Molec. gen. Genet. 138, 233--242 (1975) © by Springer-Verlag 1975

Isolation of Cold-sensitive Chinese Hamster Cells Rosann A. Farber* and Paul Unrau Division of Genetics, National Institute for Medical Research, Mill Hill, London, England Received March 8, 1975

Summary. Six cold-sensitive variants have been isolated from Chinese hamster ovary cells by the BUdR-visible light selection technique. The properties of one of these lines have been studied in detail. This line stops dividing immediately after a shift from 39° C to 33° C though its doubling time at 39° C is only slightly longer than that of wild-type cells. The rates of DNA and protein synthesis are severely reduced at 33° C, but the rate of RNA synthesis is not significantly different from wild-type cells. This line may be defective in protein synthesis, but the results of sedimentation analysis indicate that it probably has normal ribosomal subunit assembly. Introduction Several groups have reported on the selection of heat-sensitive m u t a n t s from mammalian cells (Naha, 1969; Thompson et al., 1970; Meiss and Basilieo, 1972 ; Scheffier and Buttin, 1973 ; and others). Those which have been character. ized have defects in a variety of functions, including initiation of DNA synthesis (Smith and Wigglesworth, 1973), ribosomal R N A processing (Toniolo et al., 1973) and leucyl-tRNA synthetase (Thompson et al., 1973). I n proearyotes, the isolation of cold-sensitive mutants has provided an additional source of lines defective in essential cellular processes (O'Donovan et al., 1965; Hoffman and Ingraham, 1970; Scotti, 1968; Ginther and Ingraham, 1974; Nikiforov et al., 1974; Waskell and Glaser, 1974; and others); some of these have abnormalities which had not previously been discovered among heat-sensitive m u t a n t s (Ingraham and Neuhard, 1972). Ribosome assembly m u t a n t s occur with a particularly high frequency among cold-sensitive strains of bacteria, probably because this process is strongly temperature-dependent (Guthrie et al., 1969; Tai et al., 1969). Strains with abnormal ribosomal assembly have been found among coldsensitive mutants in Neurospora crassa (Schlitt and Russell, 1974) and Aspergillus nidulans (Waldron and Roberts, 1974a, b); however, the proportion of cold-sensitive mutants having defective ribosomes in these fungi is lower t h a n t h a t in bacteria. Some ribosomal mutants selected by other means in yeast were later shown to be cold-sensitive (Hartwell et al., 1970; Bayliss and Ingraham, 1974). A number of cold-sensitive mutants have been isolated in Drosophila (Mayoh and Suzuki, 1973; Wright, 1973), and a preliminary report suggests t h a t at least one of these m a y have an altered rate of ribosome synthesis (Falke and Wright, 1974). We report here on the isolation of several cold-sensitive variants from a mammalian cell line and on the preliminary characterization of one of them. * Reprint requests to: Clinical Genetics Division, Children's Hospital Medical Center, 300 Longwood Ave., Boston, Massachusetts 02115, U.S.A.

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Materials and Methods Cell Culture. Chinese hamster ovary (CHO) cells (Puck et al., 1958) were provided by Dr. L A. Macpherson, Imperial Cancer Research Fund, Lincoln's Inn Fields, London. Cells were grown in Eagle's basal medium supplemented with 5% foetal calf serum, non-essential amino acids, and antibiotics (penicillin, 100 units/ml; streptomycin, 100 izg/ml; and aureomycin, 50 ~tg/ml). Cells were tested for mycoplasma and found to be free of contamination. Wild-type cells were maintained at 37 ° C and cold-sensitive lines at 39 ° C.

