The Action of Mendelian Genes in Human Diploid Cell Strains '

ROBERT S. KROOTH AND ELIZABETH K. SELL2 Lawrence D. Buhl Center for Human Genetics, Department of Human Genetics, University of Michigan Medzcal School, Ann Arbor, Michigan 48104

ABSTRACT Some of the cells of every human being will grow outside the body as microorganisms. It is possible to show, in a variety of ways, that these cells resemble genetically the individual from whom they were obtained. Over 35 inherited human diseases and anomalies can now be studied in such cell lines. Human diploid cell strains, biochemically marked by one or more mutant Mendelian genes, have proven particularly useful for the study of gene action in man and for the detection of genetic changes such as mutation and somatic cell hybridization. In addition, the strains have a number of clinical applications, including the antenatal diagnosis of inherited disease. The failure of cultured human cells to display their phenotype at most loci continues to restrict their use in both genetics and medicine. There are reasons for hoping that this difficulty will eventually be solved, and some experiments bearing on the problem are already feasible.

To study genetics, one must start with to the isolation of a variant subline; in this hereditary variation. In cell culture, this case as well as in several others, an en- means one must have, as a minimum, two zyme deficiency is associated with the abil- different strains of cells which differ from ity of the cell to survive in the selective one another in at least one attribute. More- medium. Indeed, there are several general over, the difference must be hereditary over methods for preferentially favoring the somatic generations. growth of auxotrophic mammalian cells The initial problem of human cell gene- (Hooper and DeMars, '60; Kao and Puck, tics, therefore, has been to identify or gen- '68; Pollack, Green and Todaro, '68). Most erate this kind of variation. Two principal such auxotrophs may be presumed to be methods have been used to obtain lines of deficient in one or more activities. human cells that have some distinctive The second method for obtaining variant and hereditary attribute. The first method lines of human cells is quite different. One is to select infrequent cells, from a popu- begins by selecting donors (rather than lation which differs in a known way from cells) who are known to carry genes which the majority of the cells. For example, affect molecules normally present in cul- nearly all cells exposed to the synthetic tured cells (Krooth and Weinberg, '61). analogue 8-azaguanine are killed by this The Mendelian basis for the cellular pheno- agent, but, over a wide range of doses, a type is shown by developing strains from few are not (Szybalski and Szybalska, '62; various persons in the same family. inter aliae). The surviving cells are resist- There are important differences between ant to the drug, even if they are grown in the first and second method. In the first its absence for an extended period of time. method, the variant line and the line with In most cases, the resistant subline is de- which it is compared usually come from ficient in an enzyme which apparently con- the same donor. In the second method, the verts 8-azaguanine to the corresponding donors are different. It can be argued that ribotide (Brockman et al., '61; inter aliae). variants isolated by the first method are It is likely to be this ribotide, rather than 1 The originaI investisations reported in this paper the free base, which is cytotoxic. In any were supported by NIH Research grant 1-Pol-15419-03. 2 Supported by NIH Training grant 5-TOl-GM-00071- case, such an experiment frequently leads 13.

