Genetics. Expression of the human argininosuccinate synthetase gene in hamster transferents. (chromosome-mediated gene transfer/somatic cell genetics).
Proc. Nati. Acad. Sd. USA Vol. 77, No. 7, pp. 4234-4238, July 1980
Genetics
Expression of the human argininosuccinate synthetase gene in hamster transferents (chromosome-mediated gene transfer/somatic cell genetics)
LYNN D. HUDSON*, RICHARD W. ERBE, AND LEE B. JACOBYt Genetics Unit, Children's Service, Massachusetts General Hospital, and Department of Pediatrics and Center for Human Genetics, Harvard Medical School, Boston, Massachusetts 02114
Communicated by John W. Littlefield, April 25,1980
ABSTRACT The structural gene for human argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] was transferred to argininosuccinate synthetasedeficient Chinese hamster cells via metaphase chromosomes isolated-from human lymphoblast line MGL8D1, a constitutive overproducer of argininosuccinate synthetase, and from its repressible parent, MGL8B2. Argininosuccinate synthetase expression was selected for ih citrulline-containing medium, and the human origin of the argininosuccinate synthetase expressed by seven transferents was identified by isoelectric focusing. Stable transferents expressing MGL8D1 argininosuccinate synthetase fell into two classes: (i) those whose argininosuccinate synthetase activity was reduced to 10-50% by arginine, similar to the repression of argininosuccinate synthetase synthesis observed in normal human lymphoblasts, and (ii) those that constitutively expressed argininosuccinate synthetase when grown in the presence of arginine or citrulline. Two transferents from the MGL8B2 donor constitutively expressed human argininosuccinate synthetase. Three hamster revertants were isolated that constitutively expressed hamster argininosuccinate synthetase. Transferents and revertants exhibited growth-dependent changes in argininosuccinate synthetase activity, in contrast to the constant synthetase activity levels in donor lymphoblasts during growth. The isolation of stable transferents that constitutively or repressibly express argininosuccinate synthetase makes possible the analysis of regulatory signals influencing expression of the argininosuccinate synthetase gene.
arginine is present in the medium (18). Constitutive overproducers of argininosuccinate synthetase, however, have been isolated by selecting human lymphoblasts resistant to canavanine, an arginine analogue (19). Argininosuccinate synthetase has been purified to homogeneity from the constitutive overproducer MGL8D1 and its parent MGL8B2. The enzymes from the two lines exhibit identical physical and kinetic behavior, providing further evidence that MGL8D1 may be an argininosuccinate synthetase regulatory mutant (20). The objectives of analyzing argininosuccinate synthetase transferents were twofold: first, to assess whether the expression of a transferred gene is controlled primarily by the recipient cell or if regulatory sequences can be cotransferred with the structural gene and influence its expression and, second, to further characterize the constitutive argininosuccinate synthetase mutation as well as to examine the normal regulatory controls of synthetase expression. The use of stable gene transferents for studying the regulation of gene expression avoids several problems associated with cell hybrids, especially chromosome segregation, which may yield heterogeneous populations with respect to the regulatory marker of interest, and secondary interactions between the two donor genomes of hybrid cells, which may obscure regulatory signals. Transferents were isolated from Chinese hamster fibroblasts treated with MGL8D1 or MGL8B2 metaphase chromosomes. Like other hamster fibroblasts, the hamster recipient a23 does not express argininosuccinate synthetase (21), a predominantly hepatic enzyme, which condenses citrulline and aspartate to produce argininosuccinate. The a23 hamster line does express the next enzyme in the pathway, argininosuccinate lyase (EC 4.3.2.1.), which converts argininosuccinate to arginine, but without argininosuccinate synthetase activity it is unable to grow in arginine-deficient medium. Thus transferents expressing argininosuccinate synthetase were selected by their ability to use the substrate citrulline and thereby grow in arginine-deficient medium. We describe here the expression of argininosuccinate synthetase activity in stable transferents.
