INDUCTION AND ANALYSIS OF GENE MUTATIONS IN - Genetics

Symposium on Mutagenesis : XI11 Iniernationd Congress of Genetics

INDUCTION AND ANALYSIS OF GENE MUTATIONS IN CULTURED MAMMALIAN SOMATIC CELLS

ERNEST H. Y. CHU

Department of Human Genetics, Uniuersiiy of Michigan Medical School, Ann Arbor, Michigan 48104

M U C H of our knowledge concerning the molecular processes of the cell has come from studies on unicellular organisms, as a result of several properties

which suit them to experimentation. In contrast, the complex life cycles and mul- ticellular organization of differentiated higher organisms have put experiments on an entirely different time-scale and have made the biochemical analysis of any single cell type difficult. Mammalian cell biologists have long been interested in the development of tissue culture methodology that would permit the applica- tion of microbiological techniques to their experimental systems. In recent years, attempts to study the genetics of mammalian somatic cells have met with good success through a number of technical advances. Firstly, due notably to the work of T. T. Puck and his co-workers (PUCK 1972). i t has been possible, in semi- defined media, to clone single mammalian cells at high efficiency and to grow pure lines to high population densities. Mammalian cells in culture, as microorganisms, are thus single units of life that are amenable to qualitative and quantitative analysis of their genetic and biochemical properties. Secondly, experimental mutagenesis in mammalian cells was first reported independently by CHU and MALLING (1968a,b) and by KAO and PUCK (1968a,b) at the International Con- gress of Genetics in Tokyo. The ability to induce mutations in mammalian cells in culture not only increases the genetic variability that is prerequisite for genetic analysis but also permits studies in these cells of the mutation process itself. Thirdly, the formation of cell hybrids by fusion of cells carrying different genetic markers allows studies of gene expression, complementation, mapping of genes, linkage and possibly mitotic recombination. Finally, recent development of the chromosome banding techniques to identify individual chromosomes and specific chromosome regions further helps to establish, in the true sense of that term, the field of mammalian cytogenetics.

In this paper I shall briefly review the studies on mutagenesis in mamalian cells in culture. At the outset I must point out that the occurrence of persistent phenotypic variations in somatic cells could be due to either genetic or epigenetic mechanisms. Although a large body of experimental evidence to be reviewed here suggests a genetic basis for mammalian somatic cell variations, there is a con- trasting proposal that some forms of variation at least may be based on changes in gene expression rather than on alterations in genetic information. HARRIS (1971, 1973) observed that mutation rates to drug resistance or to thermal tolerance in Chinese hamster cells are essentially independent of the ploidy level; MEZGER- Genetics 78: 115-132 September, 1974

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TABLE 1

Some criteria for genetic alterations in mammalian somaiic cells

1. Random occurreiice 2. Retention of stable phenotype in thc absence of selection 3. Induction with mutagens 4. Mutagenic specificity 5. Change in the activity and physicochemical properties of specific gene product 6. Conditional lethality 7. Interallelic complementation 8. Localization of a gene in a specific chromosome region 9. Mitotic recombination

IO. Gene transfer between cells

FREED (1971, 1972 and 1974) failed to find any significant increase in spontaneous or induced mutations to drug resistance in haploid as compared to dipolid lines of frog cells. In the absence of a sexual phenomenon in somatic cells, a dis- tinction between these alternative interpretations is difficult and requires rigorous investigations. For operational purposes, I shall use the term “mutant” in- stead of “variant.” The central question to which I shall address myself in this presentation is: “Are the phenotypic variations observed in mammalian somatic cell populations true gene mutations?” Short of any direct evidence for changes in the nucleotide sequence, there are a number of other criteria (Table 1) that most geneticists would consider as indications for an alteration in the genetic material. In the following discussion, I shall return to these various criteria in assessing the nature of somatic cell mutants.

Selection for Genetic Markers in Cultured Mammalian Cells

The kinds of genetic markers that are recognizable in mammalian cell cultures may include morphological, biochemical, serological and conditional lethal mutants (CHU 1971a). Isolation of mutants may involve either nonselective or selective techniques. In the non-selective category one may either isolate as many colonies as feasible and test them individually, or use a mechanical device for replica plating (GOLDSBY and ZIPSER 1969; SMITH and CHU 1973). The latter approach should demonstrate that mutations occur in the absence of selective agent and therefore are not adaptive changes.