J. CELL. PHYSIOL.,76: 311-330. 311 312 ROBERT S. KROOTH AND ELIZABETH K. SELL likely to differ from the parental line at cell is probably related to the biology of only a single locus. On the other hand, the cells. Since homonuclear cells, unlike when a variant isolated by the second heteronuclear ones, usually do not clone method is compared with a strain isolated with high efficiency, it can be difficult to from a normal donor, the two strains prob- select an infrequent cell from the popula- ably differ at many loci, for the genetic tion. The variant cell may not be able to polymorphisin of human populations is generate a colony when all the other cells very rich (Harris, '66; Lewontin, '67). The have been eliminated by the selective sys- advantage of the second method of course tem. Human heteronuclear lines, though is that one can prove by family studies that the cell attribute is hereditary over the they usually can be cloned with high ef- germ line, as well as over the somatic one. ficiency, are rarely isolated. Hence, the From such studies, one' can frequently con- probability of developing a heteronuclear clude that the attribute is controlled within line from a donor of an appropriate geno- each family by a single locus. If cell strains type is small. from several different unrelated patients A disappointing feature of cultured hu- all differ from normal strains in the same man cells is that they do not usually per- way, one can argue that normal variation form the tissue-specific functions of the at loci other than the one being studied is organ from which they were isolated. The irrelevant. reason for this phenomenon is unknown, As a rule, each method involves the use and a detailed discussion of the matter is of a different kind of cell. The first method outside the limits of this review, However, has been used successfully with heteronu- it seems likely that there are at least two clear cell lines, while the second method separate mechanisms involved. In the case employs homonuclear lines. The contrast- of human heteronuclear lines, the apparent ing properties of these two kinds of cell failure of the cells to perform tissue-speci- lines have been reviewed elsewhere fic functions may be related to the ana- (Krooth, Darlington and Velazquez, '68). plasia of the carcinomas from which they Their main features, which are relevant were isolated. In addition, most human to this discussion, are as follows: Human heteronuclear lines appear to have been heteronuclear lines show marked variation developed from carcinomas of the cervix, from cell to cell in chromosome comple- and have not been tested for their ability ment, are capable of indefinite growth in to perform a biochemical function peculiar vitro, and usually clone with high em- to the uterine cervix. In the case of human ciency. Human heteronuclear lines are homonuclear cell lines, the mechanism rarely isolated. Indeed, recent evidence is likely to be different. These cells have a suggests that many of the lines commonly fusiform morphology and engage in the used in experiments. and thought to have de novo synthesis of collagen (Green, independent origins, may in fact all be Goldberg and Todaro, '66) and mucopoly- derived from a single donor (Gartler, '67). saccharides (Fratantoni et al., '68). Hence, Most human heteronuclear lines seem to it seems likely that they are descended have been isolated from tissue involved in from the fibroblasts of intact tissues. Ap- a cancerous process. parently, with existing methods only this Human homonuclear lines, on the other type of human cell is capable of sustained hand, usually show very little variation growth in culture - at least in the case of from cell to cell in chromosome comple- normal solid tissues biopsied subsequent to ment - no more than is found in most in- birth. Perhaps the same is true of other tact tissues. They grow in vitro for only animals. The type of cell recovered in cul- about six months to a year, and are dif- ture seems surprisingly independent of the ficult to clone with high efficiency except organ whose tissue is sampled. Hence, it is perhaps very shortly after their isolation likely that the human cell which grows in from the explant. An important feature vitro is a fibroblast - a stromal element of homonuclear lines is that they can easily a It must be emphasized that the rules which govern be developed from virtually any don~r.~ the biology of cultured human cells do not necessarily The reason that the two methods have apply to the cells of other mammalian species (Todaro and Green, 63; Green and Todaro, '63; Bayreuther, each been used with a different kind of 64; Herzenberg, '64). MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 313 which is ubiquitous. It is present in all or- duria (Krooth, '64b,'66a), citrullinemia gans but is the characteristic cell in none.4 (Tedesco and Mellman, '67), arginosuc- When we began to study gene action in cinic aciduria (Shih, Littlefield and Moser, human diploid cells, we tentatively formu- '69) and the Lesch-Nyhan Syndrome (Felix lated a rule that if an enzyme or other and DeMars, '69; Siniscalco et al., '69; inter molecule were ubiquitous -if it were aliae). In the case of this latter condition, present in all tissues - it would be found a medium which will select in favor of mu- in cultured homonuclear cells. The pres- tant homozygous cells, and against normal ence of the molecule would presumably cells, has also been developed (Albertini then not depend on which way the cell had and DeMars, '70). differentiated (Krooth and Weinberg, '61; Krooth, '65). In practice, no enzyme has Examples of gene action in diploid been studied in all tissues, and we used the cell strains rule-of-thumb that if the enzyme were a. Galactosemia present in peripheral blood cells and a Galactosemia is a rare autosomal reces- parenchymatous organ other than spleen, sive disease. Affected patients have acute it was likely to be ubiquitous and therefore milk intolerance during infancy, cataracts, present in cultured cells (Krooth, '64a, '65). and other signs and symptoms (Issel- As far as is known, this rule still seems bacher, '66). The condition is treated by to hold, but its converse does not. Some withholding milk from the diet, and if un- molecules which are not ubiquitous are treated is frequently fatal. indeed present in cultured cells (Uhlen- The erythrocytes and other tissues of dorf and Mudd, '68), perhaps because the affected patients have no detectable ac- human homonuclear cell is descended from tivity for galactose-1-phosphate :UDP glu- a fibroblast - a specialized cell which per- cose uridyltransferase (cf. Isselbacher, forms its own peculiar functions. At pres- '66). This enzyme catalyzes the second ent, it seems unlikely that the cultured step in the Leloir pathway - a pathway human homonuclear cell is "dedilferen- which effects the conversion of galactose tiated," i.e., derived from parenchymatous to glucose. cells that have lost the distinctive proper- Cultured cells from mutant homozygous ties which give them their identity. persons are unable to liberate C''O0, from In table 1, we have listed many of the gala~tose-l-C'~,whereas cells from normal mutant Mendelian genes which have been and heterozygous persons can (Krooth and shown to affect specific molecules in the Weinberg, '61). Similarly, when cell ex- cultured human cell.5 There are at present over 35 such genes, and they probably re- 4Moore and his associates have recently demon- strated that lymphoblastic cell lines can frequently be flect mutations at 30 different loci. Seven isolated from the peripheral blood of normal persons of the genes are on the X-chromosome, (Moore and McLimans, '68; Moore, Porter and Huana, '69). These lines are diploid, grow in vitro indefi- while the remainder are autosomal. The fitely, and synthesize immunoglobulins (Fahey et al., 66). Hence, another class of human cells and perhaps clinical phenotypes associated with these another class of tissue-specific loci are now available genes vary from quite severe disorders, for microbiological experiments. Very recently, Men- delian mutations (at 2 different loci). affecting the such as the Lesch-Nyhan Syndrome or phenotype of these cells have been described (Conover cystinosis, to biochemical traits like the et al., '70; Choi and Bloom, '70). 5Table 1 is of course a summary of the work of electrophoretic variants of glucose-6-phos- many different laboratories. A bibliography for each of the entries in table 1 is given elsewhere (Krooth, phate dehydrogenase or lactic acid dehy- '69a) except for the most recently described cellular drogenase that produce no known signs or phenotypes. These are deficiency (Mayes and Guthrie, '68; Sell, '70). Gaucher's djsease (Uhlen- symptoms. The mutant cellular phenotypes dorf and Brady 70) GM2 Gangliosidosis Type I (O'Brien and Okid?, '6b; Pinsky, Callahan and Wolfe, associated with the genes listed in table 1 '68), GM2 Gangliosidosis Type I1 (O'Bnen et al., 701, include disorders of carbohydrate, amino GM2 Gangliosidosis Type 111 (ibid. ), GMi Gangliosido- SIS Tvpe I (Sloan et a?., '69), GMi Gangliosidosis Type acid, lipid and nucleotide metabolism. For I1 (O'Brien et al., 70; Wolfe et al., 70). gtyco- five of the genes, selective media which gen storage disease Type 111 (Justice et al., 69), Scheie's Syndrome (Wiesmann and Neufeld, '70),San- prevent the growth of mutant homozygous filippo Syydrome (ibid.), and, xeroderma pigmentosa (Cleaver, 68; Setlow et al., 69). In addition, there cells have been formulated. These genes has been important new work with several of the are the ones for galactosemia (Krooth and mucopolysaccharidoses such as Hurler's Syndrome and Hunter's Syndrome (Fratantoni, Hall and Neu- Weinberg, '61; Krooth, '66a), orotic aci- feld, '68; Fratantoni, Hall and Neufeld, '69). 314 ROBERT S. KROOTH AND ELIZABETH K. SELL

TABLE 1 Some genetic abnormalities that affect specific molecules present in the cultured human homonuclear cell

Disease or variant Affectedmolecule Genetics “A” electrophoretic variant of Glucose-6-phosphate Sex-linked co-dominant glucose-6-phosphate dehydrogenase dehydrogenase (G-6-PD) Acatalasia I (Japanese variant) Catalase Autosomal recessive Acatalasia I1 (Swiss variant) Catalase Autosomal recessive Arginosuccinic aciduria Arginosuccinase Autosomal recessive Branched-chain ketonucia Uncertain Autosomal recessive Citrullinemia Arginosuccinate synthetase Autosomal recessive Cystinosis Uncertain Autosomal recessive Electrophoretic variant of Phosphoglucomu tase Autosomal co-dominant phosphoglucomutase Electrophoretic variant of Lactic acid dehydrogenase Autosomal co-dominant lactic dehydrogenase Electrophoretic variants of 6-phosphoglucuronic acid Autosomal co-dominant 6-phosphoglucuronic acid dehydrogenase Familial nonspherocytic Glucose-6-phosphate Sex-linked recessive hemolytic anemia dehydrogenase GMI Gangliosidosis Type I Absence of B-galactosidase Autosomal recessive ( Generalized Gangliosidosis ) A, B, C GM1 Gangliosidosis Type I1 Absence of B-galactosidase Autosomal recessive (Juvenile GMi B and C Gangliosidosis ) GM2 Gangliosidosis Type I Absence of hexosaminidase A Autosomal recessive (Tay-Sachs Disease) GM2 Gangliosidosis Type I1 Absence of hexoaminidase Autosomal recessive (Sandhoffs Disease) A and B GM2 Gangliosidosis Type I11 Partial deficiency of Autosomal recessive (Juvenile GMz hexosaminidase A Gangliosidosis ) G-6-PD deficiency Glucose-6-phosphate Sex-linked recessive (Mediterranean type) dehydrogenase G-6-PD deficiency Glucose-6-phosphate Sex-linked recessive (Negro type ) dehydrogenase Galactokinase deficiency Galactokinase Autosomal recessive Galactosemia Uridine diphosphogalactose Autosomal recessive transf erase

