEPC)
Phosphoglucomutase-i (PGM1)
Soluble malate dehydrogenase (MDHs)
Soluble NADP-dependent isocitrate dehydrogenase (1CDs)
Acid phosphatase (ACP0
Phosphoglucomutase-2 (PGM0
N-Acetylhexosaminidase ,6' (HEXfl)
Soluble malic enzyme (MEs)
fl-Glucuronidase (fl GUS)
Adenylate kinase-1 (AK0
Adenylate kinase-3 (AK3)
Soluble aconitase (ACON s)
Soluble glutamate oxaloacetate transaminase (GOT s)
Lactate dehydrogenase A (LDHA)
Lactate dehydrogenase B (LDHB)
Peptidase B (PEPB)
Esterase D (ESD)
Purine-nucleoside phosphorylase (NP)
Mannosephosphate isomerase (MPI)
Pyruvate kinase-3 (PKM~)
Mitochondrial isocitrate dehydrogenase (ICDM)
Adenine phosphoribosyltransferase (APRT)
Galactokinase (GALK)
Peptidase A (PEPA)
Glucosephosphate isomerase (GPI)
Adenosine deaminase (ADA)
Superoxide dismutase-A (SODA)
Mitochondrial aconitase (ACONM)
Diaphorase-1 (DIAO
Glucose-6-phosphate dehydrogenase (G6PD)
EC no.
Marker for
human
chromosome
number
4.2.I.II
3.4.11.2.7.5.1
I
l
I
1.1.1.37
2pt
I. I. 1.42
2 q'~
3.1.3.2
2.7.5-I
3.2. 1.3O
1.I.1.40
2p
4
5 q
6
3.2.1.31
2.7.4.3
2.7.4.3
4.2.1.3
7
9q
9P
9P
2.6.1.1
1.1.1.2 7
I.I.1.2 7
3.4.I1.-
I0
11
12
12
3.I.i.1
13
2.4.2,1
5.3.1.8
2. 7 . I . 4 0
2.4.2. 7
2.7.1.6
14
I5
15
15
16
17
3.4.11.5.3.1.9
3.5.4-4
18
19
2o
1.15.I.1
4.2.1.3
21
22
1.6.4.3
I, 1.1-49
22
X
I . I . 1.42
* The information in this table is derived from the Winnipeg Conference (1977).
t P and q refer to the short and long arms of chromosomes, respectively.
m e d i u m c o n t a i n i n g d i p h t h e r i a toxin at o.oI Lf/ml. These subclones are suffixed D T R , for
example CTP34-DTR2.
Interferon inductions. Interferon was induced in h u m a n , mouse and h a m s t e r cells and
in h u m a n - m o u s e and h u m a n - h a m s t e r h y b r i d cells in confluent m o n o l a y e r cultures (I × Io ~
to I × IO7 cells in 5o or IOO m m plastic dishes) by infecting with N D V strain F ( I × I o - 4 H A U /
cell) for I h at 37 °C. The virus fluids were then removed and the cultures i n c u b a t e d for a
further 2o to 23 h in m a i n t e n a n c e m e d i u m (3 to 4 ml) which contained 2 ~ (v/v) FBS. T h e
interferon-containing fluids were harvested and dialysed for 5 days at p H 2.o at 4 °C a n d
12 h at p H 7'o before assay. The h u m a n - m o u s e hybrids were also induced with (i) N D V
strain Texas ( I o or Ioo p,f.u./cell) or (ii) Sendai virus (3 × to -4 H A U / c e l l ) .
Interferon assays. H u m a n interferon was assayed in H E F cells, mouse interferon in L - A 9
cells and Chinese h a m s t e r interferon in Chinese h a m s t e r a3 cells. T h e assay was a modification o f the inhibition o f nucleic acid synthesis ( I N A S ) m e t h o d ( M c W i l l i a m et al. 1971)
described fully for chick interferon by A t k i n s et al. 0974). The interferon titre is expressed
as the reciprocal o f that dilution which reduced i n c o r p o r a t i o n o f label into virus R N A
by 5o~o (INASs0).
The research reference s t a n d a r d for h u m a n leucocyte interferon (69/I9), defined to
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15§ ] t6
.
.
.
.
.
.