Selection of Cold-sensitive Variants. The procedure used for selection of cold-sensitive lines was an adaption of the BUdR-visible light method as used by Scheffler and Buttin (1973) for obtaining heat-sensitive mutants. Wild-type cells were treated at 39 ° C (permissive temperature) with ethylmethane sulfonate (EMS) for 16 h at a concentration which resulted in 50% survival (150 ~g/ml). They were kept at 39 ° C for 48 h to allow for the expression of mutations (Chu and Mailing, 1968). They were then replated at a density of 5 × 105 cells/ 50 mm Petri dish. The cells were allowed to attach to the dishes overnight and were then placed at the restrictive temperature (33 ° C). After 12 h they were treated with bromodeoxyuridine (BUdR, 15 t~g/ml) and fluorodeoxyuridine (FUdR, 0.2lzg/ml) for 2 4 h ; they were subsequently exposed to long wavelength UV light (365 am) at a distance of 2 inches for 30 rain in phosphate-buffered saline (PBS). The medium was replaced, and cells were returned to 39 ° C for 96 h, by which time numerous colonies were present on the plates. The cells were pooled and replated, and the selective procedure was repeated. Colonies which appeared after the second treatment were picked up from stainless steel cloning cylinders and tested for the ability to grow at 33 ° C. Cold-sensitive lines were cloned in Microtest plates (Falcon) in order to eliminate any contaminating wild-type cells. All subsequent experiments were done on pm~fied subclones. Macromolecular Synthesis. Cells were plated at a density of 5 × 104/50 mm Petri dish (wild-type) or 1 × 10~/30 mm Petri dish (mutant) and were incubated overnight at 39 ° C. They were switched to 33 ° C and pulsed for 30 min at different time points with either of 3 labelled precursors (aH-thymidine, 2 ?Ci/ml, 2 Ci/mmol; ~H-uridine, 2 tzCi/ml, 5 Ci/mmol; or 8H-leucine, 10 ~Ci/ml, 1 Ci/mmol). Incorporation of label under these conditions was linear with time for up to 1 h. At time 0 the rates of incorporation at 39 ° C were determined. The amount of labe ! incorporated into macromolecules was determined by rinsing each plate with PBS containing 100 ~tg/ml of each unlabelled precursor and then adding ice-cold 10% trichloroacetie acid (TCA). The precipitates were scraped off the plates with a silicon-rubber stopper and washed onto Whatman GF/C discs with 3 changes of 10% TCA, 3 of 5% TCA, and 3 of 96 % ethanol. Radioactivity precipitated onto the filters was counted in toluene-based scintillation fluid i n a liquid scintillation counter. At each time point cells from replicate plates were trypsinized and counted with a haemocytometcr. The amount of radioactivity incorporated was corrected for cell number. All determinations were done in duplicate. Sucrose Gradient Sedimentation Analysis o/ Ribosomes. Cells were seeded into bottles and maintained at 39 ° C for 24 h. They were then switched to 33 ° C for 45 h. During the last 21 h they were labelled with SH-uridine (1-3 ~Ci/ml, 5 Ci/mmol) or z4C-uridine {0.05 tzCi/ml, 60 mCi/mmol). The cells were harvested by trypsinization, washed once with PBS and once with 50 relY[ tris(hydroxymethyl)aminomethane (Tris)-HC1, p H 7.8, 12.5 raN[ MgC12, 80 mM KC1 (medium A, Martin e$ al., 1971). They were frozen and thawed 3 times in liquid nitrogen and debris was removed b y centrifugation at 5000 × g at 4 ° C for 10 rain. The supernatants were adjusted to a concentration of 880 mM KC1, and 20 raN[ 2-mercaptoethanol was added (medium B, Martin et al., 1971). Extracts were centrifuged in linear sucrose gradients (15-30% ribonuclease-free sucrose in medium B) in a Beckman ultracentrifuge using an SW56 rotor at 4 ° C and 40 000 rpm for 4x/2 h. 3OS subunits of E. coli ribosomes for use as markers in the gradients were provided by Dr. A . H . Scragg, Division of Biochemistry in this Institute. Two-drop fractions were collected on squares of Whatman 3MM chromatography paper, which were washed twice in 5% cold TCA:and twice in 96% ethanol, dried, and counted in teluene-based scintillant in a liquid scintillation counter.