Gaucher disease Glucoceribosidase Autosomal recessive Glycogen storage disease Lysosomal a-1,4-glucosidase Autosomal recessive (Cori type 11) Glycogen storage disease Amylo-1-6-glucosidase Probably autosomal (Type 111 ) recessive Homocystinuria Cyst athionine synthet ase Autosomal recessive MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 315

TABLE 1 (continued)

Disease or variant Affected molecule Genetics Hunter’s syndrome Uncertain (Macromolecule Sex-linked recessive which influences cellular con- tent of mucopolysaccharides) Hurler’s syndrome Uncertain (Macromolecule Autosomal recessive which influences cellular con- tent of mucopolysaccharides) ‘T-cell disease” Uncertain ( beta-glucuronidase Probably autosomal activity decreased; mucopoly- recessive saccharide content increased) Infantile metachromatic Arylsulfatase A Autosomal recessive leu kodystrophy Lesch-Nyhan syndrome Hypoxanthine: guanine:xan- Sex-linked recessive thine phosphoribosyltrans- ferase Madison electrophoretic Glucose-6-phosphate Sex-linked co-dominant variant of glucose-6- dehydrogenase phosphate dehydrogenase Methylmalonic aciduria Uncertain (Defective Uncertain Vitamin Bia synthesis) Niemann-Pick disease Uncertain (Increased cellular Autosomal recessive level of sphingomyelin) Orotic aciduria Orotidine-5’-monophosphate Autosomal recessive pyrophosphorylase and orotidine-5’-monophosphate decarboxylase Ref sum’s disease Phytanic acid a-oxidase Autosomal recessive Sanfllippo-Syndrome Uncertain (Macromolecule Autosomal recessive which influences cellular con- tent of mucopolysaocharides) Scheie Syndrome Uncertain (Macromolecule Autosomal recessive which influences cellular con- tent of mucopolysaocharides) Xeroderma pigmentosa Uncertain (Defective excision Autosomal recessive repair of DNA) perhaps an endonuclease- tracts are assayed, the mutant homozygous die in galactose, and the discrimination strains - unlike normal or heterozygous between the cellular phenotypes on the ones -have little or no activity for the basis of growth is obscured.‘ The minimum affected enzyme (Russel, ’69; Mayes and population density at which the selective Guthrie, ’68; Tedesco and Mellman,’69). system works varies from experiment to Galactosemic cells will not grow in a experiment, but a confluent monolayer of medium in which galactose is the sole galactosemic cells will invariably die in hexose present, under circumstances in galactose, whereas a confluent monolayer which normal and heterozygous cells will of normal or heterozygous cells will in- (Krooth and Weinberg, ’61; Krooth, ’66a). variably survive. The dependence of the However, the selective system for this lo- selective system for this locus on cell popu- cus works consistently only when the cells lation density may be related to the fact are at a high population density. At low that the specific activity of the population density, a significant fraction 6 This effect can be minimized somewhat by meth- of normal or heterozygous cells frequently ods described elsewhere (Krooth, ’66). 316 ROBERT S. KROOTH AND ELIZABETH K. SELL cell protein progressively increases with disorder of . Affected cell population density (Russel, '69 ). children produce uric acid at an abnor- The competence of a culture to grow in mally rapid rate, and have symptoms galactose can be predicted from its ability secondary to hyperuricemia. They also to grow in reduced quantities of glucose. show evidence of deranged function of the Repeated experiments have shown that central nervous system, and some of the only in circumstances where the cells are patients have an anemia as well. capable of growth in a medium containing Diploid cell strains (Seegmiller, Rosen- 5 mg per cent glucose will they grow in bloom and Kelley, '67; Friedman, Seeg- galactose. miller and Subak-Sharpe, '69) from af- Of course, a system which requires a fected persons have about 1% of normal high cell population density to assess geno- type is of little use for the purpose of se- activity for hypoxanthine :guanine :xan- lecting from the population a rare galacto- thine phosphoribosyltransferase (HGPRT- semic cell that may have changed geneti- ase). This enzyme (fig. 1) normally serves cally due to mutation or some other cause. the function of converting a purine catabo- However, even with this limitation, such lite (hypoxanthine) to a ribotide (- a system has a certain advantage over en- 5'-monophosphate). The ribotide in turn zyme assays or histochemical methods for inhibits the first enzyme of purine synthe- determining phenotype. The culture, if sis - the amidotransferase - and there- heterozygous or normal, is not lost to the fore perhaps the rate at which purines investigator by the procedure of detennin- are formed. In addition, HGPRTase cata- ing its phenotype. Hence, the system can lyzes the conversion of guanine to guanine- be used to screen clones (but not to isolate 5'-monophosphate, a ribotide which also them) on which other procedures can be inhibits the amidotransferase of purine subsequently performed. synthesis (cf. Seegmiller, Rosenbloom and Another curious feature of the selective Kelley, '67). system for the galactosemia locus is that, One theory of the pathogenesis of the at least in our hands, it seems to work Lesch-Nyhan Syndrome holds that in the consistently only when the medium con- absence of activity for the affected enzyme, tains dialyzed human sera. It is often not purine synthesis accelerates since the cell possible to demonstrate the hexose-depen- is now less sensitive to the size of the dence of human diploid cell strains in purine pool. The synthesized medium containing dialyzed fetal calf sera. is catabolized to hypoxanthine, but very Not only do galactosemic cells sometimes little of the hypoxanthine is converted grow in galactose, but cells of all genotypes back into inosinic acid because of the en- frequently grow in hexose-free medium. zyme deficiency. Hence, the steady state When first placed in hexose-free medium, concentration of inosinic acid in the cell the cells usually cease growth for a few is reduced, and the amidotransferase is days, and a minority of them may even released from feedback inhibition. For this die, but then, mysteriously, the culture reason (according to the theory) the recovers and growth resumes. Albertini velocity of purine synthesis is accelerated and DeMars ('70) have recently com- (Rosenbloom et al., '68; Seegmiller, '69). mented on the way in which fetal calf Another possibility (Rosenbloom et al., serum appears to interfere with another '68) is related to the fact that PRPP (5- selective system for diploid cell strains. In phosphoribosyl-1-pyrophosphate)may be a any event, the selective system for the rate-limiting in purine synthesis. galactosemic locus, using dialyzed human In the Lesch-Nyhan Syndrome, the af- sera, enables one to determine cellular fected enzyme catalyzes a reaction which phenotype when the cells being tested are consumes a significant amount of this at a high population density. compound, and hence one might expect the cellular level of PRPP to be elevated. That b. The Lesch-Nyhan Syndrome and related indeed is the case (Rosenbloom et al., '68; conditions. Greene, Boyle and Seegmiller, '70), and it The Lesch-Nyhan Syndrome (Nyhan et may be that the increase in PRPP available al., '67; Seegmiller, '69) is a rare X-linked to serve as a substrate for the amidotrans- MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 317

5-Phosphori bosyl-I-Pyrophosphote (PRPP) + Glutornine ...... ,_..'* .... .*. '.. h ,f.... 5-Phosphoribosyl-I-Amine .. 0 ,*+ '...:%o Glycine ....f', *...i" .7.. * : .. 4 8.. % .":.' '; ... .9 ......