.
I400
o
0
°400
.7oo
* The amounts of interferoninducedby NDV-F in parentalcell lines of human-mousehybridswere: HEF (human), 800 units/ml/xo° cells; L-A9(mouse),
Ioooo units/ml/loncells; PGI9 (mouse),50000units/ml/IO6cells.
i Top right sectionof boxes: human chromosomesare expressedas the percentageof each in the total number of metaphasefiguresanalysed.Lowerleft.
section of boxes: isozymesare expressedas: +, present; 4-, trace amount; --, absent; NT, not tested.
:l:Chromosomeidentificationonly.
§ Isozymeidentificationonly.
FIAK~+AK3--.
o
,4
~
o 47300
~ ~
N°E~'cr°~
ucer:4 _ ~ NTIS°+~ _~ NT~NT~_~ +~ +~ +~ +~ +~ _~ ~T~+ ~
2500
lZ
96670
16
4.0
4200
315o
HL57 4 ~Q +~NT~NT~-~NT~NT~ NT~- ~ ~ --~Q+ ~ - ~ - ~ +~ +~ - ~ --~ _+~+ ~ -~NT~-~
- ~ NT~NT~+~ +~NT~ NT~~[~ --~ + ~ +~ ~ + ~ +~ +~ + ~ +~ +~ +~ N~67T67
NT~+~
.
HL42 5
\o I\o !\ .
+~ ~T~oo~T~oo]\ i'~oo-+~ N~TO~T~- ~ NT~- ~ - ~ - ~ - ~ - ~ NT~NT~N~OT~O o ~ ~ ~ ~
HL2o 3
670
ZlO
Interferonformed
(units/ml/Io6cells)
17 18 I 19 zo zl 2z X Human Mouse
HL15 5 + ~ + ~ ~T~NT~+~NT~+~ NT~+~ - ~ + ~ + ~ - ~ + ~ +~x +~ -+~ +~ +'+~+~ +~ - ~ +~
HL53 4 + ~ +~ ~T~.NT~+~ NT~NT~NT~+ ~ +~ + ~ +x~ _~ _ ~ +~ +~ ~ ~ ~ ~ ~ ~ ~
Hybrid
Humanchromosome~
cell
line Passage
number 1 2 I 3++ 4~ I 5 6 7 8:[ 9 1o I~ 12 13§ 14§
ND V-F induction*
Table 3. Human chromosomes of primary human-mouse hybrid cell lines and their production of human and mouse interferons after
taJ
%
¢b
¢b
~°
314
A. M E A G E R A N D O T H E R S
contain 5ooo standard units/mt, had a titre of 5ooo units in H E F by the INASs0 method.
Similarly the research reference standard for mouse interferon (Goo2-9o2-o26) containing
6ooo units of activity, had a titre of 6ooo in L-A9 cells. H u m a n and mouse interferon titres
are given in this paper in reference research units. Amounts of Chinese hamster interferon
are expressed in arbitrary units.
Diphtheria toxin sensitivity. Parental cells and hybrid clones were seeded into small glass
vials and, when confluent, treated for 20 h at 37 °C with diphtheria toxin, Io -~ Lf/ml to
lo Lf/ml in o'4 ml maintenance medium. Control and treated cultures were then incubated
with 3H-leucine (l #Ci/vial) added in o.z ml PBS. After a further 3 h incubation at 37 °C
the cell sheets were washed twice with 5 ~ (w/v) trichloroacetic acid (TCA) and once with
absolute ethanol, dried, dissolved in o-I ml of Soluene (Packard Instruments) : toluene
mixture 0 : z ) and counted in 2"5 ml acidified toluene scintiltant. Diphtheria toxin sensitivity is expressed as Lf/ml needed to give 5o ~ inhibition of protein synthesis.
Antibody neutralization tests. Antibody dilutions were added to known concentrations
of human interferon derived from leucocytes, fibroblasts (FS4 or HEF) or hybrids and were
incubated for 2 h at 3o °C to allow neutralization. The interferon remaining after the antibody treatment was assayed in H E F cells as previously described. Antibody titres are
expressed as the lOgl0 of the reciprocal of that antibody dilution which neutralized IO
reference units of human interferon.