Functional genes can be transferred to mammalian cells and stably transmitted to progeny cells when either metaphase chromosomes (1-11) or DNA (12-16) is incubated with recipient cells. Stable transferents appear to have integrated the donor gene into their genomes with no apparent site specificity (7-9, 13). The integrated DNA, estimated to represent less than 0.25% of the haploid donor genome in chromosome transfer experiments (4-6) and approximately 3.4 kilobases for the viral thymidine kinase gene (12, 13), is large enough to include regulatory sequences adjacent to the structural gene sequence(s). These features of gene transfer make it attractive for analyzing regulatory signals influencing gene expression, as well as for mapping these regulatory sequences.
We have transferred the human gene for the urea cycle enzyme argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5], which has been localized to the distal portion of the long arm of chromosome 9 (17), to enzyme-deficient hamster fibroblasts and examined the regulation of the argininosuccinate synthetase expressed in transferent cells. The synthesis of argininosuccinate synthetase in human lymphoblast line MGL8B2 is normally repressed when
MATERIALS AND METHODS Cells and Culture Conditions. The hamster fibroblast line a23 (22) was the generous gift of B. Carritt. The human lymphoblast line MGL8B2 is a twice reisolated derivative of MGL8 (18) and is the parent of the canavanine-resistant variant MGL8D1 (19). Both of these lines are 47,XY,+13, and the number 9 chromosomes in each appear normal with Giemsa banding. The transferent lines isolated from a23 cells treated
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Present address: Section of Microbiology and Molecular Biology, Division of Biology and Medicine, Brown University, Providence, RI 02912. t To whom reprint requests should be addressed. *
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with MGL8D1 chromosomes are named Ti through T5, and those from MGL8B2 are named T6 and T7 (Table 1). Revertant lines derived from a23 cells are named RI through R3. Cells consistently were negative for mycoplasma infection when tested by bioassay and DNA staining. Cells were cultured in arginine-free Eagle's minimum essential medium (GIBCO) supplemented with nonessential amino acids, 10% fetal calf serum (Microbiological Associates; contains approximately 2 MM arginine), and either 0.6 mM L-arginine-HCl or 0.6 mM L-citrulline. The a23 line was maintained in arginine-containing medium, and all other lines in citrulline medium. Transferents were initially selected in citrulline medium containing 1 mM ouabain to select against any human cells that may have been present in the donor chromosome preparation. Chromosome Isolation and Transfer. Metaphase chromosomes were isolated by a modification of the Maio and Schildkraut method (23). Exponentially growing lymphoblasts were treated with Colcemid (GIBCO) at 0.11 Mg/ml for 16 hr to produce a population in which over 70% of the cells were in metaphase. Approximately 108 cells were harvested, lysed in 20 mM TrisHCI (pH 8.0) containing 2 mM CaCla and 1% Nonidet P40 (Bethesda Research Laboratories, Rockville, MD), and layered over 1 M sucrose containing 20 mM Tris-HCl (pH 7.4), 2 mM CaCl2, and 0.2% Nonidet P40 at 4VC. After centrifugation at 100 X g for 10 min, the upper chromosomecontaining layer was removed and layered again over sucrose. Chromosomes were pelleted by centrifugation at 1000 X g for 30 min. The pelleted chromosomes were resuspended in 20mM TrisHCI (pH 7.4) containing 2 mM CaC12 and centrifuged at 1000 X g for 15 min; this step was repeated twice to remove detergent and remaining cellular debris. Chromosome uptake was facilitated by precipitating DNA with CaC12 (24) as described by Sabourin and Davidson (11). Aliquots of precipitated chromosomes containing approximately 40 Mg of DNA as measured by diphenylamine assay. (25) of acid-extracted DNA (26) were added to 100-mm dishes containing 2 X 106 a23 cells. After 30 min of absorption, 9 ml of argine medium was added and the dishes were incubated for 12 hr before the chromosome mixture was replaced with fresh arginine medium. The recipient cells were plated in citrulline/ouabain medium at a density of 105 cells per 100-mm dish, 24 hr after exposure to chromosomes. The cells were refed with the selective medium every 3-4 days. After approximately 4 weeks, colonies were subcultured in citrulline medium. Only one clone was picked from each dish to ensure the independent origin of each presumptive transferent. Isoelectric Focusing -of Argininosuccinate Synthetase. Argininosuccinate synthetase was focused in 8 X 0.5 cm gels composed of 4.85% (wt/vol) acrylamide (Bio-Rad), 0.15% (wt/vol) N,N'-methylenebisacrylamide (Bio-Rad), 2.5% (vol/vol) Pharmalytes 8-10.5 (Pharmacia), 10% (vol/vol) glycerol, 0.1% (vol/vol) N,N,N',N'-tetramethylethylenediamine (Eastman), and 0.0225% (wt/vol) ammonium persulfate (Bio-Rad). Prior to casting, the acrylic acid present in the acrylamide solutions was bound by a mixed-bed resin (Bio-Rad), which was subsequently removed by filtering the solution through Whatman 54 H filter paper. Each presumptive transferent and revertant line was analyzed by isoelectric focusing on several occasions. Freshly harvested cells were sonicated in 20%6 (wt/vol) sucrose and 4% (vol/vol) ampholytes and the 100,000 X g supernatant was applied to the gels. Liver from an LSH hamster (Charles River Labs) was homogenized in 4 vol of 0.25 M sucrose and dilutions of the 100,000 X g supernatant were focused in parallel gels. The gels were run at 400 V for 2 hr with 10 mM ethylenediamine as the cathode solution and 10 mM Hepes as the anode solution. For pH determina-
Proc. Nati. Acad. Sci. USA 77 (1980)
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tions, gels were sliced into 1-mm sections and agitated in 10 mM NaCl. Argininosuccinate Synthetase Assay. Argininosuccinate synthetase was assayed by the radiochemical method of Schimke (27) as modified by Irr and Jacoby (18); 1 unit of enzymatic activity equals formation of 1 nmol of product per hr. For assay of synthetase in gels, each 1-mm slice was added to 0.2 ml of 0.1 M Tris.HCI (pH 8.5), and the assay was carried out as described except that the assay time was increased to 2 hr to facilitate diffusion through the gel. Chromosome Analysis. Cells were Giemsa banded by a modification of Seabright's technique (28). At least 20 metaphases per line were counted. RESULTS Isolation and Characterization of Transferents. After 4 weeks of selection in citrulline/ouabain medium, clones present in chromosome-treated and untreated control cultures were subcultured into citrulline medium and grown for argininosuccinate synthetase assay. Apparent revertant clones arose from the untreated a23 cells at a frequency of 5-7 X 10-6. Most of these revertants gradually lost the ability to grow in citrulline and were discarded 1-2 months after their isolation. Less than 5% of the clones grew well in citrulline for many months and expressed argininosuccinate synthetase activity. Clones appeared in chromosome-treated a23 dishes at the same frequency, 5-7 X 10-. The majority of these clones grew slowly in citrulline medium and exhibited low levels (0.1-5 units) of argininosuccinate synthetase activity. Over several months these lines lost their argininosuccinate synthetase activity and consequently their ability to grow in citrulline medium. Some of these unstable clones may have represented cells that were rapidly segregating the donor genome, but most were probably a23 revertants. The rare stable clones that continued to grow well in citrulline medium and to express argininosuccinate synthetase were reisolated in citrulline medium, and the origin of the argininosuccinate synthetase expressed was tested by isoelectric focusing. The frequency of these transferents, a23-like cells that expressed human argininosuccinate synthetase, was 5-6 X 10-7. Hamster and human forms of argininosuccinate synthetase were discriminated by isoelectric focusing on pH 8-10.5 gels (Fig. IA). The source of the hamster enzyme was a revertant of a23 named RI. The hamster synthetase (Fig. IC) had a higher pI than did the human form (Fig. 1B). Both hamster liver synthetase and synthetase from R2, another hamster revertant isolated from a control dish, focused in a position identical to that of RI. The source of the human enzyme was MGL8D1, which consistently yielded a single peak of activity that focused at about pH 9.0. Importantly, the isoelectric focusing profile of the constitutive overproducer MGL8D1 was identical to that of the parent MGL8B2, thus providing additional evidence that the two lymphoblast lines express the same structural gene for argininosuccinate synthetase. Mixtures of MGL8D1 and either hamster liver or revertant fibroblast lysate yielded two peaks of synthetase activity, one at pH 9.0 and the other at pH 9.5 (data not shown). The presumptive transferent T3 (Fig. 1D) displayed an isoelectric focusing profile that was similar to that of the human chromosome donor (Fig. iB); none of the argininosuccinate synthetase present in these cells migrated to the hamster position. Ten cell lines were characterized by the method of isoelectric focusing (Table 1). Seven transferent lines expressed the human synthetase but not the hamster enzyme, indicating that, if any human regulatory elements were cotransferred with the structural gene, these elements did not activate the dormant structural gene for hamster synthetase.