Cold-sensitive CHO Cells

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Results Selection o/Mutants Cold-sensitive lines were selected b y a BUdR-visible light reverse selection technique (see Materials and Methods). After two cycles of selection, nine colonies were picked from different dishes. Six of these clones failed to grow at 33 ° C. Since these lines were all isolated in a single experiment, it is possible t h a t some or all of them have a common origin; however, they can be categorized into three groups on the basis of their growth properties and degrees of genetic stability. Two of the variants, cs-8 and cs-9, stop growing immediately upon shiftdown to 33°C and have very high reversion frequencies (between 1 × 10 -a and 1 × 10-a). Two others, cs-2 and es-3, undergo one population doubling at 33°C before ceasing growth and have much lower reversion frequencies (less than 2.5 × 10-8). The remaining two, cs-4 and cs-7, stop dividing immediately after they are shifted to 33 ° C and also have lower reversion frequencies. The properties of cs4-D3, a subclone of the cold-sensitive line cs-4, have been analyzed in detail.

Properties o/cs 4-D 3 Growth at Di//erent Temperatures. Fig. 1 illustrates the growth of wild-type CHO cells and the m u t a n t cs4-D3 at 33 ° C and 39 ° C and at intermediate temperatures. The cells were plated into a series of 50 m m Petri dishes and incubated overnight at 39 ° C. They were switched to the appropriate experimental temperature and the numbers of cells in duplicate plates were counted at each time point. The wild-type cells exhibit similar growth rates at all four temperatures. The doubling time at 39 ° C is approximately 101/2 h and at 33 ° C, 14 h. The mut a n t line undergoes very little increase in cell number at 33 ° C, although its doubling time at 39 ° C (approximately 121/2 h) is only slightly longer t h a n t h a t of the wild-type cells. The growth o f cs4-D3 at 33 ° C is not density dependent. Cells plated at densities ranging from 5 × 104 to 4 × 105 per 50 m m dish (2.5 × 10a-2.0 × 10 a cells per cm ~) fail to grow at 33 ° C. The cloning efficiency of cs4-D3 at 39 ° C is 27.3%, relative to 51.2% in the wild-type cells. Viability Loss at 33 ° C. A high proportion of cs4-D3 cells remain viable after incubation at 33°C for several days. Bottles containing equal numbers of cells were removed from incubation at 33°C at daily intervals after shiftdown. One thousand cells were plated into each of two 90 m m Petri dishes, and the plates were incubated at 39 ° C. Plates were Stained with Giemsa after one week, and colonies were counted. As can be seen in Table 1, as m a n y as 83 % of the cells attached to the surface of the bottles after 5 days remained viable. Test ]or Auxotrophy. The cells were tested for their ability to grow in a very rich medium, medium 199 (Morgan et al., 1950) with 10% foetal calf serum, at 33 ° C. They remained cold-sensitive under these conditions, indicating t h a t t h e y are probably not conditional auxotrophs. tCeversion Frequency. Soon after subcloning, cells were plated at a density of 2.43 × 105 per 90 m m Petri dish and incubated at 33 ° C for one week. The plates were stained with Giemsa, and colonies were counted. The frequency of revertants was 9 × 10 -7.

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Table 1. Loss of viability of cs4-D3 at 33° C Day at 33° C

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Karyology. Metaphases of cs4-D3, as well as the other cold-sensitive lines, have been examined. The cells have near-diploid chromosome n u m b e r s , as do t h e p a r e n t CHO cells. Synthesis ol Macromolecules. The rates of synthesis of DNA, RNA, a n d prot e i n i n cs4-D3 a n d wild-type cells, as d e t e r m i n e d b y 3 0 - m i n u t e pulses with labelled precursors, are illustrated i n Fig. 2. The rates of i n c o r p o r a t i o n of all three precursors i n wild-type a n d m u t a n t cells are s h a r p l y reduced after shiftd o w n to 33 ° C. The r e d u c t i o n i n the rate of D N A synthesis of t h e wild-type cells is p a r t i c u l a r l y striking, although t h e level increases w i t h i n a few hours to ap-