Guanosine Phosphoiibosylfronsferase lnosine A den o si ne - P Adenine

Xonthine

*Azaserine block Uric IAcid Fig. 1 Purine synthesis and catabolism in man (from Seegmiller, Rosenbloom and Kelley, '67). ferase is partly- or even mainly- re- Nyhan cells fail to grow in HAT medium sponsible for the acceleration of purine presumably because their deficiency for synthesis. Other possibilities, however, are Z-IGPRTase prevents them from converting also admissible (Rosenbloom et al., '68). the hypoxanthine in the medium to ino- Diploid cell strains from persons a€- sinic acid. fected with this disease have about three On the other hand, purine analogues, times the normal level of adenine phos- such as 8-azaguanine and 6-mercapto- phoribosyltransferase (Seegmiller, Rosen- purine, which prevent the growth of nor- bloom and Kelley, '67). Recent evidence mal cells might be expected to have little suggests that the accumulation of PRPP or no effect on Lesch-Nyhan cells (Seeg- inside the cell (referred to above) may miller, Rosenbloom and Kelley, '67). These serve to stabilize this enzyme (Greene, fraudulent purines are apparently mis- Boyle and Seegmiller, '70). It is also pos- taken by the enzyme (HGPRTase) for sible that still a third enzyme, inosine utilizeable ones and seem to affect cellular , may be affected in the Lesch-Nyhan metabolism after they have been converted Syndrome, but evidence on this point is to the corresponding ribotides. The ribo- still being accumulated (Friedman, Seeg- tides then probably inhibit the amido- miller and Subak-Sharpe, '69; Payne et transferase, and decelerate the velocity of al., '70). purine synthesis. It has recently been Diploid cell strains from mutant hemi- shown that 6-mercaptopurine slows purine zygous males are unable to grow in "HAT synthesis in normal cells but not in Lesch- medium, whereas strains from normal per- Nyhan cells (Seegmiller, Rosenbloom and sons grow readily in this medium (Sinis- Kelley, '67). Moreover, 8-azaguanine does calco et al., '69). HAT medium contains not inhibit the growth of Lesch-Nyhan hypoxanthine, amethopterin, and thymi- cells and indeed can be used to select cells dine. Amethopterin blocks both de novo deficient in HGPRTase out of a population purine synthesis and the de novo synthesis of normal diploid cells (Albertini and De- of thymidylic acid. Hence, in the presence Mars, '70). of amethopterin, normal cells require both Lesch-Nyhan cells from at least one thymidine and hypoxanthine. The Lesch- patient have other abnormal nutritional 318 ROBERT S. KROOTH AND ELIZABETH K. SELL requirements. The cells will not grow of all new cases should be fresh mutations. unless the medium is supplemented with These new mutations need not all produce either adenine or unusually high concen- the disease by the same mechanism. The trations of folic acid (Felix and DeMars, structure of the enzyme may conceivably '69). The requirement for adenine prob- be altered in different ways, or even, in ably reflects the fact that the mutant cells some instances, through a gene substitu- are starved for purines, even though they tion whose effect on catalytic activity is are producing purines at an accelerated less direct. In any case, such an explana- rate. Presumably, in the absence of HG- tion may account for the clinical differ- PRTase, most of the inosinic acid synthe- ences between patients, such as the pres- sized is catabolized to oxypurines and is ence or absence of anemia. It may also unavailable for DNA and RNA synthesis. account for certain paradoxes, such as Adenine, when converted to AMP by the the fact that some strains will not grow adenine PRTase, can be subsequently con- in HAT medium while the growth of others verted to inosinic acid, and thus should is stimulated by hypoxanthine,' or even serve to increase the steady-state concen- the possible variation between patients in tration of this vital metabolite. Folic acid whether inosine kinase activity is affected may be a rate-limiting factor in purine by the gene (Friedman, Seegmiller and synthesis. When the medium is supple- Subak-Sharpe, '69; Payne et al., '70). mented with very high levels of folic acid, Apart from the Lesch-Nyhan Syndrome, the Lesch-Nyhan cells might accelerate several other disorders associated with de- purine synthesis even further. Such an ficient HGPRTase activity have been de- acceleration would in turn tend to increase scribed (Kelley et al., '67; Kelley et al., the cellular concentration of inosinic acid. '69). In these conditions, the enzyme de- A third and even more surprising nutri- ficiency is less complete than in the Lesch- tional feature of this strain of cells is that Nyhan Syndrome. Clinically, the patients their growth is very significantly stimu- are characterized by hyperuricemia, gouty lated by high concentrations of hypoxan- arthritis, and in some cases evidence of thine - the substrate of the affected en- mild spinocerebellar disease. The clinical zyme. There is, on the face of it, no features and the enzyme deficiencies are immediate explanation for this paradoxi- familial, and have thus far been reported cal result; one might argue that the af- only in males. The family data are con- finity of the enzyme for substrate has been sistent with X-linkage of the relevant locus altered by a structural mutation, so that or loci. Studies of the heat-stability of the abnormally large quantities of substrate HGPRTase activities recovered from af- are required to saturate the enzyme. Al- fected patients suggest that the enzyme ternatively, the fact that hypoxanthine molecules are abnormal, and that the ab- stimulates purine synthesis, in at least one normality differs from family to family. mutant strain, might be the explanation These less complete enzyme deficiencies (Rosenbloom et al., '68). can be detected in cultured fibroblasts In the case of a severe sex-linked dis- (Kelley et al., '69). In addition, fibroblasts ease, like the Lesch-Nyhan Syndrome, from two male, gouty patients - who are patients from different families may not not members of the same family - appear always be comparable. If every patient to be relatively insensitive to feedback with the disease fails to reproduce, one- inhibition by exogenous purines (Hender- third of the genes will be eliminated at son et al., '68). Cells from these patients each generation. The reason for this is do not decelerate purine synthesis, as that all the mutant genes are on X-chro- readily as normal cells, when incubated in mosomes, and approximately one-third of a purine-containing medium. Yet the cells X-chromosomes are in males. Therefore, formed purine nucleotides from hypoxan- one-third of the mutant genes will be in thine at a normal rate, implying that males, and the males will presumably be HGPRTase was not deficient. Hence, it is eliminated by natural selection. Now if the population is in equilibrium with respect to the frequency of the disease, one-third MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 319 conceivable that in one or both of these thesis. This anomaly in turn has proven, patients the amidotransferase - the first in each case, to be due to a deficiency or enzyme of the synthetic sequence - is abnormality of an enzyme which fibroblasts abnormal. If indeed the amidotransferase contain. is affected, the abnormality would pre- sumably be one which diminishes the c. Orotic acidziria inhibitory effect of purine ribotides on the Orotic aciduria is a rare autosomal re- catalytic activity of the enzyme. cessive disorder which is characterized by It is of interest in this connection that failure to grow, pancytopenia, and the ex- there are at least three different diseases cretion of extremely large quantities of which lead to an overproduction of uric orotic acid (Smith, Huguley and Bain, '66). acid and which can be recognized and Diploid cell strains from affected persons studied in cultured human fibroblasts - a (Krooth, '64b; Howell, Klinenberg and type of cell that in fact lacks xanthine Krooth, '67) have about 1% of normal oxidase activity and therefore cannot syn- activity for the final two sequentially act- thesize uric acid. The reason for this sur- ing in the synthesis of uridine-5'- prising state of affairs is that in these monophosphate (UMP). The enzymes (fig. diseases the more basic lesion is an ab- 2) are orotidine-5'-monophosphate (OW) normally rapid rate of de nwo purine syn- pyrophosphorylase and OMP decarboxyl-