Genetics: Hudson et al.
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Proc. Natl. Acad. Sci. USA 77 (1980)
z
Table 2. Stability of the transferred phenotype in transferents grown in nonselective medium Generations in Relative plating Cell line arginine efficiency T1
0 31 74
1.10 1.02 1.03
T2
0 23 67
1.12 1.02 1.16
T3
0 22 65
0
fa
-a
0-
b
0.90 0.71 1.19 Cells were cloned in either arginine or citrulline, n = 4 plates per treatment. The relative plating efficiency is the ratio of the mean number of clones observed in citrulline medium to the mean number observed in arginine medium. The absolute plating efficiency of transferents was approximately 90%o.
x
F._ u
._
Ix
8
10 12 14 16 18 20 22 24 Gel slice
FIG. 1. Discrimination of "amster and human argininosuccinate synthetase by isoelectric focusing. Lysates from freshly harvested MGL8D1 (B), R1 (C), or T3 (D) cells were focused on disc gels containing pH 8-10.5 ampholytes (A). The gels were sliced for argininosuccinate synthetase assay or for pH determination.
The chromosomes of these lines were examined by Giemsa banding. The a23 subline used as the recipient in these transfers had 42 chromosomes, twice the usual number. The seven transferents had median chromosome numbers of 40 (T2 and T5), 41 (T3, T4, T6, and T7), or 42 (T1), and the three revertants had median numbers of 39 (Ri) or 42 (R2 and R3) chromosomes. No unusual marker chromosomes or minute chromosomes were observed consistently in any of the lines. Thus all the transferents and revertants had chromosome complements similar to the parental line. Stability of Transferents. Studies of the regulation of argininosuccinate synthetase expression in transferents required a homogeneous population of transferents in which the donor synthetase gene was stably inherited. The transferent cells that had been isolated, reisolated, and maintained for several months in citrulline medium were tested for the stability of argininosuccinate synthetase gene expression. Transferents were grown for over 70 generations in the nonselective arginine medium and periodically cloned in nonselective and selective medium. As shown in Table 2, the transferents had equivalent plating Table 1. Origin of argininosuccinate synthetase expressed by transferents and revertants Chromosome Argininosuccinate Cell lines donor synthetase MGL8D1 Human T1, T2, T3, T4, T5 MGL8B2 Human T6, T7 None* Hamster R1, R2 R3 MGL8B2 Hamster Origin of argininosuccinate synthetase was determined by isoelectric focusing profile as shown for T3 and R1 in Fig. 1. * R1 and R2 were isolated from control cultures of a23 cells that were not incubated with human chromosomes.
efficiencies in citrulline and arginine long after the selection pressure was removed, indicating that these lines had stably integrated the synthetase gene. Regulation of Argininosuccinate Synthetase Expression. The levels of argininosuccinate synthetase activity expressed by transferents grown for 4-6 days in citrulline or arginine medium are shown in Table 3. These levels were unchanged over a period of 6 months. For example, T1 exhibited 91 units/mg of protein in citrulline medium and 28 units/mg of protein in arginine medium when assayed 3 months after chromosome transfer; after continued growth in citrulline Table 3. Argininosuccinate synthetase activity in transferents and revertants grown in citrulline or arginine medium Argininosuccinate synthetase, nmol product/mg protein per hr Cell line Citrulline Arginine Parents a23
MGL8D1 MGL8B2 Transferents MGL8D1 donor T1 T2 T3 T4 T5 MGL8B2 donor T6 T7
220 22
._
_ *C
c 20I ==401
a
.r-
B. T3
.£ .
.
c
-
._
-
C
0 1
2
3
4
Time, days
FIG. 2. Argininosuccinate synthetase activity in transferents shifted from arginine to citrulline medium. T1 (A) or T3 (B) cells were grown in arginine medium until nearly confluent and were refed with either citrulline (-) or arginine (&) medium at 0 hr. Cells were harvested daily for assay of argininosuccinate synthetase.