Chromosome analyses. At or close to the passage levels used in interferon inductions,
hybrid cell cultures were treated with colcemid (o. l #g/ml) for 2 to 4 h at 37 °C- Cells were
then harvested by trypsinization and swollen in 25 ~o (v/v) FBS in water or o'o75 M-KCI at
37 °C for lo min. They were fixed using absolute ethanol/acetic acid (3: I) at 4 °C overnight,
and suspended in fresh fixative. Drops were placed on wet glass microscope slides. The
preparations were banded either by heating to 8o °C in Sorenson's buffer, pH 6"8, for 9o min
(Bishun et al. 1975), or left for I week and trypsinized (Seabright, I97I). Banded metaphase
chromosomes were stained with 7 ~ Giemsa or l ~ Leishmans. Fifteen to twenty-five
metaphase figures were examined for each hybrid clone and photographed through a
Reichart or Zeiss microscope.
lsozyme analyses. The enzymes listed in Table 2 were used as markers for individual
human chromosomes. They were tested by routine electrophoretic methods (Harris &
Hopkinson, i976, I977) except that N-acetylhexosaminidase fl (HEX fl) was assessed by
immunodiffusion (Swallow et al. 1977).
RESULTS
Production of interferon by human-mouse cell hybrids'
Forty-two primary human-mouse hybrid cell lines were tested for human interferon production with viruses as inducers. Eight of these hybrids produced human interferon (Table 3)
and could be grouped into two classes on the basis of the amounts formed in response to
NDV-F: '(i) high p r o d u c e r s - p r o d u c i n g more than 2o0 units/ml/1o 8 cells, that is H L I 5 ,
HL53 and CTP34 and (ii) low producers - producing less than zo units/ml/1o 6 cells, that is
HLzo, HL35, HL4u, HL57 and CTP41. Similar results, though lower titres, were obtained
with the two other virus inducers NDV-Texas and Sendal virus. Both classes progressively
lost the ability to produce human interferon on continued passage.
All hybrid lines tested, both producers and non-producers of human interferon, produced
mouse interferon in response to the three inducing viruses, NDV-Texas, N D V - F and
Sendal virus. The amounts of mouse interferon produced by the hybrids varied but in
general were much greater than the amounts of human interferon (Table 3); the mouse
interferon produced no antivirat response in H E F cells.
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Genetics of human interferon
315
Table 4- Comparison of human interferons from leucocytes, fibroblasts and hybrid human-
mouse cells." neutralization with antisera and activity on cat lung cells
Antiviral activityi" in INASso
assay on:
Neutralization titre* with antiserum
raised against :
A
f
Source of interferon
Leucocytes (P-IF)
Fibroblasts (HEF cells)
Fibroblasts (FS4 cells)
Hybrid cells
(i) CTP34
(ii) HL15
(iii) HL53
(iv) HL2o
(v) HL57
Leucocyte
interferon
5"3
3"5
3"5
3"3
3"3
3"5
3"5
3'5
~
Fibroblast(FS4)
interferon
< 1.o
3"0
3-o
3-0
3-0
NT$
NT
NT
f-
HEF
(units/ml)
6300
3200
63o
iz6
159
80
I z6
90
Cat lung cells
(units/ml)
126oo
<
I00
<
IO0
<
IO
<
10
<4
<
IO
<4
* Expressed as the log10of the reciprocal of that antibody dilution causing neutralization of approximately
io reference research units of human interferon.
l" Reciprocal of dilution at which Semliki Forest virus RNA synthesis was reduced to 50 ~ of the control
value.
$ Not tested.
Characterization of human interferon made by hybrids
The human interferons made by the eight hybrids were neutralized with sheep antileucocyte interferon in comparison with reference preparations of leucocyte and fibroblast
interferons. The results are given in Table 4. Approximately Ioo-fold greater concentrations of anti-leucocyte interferon serum were required to neutralize fibroblast interferon
than to neutralize leucocyte interferon. Human interferon made by the hybrid cells was
neutralized by approximately the same concentration of antiserum as the control fibroblasts.
Two of the hybrids, CTP34 which has a human lymphocyte parent, and HL15 which has
a human fibroblast parent, were also tested with anti-fibroblast interferon. They also
resembled the control fibroblasts in having human interferon which was neutralized by
much lower concentrations of anti-fibroblast interferon serum than leucocyte interferon
(Table 4).
