- Email: [email protected]
Friend cell variants temperature-sensitive for growth
Copyright 0 1980 by Academic Press, Inc. All rights of reproduction I” any form reserved 0014.4X27/80/040439-06$02.00/O
Friend cell variants temperature-sensitive for growth DAVID CONKIE, BRYAN D. YOUNG and JOHN PAUL, Beatson Institute for Cancer Research, Wolfson Laboratory ifor Molecular Pathology, Glasgow G61 IBD, Scotland Summary. Friend erythroleukemia cells, thermosensitive for growth, have been isolated by a novel selection procedure employing hypoxanthine, aminopterin and bromodeoxyuridine (HAB) with nearvisible light. This reagent eliminates both wild-type cells replicating at the non-permissive temperature of 39°C and cells lacking thymidine kinase activity unable to incorporate bromodeoxyuridine (BUdR), the lethal constituent of HAB. Clones growth arrested at the non-permissive temperature have a temperaturesensitive hefect in progression through Gl of the cell cycle. At permissive temperatures these clones have a karyotype similar to that of wild-type cells and are inducible for synthesis of hemoglobin. Clones which have survived the selection by means of an extended generation time are almost tetraploid at permissive temperatures, are larger than wild-type cells and are inducible for hemoglobin synthesis. At 39°C these cells are defective in accurate mitotic division. This results in a population of cells heterogeneous in size, having chromosome complements ranging from less than the mouse diploid number to approx. 150 chromosomes/ cell. In the latter giant cells, not all nuclei are in mitosis at any one time. Such cells may be defective in cytokinesis. The two distinct classes of ts variant obtained should be useful for I. the study of whether induction of hemoglobin synthesis is cell-cycle dependent; 2. mapping the chromosomes important in controlling accurate mitotic division.
The question of whether induction of hemoglobin synthesis in erythropoietic cells is dependent upon some prior cell-cycle event has been reviewed by Harrison et al. [l]. Many of the experimental approaches used have depended on the growth arrest of cells by drugs or amino acid deprivation prior to treatment with inducers of hemoglobin synthesis. Such methods can be criticised on the grounds of the possible side effects of 29-801819
the growth-arresting procedures and so, in order to eliminate this problem as far as possible, we have isolated conditional lethal variants of Friend cells, thermosensitive for growth. The method has provided a variety of independent clones which can be classified as either cells having a ts defect in Gl progression or cells defective in accurate mitotic division and cytokinesis. Materials
and Methods
Clone 707 Friend cells were freshly established from frozen stocks by culturing at 33.X in Ham’s F12 medium deficient in thymidine and hypoxanthine and suonlemented with 15% fetal bovine serum, a ten-fold in&eased concentration of folic acid and excess essential amino acids (equivalent to adding Eagle’s MEM amino acids). Friend cells were also used which had been cultured continuouslv, with two passages per week, in modified HAM’s F12 over a-period of 6 months at 33S”C. Their generation time at 33S”C was 20 h, and at 39°C (the non-permissive temperature used in selection experiments) 11 h. N-methyl-W-nitrosoguanidine (MNNG), bromodeoxyuridine, hypoxanthine, thymidine and aminopterin were obtained from Sigma and the fluorescent dye Hoechst 33258 from Hoechst, UK. In order to test clonal growth at more than one temperature simultaneously, cell populations were cloned
r
3
0
1
I
I
2 3 Days ,n culture
1 4
Fig. 1. Growth curves of wild-type cells at 33.5-C (O---O); at 39°C (O-O); and of ts cells at 33.X (0- - -0); and at 39°C (O-O). Erp Cell Res 126 (1980)