Three selective methods have been applied to obtain mutants in mammalian cell cultures (cf CHU 1971a). Firstly, by mass selection mutants have been isolated in a number of mammalian cell lines that are resistant to various antimetab- olites (see reviews by GARTLER and PIOUS 1964; KROOTH, DARLINGTON and VEL~ZQUEZ 1968). Secondly, the lethal growth method involves the creation of a situation in which the normal cells grow and are killed, leaving behind the non- growing mutants, which may then be rescued. PUCK and KAO (1967) and KAO and PUCK (1968b) successfully isolated a series of auxotrophic mutants in Chi- nese hamster cells by applying the principle of the lethal growth method. In their experiments, the growing nomnutant cells, in a nutritionally deficient me-

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MUTATIONS IN MAMMALIAN CELLS 117

dium containing 5-bromodeoxyuridine (BUdR) , incorporated the thymidine analog and later were preferentially eliminated because of their increased sensitivity to light. We have shown, however, that the combined treatment of cells with BUdR and visible light is itself a highly mutagenic process (CHU, SUN and CHANG 1972). THOMPSON and co-workers (1970) have isolated temperature- sensitive mutants in mouse L-cells and in Chinese hamster cells. The procedure they used involves the growth of cells at an elevated temperature and the differ- ential killing of wild-type cells with radioactive thymidine. The third method for mutant selection is modeled after the “thymineless death” technique for selecting mutants in bacteria. DNA synthesis in mammalian cells may be blocked by treating cells with folic acid antagonist, such as aminopterin; under these conditions, amino acid prototrophs are killed because of abnormal growth, while the auxotrophic mutants requiring amino acids for growth remain alive. By this method, glutamine-requiring mutants have been isolated in HeLa cells ( DEMARS and HOOPER 1960) and in Chinese hamster cells (CHU et al. 1969).

Table 2 shows a partial list of genetic markers that have been developed in human and other mammalian cells. The reader is referred to other reviews ( GARTLER and PIOUS 1964; KROOTH, DARLINGTON and VELAZQUEZ 1968; THOMP- SON and BAKER 1973) for citations of the earlier papers. In some instances, the random nature of the mutational events was confirmed by the LURIA-DELBRUCK fluctuation analysis (LURIA and DELBRUCK 1944) and estimates of spontaneous mutation rates were made. It is also generally true that the mutant phenotypes were stable and persistent in cell progeny in the absence of selection.

Table 3 lists the mutants obtained thus far in Chinese hamster cell lines. The group of auxotrophic mutant lines on the right side of the table have been iso-

TABLE 2

Some genetic markers developed in cultured mammalian cells that respond to selection

Species Marker

Human Purine analog resistance Aminopterin resistance Virus resistance Glutamine dependence

Monkey Temperature sensitivity Mouse Purine, pyrimidine analog resistance

Aminopterin resistance Radiation resistance Asparagine independence Temperature sensitivity

Pig Purine analog resistance Aminopterin resistance

Rat Asparagine independence Chinese hamster Purine, pyrimidine analog resistance

Cytosine arabinoside resistance Temperature sensitivity Auxotrophy

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118 E. H. Y. CHU

TABLE 3

Mutants isolated from Chinese hamster cell lines

1. Drug resistance azgs + azgr

(HGPRT+) (HGPRT-) daps + dapr

(APRT+) (APRT-) bus e bur

(X+) (TK-) ara-Cr aptr aman' col'

2. Conditional lethality ts gal-,,

3. Auxotrophy

gln- pro- gb- glSrk,B,C,D hYP- ade-*,B urd- ino- (gly,hyp,tdr) - (ade,tdr) - gal- (gly,ade,tdr) -

ser

Abbreviations: azg, 8-azaguanine; dap, 2,6-diaminopurine; bu, 5-bromodeoxyuridine; ara-C, cytosine arabinoside; apt, amethopterin; aman, a-amanitin; col, colchicine; ts, temperature sensitive; gln, L-glutamine; gly, glycine; hyp, hypoxanthine; urd, uridine; tdr, thymidine, gal, galactose; pro, proline; ade, adenine; ino, inositol; ser, serine.