COOH COOH I I \ ASPARTATE TRANSCAR BAMYLASE- D IHYDROOROTASE

PHOSPHATE co COOH AS PARTATE H

H D IHYDROGROTI C ACID

0 IHY DROOROTI C I DEHYDROGENASE OH

OROTlDlNE -5: MONOPHOSPHATE Nc\ [OROTIDINE -$ MONOPHOSPHATEl DECARBOXYLASE N CH PYROPHOSPHORYLASE -- I II MG++, PRPP i II co C-EOOH

\N/ I H OROTIC ACID Ribose - 5 - Monophosphate Ribose - 5 - Monophosphate URlDlNE -5'- MONOPHOSPHATE OROTlDlNE -5'- MONOPHOSPHATE

IUMP) (OMPI Fig. 2 The pathway of de novo pyrimidine synthesis in human diploid cell strains. 320 ROBERT S. KROOTH AND ELIZABETH K. SELL ase. The other enzymes peculiar to the inhibits OMP decarboxylase - the final purine pathway appear to be unaffected enzyme of UMP synthesis (Cihak and by the gene (Krooth, '64b; Howell, Klinen- Sorm, '67; Cihak, Vesely and Sorm, '68; berg and Krooth, '67; Smith, Sullivan and Ma and Krooth, '70j. Huguley, '61; Wuu and Krooth, '68). He- When cells are grown in the presence terozygous persons have only 20% of nor- of one of these inhibitors and the mal activity for the two affected enzymes of the pathway, as uridine or cytidine, the in their erythrocytes (Smith, Sullivan and inhibitor has no effect on growth. However, Huguley, '61; Fallon et al., '63). Heterozyg- the cells increase their specific activity for ous cell strains also have intermediate the final two enzymes of the sequence but levels of activity, although the value, as a not for at least two of the enzymes which act earlier (Pinsky and Krooth, '67a,b; percentage of normal, is somewhat vari- Wuu and Krooth, '68). The increase in able (Krooth, '64). specific activity is frequently over 100-fold Diploid cell strains from affected per- in the case of mutant homozygous strains sons will grow in a minimal medium, but (Krooth, '69b), which develop heterozyg- their growth is stimulated if the medium ous or normal levels of activity for both contains uridine or cytidine (Krooth, '64b). affected enzymes. In the case of normal However, if a purine or purine riboside is and heterozygous cells, the absolute level added to the medium, the mutant homozyg- of increase in specific activity for the two ous cells cease growth after about 72 enzymes is somewhat greater than in the hours, whereas normal or heterozygous case of mutant homozygous cells, but the cells are unaffected. The growth of the mu- proportionate increase is considerably less tant homozygous cultures is not inhibited (Krooth, '69b). by purines or purine ribosides if the me- The mechanism by which these inhibi- dium also contains uridine or cytidine tors stimulate cells to develop increased (Krooth, '64b). It can be shown that in levels of enzyme activity is unknown. How- the presence of purines or purine ribosides ever, the present evidence suggests that the specific activity of the cell protein for they act, at least in part, by causing an at least one of the two enzymes (OMP de- intermediate in the pathway - perhaps carboxylase) affected by the gene is re- orotic acid - to accumulate inside the duced by about three-fold in mutant homo- cell. This intermediate, in turn, is likely zygous strains (Krooth, '64b). The specific to be the active molecule which induces or activity of the cell protein for this enzyme stabilizes the two enzymes affected by the is not affected by purines or purine ribo- gene (Krooth, '69b, '70). In Saccharomyces sides in the case of normal and heterozyg- ceversiae, the final three enzymes of the ous cells. sequence are induced or stabilized by the When cell strains of any genotype are substrate of the first of these enzymes - grown in the presence of certain inhibitors dihydroorotic acid (Lacroute, '68). of UMP synthesis, they develop increased The reason two enzymes are affected in levels of activity for both the enzymes af- orotic aciduria is not known. In one re- fected by the mutant gene (Pinsky and ported patient, only the final enzyme is Krooth, '67a,b; Wuu and Krooth, '68; affected (Fox, OSullivan and Firkin, '69). Krooth, '69b, '70). The inhibitors are bar- However, the other cases have invariably bituric acid, 5-azaorotic acid, and 6-azauri- been deficient in both OMP pyrophos- dine. The first two of these compounds are phorylase and OMP decarboxylase. One specific and competitive inhibitors of, re- possibility is that the two catalytic activi- spectively, the third and fourth enzymes ties reside in a single protein. However, of the UMP sequence.' The metabolic de- this seems unlikely, for at least in the case rivative of 6-azauridine, 6-azauridine-5'- of calf thymus, the activities can be sepa- monophosphate, is a competitive inhibitor rated by starch gel electrophoresis (Kasbe- of the final enzyme of the sequence (OMP kar, Nagabhushanam and Greenberg, '64). decarboxylase). It now seems likely, how- Among the other possibilities would be ever, that all these compounds, not just 6-azauridine, are converted by the cell to 8 These enzymes are dihydroorotic acid dehydrogen- ase and orotidine-5'-monophosphate pyrophosphorylase the corresponding ribotide which in turn (fig. 2). MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 32 1 that the two enzymes are encoded in a low levels of the halenzymes of the UMP single operon (Fallon et al., '63; Krooth, sequence. Indeed, there are reasons for '64) - if indeed such genetic units exist suspecting that some differentiated mam- in eucaryotic cells. Alternatively, the two malian cells may normally liberate inter- enzymes might each contain an identical mediate for the use of the cells around subunit whose structure was affected by them (Krooth, '69b). the mutant gene. A fourth, and in some ways attractive possibility, is that the two d. Xerodemza pigmentosa enzymes are normally aggregated into a Xeroderma pigmentosa (Rook and Wells, multimolecular complex. Such complexes '68) is an autosomal recessive disease uniting the enzymes of a single metabolic characterized by photophobia, hypertrophic sequence, have frequently been observed changes in the skin, and frequently skin in eucaryotic cells (Reed and Oliver, '68; cancer. AfEected persons develop unus- Gross, '69; Fink, '70). In these organisms, ually severe freckling and other disagree- a mis-sense mutation in one enzyme some- able skin changes when they are exposed times affects the catalytic activity of the to sunlight. Dermatology textbooks (Lewis, complex for a number of enzymes (cf. '52) state that the destructive wavelengths Fink, '70). The advantage of such com- for these patients are in the ultraviolet plexes for the cell is believed to be related range. to the fact that the complexes are theoreti- Diploid cell strains from persons who are cally capable of channeling metabolic in- mutant homozygous are unusually sensitive termediates. Intermediates can be directed to ultraviolet light (Gartler, '64). Bio- towards subsequent enzymes in the metab- chemical studies reveal that, following UV- olic sequence, which form part of the ag- exposure, the mutant homozygous cell cul- gregate, rather than released after each tures release an abnormally small propor- reaction to diffuse throughout the cell tion of their total DNA into the acid-soluble (Davis, '67; Reed and Oliver, '68; Gross, cellular pool (Cleaver, '68; Setlow et al., '69; Fink, '70). In the case of human cells, '69). It is possible, therefore, that the precursors in the UMP pathway might tend mutation affects an endonuclease which to stabilize an aggregate containing the excises segments of DNA containing thy- final two enzymes of the sequence; the mine dimers (Setlow et al., '69). Photore- effect might be particularly pronounced if activation - or "dark repair" - involving the aggregate were abnormally unstable the chemical repair of thymine dimers in because of the inclusion of a mutant en- situ does occur in mammalian cells zyme among its component molecules. (Painter, '70). Hence, it is almost certain Another possible pathogenesis for orotic that in this disease, the mechanism of aciduria, in which we have been especially excision repair is disturbed. interested, is as follows: As noted above, This disorder, incidentally, had until re- the evidence suggests that cells of all geno- cently been considered a skin disease al- types develop increased levels of activity though in some patients, who have had for the two enzymes affected by the gene extensive exposure to sunlight, the cornea in response to the accumulation inside the and other external tissues of the eye be- cell of an intermediate in the pathway. The came involved. The cell culture studies mutant gene might, therefore, act by af- suggest that the skin is primarily affected fecting a cellular enzyme which normally because it is the only tissue normally ex- confers upon the cell the ability to retain posed to ultraviolet light. an intermediate (Krooth, '70). Such en- zymes, affecting intermediates in the UMP e. The single active X-theory pathway, apparently exist in eucaryotic The single active X-theory (Lyon, '68) forms (Lacroute, '68). On this view, the in its simplest form is based on the follow- deficiency in the two enzymes of UMP syn- ing premises : thesis would not be the primary biochemi- 1. Each female mammalian zygote con- cal lesion. Rather, the diffusion of inter- tains two X-chromosomes - one derived mediate out of the cell would lead to from the father and the other from the pyrimidine starvation, and, secondarily, to mother. 322 ROBERT S. KROOTH AND ELIZABETH K. SELL