440
Preliminary notes
Table 1. Summary of the ts clone characteristics Generation time (hours)
Karyotype (chromosomes/ cell)
(a) 21 clones grow in suspension; 2 grow as a monolayer
20
3740
(b) 3 clones grow as suspension cells larger than wild-type
20
55-80
cc
Not applicable. Cells growth arrested 30-150
Morphology Permissive
iemperuture
Non-permissive
Haemoglobin synthesis
Reversion frequency
19 inducible 1 non-inducible 1 constitutive
1.0-3.5x 10-7
(33.5”C)
temperuture
(c) 21 clones in suspension; 2 adherent
3 clones inducible
Not tested
Cell-cycle defect
(39°C)
(d) 3 clones grow as suspension 48 cells heterogeneous in size; 15% cells are giant multinucleates
at 33.5”C by inoculating into a number of microtitre plates (Linbro, Flow Labs.) 70 cells per 96 wells, using a 96-channel automatic pipetting device (Dynatech). When the cloned cells had replicated sufficiently, usually over 7-10 days, replica plates were prepared from each master microtitre plate using a flexible microtitre transfer plate (Dynatech). The fidelity of transfer was found to be 85 %. Karyotype analysis was performed as previously described [2]. Incorporation of [SH]thymidine (Radiochemical Centre, Amersham; spec. act. 40 Cilmmol) into DNA was as described by Paul & Conkie [3]. For flow cytometry, cells 5X lo6 were fixed in 70% methanol at 4”C, and then resuspended in 2 ml of chromomycin A3 (Sigma) (20 pg/ml dissolved in 0.015 M magnesium chloride). The cellular DNA distribution was obtained by sorting 40000 cells/ sample in a Becton Dickinson FACS II cell sorter using an exciting wavelength of 457 nm. DNA histograms were analysed using the non-linear least squares technique described by Fried [4].
Cells arrest in Gl Defect in accurate mitotic division and in cytokinesis
Results Isolation of Friend cells temperaturesensitive (ts) for growth. The method for isolation of conditional lethal mutants thermosensitive for growth was modified from that described by Puck & Kao [5] for isolation of auxotrophic mutants and adapted by Naha [6] for isolating conditional lethal (ts) mutants of BSC-1 cells. Several 100 ml cultures of Friend cells (2X lo6 cells/ml) were mutagenised for 10 min with 0.5 pg/ml MNNG. This reduced the cloning efficiency of the cells to 70% that of untreated cultures. The cultures were incubated at 33S”C for a further 5 days
Fig. 2. DNA distribution in ts cells cultured (a) 36 h at 33.X, or(b) 36 h at 39°C.
Preliminury notes
3. Growth curves of ts cells incubated at 39°C (O-O) or at 33.X (O---O). Arrows indicate the times at which individual cultures were shifted from 39 to 33.5”c.
Fig.
with daily medium changes to permit expression of mutant phenotypes. The cultures were then incubated at 39°C for 18 h to arrest the growth of any cells thermosensitive for replication (ts cells). In order to select against wild-type cells which replicate normally at 39°C and against cells lacking thymidine kinase unable to incorporate BUdR, the cells were incubated for 48 h at 39°C in lop4 M hypoxanthine, 4x 10e7 M aminopterin and lop5 M bromodeoxyuridine (HAB). During the last 3 h of this incubation the Hoechst fluorescent dye 33258 was added at a final concentration of 1 (ug/ml and then the cells, resuspended in phosphate-buffered saline, were placed over a fluorescent tube (Atlas 15 W, 6500 K) for 1 h. The dye enhances the effect of near-visible light on chromosomes which have incorporated BUdR [7]. The cultures were then incubated at 33.5”C in fresh medium without aminopterin or BUdR-but containing 1.6~ lop5 M thymidine and 10e4 M hypoxanthine to permit replication of ts survivors. The entire selection procedure was then repeated four times.