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Fig. 2 a - - c . Rates of synthesis of macromolecules at 33 ° C at different times after shift-down, relative to the rate at 39 ° C (time 0). Cells were pu]se-labe]led for 30-m~nute intervals beginning at each designated time point. Each point represents the average of 2 determinations. (al DNA; (b) R N A ; (c) protein, o wild-type; • cs4-D3

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Fig. 3. Ribosomal subunit sedimentation patterns from a mixture of wild-type and cs4-D3 cells grown at 33° C for 45 h and labelled during the last 21 h of that period. Wild-type cells were labelled with 14C-uridine and cs4-D3 with 3H-uridine. Positions of the peaks were determined by comparison with gradients of wild-type cells which were co-sedimented with an E. coli 30S ribosomal subunit marker, o wild-type; • cs4-D3

proximately 60% of the level at 39 ° C. This decreased rate of DNA synthesis is consistent with the results of Saladino and Johnson (1974) on the effect of temperature on DNA synthesis in Chinese hamster cells; they found that the rate at 33.5° C was approximately 50% of that at 39.5 ° C. The wild-type cells behave as if they are undergoing a temperature shock, followed by an adjustment to the low temperature. This initial rapid reduction in the rate of DNA synthesis was observed several times in independent experiments and also occurs in RNA and protein synthesis, although to a lesser extent. A similar observation was made for DNA synthesis upon shift-up of a mouse cell line from 33 ° C to 39 ° C (Sato and Shiomi, 1974). DNA synthesis in the mutant line is almost completely shut off by 24 h at 33 ° C, as would be expected of any cells which survived the selection procedure. The rate of protein synthesis is also decreased in the mutant at the restrictive temperature; within 8 h at 33°C it is reduced to 15-20% of the rate at 39 ° C, compared to a reduction of approximately 50% in the wild-type cells. The relative rate of R N A synthesis is not significantly different from wild-type at 33 ° C over a period of 48 h.

Sedimentation Analysis o/ Ribosomes. Sucrose gradient sedimentation analysis of ribosomal subunits from cs4-D3 was undertaken in order to determine whether this line might be defective in ribosomal subunit assembly. The gradients were run under conditions of high salt to effect a complete dissociation of 80 S ribosomes into 60S and 40S subunits (Martin et al., 1971). ~ig. 3 is an example of a gradient of subunit particles from wild-type cells and cs4-D3, 45 h after shift-down to 33 ° C. There is no evidence for the accumulation of any defective

Cold-sensitive CHO Cells

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subunit precursors, and the ratios of the 60 S to 40 S peaks are not significantly different. The only difference between the mutant and wild-type patterns is in the relative excess of small (4-5 S) RNA from the mutant cells. Discussion

