Chapter 1 7

Rapid Biochemical Screening of Large Nzlmbers o f Animal Cell Clones MICHAEL BRENNER,' RANDALL L. DIMOND,2 AND WILLIAM F. LOOMIS

I. Introduction

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11. Mutagenesis and Mutation Frequency 111. Screening . . . . . IV. ClosingComments . . .

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1. Introduction Analysis of mutant strains has consistently played a key part in deciphering metabolic patterns operating in simple organisms such as bacteria and yeast. The use of mutants in studies of more complex organisms has been limited, however, primarily because of the difficulties involved in obtaining the desired mutants. Many of these difficultiescan be eliminated by working with animal cells in culture rather than with whole organisms. But even so, only in certain instances has it been possible to devise selection procedures for mutant isolation (for reviews, see Basilico and Meiss, 1974; Kao and Puck, 1974; Naha, 1974; Shapiro and Varshaver, 1975); many mutant types still cannot be isolated by selective techniques because the function of the gene of interest is unknown, or because, as for most developmental functions, the activity of the gene is not important for the maintenance of the cell in culture. In these latter cases mutants can be conveniently obtained only

' Biological Laboratories. Harvard University, Cambridge, Massachusetts. 'Department of Biology, MassachusettsInstitute ofTechnology, Cambridge, Massachusetts. 'Department of Biology, University of California, San Diego, La Jolla, California. 187

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if efficient screening methods are available. In a recent article(Brenner et al., 1975) we presented a procedure that should help meet this need in many cases. We described the construction and operation of a “multipipette” which can be used to clone cells into plastic multitest trays having 96 individual wells. Using this simple apparatus a team of two persons can fill more than 400 trays in an hour (40,OOO wells). Once clones have grown in the wells, the multipipette can be used to add reagents for colorimetric enzyme assays. We have used this procedure successfully to screen for several developmental mutants of the cellular slime mold Dictyostelium discoideum (Dimond et al., 1973, 1976; Dimond and Loomis, 1976; Free and Loomis, 1974). In this chapter we discuss more fully the application of this methodology to obtaining mutants of animal cells.

11. Mutagenesis and Mutation Frequency In order for mutants to appear with a sufficient frequency that they may be isolated by screening procedures, it is necessary to mutagenize the cells. In our work with Dictyostelium discoideum, we found that mutagenizing to a survival rate ofO. 1%yielded gene-specificmutants with a frequency of about lo-’ (Dimond et al., 1973; Loomis et al., 1977). Since this organism has a small DNA content and is haploid, one might question whether gene-specific mutations may be obtained with a similar frequency in more complex diploid animal cell lines. Fortunately, however, neither the DNA content nor the diploid state of animal cells influences the frequency of mutations that will be found for a given level of survival; the only relevant parameter is the number of genes essential for cell viability. For example, suppose that 104 genes are essential for cellgrowth. [There are nodataavailable on the number of genes essential for viability of any animal cell line. Genetic arguments have been made to suggest that for some higher animals (e.g., man) the number of genes essential for the entire organism is on the order of lo5 (reviewed in Lewin, 1974). The estimate here of 104 genes essential for a single cell type therefore seems reasonable, especially in view of the fact that many simple eukcaryotes, and even nematodes and Drosophila survive with no more than 5 x lo3genes (Bishop, 1974; Brenner, 1974).] From the Poisson equation one can calculate that mutagenesis to a survival rate of yields an average number of6.9 lethal mutations per cell. [This assumes that killing is due to mutational events. This assumption led to reasonably accurate estimates for mutation frequencies in our studies of D. discoideum using N-methyl-N‘-nitro-N-nitrosoguanidine (Dimond et al., 1973; Loomis et al., 1977), but may not hold for other mutagens or other cell systems