lated from Chinese hamster ovary (CHO) cells in PUCK'S laboratory (KAO and PUCK 1967, 1968b, 1969, 1971; JONES and PUCK 1973). Purine-requiring mutants have also been obtained in the CHO cells by TAYLOR, SOUHRADA and Mc- CALL (1971). Mutant clones resistant to a-amanitin (CHAN, WHITMORE and SIMINOVITCH, 1972) and to colchicine (LING and THOMPSON, cited in THOMPSON and BAKER, 1973) have been isolated from the CHO cells. A number of amethopterin-resistant mutants have been isolated in another Chinese hamster cell line (DC-3F) and its derivatives (ALBRECHT, BIEDLER, and HUTCHISON. 1972). The rest of the listed mutants have been isolated from Chinese hamster lung (V79) cells in my laboratory. It should be noted that both nonspecific (SMITH and CHU 1973) and locus-specific (SUN, CHANG and CHU 1974 and unpublished data) temperature-sensitive mutants have been isolated in the hamster cells.

Intercistronic complementation was detected in cell hybrids formed between independently isolated mutants of different (e.g., gln- x azg') or similar (e.g., g lyA X gly-B) function (CHU et al. 1969; KAO, JOHNSON and PUCK 1969; KAO, CHASIN and PUCK 1969), leading to the conclusion that nearly all the mutants so far examined behave like recessive characters.

Among the drug-resistant markers are those that are resistant to 8-azaguanine (deficient for hypoxanthine guanine phosphoribosyltransferase, HGPRT) , to 2, 6-diaminopurine (deficient for adenine phosphoribosyltransferase, APRT) and to 5-bromedeoxyuridine (deficient for thymidine kinase, TK) . Selective media have been devised to obtain forward mutations to resistance and reversions to analog sensitivity ( SZYBALSKI, SZYBALSKA and RAGNI 1962; LITTLEFIELD 1964; CHU et al., 1969; KUSANO, LONG and GREEN 1971 ) .

A question may be raised concerning the detection of recessive mutant characters in essentially diploid mammalian cells. The assumption that one can de- tect this kind of mutants has involved the postulate of hemizygosity of the in-

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volved chromosome, pre-existing heterogygosity, or X-linkage of the markers used (CHU 1971a). In established heteronuclear lines, such as the Chinese hamster V79 and CHO cell lines, aneuploidy or deletion of specific chromosomal regions may have occurred through mitotic disjunctional errors or structural rearrangements. KAO and PUCK (1968b) suggested that the successful isolation of recessive mutations in diploid cells may depend on the existence of specific monosomies. CHASIN ( 1972) studied marker segregation in homospecific hybrids between different hamster cell mutants and found a lack of linkage for the wild- type alleles of at least three recessive mutations (HGPRT-, APRT-, and gly-* or glTB). The result would require the existence of at least three different monosomies or deleted chromosome regions in the CHO cell line.

Autosomal recessive mutations may be detectable in the heterozygous state. On the basis of partial resistance to 2,6-diaminopurine7 RAPPAPORT and DEMARS (1973 a,b) were able to distinguish normal human skin fibroblasts in culture from those derived from presumptive heterozygotes at the autosomally- linked APRT locus. From these heterozygous cells, forward mutations to resistance to higher concentrations of diaminopurine (presumably homozygous re- cessives at this locus) were obtained. By similar reasoning, CLIVE et al. (1972) obtained spontaneous forward an reverse mutations at the TK locus in mouse lymphoma L5178Y cells. Table 4 summarizes the results of these workers on spontaneous rates of forward and reverse mutations as well as the relative activity of thymidine kinase in the lymphoma cells. The postulated genotypes appear to explain reasonably well the observed phenotypic changes at the frequen- cies indicated, but there is no proof of the validity of this explanation. The same conclusion may be drawn from a similar study on TK-deficient mutants in Chi- nese hamster cells (ROUFA, SADOW and CASKEY 1973).