Several cold-sensitive variants were isolated from a Chinese hamster cell line by methods similar to those used by a number of workers from the isolation of heat-sensitive mammalian cell mutants. One of these mutants has been tested for defects in ribosome assembly and appears to be normal. Bacterial ribosomal subunit assembly mutants have altered ratios of 50 S to 30 S subunits and accumulate abnormal precursor particles at the restrictive temperature (Guthrie et al., 1969; Tai et al., 1969). The mammalian cell mutant examined here has a normal ratio of 60 S to 40 S particles and does not contain any abnormal precursors at 33 ° C. Studies on the ribosomes of the remaining cold-sensitive lines are in progress. Ohlsson-Wilhelm et al. (1974) have also published a preliminary report on the isolation of cold-sensitive CHO cells which apparently have normal ribosomes. There are other approaches which may be more productive for the selection of ribosomal mutants in mammalian cells. Resistance to antibiotics which interfere with ribosome function would be one obvious criterion (Davies and Nomura, 1972). Puromycin-resistant variants of mammalian cells have been obtained, but these have not been characterized (Lieberman and Ore, 1959; Harris, 1967); they may have altered permeability to the drug, as do puromycin-resistant frog cells (Metzger-Freed, 1971). Attempts to obtain cycloheximide-resistant mutants are in progress in our laboratory. I t may also be possible to enrich for ribosomal mutants among temperature-sensitives by using high specific activity tritiated amino acids to kill cells which undergo protein synthesis at the restrictive temperature, as suggested by Thompson et al. (1973). Finally, it has recently been shown that a high proportion of revertants of a tRNA synthetase mutant in E. coli have abnormal ribosomes (Wittmann and StSffler, 1974); a similar approach might be used in CHO cells, since a well characterized temperature-sensitive leueyl-tl~NA synthetase mutant exists (Thompson et al., 1973). Although the cold-sensitive line cs4-D3 has no obvious ribosomal defect, it may be mutant in some other aspect of protein synthesis. The principle evidence for this is the reduced rate of incorporation of amino acids into protein at 33 ° C. DNA synthesis is also inhibited, but this behaviour would be expected of a protein synthesis mutant, since DNA synthesis stops in the presence of protein synthesis inhibitors in mammalian cells (Gautschi and Kern, 1973). It is difficult in the ease of es4-D3 to determine whether protein synthesis inhibition precedes that of the DNA, since wild-type cells also undergo a rapid reduction in the rate of macromolecular synthesis after shift-down to 33 ° C. It is perhaps surprising that the rate of I~NA synthesis is not significantly different from wild-type in this mutant at the restrictive temperature, since RNA synthesis has been shown to be coupled to protein synthesis in mammalian cells in the presence of protein synthesis inhibitors (Willems et al., 1969; Craig and Perry, 1970) and under conditions of amino acid or serum deprivation (ttershko et al., 1971; Fan et al.,

240

R.A. Farber and P. Unrau

1973; Morhenn et al, 1974). I t is possible t h a t a decrease in the rate of protein synthesis to approximately 40% of the wild-type level is n o t in itself sufficient to shut off R N A synthesis. One other piece of evidence which points to a possible protein synthesis defect is the high ratio of small R N A to ribosomal R N A in cs4-D3 relative to wild-type cells at the restrictive temperature. Willis et al. (1974) have shown that, in the absence of protein synthesis, the synthesis of 4 S R N A is increased, whereas ribosomal R N A synthesis is reduced. The actual rate of ribosomal R N A synthesis in cs4-D3 has n o t y e t been determined. Polysome profiles will be analyzed in order to find out which component of the protein synthetic machinery is affected in this m u t a n t . I t is alternatively possible t h a t the protein synthesis defect is a secondary effect of some other t y p e of cellular abnormality. I t is likely t h a t cold-sensitive m u t a n t s of m a m m a l i a n cells will have abnormalities in a wide range of functions, as t h e y do in procaryotes. T h e y should be useful for the analysis of essential cellular processes, especially since t h e y m a y uncover defects different from those which are already available a m o n g heat sensitive m a m m a l i a n cell lines. Acknowledgements. We would like to thank Dr. Robin Holliday for many helpful discussions. R.A.F. is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.