Further characterization of the interferon produced by the hybrid cells was made by
comparing the response of human diploid cells and cat lung cells to the interferon. Cat lung
cells are more sensitive to human leucocyte interferon than are human diploid fibroblasts,
whereas the reverse is true for human fibroblast interferon (Desmyter & Stewart lI, I976).
As shown in Table 4, interferons derived from the hybrid cells gave much higher antiviral
titres in the human fibroblast line, HEF, than in cat lung cells and thus again resembled
human fibroblast interferon.
Karyotype and isozyme analysis of the primary hybrids
Karyotypes of l I primary hybrids, including all the interferon-producing clones, were
analysed on cells separated by two passages or less from the cells used in tests for interferon
induction. Isozyme analyses were also made on cell extracts derived from 13 hybrids at the
same passage level used for karyotype analysis. Human chromosomes 3, 4 and 8 were
usually identified by chromosome banding procedures alone; chromosomes I3, 14 and I5
were identified by isozyme tests alone, because of their marked similarity to mouse chromosomes with the banding techniques used. The data are shown in Table 3. The three 'high
producer' hybrid clones, CTP34, HLI5 and HL53, were shown to have many human
chromosomes in common, and all contained chromosomes 2 and 5 in a high percentage of
II
VIR
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45
316
A. MEAGER AND OTHERS
Table 5.
Sensitivities of human-mouse cell hybrids and parent lines to diphtheria toxin*
Cell line
Parental
Hybrid
Human
interferon
production
+
HEF
LA9
PGI9
-
-
-
HLI5
HL53
HL8
HL54
CTP34
HL6o
DUR4
HL42
HL24
HLlo
HORP9
HL44
HL35
CTP41
HL6
HL2o
HL57
HL22
HORPI4
Sensitivity to
diphtheria toxin
Lf/mlt
o.oool 2
> IO
0'25
+(H)
+ (H)
- -
-+ (H)
--+(L)
---÷(L)
4-(L)
-~(L)
F(L)
-
>
>
>
>
>
Presence of
HEX fl and/or
chr. 5
+
Mouse
Mouse
o'oolo
o'ooJo
0"0025
o.oo32
0-0050
0"0063
0"0063
o'olo
0'020
0'050
0.050
0'063
0"32
0"80
l.o
l.o
I.o
I'O
I'o
+
q-~
NT
÷
4- :l:
÷
+
- -
--~
--4---:~
--q--
* Abbreviations: H, high producer line; L, low producer; NT, not tested.
t Concentration giving 5o ~ inhibition of protein synthesis.
z~These hybrids were tested for HEX/3 after four passages beyond the passage when they were tested for
diphtheria toxin sensitivity; induction of these later passage hybrids with NDV-F confirmed they were
non-producers of human interferon.
their cells. C h r o m o s o m e 9 was also present in these three hybrids but absent in all the lowand non-producers.
Sensitivity of hybrids to diphtheria toxin
The sensitivity to diphtheria toxin, measured in terms o f effects on protein synthesis, o f 19
hybrid clones and o f the parent h u m a n ( H E F ) and mouse (LA9 and P G [ 9 ) cells was c o m pared. H u m a n e m b r y o fibroblasts ( H E F ) were extremely sensitive to diphtheria toxin,
whereas the m o u s e lines LA9 and P G t 9 were relatively insensitive (Table 5)- The sensitivities o f hybrids generally fell between those o f h u m a n and m o u s e cells. T h e r e was n o
correlation between diphtheria toxin sensitivity and h u m a n interferon p r o d u c t i o n , t h o u g h
diphtheria toxin sensitivity correlated well with the presence o f H EX [] a n d / o r c h r o m o s o m e 5.