441
In order to test for the ts phenotype, the selected populations were cloned and replica plated. One of the replicates was incubated at 39°C and the other at 33.5”C. Clones growing up at 33.5”C but not at 39°C were considered to be, in the first instance, thermosensitive for growth. One drawback with this method is loss of viability of variants exposed to non-permissive conditions. Thus a quantitative recovery of variants was not obtained. Nevertheless, from the abundance of clones growing only at 33.5”C, after five rounds of selection, it was established that 2.5-5% of the surviving cells were both ts for growth and sufficiently viable to produce clones after the repeated 2&-day periods of growth arrest. Wild-type cells incubated at 33.5 or 39°C for 48 h with 10-j M BUdR and treated for the final 3 h with Hoechst 33258 and then near-visible light had a subsequent cloning efficiency at 33.5”C ~0.02% of that of cells cultured without the BUdR treatment. Similarly, ts cells treated in the same way with BUdR at 33.5”C had a relative cloning efficiency ~0.02%. By contrast, ts cells growth arrested by a preincubation of 18 h at 39°C were then fairly resistant to the 48 h BUdR treatment at 39°C. Such cells had a subsequent 33.5”C cloning efficiency 62.5 % that of ts cells cultured in the same way but without the BUdR treatment. These results suggest that BUdR incorporation into replicating cells was the basis for the selective action of the HAB reagent. Initial classi~cation. From three independent mutagenised cultures subjected to the HAB selection procedure, 2 500 clones were screened at the permissive and non-permissive temperatures. Of these, 26 clones, growing at 33.5”C but not at 39”C, were analysed further. On raising the incubation temperature of
442
Preliminary notes
these clones, several distinct phenotypes became evident. For the majority of the 26 clones, growth was arrested at the nonpermissive temperature of 39°C whereas the generation time at 33.5”C was 20 h like that of the wild-type cells (fig. 1). Twentyone of these clones grew in suspension whereas two clones grew as attached monolayers during routine passage (table 1 a). The suspension cells resembled wild-type cells in morphology and karyotype, having a modal number of 38-39 chromosomes including four biarmed chromosomes. The reversion frequency of representative mutants from each of the three independent selections varied from 1.0-3.5X 10P7. All of these clones were inducible with a variety of inducers at the permissive temperature, except for one clone which was noninducible and a second which synthesised hemoglobin constitutively (up to 45% cells benzidine-positive) over a 6 week period. Whether or not this clone contains a subpopulation of spontaneously inducing terminal cells has yet to be investigated. In order to determine the phase of the cell cycle in which the ts cells were arrested, the DNA content of cells cultured for 36 h at 39°C was established by flow cytofluorometry. About 60% cells accumulated in Gl , 30 % in S and 10 % in G2/M, whereas in permissive conditions about 25 % of cells were in Gl , 65 % in S and 10% in G2/M (fig. 2). Of the small proportion of cells in G2 at 39°C about 50 % were binucleate, presumably having a Gl content of DNA/nucleus. Essentially a similar result was obtained for each of the 21 suspension clones in which up to 75% cells were blocked in Gl after 48 h at 39°C (table 1 c). The incorporation of [3H]thymidine during a 1 h pulse by cells incubated 6, 12 and 40 h at 39°C was 27, 12 and 0.05% that of Eup Cr//Rr\126(1980)
wild-type cells in the same conditions, confirming that the arrested cell population does not enter DNA synthesis to any significant extent at 39°C. Viability of cells growth-arrested at 39°C was estimated from the growth curves of cells released from the Gl block and subsequently incubated at 33.5”C. After 24 or 48 h at 39°C the cells grew with initial generation times of about 24 or 40 h respectively (fig. 3). After 3 or 4 days at 39°C the poor initial recovery of cell growth suggested almost complete loss of viability. Contrasting the Gl-defective ts clones, three clones which were not strictly temperature sensitive for replication appear to be defective in accurate mitotic division. The cells had a generation time of 20 h at 33°C and were larger than wild-type cells. Karyotype analysis showed they were pseudotetraploid, having a modal chromosome number of 66-70 including 4-7 biarmed chromosomes (table 1 b). At 39°C these clones had a generation time of 48 h (table 1 d), and the cell population was then heterogeneous containing about 15 % giant multinucleate cells of up to 150 pm in diameter. The majority of cells had a chromosome complement between 50 and 65 with O-6 biarmed chromosomes; but about 17% of dividing cells had less than the mouse diploid complement whereas about 18 % had 100-150 chromosomes/cell. Of the latter multinucleate giant cells only a few of the nuclei were in mitosis at any time suggesting a lack of cell-cycle synchrony between the nuclei of a single cell. In order to investigate the viability of the heterogeneous population after 4 days of culture at 39°C the cells were cloned in methocel for 3 weeks at 335°C. Only 9 % of the cells formed colonies and these had modal chromosome complements varying between 60 and 71. Thus only cells with a