References Bayliss, F.T., Ingraham, J . L . : Mutation in Saccharomyces cerevisiae conferring streptomycin and cold sensitivity by affecting ribosome form and function. J. Bact. 118, 319328 (1974) Chu, E. H. Y., Malling, It. V.: Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro. Proc. nat. Acad. Sci. (Wash.) 61, 13061312 (1968) Craig, N.C., Perry, R.P.: Aberrant intranucleolar maturation of ribosomal precursors in the absence of protein synthesis. J. Cell Biol. 45, 554-564 (1970) Davies, J., Nomura, M.: The genetics of bacterial ribosomes. Ann. Rev. Genet. 6, 203-234 (1972) Falke, E.V., Wright, T. R.F.: Ribosome assembly defective cold sensitive mutants in Drosophila melanogaster. (abstr.) Genetics 77, s21 (1974) Fan, K., Fisher, M. K., Edlin, G.: Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells. Exp. Cell Res. 82, 111-118 (1973) Gautschi, J. R., Kern, R. M.: DNA replication in mammalian cells in the presence of cycloheximide. Exp. Cell Res. 80, 15-26 (1973) Ginther, C. L., Ingraham, J. L.: Cold-sensitive mutant of Salmonella typhimurium defective in nucleosidephosphokinase. J. Bact. 118, 1020-1026 (1974) Guthrie, C., Nashimoto, H., lSIomura, M.: Structure and function of E. coli ribosomes. VIII. Cold-sensitive mutants defective in ribosome assembly. Proc. nat. Acad. Sci (Wash.) 63, 384-391 (1969) Harris, M.: Phenotypic expression of drug resistance in cell cultures. J. nat. Cancer Inst. 38, 185-192 (1967) Hartwell, L. H., McLaughlin, C. S., Warner, J. R. : Identification of ten genes that control ribosome formation in yeast. Molec. gen. Genet. 109, 42-56 (1970) Hershko, A., Mamont, P., Shields, R., Tomkins, G. M.: Pleiotypic response. Nature (Lond.) New Biol. ~32, 206-211 (1971) Hoffman, B., Ingraham, J. L. : A cold-sensitive mutant of Salmonella tylohimurium which requires tryptophan for growth at 20° C. Biochim. biophys. Acta (Amst.) 201, 300-308 (1970)