Diphtheria toxin-resistant subclones of primary hybrids
Eleven subclones o f C T P 3 4 were isolated, after selection with diphtheria toxin in t w o
separate experiments and were screened for h u m a n interferon p r o d u c t i o n using N D V - F
as inducer. C T P 3 4 - D T R I and C T P 3 4 - D T R z to D T R I I were isolated on two different
occasions. Seven o f the eleven subclones were high producers like the parental line; one was
a low p r o d u c e r and three were n o n - p r o d u c e r s (Table 6). The h u m a n interferon p r o d u c e d
was shown to be the fibroblast type o f interferon by a n t i b o d y neutralization tests (data n o t
shown). K a r y o t y p e and isozyme analyses (data c o m b i n e d ) on these subclones (Table 6)
showed that none contained c h r o m o s o m e 5- The three non-producers, C T P 3 4 - D T R 5 ,
- D T R 7 and - D T R 8 had also lost c h r o m o s o m e 9 in contrast to the high p r o d u c e r subclones
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DTR2
DTR3
DTR4
DTR5
DTR6
DTR7
DTR8
CTP34
CTP34
CTP34
CTP34
~P34
CTP34
CTP34
+
-
DTRI i
sc4
sc9
CTP34
CTP34
HLI5
HL53
+
+
+
+
+
+
--
-
-
--
--
--
--
--
-
-
--
---
+
(_)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
--}-
+
+
+
+
+
I
(--)i(--)
+
+
+
+
-
--
--
--
--
--
+
+
-b
+
+
+
+
+
+
+
+
+
-
-
--
-
--
-
--
---
--
-
(_+)~_+)
+
(_+)
- ( _ + ) +
--
---
+
+
--b
+
+
+
i_+)§-
+
+
+
+
~+)
-
-,-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
--
--
-
-
-
-
--
-
--
---
--
+
+
+
+
--k
+
+
+
+
+
+
+
+
+
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+
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(_+)
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--
-
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+
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--
-
+
+
--
+
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+~:
+
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+
+
+
+
+
(+)
+
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+
+
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+
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+
+
+
+
+
+
+
+
+
+
+
-{- ( + )
+
+
+
+
+
+
+
+
+
+
* H u m a n i n t e r f e r o n i n d u c e d w i t h N D V - F a n d e x p r e s s e d a s u n i t s / m l / l o n cells.
f Chromosome identification only.
:~ I s o z y m e i d e n t i f i c a t i o n o n l y .
§ ( 4 - ) , C h r o m o s o m e p r e s e n t in less t h a n Io % m e t a p h a s e s e x a m i n e d o r t r a c e i s o z y m e a c t i v i t y .
-
sc3b
HL53
-
+
sci4
sc3a
HLI5
+
--
+
--
DTR9
--
DTRIo
+
--
CTP34
+
--
CTP34
+
+
-
+
+
+
+
-
--
--
--
--
DTRI
CTP34
Human chromosome
"-
+
+
+
+
-
--
-
-
-
--
-
--
--
--
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
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-
+
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+
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+
+
+
+
+
+
(+)
(+)
+
+
+
+
~
o
45
9
I9O
<2
238
256
740
o
o
168
o
2t
~o6
445
740
830
Human
interferon
formed
1, (5), 7, 9, ( I 0 ) , I7,
18, 20, 22
(Io)
l, 9, 0 5 ) , zo
(7)
2, 9, I5, i 8
5
5, I 5 , ( I 8 )
5, 15, 18
5,9
5,9
5, I5
5,(6),9, I5,(16),(X')
5, 6, (9), ~5, ( X )
5
5
5, 15, t 8
-
Human chromosomes
segregated from
parental hybrid
Human chromosomes and human interferon production* by random and diphtheria toxin-resistant subclones of primary humanmouse hybrids
Subclone
6.
UFP34
Cell
line
Table
5"
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NT
46.6.I
C123
+
+
+
+
+
+
+
+
+
+
+
+
+
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-
+
AK',+
AKa +
ACONs+
AKt+
AK3ACONs+
+
+
AKI+
AKz +
+
:AK~AK3 -+
AKI+
AK~ +
+
AKt+
AK3 +
+
AKt+
AK~+
AKI+wk
+
AK3 + wk
+
AKI+
AK8 +
+
AK~+
AK3 +
+
AK~+
AK3+
+
+
+
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
+
--
+
+
-
--
-
+
NT
NT
NT
NT
I
+
+
+
NT NT NT NT
NT NT NT NT
NT NT NT NT
NT NT NT NT
NT NTNTNT
NT NT NT
NT NT NT NT
+
+
+
--
--
NT
bit
+
+
+
+
-- I N T N T N T N T
--
NTI -
NT'-
NT
NT
NT
NT
-
-
-
+
+
+
+
-
+
+
+
+
qT NT
NT
+
+
+
+
NT NT
+
+
NT
NT NT NT
NTINT
NT
qT NT
NT'NT
NT
NT NTNT
NT NT NT
NT
* Abbreviations: A K , , adenylate kinase-I ; AKa, adenylate kinase-3; A C O N s , soluble aconitase; NT, not tested; wk, weak.