X-linked recessive mutations may be expressed because of the functional haploidy of this chromosome in male animals. X-linked recessive mutations in somatic cells from female mammals can also be expressed in clones in which the normal-allele-bearing X chromosome is genetically inactivated. In the Lesch- Nyhan syndrome in man, the gene for HGPRT is X-linked (KELLEY and WYN- GAARDEN 1972). In human diploid fibroblast cultures, spontaneous mutants at the HGPRT locus whose properties resembled the cells from Lesch-Nyhan pa-

TABLE 4

The raies of forward and reverse mutations at the thymidine kinase ( T K ) locm of mouse lymphoma (L5178Y) cells (data taken from CLIVE et al. 1972)

5x10-11$ 6x10-9

1 . 2 ~ 10-7

Spontaneous mutation rate* TK+/+ (BUdRS) 3 TK-/- (BUdRr) + TKf/- (BUdRs)

Thymidine kinase activity+ 61 i: 3.6 0 27 t 1.3s

* Mutations/locus/generation. + ,ppmoles thymidine phosphorylated/lO6 cells/min incubation. $ One mutant only.

Nine independent revertants.

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120 E. H. Y. CHU

tients have been selected (DEMARS 1971; DEMARS and HELD 1972; VAN ZEE- LAND, VAN DIGGELAN and SIMMONS 1972). The same gene is probably X-linked in the Chinese hamster, since azaguanine resistance segregates from cell hybrids with the X-linked marker GGPD ( WESTERVELD et al. 1972).

Among the HGPRT mutants isolated from mammalian cells of various species origin, the phenotypic resistance to the analogs of hypoxanthine and guanine is usually accompanied by a deficiency in the HGPRT activity. DEMARS and HELD (1972) detected no electrophoretic differences between the HGPRT activities of normal and mutant human cell strains, but they observed in some mutants other qualitative alterations of the enzyme (such as thermolability, PRPP dependence, NaF sensitivity, altered pH opimum) . Two lines of evidence support the mutational model of azaguanine resistance (HGPRT-) in Chinese hamster cells. BEAU- DET, ROUFA and CASKEY (1973) used highly specific rabbit antibody directed against purified Chinese hamster HGPRT to examine cell lines containing mutations and reversions at the HGPRT locus. They detected both CRM+, HGPRT- lines and reversion of CRM-, HGPRT- cell lines to various independent levels of CRM and enzyme activity. This result indicates that the defective enzyme deficient for catalytic activity but not the immunological properties were produced in cells with missense mutation at the HGPRT locus. SEKIGUCHI and SEKIGUCHI ( 1973) demonstrated possible interallelic complementation in hybrid cells formed between hamster mutant clones deficient in HGPRT activity. This result suggests, but does not prove, the hypothesis of conformation correction between defective enzyme subunits in complementing hybrids. Whether the hamster HGPRT is a multimer is not known, but there is evidence (ARNOLD and KELLEY 1971) that human erythrocyte HGPRT is composed of two identical subunits.

There are several additional lines of evidence that altered specific gene products (proteins) are produced in mutant mammalian cells which arose either spontaneously or after mutagenic treatment. ALBRECHT, BEIDLER and HUTCH- ISON (1 972) showed that the amethopterian-resistant mutants isolated in Chinese hamster cell lines (DC-3F and DC-3F8) had structurally altered dihydrofolate reductase, as compared to the parental enzyme. Mutants of Chinese hamster CHO cells resistant to a-amanitin have been shown to contain an altered form of DNA- dependent RNA polymerase I1 ( CHAN, WHITMORE and SIMINOVICH 1972). More recently, another mutant in the same cell line, which is temperature-sensitive for protein synthesis, has been shown (THOMPSON, HARKENS and STANNERS 1973) to be specificially defective in vivo in its ability to charge tRNA with leu- cine. Cytoplasmic extracts of this mutant exhibited temperature-sensitive leucyl- tRNA synthetase activity. It is, therefore, highly likely that the mutant has a structural alteration in the leucyl-tRNA synthetase. Finally, starch gel electrophoresis studies of 26 isozymes in long-term lymphoblastoid lines by POVEY and associates (1973) demonstrated that in certain clonal sublines there are spontaneous changes in the electrophoretic patterns of APRT and pepsidase D (Pep D) . These workers further showed that after treatment of lymphocytes with either a chemical mutagen (MNNG) or ultraviolet light, different apparent mutations

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occurred at the Pep D, Pep A and phosphogluconate dehydrogenase (PGD) loci. Particularly interesting is an MNNG-induced PGD mutant clone in which the enzyme exhibited changed electrophoretic mobility and increased thermal stability, deflecting possibly the result of amino acid substitution in the enzyme mole- cule.