Preliminary notes chromosomal constitution similar to that of the stable chromosome complement at 33.X were able to produce clones, suggesting giant cells and cells with less than about 60 chromosomes were non-viable. Alternatively, during the 3 week cloning period at permissive temperature, giant cells may have undergone reversal of a defect in cytokinesis to produce viable cells having the stable karyotype. Discussion This report is as far as we know the first to contain a description of Friend cells temperature-sensitive for (1) entry into the DNA synthesis phase of the cell cycle, and (2) accurate mitotic division, including cytokinesis. Initially, uncloned ts lines were derived by repeated selection at 39°C in 10 pCi/ml [3H]thymidine. However, growth of these lines was only transiently temperaturesensitive, the ts phenotype being lost within two passages following selection. Furthermore, the cultures were then HAT-sensitive, suggesting overgrowth by TK- cells no longer ts for growth. TK- cells arise in Friend cell populations at a rate of 2.1 x 10P6/cell/generation (Ivor Hickey, personal communication), whereas in L5178Y populations the rate is 5 x IO-“/cell/generation [8]. This may explain why [3H]thymidine, or BUdR alone, previously employed successfully in a number of isolations (reviewed by Basilic0 [9]) could not readily be used in the isolation of ts Friend cells. Therefore the basis for the selection system devised was 2-fold. In addition to selecting against replicating wild-type cells hypersensitive to light because of BUdR incorporation, aminopterin was used to select against TK- cells. Furthermore, in the presence of aminopterin the wild-type cells
443
were under pressure to incorporate the thymidine analogue in the absence of the de novo synthetic pathway. Since BUdR acts as a mutagen in mammalian cells [lo] this may have complemented the effects of MNNG. For the majority of the ts Friend cell clones, the cells survived the selection procedure by avoiding incorporation of BUdR, the lethal constituent of HAB. After about 40 h at the non-permissive temperature the cell population acquired a Gl DNA content and failed to incorporate [3H]thymidine while retaining viability, suggesting that these clones have a primary ts defect in Gl progression. However, the results do not distinguish between a ts defect throughout Gl and a function defective at a specific point in Gl as described by Basilic0 [9], Naha et al. [ll] and Floros et al. [12]. Regardless of this incomplete characterisation, Gl-arrested Friend cells should be useful when studying whether differentiation in response to inducers is cell-cycle dependent. Other mutants have been isolated in which a lengthened generation time of 48 h, equivalent to the time of exposure to BUdR during selection may be the basis for surviva1 . At the non-permissive temperature, these cells appeared to have aberrations of the mitotic process resulting in heterogeneity in cell size and DNA content. In addition to small cells, giant multinucleate cells were observed at 39°C representing about 15% of the population. A similar mutant, isolated by Hatzfeld & Buttin [ 131 from CHO cells, had a majority (up to 63%) of multinucleate cells after 48 h at 39°C together with a significant fraction of cells less than onethird the volume of control cells. Another ts cell defective in cytokinesis produced mainly binucleate cells [14]. Contrasting the
444
Preliminary
notes
mutant isolated by Hatzfeld & Buttin [13], the nuclei of the Friend giant cells were not synchronised with respect to the cell cycle. Multinucleate cells could not be cloned at 33 .YC suggesting that they were inviable or that the defect in cytokinesis could be reversed to yield viable cells with lower chromosome numbers. In these multinucleate cells it would be of interest to measure the activity of ornithine decarboxylase, the enzyme regulating polyamine synthesis, since polyamines may have a role in cell division and cytokinesis [ 151. In addition, these cells may be useful in studies aimed at assigning to specific chromosomes the role of controlling accurate mitotic division. This work has been supported by grants from the Medical Research Council and the Cancer Research Campaign. We gratefully acknowledge the excellent technical assistance of Janis Fleming, Rory Sillars and David Tallach.