t Except for the parent hybrid 4 6 . 6 . I, the presence o f h u m a n c h r o m o s o m e s was tested by isozyme analyses alone.
1: Induced with N D V - F .
§ N o isozyme tests for c h r o m o s o m e s 3 a n d 8 h a d been developed when this work was carried out.
II The X linked marker, h u m a n glucose-6-phosphate dehydrogenase ( G 6 P D ) was absent in 4 6 . 6 . l a n d its subclones.
NT
46.6.*
Cll 3
+
+
NT
NT
+
NT
+
+
NT
NT
+
NT
-
+
NT
-
+
NT
46.6.I
scl
46.6.I
sc2
46.6.I
sc3
46.6.1
sc 5
46.6.I
sc7
46.6.t
sc8
46.6.I
sc9
46.6.I
sclo
+
+
_..._.x
H u m a n chromosome]"
+
- -
--
--
0
I4O
7I
I67
375
37
420
-- -
230
84
o
Human
135
22
3Z
5°
25o
2oo
4O
5o0
200
5°
,o
100-200
Hamster
Interferon formed~
( u n i t s / m l / I O 6 cells)
--
- -
-II
+
(X; T)
Human chromosomes and interferon production in human-Chinese hamster cell hybrids*
46.6.1
Hybrid cell line
T a b l e 7.
r~
0
t'el
>
>
t..O
Genetics of human interferon
319
which had retained chromosome 9. The low producer subclones CTP34-DTR4 had lower
levels of the marker enzymes for chromosome 9 (AK1 and AK3) thus suggesting the correlation between ability to produce human interferon and chromosome 9.
A further 13 diphtheria toxin resistant subclones were established from H Lt5 and HL[ 5
sc9. These, however, were less informative than the CTP34-DTR subclones; eight of them
retained the human HEX fl marker, although chromosome 5 was not identified by karyotype
analysis, suggesting a translocation involving chromosome 5 in these hybrids. All these
hybrids produced high levels of human interferon and contained chromosome 9 isozyme
markers, AK1 and AKz, with one exception; this non-producer (HLI5 sc9 D T R I ) had lost
HEX fl and also chromosomes 5, 6, 9, TI, 12 and [6.
Random subclones of primary hybrids
Twenty randomly selected subclones derived from the high producer hybrids, that is
CTP34, HLI5 and HL53 were induced with NDV-F (Table 6). Only three low producer/
non-producer subclones were identified. Five subclones were analysed in detail (Table 6).
The only chromosome lost from the low producer/non-producer subclones which was
common to all three subclones [CTP34 sc4, HLI5 sct4 and HL53 sc3b (passage 6)] was
chromosome 9.
Human-Chinese hamster hybrids and their subclones
A human-Chinese hamster hybrid, 46.6. I, containing human chromosomes 5 and 9
among others, but lacking chromosome 2, produced human interferon after induction with
NDV-F (Table 7). This behaved as human fibroblast interferon in neutralization tests with
specific anti-interferon antisera and when assayed on heterologous cells (data not shown).
Chinese hamster interferon was also induced in the hybrid (Table 7), although the parental
line E36 was relatively poorly inducible, yielding only 4 to Io units/ml/~o 8 cells.
From eight subclones of 46.6. I it was found that one, sci, had lost the capacity to produce human interferon (Table 7) and it was found that it had also lost chromosome 9
isozyme markers, AK1 and AKa, whereas the chromosome 5 isozyme marker, HEX/~ was
retained. All the remaining seven subclones which produced human interferon retained both
chromosome 5 and 9 isozyme markers, though the 9 isozyme markers were weak in the
subclone, sc7, which produced the least human interferon.