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Ingraham, J. L., Neuhard, J. : Cold-sensitive mutants of Salmonella typhimurium defective in uridine monophosphate kinase (pyrH). J. biol. Chem. 247, 6259-6265 (1972) Lieberman, I., Ore, P.: Isolation and study of mutants from mammalian cells in culture. Proc. nat. Acad. Sci. (Wash.) 45, 867-872 (1959) Martin, T. E., Wool, I. G., Castles, J. J. : Dissociation and reassoeiation of ribosomes from eukaryotie cells. In: Methods in enzymology (K. Moldave and L. Grossman eds.), vol. 20, p. 417-429. New York: Academic Press 1971 Mayoh, It., Suzuki, D. T. : Temperature-sensitive mutants in Drosophila melanogaster. XVI. The genetic properties of sex-linked recessive cold-sensitive mutants. Canad. J. Genet. Cytol. 15, 237-254 (1973) Meiss, H. K., Basilico, C. : Temperature sensitive mutants of BHK 21 cells. Nature (Lond.) New Biol. 289, 66-68 (1972) Metzger-Freed, L. : Puromycin resistance in haploid and heteroploid frog cells: Gene or membrane determined ? J. Cell Biol. 51, 742-751 (1971) Morgan, F., Morton, H. J., Parker, R. C. : Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proe. Soc. exp. Biol. (N.Y.) 78, 1-8 (1950) Morhenn, V., Kram, R., Hershko, A., Tomkins, G.M.: Studies on amino acid control of cellular function. Cell 1, 91-94 (1974) Naha, P.M.: Temperature sensitive conditional mutants of monkey kidney cells. Nature (Lond.) 228, 1380-1381 (1969) Nikiforov, V. G., Kalyaeva, E. S., Velkov, V. V.: Cold sensitive mutant of E. coli with altered RNA polymerase. Molec. gen. Genet. 180, 1-7 (1974) O'Donovan, G.A., Kearney, C. L., Ingraham, J. L. : Mutants of Escherichia coli with high minimal temperatures of growth. J. Bact. 90, 611-616 (1965) Ohlsson-Wilhelm, B.M., Freed, J . J . , Perry, R . P . : Somatic cell genetics: Cold sensitive variants (abstr.). J. Cell Biol. 68, 248a (1974) Puck, T. T., Cieciura, S. J., Robinson, A. : Genetics of somatic mammalian cells. III. Longterm cultivation of euploid cells from human and animal subjects. J. exp. Med. 108, 945-956 (1958) Saladino, C.F., Johnson, H.A.: Rate of DNA synthesis as a function of temperature in cultured hamster fibroblasts (V-79) and tteLa-S3 cells. Exp. Cell Res. 85, 248-254 (1974) Sate, K., Shiomi, T.: Isolation of temperature-sensitive mutants from murine leukemic cells (L5178¥). Exp. Cell Res. 88, 295-302 (1974) Scheffler, I. E., Buttin, G. : Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies. J. cell Physiol. 81, 199-216 (1973) Schlitt, S. C., Russell, P. g. : Neurospora crassa cytoplasmic ribosomes: Isolation and characterization of a cold-sensitive mutant defective in ribosome biosynthesis. J. Bact. 120, 666-671 (1974) Seotti, P. D.: A new class of temperature conditional lethal mutants of bacteriophage T4D. Mutation Res. 6, 1-14 (1968) Smith, B. J., Wigglesworth, N. M. : A temperature-sensitive function in a Chinese hamster line affecting DNA synthesis. J. cell. Physiol. 82, 339-348 (1973) Tai, P. C., Kessler, D. P., Ingraham, J. : Cold-sensitive mutations in Salmonella typhimurium which affect ribosome synthesis. J. Bact. 97, 1298-1304 (1969) Thompson, L. H., Harkins, J. L., Stanners, C. P. : A mammalian cell mutant with a temperature-sensitive leucyl-tRNA synthetase. Proc. nat. Acad. Sei. (Wash.) 70, 3094-3098 (1973) Thompson, L. It., Mankovitz, R., Baker, R.M., Till, J.E., Siminoviteh, L., Whitmore, G. F. : Isolation of temperature-sensitive mutants of L-cells. Proc. nat. Aead. Sci. (Wash.) 66, 377-384 (1970) Toniolo, D., Meiss, It. K., Basilico, C.: A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells. Proc. nat. Acad. Sci. (Wash.) 70, 12731277 (1973) Waldron, C., Roberts, C. F. : Cold-sensitive mutants in Aspergillus nidulans. I. Isolation and general characterization. Molee. gen. Genet. 184, 99-113 (1974a) Waldron, C., Roberts, C. F.: Cold-sensitive mutants in AspergiUus nidulans. II. Mutations affecting ribosome production. Molee. gen. Genet. 134, 115-132 (1974b)

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Waskell, L., Glaser, D. A.: Mutants of E. coli with cold-sensitive DNA synthesis. J. Bact. 118, 1027-1040 (1974) Willems, M., Penman, M., Penman, S. : The regulation of RI~A synthesis and processing in the nucleolns during inhibition of protein synthesis. J. Cell Biol. 41, 177-187 (1969) Willis, M. V., Baseman, J. B., Amos, H. : Noncoordinate control of RNA synthesis in eucaryotic cells. Cell 3, 179-184 (1974) Wittmarm, H. G., StSffler, G.: Altered $5 and $20 ribosomal proteins in revertants of an alanyl-tRNA synthetase mutant of Escherichia coli. Molec. gem Genet. 134, 225-236 (1974) Wright, T. R. F. : The recovery, penetrance and pleiotropy of X-linked cold sensitive mutants in Drosophila. Molee. gem Genet. 122, 101-118 (1973) C o m m u n i c a t e d b y B. A. Bridges Dr. Rosann A. Farber Clinical Genetics Division Children's Hospital Medical Center 300 Longwood Ave. Boston, Massachusetts 02115 U.S.A.

Dr. Paul Unrau Biology Branch Chalk River Nuclear Laboratories Chalk River, Ontario Canada

Isolation of cold-sensitive Chinese hamster cells.

Six cold-sensitive variants have been isolated from Chinese hamster ovary cells by the BUdR-visible light selection technique. The properties of one o...
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