References P R. Conkie. D. Rutherford. T & Yeoh. (BSCB G, Stem cells and tissue homeostasis svmn. 2) (ed B T Lord. C S Potten & R J Cole) p. 241. Cambridge University Press (1978). 2. Paul, J, Cell and tissue culture, 5th edn. Churchill Livingstone (1975). 3. Paul, j & Conkie, D, Exp cell res 77 (1973) 105. 4. Fried. J. Commuters and biomed res 9 (1976) 263. 5. Puck; T T &-Kao, F, Proc natl acad sci ‘US 58 (1967) 1227. Naha, PM, Nature 223 (1969) 1380. 67: Stetten, G, Davidson, R L & Latt, S A, Exp cell res 108 (1977) 447. 8. Clive, D, Flam, W G, Machesko, M R & Bernheim, N J, Mutat res 16 (1972) 77. 9. Basilica, C, Adv cancer res 24 (1977) 223. 10. Chu, E H Y, Sun, N C & Chang, C C, Proc natl acad sci US 69 (1972) 3459. 11. Naha, P M, Meyer, A L & Hewitt, K, Nature 258 (1975) 49. 12. Floras. J. Ashihara. T & Baseraa. R. Cell biol int repts 2(1978) 259. 13. Hatzfeld, J & Buttin, G. Cell 5 (1975) 123. 14. Smith, R J & Wigglesworth, N M, J cell physiol80 (1972) 253. 15. Sunkara, P S, Rao, P N, Nishioka, K & Brinkley, B R, Exp cell res 119 (1976) 63. 1. Harrison,
Received April 19, 1979 Revised version received November Accepted November 22, 1979 Exp Cell Res 126(19801
13, 1979
Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form rererved 0014.4R27/80/04044405$02.00/0
Distribution
of repetitive DNA sequences in
Vi& faba chromosomes R. E. ROWLAND,l Botuny Depurtment, Victoria University of Wellington, Wellington, Neal Zealand Summury. In situ hybridization of labelled complementary RNA transcribed from whole DNA to metaphase chromosomes indicates the presence of repetitive DNA in both euchromatin and heterochromatin of the Viciafaba genome.
The development of in situ hybridization has demonstrated that certain heterochromatic sites are rich in repetitive DNA sequences [l-3]. In situ hybridization studies in Viciafaba have shown that satellite DNA is distributed diffusely throughout all the chromosomes [4]. It is unusual to find such non-localization of a satellite DNA component on the chromosomes of higher organisms. Scilla sibirica (Liliaceae) satellite DNA, for example, is strongly localized on the chromosomes [4]. The present investigation was undertaken to establish the relationship between the localization of heterochromatin and repetitive DNA in Vicia faba chromosomes by hybridizing labelled cRNA transcribed from whole DNA. The conditions were such as to allow hybridization of reiterated sequences only. Materials and Methods Isolation of DNA. Whole DNA was extracted from bean seedlings according to the method of Marmur [5]. For final puhfication the DNA was pelleted by preparative centrifugation for 4 h at 200000 g. The pellet was dissolved in O:l X SSC. Beckman Model-E uhracentrifuge analysis of the DNA was performed in CsCl using P&dot&as aeruginosa DNA (1.726 g/cm”) as a marker. Calculation of buoyant density was made according to Vinograd & Hearst [6]. DNA transcription. The method of transcribing hiahlv radioactive RNA complementarv to DNA was essentially that described by Pardue et al. [7]. Approx.