Two further human-Chinese hamster hybrids (Ch 3 and C123), known to be very similar
in their complement of chromosomes were induced with NDV-F. Only Clr3 produced
human interferon (Table 7) which was shown with specific anti-interferon antisera to be
predominantly fibroblast. Both Ch3 and C123 produced small amounts of Chinese
hamster interferon (Table 7), although the parental Chinese hamster cell line, Wg3h, of
these hybrids could not be induced to form hamster interferon. Isozyme analysis showed
that C123 differed from Clt3 only in that the markers AK3 and aconitase (ACONs) for
the short-arm of human chromosome 9 were lost (Table 7).
DISCUSSION
We have found that the human interferon produced by our human-mouse hybrids was
of the fibroblast type irrespective of whether the human cell parent was of lymphoid
(CTP34 series) or fibroblast (HL series) origin. The response of the interferon-producing
system of the hybrid cells to virus inducers thus appears to be controlled by the mouse
fibroblast cell parent.
Knight (I976) has recently shown that human fibroblast interferon is a monomeric glycoprotein of mol. wt. 24ooo. This indicates that a single structural gene codes for the interferon polypeptide, though the products of other genes are probably responsible for the posttranslational glycosylation. In order to analyse the segregation of interferon production and
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320
A. M E A G E R A N D O T H E R S
the human chromosomes it is necessary to take into account that the human interferon assay
is considerably more sensitive than the karyotype and isozyme analytical tests and is
therefore capable of detecting interferon produced by a small minority population of cells
within a culture. We therefore only considered as producers those hybrids which yielded
more than 2o units/ml/to 6 cells (about 5~o of the yield of high producers) as this corresponds to the greatest sensitivity expected of the karyotype and enzyme analysis. From Tables
3 and 6 it can be seen that, using this criterion, chromosomes t to 8, IO to 22 and X can be
excluded as candidates for the localization of a single gene coding for interferon. Analysis
of the hybrid subclones shows also that interferon production does not correlate with the
presence of chromosomes 2 and 5 together. However, the data are compatible with the
assignment of a single gene to chromosome 9. Our data are difficult to reconcile with those
of previous authors. Tan et al. (1974) did not find chromosome 9 in any of their hybrids.
However, all the hybrids studied by Tan et al. 0974) and also by Morgan & Faik 0977)
produced relatively little human interferon compared with those in our study, which may
have been produced by a subpopulation of cells which did contain chromosome 9Tan (1977) recently published data based on studies with human aneuploid cells from
which he suggests that the assignment of the interferon gene to the long-arm of chromosome
5 has been confirmed. He isolated cells containing multiple copies of the short- and longarms of chromosome 5 present in aberrant 'marker' chromosomes, and found that only
those cells which have more long-arms than short-arms make high levels of interferon.
However, the karyotype analysis of these cells has shown them to contain many other
aberrant 'marker' chromosomes and these could contain genes that have an effect on
interferon production.
The results obtained from our CTP34 D T R subclones which have lost chromosome 5 but
still produce large amounts of human interferon suggest that this chromosome contains
neither structural nor regulatory genes for human interferon, though we cannot exclude a
translocation involving a small section of chromosome 5- To date, we cannot rule out
involvement of chromosome 2 in human interferon production in h u m a n - m o u s e hybrids,
since chromosome 2 occurs in all the hybrids which produce human interferon. However, in
agreement with Morgan & Faik (z977), we found that chromosome 2 is not required for
human interferon production by human-Chinese hamster hybrids.
In conclusion, analysis of our human-rodent hybrids has provided evidence for the
assignment of the human fibroblast interferon structural gene, IfF, to chromosome 9. Data
from the human-Chinese hamster hybrid C123 and the human-mouse hybrid HL42
(Table 3 and 7) indicate that l f F may be located on the short-arm of chromosome 9, since
no AKa could be detected in these hybrids and this is coded for by a gene believed to be on
the short-arm (Winnipeg Conference, I977).
We thank the Medical Research Council for programme grant support and for support
for J.R.W. We are also grateful to Dr E. Jones, Dr E. Solomon and Dr R. Buckland for
supplying some of the cell hybrids used in this study, and Ms Lorraine Evans and Mr S.
Jeremiah for technical assistance.
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