2. Early in development one X-chromo- ments have used a total of five sex-linked some in each female cell becomes inactive. genes which are probably at three different The inactive X does not function, although loci. In every case so far, at least the it continues to replicate when the cell second prediction has been met. When divides. tested, the first prediction was also satisfied 3. The maternal and paternal X-chromo- in one case, and an ambiguous result was some have an equal probability of being the obtained in another. It has also been shown inactive one in any given cell. that the second prediction is not met in 4. The particular X-chromosome inacti- the case of five autosomal genes tested. In vated in a cell continues to be inactive in other words, clonal cell populations from all the descendants of that cell. females heterozygous for each of these These premises generate two predictions genes displayed the heterozygous pheno- about the behavior of diploid cell strains type in every case. The mutant genes were developed from females who are heterozyg- the ones causing electrophoretic variants ous for X-linked genes. of phosphoglucomutase (Davidson et al., '1. Mass cultures should consist of two '65; Sigman and Gartler, '66), lactic acid cellular populations. One population of dehydrogenase (Davidson et al., '66) and cells should resemble the wild-type and 6-phosphogluconic acid dehydrogenase the other should resemble cells from mu- (Davidson et al., '66); the other mutant tant homozygous females or from mutant genes were the ones causing branched hemizygous males. chain ketonuria (Sigman and Gartler, '66), 2. When mass cultures are cloned, two and Hurler's syndrome (Danes and Beam, types of colonies should be recovered in '67a). about equal proportions : colonies whose The data described above are, in aggre- cells have the mutant phenotype and colo- gate, the strongest evidence yet collected nies whose cells have the normal pheno- to show that the single active X-theory is type. There should not be any mixed true, or at least that it applies to cultured colonies. diploid human cells for the loci studied. The second prediction seems, on the Whether the inactivation is absolutely face of it, to be more strict than the first. complete and whether it applies to all loci However, the first prediction contains two are questions which are presently under assumptions which are not included in the study in a number of laboratories. second prediction. The first assumption is that one has a method - say autoradiog- Some of the uses of genetically raphy or histochemistry - for determining marked human diploid the phenotype of individual cells. The cell strains second assumption is that cellular pheno- Genetically marked human cell strains, type is autonomous in the sense of not like genetically marked strains of E. coli, being influenced by the phenotype of have proven useful in the study of genetic other cells which are adjacent or in the change. The human diploid strains have same container. Both predictions assume been employed to demonstrate somatic cell that neither of the two cell types postu- hybridization (Weiss and Green, '67; lated by theory has been eliminated by Silagi, Darlington and Bruce, '69; Sinis- selection in vivo or in vitro. This latter calco et al., '69), the latter report showing possibility means that finding only one kind that two such strains will hybridize with of cell in mass culture or only one kind one another. Hopefully, a way will eventu- of colony after cloning does not necessarily ally be found to use human-human somatic invalidate the theory. Finding both pheno- cell hybrids for obtaining information types in a single clone is disastrous for about genetic linkage in man. Thus far, the theory. The two predictions are critical, only mouse-human somatic cell hybrids of course, only if one does not find a di- have been employed for this purpose (Boon morphism in cell strains developed from and Ruddle, '69; Klebe, Chen and Ruddle, hemizygous males or homozygous females. '70; inter aliae). Table 2 summarizes the work thus far Gene mutation also appears to have been done to test these predictions. The experi- detected in human diploid strains (Alber- MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 323

TABLE 2 Experimental tests, using heterozygous human diploid cell strains of the single active x-theory