’ Current address: Botany Department, University of Nairobi, P.O. Box 30197, Nairobi, Kenya. Printed
in Sweden
Friend cell variants temperature-sensitive for growth DAVID CONKIE, BRYAN D. YOUNG and JOHN PAUL, Beatson Institute for Cancer Research, Wolfson Laboratory ifor Molecular Pathology, Glasgow G61 IBD, Scotland Summary. Friend erythroleukemia cells, thermosensitive for growth, have been isolated by a novel selection procedure employing hypoxanthine, aminopterin and bromodeoxyuridine (HAB) with nearvisible light. This reagent eliminates both wild-type cells replicating at the non-permissive temperature of 39°C and cells lacking thymidine kinase activity unable to incorporate bromodeoxyuridine (BUdR), the lethal constituent of HAB. Clones growth arrested at the non-permissive temperature have a temperaturesensitive hefect in progression through Gl of the cell cycle. At permissive temperatures these clones have a karyotype similar to that of wild-type cells and are inducible for synthesis of hemoglobin. Clones which have survived the selection by means of an extended generation time are almost tetraploid at permissive temperatures, are larger than wild-type cells and are inducible for hemoglobin synthesis. At 39°C these cells are defective in accurate mitotic division. This results in a population of cells heterogeneous in size, having chromosome complements ranging from less than the mouse diploid number to approx. 150 chromosomes/ cell. In the latter giant cells, not all nuclei are in mitosis at any one time. Such cells may be defective in cytokinesis. The two distinct classes of ts variant obtained should be useful for I. the study of whether induction of hemoglobin synthesis is cell-cycle dependent; 2. mapping the chromosomes important in controlling accurate mitotic division.
The question of whether induction of hemoglobin synthesis in erythropoietic cells is dependent upon some prior cell-cycle event has been reviewed by Harrison et al. [l]. Many of the experimental approaches used have depended on the growth arrest of cells by drugs or amino acid deprivation prior to treatment with inducers of hemoglobin synthesis. Such methods can be criticised on the grounds of the possible side effects of 29-801819
the growth-arresting procedures and so, in order to eliminate this problem as far as possible, we have isolated conditional lethal variants of Friend cells, thermosensitive for growth. The method has provided a variety of independent clones which can be classified as either cells having a ts defect in Gl progression or cells defective in accurate mitotic division and cytokinesis. Materials
and Methods
Clone 707 Friend cells were freshly established from frozen stocks by culturing at 33.X in Ham’s F12 medium deficient in thymidine and hypoxanthine and suonlemented with 15% fetal bovine serum, a ten-fold in&eased concentration of folic acid and excess essential amino acids (equivalent to adding Eagle’s MEM amino acids). Friend cells were also used which had been cultured continuouslv, with two passages per week, in modified HAM’s F12 over a-period of 6 months at 33S”C. Their generation time at 33S”C was 20 h, and at 39°C (the non-permissive temperature used in selection experiments) 11 h. N-methyl-W-nitrosoguanidine (MNNG), bromodeoxyuridine, hypoxanthine, thymidine and aminopterin were obtained from Sigma and the fluorescent dye Hoechst 33258 from Hoechst, UK. In order to test clonal growth at more than one temperature simultaneously, cell populations were cloned
r
3
0
1
I
I
2 3 Days ,n culture
1 4
Fig. 1. Growth curves of wild-type cells at 33.5-C (O---O); at 39°C (O-O); and of ts cells at 33.X (0- - -0); and at 39°C (O-O). Erp Cell Res 126 (1980)
440
Preliminary notes
Table 1. Summary of the ts clone characteristics Generation time (hours)
Karyotype (chromosomes/ cell)
(a) 21 clones grow in suspension; 2 grow as a monolayer