Experimental Mutagenesis and Mutagenic Specificity

The next necessary evidence for gene mutation is that the mutation frequency should be increased with the dose of mutagens. Indeed, chemical mutagens (alky- lating agents, half mustards, polycyclic hydrocarbons, nitroso-compounds, prim- idine nucleoside analogs) and physical mutagens (X-rays, ultraviolet light) have been shown to increase the mutation frequency in Chinese hamster cells (CHU and MALLING 1968b; CHU 1971b; CHU, SUN and CHANG 1972; CHU et al. 1974; KAO and PUCK 1968b, 1969,1971; BRIDGES and HUCKLE 1970; ARLETT and POTTER 1971 ; ARLETT and HARCOURT 1972; SHAPIRO et al. 1972; HUBERMAN and HEIDELBERGER 1972; HUBERMAN et al. 1971; HUBERMAN, DONOVAN and DI- PAOLO 1972; DUNCAN and BROOKES 1973; GILLEN et al. 1972; ROUFA, SADOW and CASKEY, 1973), mouse lymphoma cells (Fox 1971; CLIVE et al. 1972; SUM- MERS 1973), human diploid fibroblasts (ALBERTINI and DEMARS 1970, 1973; RAPPAPORT and DEMARS 197313; VAN ZEELAND, VAN DIGGELEN and SIMONS 1972) and human lymphoblastoid cells (SATO, SLESINSKI and LITTLEFIELD 1972). We have shown that the frequency of azg' (HGPRT-) mutants in Chinese hamster cells increases as a function of the concentration (within a certain range) of ethyl methanesulfonate (CHU 1972). The frequency of X-ray-induced forward mutations at the HGPRT locus in the hamster cells was dose-dependent (BRIDGES and HUCKLE 1970; CHU 1971b).

Although it is not directly related to the present discussion on the nature of mutations, an important point should nevertheless be made concerning the rate of induced mutations. In our study, the rate of X-ray-induced forward mutations at the HGPRT locus in the hamster cells was between 4.2 X lo-' and 1.8 X per locus per roentgen ( CHU 1971 b) . In the same cell line, BRIDGES and HUCKLE (1970) estimated that the rate of X-ray-induced mutations to azaguanine resistance was 9 x per locus per rad. ALBERTINI and DEMARS (1973) showed that in cultured human diploid fibroblasts the rate of X-ray-induced HGPRT- mutation was 1.8 x per locus per roentgen. The rate of X-ray-induced forward mutations from TK+/- to TK-/- in mouse lymphoma cells was 5 X IO-' per locus per roentgen ( FLAMM, personal communication). ABRAHAMSON et al. (1973) reported that when the existing data are normalized to a common base- line in terms of the amount of DNA per nucleus, there is a remarkable consis- tancy in the rates (ranging from 1.8 to 5.6 x lo-? per locus per rad) of specific locus forward mutations induced by acute doses of ionizing radiations in various experimental organisms (from E . coli, yeast, Neurospora, Drosophila, higher plants to the mouse). The fact that the rates of X-ray-induced forward mutations in cultured human and other mammalian cells are also within the same range indicates that we might extrapolate with confidence to man the data on induced

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E. H. Y. CHU

TABLE 5

Reuertibility of azaguanine-resistant mutants of independent origin isolated in Chinese hamster cells’

Reversion mechanism

Mutant origin

Spontaneous MMS-induced EMS-induced MNNG-induced ICR-170 induced N-AcO-AAF induced X-ray induced

Total

BPS +/- SP NON

8 1 6 1 0 8 - 7 3 4 - 6 9 5 - 2 13 0 1 15 12 2 4 7 1 2 9 12 10 29

Total no. of mutants

25 18 19 20 28 25 60

I95

* Data summarized from CHU (1971b) and CHU et al., in press. Abbreviations: MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; MNNG, N-

methyl-N’-nitro-N-nitrosoguanidine; ICR-I 70, 2 methyl-6-chloro-9- [3- (ethyl-2-chloroethyl) amino-propylaminol-acridine &hydrochloride; N-AcO-AAF, N-acetoxy-2-acetylaminofluorene. BPS, base pair substitution; +/-, base pair insertion or deletion; SP, reverts only spontaneously (reversion mechanism unknown) ; NON, non-revertible (no reverse mutations obLained in all the revertibility tests).

forward mutation rates obtained in mammalian cells in uitro a5 a result of exposure of ionizing radiation.