1
2
3
4
5
6
7
Time, days FIG. 3. Cell number (A) and argininosuccinate synthetase activity (B) during growth of transferent cells. T1 cells grown in citrulline medium were plated in citrulline medium at a density of 2 X 105 cells per 100-mm dish on day 0, harvested at the indicated times, counted, and assayed for argininosuccinate synthetase activity. Cells were refed on day 4.
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repressible and constitutive transferents. Both total enzyme activity and responsiveness to arginine in the medium could be influenced by the position in the hamster genome at which integration occurred. Alternatively, the synthetase structural gene could have been amplified subsequent to the transfer process, as was suggested recently for the herpes simplex thymidine kinase gene (10). Selective amplification of the structural gene could explain the elevated synthetase activities, although it would not readily explain the existence of both repressible and constitutive transferents, particularly since the total enzyme activity was similar in both classes of transferents after growth in citrulline. Finally, there could be a human regulatory locus closely linked with the synthetase structural gene. If, for example, a regulatory locus that mediated the arginine repressive effect was present in both the normal and overproducer lymphoblast lines, then cotransfer of the structural and regulatory gene loci could have caused the arginine repressible phenotype while transfer of the structural gene alone could have caused the constitutive phenotype. These models are not mutually exclusive, and the transferent phenotypes could reflect, for example, amplification of the synthetase structural gene together with different lengths of neighboring DNA sequences that regulate synthetase gene expression. Discrimination among these mechanisms and others awaits the preparation of a DNA probe for the synthetase structural gene to determine the number of structural genes present and the DNA sequences surrounding them in both constitutive and repressible transferents. A modification of the present gene transfer system using a23 as a recipient should facilitate the detection of the appropriate human DNA sequence. The revertants of the a23 Chinese hamster fibroblast line that expressed argininosuccinate synthetase actvity are also of interest. Argininosuccinate synthetase activity has not been detected in any cultured hamster fibroblast line (21, 29, 30), although it is present in hamster epithelial cells (30) as well as in most other cultured mammalian cells. This naturally occurring enzyme deficiency presumably reflects the differentiated state of the tissue from which the cells arise or else it results from mutation in the gene or extinction of synthetase activity early in culture. Our experiments showed that this process was reversible and that synthetase activity was regained at a low frequency independently of signals from the human genome, because revertants appeared at approximately the same frequency in a23 cells treated or untreated with human chromosomes and, in addition, hamster synthetase was not present in any of the transferents expressing high levels of human synthetase. The revertant synthetase had the same isoelectric focusing point as the hamster liver enzyme and was clearly distinguished from the human enzyme. It is possible that this reversion was promoted by a gene dosage effect, because the particular a23 subline used in these experiments had twice the usual hamster genome. All the revertants and transferents, however, also had twice the usual number of hamster chromosomes, so that gene dosage alone does not readily explain the reversion. The three revertants studied in detail all expressed argininosuccinate synthetase constitutively. Regulation of argininosuccinate synthetase in mammalian liver is responsive to protein intake or arginine deprivation (31). In cultured mammalian cells, however, synthetase expression can be either constitutive (e.g., in human fibroblasts) or repressible (e.g., in human lymphoblasts). The characterization of these transferents has not defined the precise basis of argininosuccinate synthetase regulation in canavanine-resistant variants. The data do not rule out the possibility that gene amplification, analogous to that observed by Alt et al. (32) for dihydrofolate reductase, accounts for the constitutive overproduction of argininosuccinate synthetase in
Proc. Natl. Acad. Sci. USA 77 (1980)
MGL8D1. Our results are also consistent with the possibility that the canavanine resistance marks a second regulatory locus, which segregates independently from the structural gene and an adjacent regulatory site mediating arginine repression. In this model, either loss of the normal regulatory site adjacent to the structural gene or segregation of the canavanine resistance locus could produce the constitutive phenotype. The combination of further studies of selected somatic cell hybrids together with the isolation of a DNA probe for the argininosuccinate synthetase gene should allow these possible mechanisms to be distinguished. We thank Drs. L. Atkins and J. Biedler for determining karyotypes and Dr. B. Migeon for helpful criticism on the manuscript. These studies were supported by Research Grants HD06356 and GM25758, Biomedical Research Support Grant RR05486, and Training Grant HD07092 (L.D.H.) from the U.S. Public Health Service.
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