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(Hsie et al., 1975).] Thus the mutation frequency per gene is 6.9/104. If all genes are mutated independently and at the same frequency, then among the surviving clones any given nonessential gene will be mutated also with a frequency of 6.9 x Hence one mutant of interest should be isolated from about every 1500 clones screened. If, as is commonly supposed, expression of recessive mutations in diploid cells requires two independent mutational events, mutations leading to cell death would be primarily dominant mutations, or recessive mutations in genes present in only one functional copy per cell, such as on X chromosomes. Therefore the mutation frequency calculated above would refer primarily to these two classes of genes, and only these classes would yield mutations which could be readily isolated by the screening procedure. However, in the several cases where the effect of gene dosage on mutation frequencies has been determined, additional copies of a given recessive gene have decreased its mutation frequency by less than 1% of that predicted (Chasin and Urlaub, 1975). A suggested explanation for this departure from expectation is the occurrence of mitotic crossing over (Chasin and Urlaub, 1975), so it may be advisable to grow the mutagenized cells for severalgenerations before cloning. Regardless of the actual mechanism operating, however, the practical consequence of this phenomenon is that mutations in recessive genes should occur at about the frequency given above, and so should be present in sufficiently high numbers to permit their isolation by screening procedures. Often mutants are sought that are defective for a particular protein or RNA in order to determine the role of that molecule in some cellular process. These studies are complicated by the use of mutagenesis in that each clone isolated will carry multiple lesions. In the above example, should the cell line have lo5 nonessential genes, mutant clones isolated would on the average carry about 69 mutations. [No direct data are available on the number of genes expressed in animal cell lines. Based on nucleic acid hybridization experiments using messenger RNA the number of genes being translated into protein has been estimated to be up to 3 x lo4 for mouse L cells and HeLa cells (reviewed in Lewin, 1975). In tissues composed of mixtures of cell types, hybridization using total RNA (nuclear and cytoplasmic) has been used to obtain estimates of 4 to 8 x lo4genes being active in mouse liver, kidney, and spleen, and 3 x lo5 in mouse brain (reviewed in Davidson and Britten, 1973). The value of lo5 genes being active in a given cell line seems therefore a reasonable upper limit value.] Caution must be exercised, therefore, in assigning responsibility for any phenotype of a mutant to the gene whose product is being screened. This problem is partially alleviated by obtaining several independent mutants. The probability that one unscreened for mutation present in one strain will also be present

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in another is quite small; it is simply the mutation frequency itself, 6.9 x in the above example. Hence if several independently isolated mutants all display the same phenotype, there is good assurance that the mutation causing change in the gene product being screened is responsible. However, this change need not be in the gene specifying that product, it could be in some other component affecting both the screened property and the phenotypic change, such as a processing enzyme. Thus no causal relationship need exist between the screened product and the phenotype. This problem of pleiotropy can be largely eliminated through use of conditional mutants. Correspondence of the conditionality of the screened product in v i m (e.g., temperature sensitivity of an enzyme), and of the associated phenotype in vivo strongly implies that the alteration in the screened product produces the phenotypic property noted. Conditional mutants are a necessity for obtaining strains defective in essential gene functions.

111. Screening The screening procedure we have developed is particularly well adapted for obtaining mutants defective in enzymic activities which can be assayed colorimetrically; reagents may be added to the colonies with the multipipette, and enzyme activities be determined by visual screening. For multistep assay procedures, as many as five additions may be made using a multipipette that delivers 50-pl drops. As indicated in Table I, a surprisingly large number of enzymes may be assayed colorimetrically. For any specific enzyme and cell type it is necessary to ascertain whether the amount of enzyme produced by the cells in a multitest well is sufficient to permit its assay by the colorimetric procedure. In many cases it may be possible to make the assays extremely sensitive by employing cycling procedures analogous to those of Lowry (1962-1963). Before conducting some of the enzyme assays it may be necessary to wash the cells free of their growth medium. This has not been possible with D. discoidam since the cells adhere poorly to the plastic trays; the firm adherence of most animal cell lines, however, should make washing an easy matter. The trays can probably be immersed in a large container of buffer and the wash liquid shaken or aspirated from the wells (a multipipette with narrow tubing can be used to aspirate the liquid). For some enzyme assays it is necessary to lyse the cells to obtain good activity. This may be done by adding detergent or organic solvents (e.g., Triton X-100, toluene) with the assay mixture. In these cases it will first be necessary to replicate the cells to

17.