The next criterion for gene mutation is mutagenic specificity in reversion tests with known chemical mutagens. Table 5 summarizes the results on the revertibility from azaguanine resistance to sensitivity in resistant hamster cell mutants of different origin (CHU 1971b; CHU et al. 1974). It is evident that over 27% of mutants can be reverted specifically with specific mutagens. The genetic alterations at the mutant sites can be inferred as base-pair substitutions and frame-shift changes. On the other hand, the nature of the genetic alteration is unknown for those mutants which either revert spontaneously or are nonrevertible.

Galactose Negatiue Mutants in Chinese Hamster Cells

In the LELOIR pathway (LELOIR 1951; KALCKAR, BRAGANCA and MUNCH- PETERSEN 1953), galactose enters the mainstream of carbohydrate metabolism via a series of reactions which result in its conversion to glucose-l-phosphate (Figure 1). Galactose first reacts with ATP to give gal-l-p, as catalyzed by galactokinase. Gal-l -p then reacts with nucleotide uridine diphosphoglucose (UDPG) to give glucose-l-p and UDP-gal. The enzyme involved is gal-l-p uridyltransferase. UDPG can be regenerated from UDP-gal by the enzyme epimerase, a reaction requiring the coenzyme NAD. Glucose-l-phosphate is further catalyzed by phosphoglucomutase to form glucose-6-phosphate7 which in turn either enters the glycolytic pathway or is converted to glycogen.

In man, transferase deficiency galactosemia is a structural gene mutation (TEDESCO and MELLMAN 1971) and the disease conforms to autosomal mende- lian inheritance (HSIA 1969; SEGAL 1972). There are several allelic variants, in-

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galactose L: galac tokinase

galactose -1- phosphate

aalactose -1-DhosDhate uridyl transferase

uridine diphosphoglucose

,uridine diphosphogalactose

uridine diphosphogalactose-4-epimerase

NAD+

glucose-l- phosphate

phosphoglucomutase J / ^ \ glucose-6-phosphate glycogen

FIGURE 1 .-Pathways in galactose metabolism.

cluding the Duarte variant (BUETLER et al. 1965) and the recently discovered Los Angeles variant (NG, BERGREN and DONNELL 1973). Juvenile cataract may be due to a deficiency in galactokinase activity, and galactokinase deficiency is also inherited as an autosomal recessive (GITZELMANN 1967). A single case of epimerase mutation in man has recently been reported (GITZELMANN and STEIN- MANN 1973).

In this pethway, several gene loci may be involved during selection for a single altered phenotype, namely, the ability to utilize exogenous galactose. By a combined treatment of hamster cells with BUdR and near-visible light, we were able to induce 67 galactose-negative (gal-) mutants (CHU, SUN and CHANG 1972.

Our first series of experiments was tcJ determine whether the human genome can complement the hamster gal- phenotype in hybrids formed from a mutant hamster cell and a normal human cell. Somatic cell hybrids were produced between gal- mutant hamster cells and human lymphocytes (SUN, CHANG and CHU 1973). Eight hybrid clones which survived in the galactose medium were analyzed for the concurrent presence of human transferase and a specific human chromosome. Figure 2 shows the electrophoretic pattern of the transferase in both parental cells and in four of the eight hybrids. The transferases in all eight hybrids showed an identical electrophoretic pattern. The human form of the transferase can be distinguished from that of Chinese hamster by its slower anodal movement during electrophoresis at neutral pH, possibly reflecting some differences in the amino acid composition of the homologous enzymes from the two species arising in the course of evolution. Gal-l-p uridyltransferase in E . coli is a dimer and the molecular weight of each monomer is about 40,000 daltons ( SAITO, OZUTSUMI and KURAHASHI 1967). According to TEDESCO (1972), human

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124 E. H. Y. CHU

+

0 rigin

1 2 3 4 5 6 7 8 FIGU~I: ?.-zymogram showing thr activity of gal-1 -p uridy~tr;~nsferasr. Gal-2 and Gal-3