Results

Predic- Predic- Predic- Predic- Mutant phenotypes tion 1 tion 2 (cellular (clonal tion 1 tion 2 .dimor- dimor- phism) phism) Davidson, Nitowsky and Glucose-6-phosphate dehydrogenase Childs deficiency (Mediterranean variety) ('63) Davidson, Nitowsky and B-Variant of glucose-6-phosphate Childs dehydrogenase ('63) Madison variant of glucose-6-phosphate DeMars and dehydrogenase - + - Nance ('64) Danes and Danes and Hurler's syndrome (tissue culture Beam Beam phenotype: cellular metachromasia) ?3 + ('66 1 ('67) Lesch-Nyhan syndrome (tissue culture phenotype: absence of hypoxanthine: guanine phosphoribosyltransferase and failure of individual cells to incor- Rosenbloom Migeon porate 3H-guanineor 3H-hypoxanthine et al. et al. into acid-insoluble material ) + + ('67) ('68) ~~ ~ 1 The definition of the symbols is as follows: +, denotes prediction was found to apply to the speci- fied gene; 0, denotes prediction was found not to apply to the specifled gene; -, denotes prediction has not yet been tested. 2 A histochemical method for determining cellular phenotype, with respect to this gene, has recently been developed by Wajntal and DeMars ('67). Hence, it should be possible to test Prediction 1 in this case almost at once. 3The results for this locus are difficult to interpret. About half the cells of female heterozygotes have the metachromatic granules characteristic of the mutant hemizygous genotype. However, cells from one hemizygous male showed a si.milar dimorphism in mass culture, which seems to vitiate the theoretical significance of the observation on female cells. Nearly all the cells of two other hemizy- gous males who were studied contained the granules. tini and DeMars, '70). It seems likely that nancy usually yields a diploid strain of other phenomena, such as transduction fetal origin. This strain can then be used, and DNA-mediated genetic transformation, in the case of many loci, to infer the bio- will eventually be demonstrated with these chemical phenotype of the fetus (Jacobson cells. and Barter, '67; Nadler, '69; Nadler and The potential value of human diploid Gerbie, '69; Littlefield, '70; Emery, '70; cell strains in genetic analysis is very great, inter aliae). If the cellular phenotype in- partly because the biology of the cells sug- dicates that the fetus is abnormal, the par- gests that they are more likely than hetero- ents may wish, in certain circumstances, nuclear cells to reveal the normal proper- to have the pregnancy aborted. In some ties of the human cell, and partly because states, abortion on these grounds is legally of their chromosomal stability. However, permissible. Antenatal diagnosis of bio- the main advantages of diploid cell strains chemical disorders has been used primarily for genetic analysis arise from the fact in cases where a high risk of a severe that the strains can be isolated from vir- genetic abnormality is inferred either tually any donor. from previous pregnancies in the family or Perhaps the most dramatic present use from the results of studies on the somatic of diploid cell strains is in the antenatal tissues of the parents. diagnosis of hereditary disease. The culti- This technique represents an unusual vation of the cells recovered from amniotic application of cell genetics to clinical fluid during the first trimester of preg- medicine, and is likely to be widely prac- 324 ROBERT S. KROOTH AND ELIZABETH K. SELL ticed in the years ahead. Its social, moral stances, it may even be sufficient to en- and genetic implications are already the able one to determine the amino acid se- object of considerable discussion. quence of the protein. Hence, it should be Another possible use of genetically possible in some cases to determine pre- marked diploid cell strains is the study of cisely what is wrong with the affected en- complementation. By the technique of zyme. Fortunately, most of the therapeutic somatic cell hybridization or simply by the methods now in use in the treatment of formation of heterokaryocytes, it should genetic diseases do not require such de- sometimes be possible to determine tailed information about the affected en- whether two human mutations, affecting zyme. However, as our approaches to the same enzyme or cellular process, will therapy become more subtle and powerful, complement (Velazquez, '70). Information that situation could change. on this point may occasionally be useful in genetic counselling. It should also help Problems for the future considerably in understanding the precise In this review, we have been mainly mode of action of individual human genes. concerned with gene action in human Cultured homonuclear cells have several diploid cell strains. The cells of such other potential uses in diagnosis. For one strains, like all normal somatic cells, are thing, it is feasible to grow cells from a presumed to contain the complete human patient with an unknown metabolic defect genome. Yet only a fraction of the loci in both minimal and complete tissue cul- are expressed. If we could compel the cul- ture media (Krooth, '66b). If the cells grow tured human cell to display its phenotype in the complete medium but not in the at any specified locus in response to a de- minimal one, it should then be possible fined signal, we would be in a far better (in at least some cases) to infer which position to apply the technique of cell cul- molecule the cells need. This can be done ture to both scientific and practical prob- by testing the known molecules which the lems. complete medium contains and the mini- For example, we could greatly increase mal medium does not - a procedure that the number of loci to which the method is commonplace in microbial genetics. If of antenatal diagnosis might be applied. the molecule is identified, the result will Even more important, perhaps, the clini- suggest not only the probable metabolic cian would have a way of evoking the ap- location of the biochemical lesion but also pearance in cuItured fibrobIasts of en- the appropriate therapy. In actual fact, zymes normally found only in inaccessible however, no new genetic abnormalities organs such as brain or liver. It is not al- have been clearly diagnosed in this way ways easy to defend - morally and legally though several attempts have been made - the biopsy of an intra-abdominal or in- (Krooth, '66b). This approach to diagnosis tra-cranial structure for genetic studies. is likely to be worth exploring further. Even if the tissue is removed for the pur- Another use of cultured human cells is pose of diagnosis on the person biopsied, related to the fact that they can be grown it would be better to assay the enzyme or on a large scale. Today it is quite feasible protein in cultured cells from a skin speci- (though neither easy nor cheap) to pro- men than to remove tissue from an inter- duce between 300 and 600 gm of cells nal organ - a procedure that is always from only a minute biopsy. Clearly, most associated with some risk. Moreover, once patients, especially pediatric ones, would the clinician has the fibroblasts he can do be in no position to forfeit so much tissue microbiological studies which would not for diagnostic purposes. This quantity of be feasible with a whole patient or a frag- cells should yield between 50 and 100 gm ment of tissue. Hence, the number of of protein - all presumably biosynthesized hereditary diseases that can be studied by according to information the patient in- the microbiological methods would be herited. One hundred grams of protein greatly increased. In addition, cell lines should be sufficient to purify to homogene- which synthesized many human tissue- ity many enzymes of the cell, and to ana- specific molecules would probably prove lyze their tryptic peptides. In a few in- extremely useful in the manufacture of MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 325 pharmaceuticals (Tashjian, ’69) such as been found in cultured human fibroblasts growth hormone for which there is at pres- (Watts, Watts and Seegmiller, ’65; Seeg- ent no satisfactory natural source. Finally, miller, ’69). Extremely low levels of this it would be extremely valuable to be able enzyme can be detected in Aspergillus to study the molecular events that occur nidulans. In this organism, however, cells when a cell is stimulated to initiate the can be induced to develop high levels of synthesis of a tissue-specific protein it has xanthine oxidase by uric acid, the product not previously synthesized. A study of these of the enzyme (Scazzochio and Darlington, events might well reveal much about the ’68). Indeed, the control of this enzyme in molecular mechanism which underlies cell Aspergillus appears to resemble one of the differentiation. “circuits” suggested by Monod and Jacob Of course, if we thoroughly understood (’61) for the regulation of enzymes in dif- the mechanism for cell differentiation in ferentiated cells. Dr. Brunschede is pres- the first place, we would probably be able ently attempting to determine whether to guess the chemical nature of the stimuli very high intracellular levels of uric acid which are capable of activating unex- will induce xanthine oxidase in human pressed loci. In the absence of such knowl- diploid cell strains. edge, how can we try to identify the sig- A second approach involves the tech- nals to which the cell will respond? First, nique of somatic cell hybridization. In sev- of course, one must postulate that chemical eral reported experiments, hybrids have stimuli of this kind exist and can be identi- been formed between a cell line which per- fied. Given those assumptions, however, forms a known tissue-specific function and how does one proceed in the absence of a cell line which does not perform that any further information? function. In every instance, the hybrid We should like to summarize from two clones have failed to perform the func- approaches that we have been interested in. tion studied (Davidson, Ephrussi and The first approach is to select a tissue- Yamamoto, ’66; Finch and Ephrussi, ’67; specific enzyme which has the following Sonnenschein, Tashjian and Richardson, properties : ’68). Hence, it has been argued that the 1. The catalytic activity is normally cell line which fails to perform the tissue- absent from human diploid cell strains. specific function may synthesize a diffus- 2. The catalytic activity is normally ible “repressor” of the function (Davidson absent - or present in only trace amounts and Yamamoto, ’68). Certainly, this possi- - in the cells of a eucaryotic fungus (pre- bility is the simplest (but by no means the ferably a higher filamentous fungus). only) explanation for the fact that tissue- 3. The catalytic activity is inducible in specific functions have so far acted like the fungus by a known molecule. “recessive” attributes in somatic cell The experiment then is simply to chal- crosses, There are now methods which lenge the human diploid cells with the in- should work for back-crossing a hybrid cell ducer identified for the fungus cells. By line (Darlington, ’70) against the parental challenge, I mean not only to expose the line which performs the tissue-specific human cells to the fungal inducer but to function. Moreover, it should be possible ensure that the inducer gets inside them. to repeat the process - again to back-cross It is not unreasonable to suspect that the hybrid which resulted from a back- eucaryotic cells capable of differentiating cross against the cell line which performs may respond similarly to the same stimu- the tissue-specific function. If the process lus. can be repeated for a sufficient number of Dr. Horst Brunschede’ and one of us cycles, it may be possible to “titrate” the (R. S. K.) have noticed that the enzyme theoretical repressor by introducing into xanthine oxidase satisfies the three criteria the hybrid cell successive doses of those listed above. Catalytic activity for this en- genes which cause the cell autonomously zyme is found in appreciable amounts only to perform the tissue-specific function. in liver and gastrointestinal mucosa Eventually - if the idea of a repressor is (Watts, Watts and Seegmiller, ’65). No ac- 9 Of the Department of Human Genetics, University tivity, even with very sensitive assays, has of Michigan Medical School. 326 ROBERT S. KROOTH AND ELIZABETH K. SELL correct - the hybrid might perform the Human diploid cell strains, biochemi- tissue-specific function. Such an nth order cally marked by one or more mutant Men- hybrid cell could be a most useful reagent. delian genes, have proven particularly use- If the cell indeed performed the function, ful for the study of gene action in man and then it might prove possible to cross the for the detection of genetic changes such n”‘ order hybrid (or a higher order one) as mutation and somatic cell hybridization. with a diploid cell strain from any random In addition, the strains have a number of donor and obtain a line which made the clinical applications, including the ante- tissue-specific product biosynthesized ac- natal diagnosis of inherited disease. cording to the genome of the donor of the The failure of cultured human cells to strain. Such a line would of course also display their phenotype at most loci con- synthesize the product according to the tinues to restrict their use in both genetics genome it contained prior to being crossed and medicine. There are reasons for hop- with the diploid strain. However, if the nth ing that this difficulty will eventually be order hybrid were formed from a non- solved, and some experiments bearing on human species, it should in many cases be the problem are already feasible. possible chemically to resolve the two prod- LITERATURE CITED ucts. Alberini, R. J., and R. DeMars 1970 Diploid If the above experiment is to work, cer- azaguanine resistant mutants of cultured hu- tain assumptions must be true. The failure man fibroblasts. Science, in press. of the hybrid to perform a tissue-specific Bayreuther, K. 1964 Technical problems. In: function must indeed be due to a “diffus- Somatic Cell Genetics. R. S. Krooth, ed. Fourth Conference on Genetics. Josiah Macy, Jr. ible” repressor - a repressor that acts on Foundation. Univ. of Mich. Press, Ann Arbor, the genome that originally contained it as Michigan. 301 pp. well as on the genome that did not. The Boone, C. M., and F. H. Ruddle 1969 Inter- number of repressor molecules per cell specific hybridization between human and must be relatively small. It would not be mouse somatic cells: enzyme and linkage studies. Biochem. Genet., 3: 119-136. feasible to try to make 10,000 successive Brockman, R. W.,G. G. Kelley, P. Stutts and V. back-crosses. Other assumptions are also Copeland 1961 Biochemioal aspects of re- necessary. sistance to 6-mercaptopurine in human epider- However, the point of this discussion is moid carcinoma cells in culture. Nature, 191: 469-471. that experiments aimed at developing lines Choi, K. W.,and A. D. Bloom 1970 Biochemi- of cultured cells which synthesize tissue- cally marked lymphocytoid lines: establishment specific products according to the genomes of Lesch-Nyhan cells. Science, in press. of individual human donors are already Cihak, A., and F. Sorm 1967 Metabolic trans- formation of 5-azaorotale: cause of marked in- feasible. The potential value of such lines hibition of orotidine-5’-monophosphate decar- if they could be developed is enormous. boxylase. Biochim. Biophys. Acta, 149: 314-316. The initial difficulty in the use of hu- Cihak, A., J. Vesely and F. Sorm 1968 Inhibi- man diploid fibroblasts for genetic studies tion of pyrimidine biosynthesis by 5-azaorotate in mouse liver. Collection Czechoslov. Chem. was the failure of the cells to display their Commun., 33: 1778-1787. phenotype at most loci. Despite the remark- Cleaver, J. 1968 Defective repair replication of able discoveries of the past ten years, this DNA in xeroderma pigmentosum. Nature, 218: difficulty persists. Its solution - if it can 652-656. be solved- would be an enormous ad- Conover, J. H., P. Hathaway, P. R. Glade and K. Hirschhorn 1970 Persistence of phosphoglu- vance. comutase (PGM) polymorphism in long-term lymphoid lines. Proc. SOC.Exp. Biol. Med., 133: CONCLUSION 750-753. Some of the cells of every human being Danes, S., and A. Bearn 1966 Hurler’s syn- dome: a genetic study in cell culture. J. Exp. will grow outside the body as microorgan- Med, 123: 1-16. isms. It is possible to show, in a variety of 1967 Hurler’s syndrome: a genetic ways, that these cells resemble genetically study of clones in cell culture with particular the individual from whom they were ob- reference to the Lyon hypothesis. 3. Exp. Med., 126: 5Q9-522. tained. Over 35 inherited human diseases Darlington, G. A. J. 1970 Studies of mixed cul- and anomalies can now be studied in such tures of human somatic cells. Ann Arbor, cell lines. Michigan, University of Michigan. Ph.D. Thesis. MENDELIAN GENES IN HUMAN DIPLOID CELL STRAINS 327

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