RAPID SCREENING OF CLONES

ENZYMES THATCAN

TABLE I ASSAYED COLORIMETRICALLY~

BE

Enzyme class ADP producing enzymes (e.g., kinases) ATP-producing enzymes Aldolases Ammonia-producing enzymes (e.g., deaminases, amidases) Coenzyme A-producing enzymes Dehydrogenases Dihydroxyacetonephosphate-producing enzymes Esterases Glucose, glucose 1-phosphate, or glucose 6-phosphateproducing enzymes Glutamatsprcducing enzymes Glyceraldehyde 3-phosphateproducing enzymes a-Glycerol phosphateproducing enzymes Glycosidases Hydroxylases Nucleotide sugar:Acceptor transferases Nucleotide sugar pyrophosphorylases Oxidases and oxygenases Peroxidases Phosphateproducing enzymes (e.g., phosphatases) Proteases Pyruvateproducing enzymes Reductases Transaminases

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Color reaction Linked to NBTb reduction through pyruvate kinase Linked to NBT reduction through hexokinase and glucosedphosphate dehydrogenase Linked to NBT reduction through NADH Reduction of NBT; linked to NBT reduction through glutamic dehydrogenase; indophenol formation from NH, Formation of 2-nitro-5-mercaptobenzoate from 5,5'-dithiobis(2-nitro benzoate) Reduction of NBT by NADH or NADPH Linked to NBT reduction through a-glycerol phosphate dehydrogenase Many colorimetric substrates, including napthol derivatives; many products can be assayed by the addition of color reagents Linked to NBT reduction through NADPH Reduction of NBT through glutamate dehydrogenase Linked to NBT reduction through a-glycerol phosphate dehydrogenase Linked to NBT reduction through &-glycerol phosphate dehydrogenase p-Nitrophenol, naphthol derivatives, or methylumbelliferyl-linked substrates Reduction of NBT by NADH or NADPH Linked to NBT reduction by pyruvate through nucleotide kinase and pyruvate kinase Linked to NBT reduction through NADH, NADPH, or pyruvate Linked to NBT reduction Guaiacol; di-0-aniside p-Nitrophenyl phosphate; formation of colored phosphomolybdate complex Colorimetric esterase assays; assay released tyrosine with Folin-Ciocalteau reagent Reduction of NBT Reduction of NBT by NADH or NADPH Formation of hydrazones or ninhydrin derivatives of keto acids; linked to NBT reduction through NADH or NADPH oxidation (cont.)

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TABLE I (cont.)

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Enzymeclass

Color reaction

Specific enzymes not necessarily subsumed under the above classes include the following: N-Acetylglucosamine-2-epimerase Glycerol dehydratase N-Acetylglucosamine-6-phosphate deacetylase Glycogen synthetase Guanosine-5’-phosphate pyrophosphorylase Aconitase Hyaluronidase Aldose reductase Imidazolylacetolphosphate: glutamate aminoa-Amylase transferase Arabinose isomerase L-2-Keto-3-deoxyarabonate Arylsulfatase dehydratase Argininosuccinate synthetase Neuraminidase Carbonic anhydrase NAD Kinase Citratecleavage enzyme NAD Pyrophosphorylase Condensing enzyme Ornithine carbamoyltransferase Creatine phosphokinase Phenol sulfokinase Crotonase Phosphodiesterase y -Cystathionase Phosphoribosylformimino-PRAIC Cytochrome oxidase 5-Dehydroquinase ketolisomerase Proline oxidase Dihydrodipicolinic acid Rhodanese synthetase Fructose 1.6-diphosphatase RibuloseS-phosphate-4epimerase Fumarase Glucosamine-6-phosphate Serine transhydroxymethylase deaminase Thiol-disulfide transhydrogenase Glucosamine phosphate isomerase Glutamine synthetase Tyrosinase y -Glutamyltranspeptidase UDP-N-acetylglucosamine2’-epimerase; UDPG-4-epimerase; UDPG: fructose transglucosylase;UDP-Glucuronyltransferase “Culled from various volumes of “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.). Academic Press, New York; and “Methods of Enzymatic Analysis” (H. U. Bergmeyer, 4.). Academic Press, New York. bNBT = nitro blue tetrazolium. It is reduced to form a mauve precipitate by NADH, NADPH, and several keto acids. It can be used to visualizeeither the oxidation or reduction of nicotinamide adenine dinucleotides. It may be possible to increase sensitivity by using the dinucleotide generated in the primary reaction to catalyze reduction of NBT in a second reaction in the presence of an excess of an oxidizable substrate (Lowry, 1962-1963).