(channels 1 and 2) are galactose-nrgatire hamstrr crll mutants; UM-10 and P3.1 (channels 7 and 8) are human lymphocyte cell linrs; channels 3 to 6 are man-Chinrsr hamstrr cell hybrids (taken from SUN, CIIANG nntl Ctru 1974). -

transferase is a mu1 timer. The human-Chinese hamster cell hybrid clones showed both parental transferase bands as well as an enzymologically functional heteropolymer band at an intermediate position (Figure 2). This heteropolymer band was formed presumably by aggregation of human and hamster transferase mon- omers. An artificial mixture of human and hamster cell extracts showed on the zymogram only the characteristic bands of both species. These results indicate that the hamster transferase is also a multimer. at least a dimer.

All the hybrid clones were maintained in galactose medium. One clone, GJ-1, had only 21 chromosomes. The Giemsa-banding of the hamster parent, Gal-2, is compared with the hybrid GJ-1 (Figure 3). Karyotype analysis of 19 hybrid cells shows that all contained a single human chromosome. A2. No other human chromosome could be detected in this particular hybrid. It seems likely that the human structural gene for tranferase is on chromosome A2 (SUN, CHANG and CHU 1974).

Our next series of experiments was to see if different gal- mutants can complement each other with respect to galactose utilization in hamster-hamster cell hybrids. The results are illustrated in Figure 4. At the time of the fusion experi- ment, the spontaneous reversion frequency of different gal- mutants was from 10" to Positive complementation was indicated by a significant increase in the number of surviving colonies in the test plate, by the large cell size in each colony and by chromosome analysis. The frequency of hybrid formation was about 1 O-9.

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MUTATIONS I N MAMMALIAN CELL. 125

- 4

L"

a t d i 0

LI

e d

-\ * a

1 2 - 3

FIGURE 3.-(a) The karotyye of a cell from one of the hamster parental rrll populations, Gal-2. (h) The karyotype of a man-hamster cell hybrid (GJ-1) which contains one complete set of the hamstrr chromosomes and a single human chromosome A2 (in box). (c) Examples of human cliromosomrs A2 from different sources.

On the basis of the pattern of complementation in pairwise combinations among 27 independent derived gal- mutants, a complementation map was con- structed, as shown in Figure 5. The map is linear and two-ended and consists of seven complementation units. It may be assumed that the order of the segments in the complement map is the same as the order of the corresponding mutational sites in the genetic map. Certain gal- mutants are represented by one complementation unit while others involve two such units. No mutant has as yet been found to involve more than two units. I t may be concluded that bromouracil mutagen-

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126 E. H. Y. CHU

FIGURE l.-Complcmentiitiori pattrrn as incliratrcl hy the formation of large hybrid cell C O ~ O -

nirs in certain combinations of galactow-negative mutants and the absencr of such colonies in other combinations. The mutant numhcis are labeled at the sides of rach row and column of platrs. Thc few small colonirs zhown in plates with noncomplementing combinations are revertants. The bottom row of plates was inoculatrtl with the Same number of (4 x 101) of cells of rach mutant but without the addition of viruz. T h r inwrtrd photomicrographs show the com- plrmrnting tetraploid hybrid crlls az compnrrd to diploid revertant cells.

32

2.19.35

50.51

60.68.71.73

1.9.46.47.48.49 58.63.64.65.66.70

3.18

6

FIGURE 5-Complementntion map in crll hybrids formed among 27 galactose-negative mutant clones of Chinese hamster crlls.

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esis probably produced in this case mainly missense mutations and not gross de- letions.

In contrast to the parental cells, gal- mutants could not utilize exogenous galactose, mannose, fructose, galactose-1 -phosphate, glucose-l-phosphate or glucose-6- phosphate. Permeation of galactose into mutant cells appeared unimpaired. In intra- and interspecific cell hybrids, the gal- mutation behaved like a recessive character. The pleiotropic nutritional response was due not to deficiency of any one specific enzyme in the Leloir pathway but to significant reduction in the activities of phosphoglucomutase, NADP-dependent isocitrate dehydrogenase and perhaps other enzymes. We postulate that the change from gal+ to gal- phenotype in hamster cells could be due to single gene mutations at a regulatory gene locus, or at a yet unknown locus with enzymic defect that causes secondary metabolic imbalances (SUN, CHANG and CHU, manuscript in preparation). The mutant protein involved could be either a regulatory protein or an enzyme. The present result of allelic complementation can be interpreted by the hybrid protein hypothesis (CATHESIDE and OVERTON, 1958; CRICK and ORGEL, 1964). According to this hypothesis, interallelic complementation occurred through co- aggregation probably with conformation correction of polypeptide chains.

Finally, we have obtained three clear-cut temperature-sensitive, locus-specific gal- mutants. The existence of ts mutants indicates that missense mutation at the postulated regulatory or enzyme locus might have occurred in the hamster cells. Formal proof that some ts mutants are indeed missense mutants has been pro- vided by WITTMANN and WITTMANN-LIEBOLD (1966) with tobacco mosaic virus, in which the changes in amino acid sequence of the viral polypeptide has been determined for several ts mutants. Furthermore, several cases of mutant proteins in which the amino acid substitution has been determined exhibit changed physiochemical properties in vitro. For example, hemoglobin Ann Arbor (RUCKNAGEL, BRANDT and SPENCER 1971) as a single amino acid substitution in the alpha chain shows a changed thermal stability in vitro, as compared to normal hemoglobin A (R. ABRAHAMSON, personal communication). Another example of a possible missense mutation is the human erythrocyte carbonic anhydrase I which is composed of a single chain of 259 amino acids in a known sequence. In different human populations a total of nine electrophoretic variants of human erythrocyte carbonic anhydrase have been discovered, in five of which the specific amino acid substitution has been characterized. OSBORNE and TASHIAN (1974) have shown recently that two of the purified mutant enzymes in which the substitution is known exhibited different thermal inactivation curves in vitro, as compared to the normal enzyme. Since single amino acid changes in the protein could result in a characteristic change of its thermal stability, the implication is that the t s mutants we discovered could be the result of amino acid changes in the proteins reflecting missense mutations.

SUMMARY A N D CONCLUSIONS

Despite considerable work it is in many instances unclear whether the mutant” cell lines which can be isolated from mammalian cells in culture are ( 6

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due to genetic or epigenetic mechanisms. Furthermore, where the phenomenon seems to be genetic, the nature of the change has usually remained obscure. The central purpose of this paper has been to review available evidence and to assess whether consistent phenotypic variations in mammalian somatic cell populations in culture are true gene mutations.

By the efforts of many investigators, selective procedures have been devised to isolate mutant clones with a variety of changed phenotypes such as nutritional requirements, drug resistance, and conditional lethality. By a number of criteria, these isolated mutant clones appear to fulfill the properties of gene mutations. These criteria for genetic alterations include random occurrence, retention of stable phenotype in the absence of selection, induction with mutagens, mutagenic specificity, conditional lethality, interallelic complementation and changes in the activity as well as the physiochemical properties of specific gene products. To be sure, some of the supporting evidence is circumstantial and indirect, and not all criteria were met in every instance of the observed somatic variation. Nevertheless, experimental evidence is reasonably strong in a number of cases to conclude that we are dealing with gene mutations. One such example cited is our recent study on the galactose-negative mutants in Chinese hamster cells in which temperature-sensitive mutations and interallelic complementation have been demonstrated.

Similar to the situation that existed some thirty years ago when the mutational or adaptational origin of bacterial variants was debated, the question of the nature of variants in somatic mammalian cells is confronted with doubts and uncertain- ties. There is published information on the drug resistance in mammalian cells which is difficult to explain in conventional genetic terms, yet evidence, as summarized in this paper, is persuasive that many, if not all, markers are governed ultimately by information in the cellular genome. Of course, other alternative possibilities have not been ruled out. For instance, drug resistance may be a special case which might reflect the occurrence of cytoplasmic as well as nuclear inheritance. A resolution of this apparent conflict between the genetic and epigenetic interpretations for the origin of variation in mammalian cells in culture requires further careful and rigorous investigations.

I wish to thank DRS. C. C. CHANC and N. C. SUN for their invaluable contributions to the original work mentioned herein and to DRS. A. D. BLOOM, J. V. NEEL, E. SHAMAY, and R. E. TASHIAN for discussions and review of the manuscript. This research was supported by grants GB 34302 and GB 37100 from the National Science Foundation.

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INDUCTION AND ANALYSIS OF GENE MUTATIONS IN - Genetics

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