a second set of trays. In our prior article (Brenner et al., 1975)we described a replicator that can be used for this purpose. In addition to colorimetric screening, assays employing radioactive substrates may be feasible in some instances; procedures have been described for flushing cells from microtest wells with trichloroacetic acid and collecting the precipitate or supernatent for scintillation counting (Harrison et al.,

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1974). The applicability of this methodology might be expanded by using a suitable buffer to harvest the cell assay mixture followed by batch processing of the supernatant with ion-exchange resin. Finally, screening need not be limited to enzymic activities; for example, morphological variants may also be isolated. One of us (W. F. Loomis) has used cloning of mutagenized muscle cells into multitest trays to isolate mutants defective in fusion (Loomis et al., 1973).

IV. Closing Comments The methodology we describe here allows any clonable cell line to be rapidly and inexpensively screened for mutations. The initial investment for the multipipette, replicator, and microtest plates is small, and all may be reused repeatedly (we resterilize the microtest plates with ethylene oxide, facilities for which are available at most medical schools and hospitals). From our experience with the screening protocol, we estimate it should take under 40 man-hours to process 10,OOO animal cell clones. This includes the time required for mutagenesis, cloning, replicating, washing, adding substrates, and screening. Above we calculated that only about 1500 clones should have to be screened to obtain any desired mutant. Even if this frequency is low by a factor of 10, it would still require but a few man-weeks of labor to obtain the mutants. Thus through this technique it should be possible to greatly expand the range of mutant types that can be isolated for the study of animal cells in culture.

REFERENCES Basilico, C., and Meiss, H. K. (1974). Methods Cell Biol. 8, 1-22. Bishop, J. 0. (1974). Cell 2, 81-86. Brenner, M., Tisdale, D., and Loomis, W. F., Jr. (1975). Exp. Cell Res. 90, 249-252. Brenner, S. (1974). Genetics 77,71-94. Chasin, L. A., and Urlaub. G. (1975). Science 181, 1091-1093. Davidson, E. H., and Britten, R. J. (1973). Q.Rev. Biol. 48, 565-613. Dimond, R. L., and Loomis, W. F. (1976). J. Biol. Qlem. 251, 2680-2687. Dimond, R. L., Brenner, M., and Loomis, W. F., Jr. (1973). Proc. Nutl. A d . Sci. U.S.A. 70,3356-3361. Dimond, R. L., Farnsworth, P..and Loomis, W. F. (1976). Dev. Biol. 50, 169-181. Free, S., and Loomis, W. F. (1974). Biochimie 56, 1525-1528. Harrison, M. R., Thurman, G . B., and Thomas, G. M. (1974). J. Immunol. Methods 4.11-16. Hsie, A. W., Brimer, P.A. , Mitchell, T. J., and Gosslee, D. G. (1975). Somatic Cell Genet. 1. 247-26 1. Kao, F., and Puck, T. T. (1974). Merhods Cell Biol. 8,23-39. Lewin, B. (1974). “Gene Expression,”Vol. 2, pp. 148-149. Wiley, New York.

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Lewin, B. (1975). Cell 4, 77-93. Loomis, W. F., Wahrman, P., and Luzzati, D. (1973). Proc. Natl. A d . Sci. V.S.A. 70, 425-429, Loomis, W. F., Dimond, R. L.. Free, S. J., and White, S.(1977). In “Microbes as Model Systems for Development” (D. O’Day and P. Horgan, 4 s . ) . Marcel Dekker, New York. Lowry, 0. H. (1962-1%3). Harvey Lect. 58, 1-19. Naha, P. M. (1974). Methods Cell Biol. 8, 4146. Shapiro, N. I., and Varshaver, N. B. (1975). Methods cell Biol. 10,209-234.

Rapid biochemical screening of large numbers of animal cell clones.

Chapter 1 7 Rapid Biochemical Screening of Large Nzlmbers o f Animal Cell Clones MICHAEL BRENNER,' RANDALL L. DIMOND,2 AND WILLIAM F. LOOMIS I. Intr...
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