20
3740
(b) 3 clones grow as suspension cells larger than wild-type
20
55-80
cc
Not applicable. Cells growth arrested 30-150
Morphology Permissive
iemperuture
Non-permissive
Haemoglobin synthesis
Reversion frequency
19 inducible 1 non-inducible 1 constitutive
1.0-3.5x 10-7
(33.5”C)
temperuture
(c) 21 clones in suspension; 2 adherent
3 clones inducible
Not tested
Cell-cycle defect
(39°C)
(d) 3 clones grow as suspension 48 cells heterogeneous in size; 15% cells are giant multinucleates
at 33.5”C by inoculating into a number of microtitre plates (Linbro, Flow Labs.) 70 cells per 96 wells, using a 96-channel automatic pipetting device (Dynatech). When the cloned cells had replicated sufficiently, usually over 7-10 days, replica plates were prepared from each master microtitre plate using a flexible microtitre transfer plate (Dynatech). The fidelity of transfer was found to be 85 %. Karyotype analysis was performed as previously described [2]. Incorporation of [SH]thymidine (Radiochemical Centre, Amersham; spec. act. 40 Cilmmol) into DNA was as described by Paul & Conkie [3]. For flow cytometry, cells 5X lo6 were fixed in 70% methanol at 4”C, and then resuspended in 2 ml of chromomycin A3 (Sigma) (20 pg/ml dissolved in 0.015 M magnesium chloride). The cellular DNA distribution was obtained by sorting 40000 cells/ sample in a Becton Dickinson FACS II cell sorter using an exciting wavelength of 457 nm. DNA histograms were analysed using the non-linear least squares technique described by Fried [4].
Cells arrest in Gl Defect in accurate mitotic division and in cytokinesis
Results Isolation of Friend cells temperaturesensitive (ts) for growth. The method for isolation of conditional lethal mutants thermosensitive for growth was modified from that described by Puck & Kao [5] for isolation of auxotrophic mutants and adapted by Naha [6] for isolating conditional lethal (ts) mutants of BSC-1 cells. Several 100 ml cultures of Friend cells (2X lo6 cells/ml) were mutagenised for 10 min with 0.5 pg/ml MNNG. This reduced the cloning efficiency of the cells to 70% that of untreated cultures. The cultures were incubated at 33S”C for a further 5 days
Fig. 2. DNA distribution in ts cells cultured (a) 36 h at 33.X, or(b) 36 h at 39°C.
Preliminury notes
3. Growth curves of ts cells incubated at 39°C (O-O) or at 33.X (O---O). Arrows indicate the times at which individual cultures were shifted from 39 to 33.5”c.
Fig.
with daily medium changes to permit expression of mutant phenotypes. The cultures were then incubated at 39°C for 18 h to arrest the growth of any cells thermosensitive for replication (ts cells). In order to select against wild-type cells which replicate normally at 39°C and against cells lacking thymidine kinase unable to incorporate BUdR, the cells were incubated for 48 h at 39°C in lop4 M hypoxanthine, 4x 10e7 M aminopterin and lop5 M bromodeoxyuridine (HAB). During the last 3 h of this incubation the Hoechst fluorescent dye 33258 was added at a final concentration of 1 (ug/ml and then the cells, resuspended in phosphate-buffered saline, were placed over a fluorescent tube (Atlas 15 W, 6500 K) for 1 h. The dye enhances the effect of near-visible light on chromosomes which have incorporated BUdR [7]. The cultures were then incubated at 33.5”C in fresh medium without aminopterin or BUdR-but containing 1.6~ lop5 M thymidine and 10e4 M hypoxanthine to permit replication of ts survivors. The entire selection procedure was then repeated four times.
441
In order to test for the ts phenotype, the selected populations were cloned and replica plated. One of the replicates was incubated at 39°C and the other at 33.5”C. Clones growing up at 33.5”C but not at 39°C were considered to be, in the first instance, thermosensitive for growth. One drawback with this method is loss of viability of variants exposed to non-permissive conditions. Thus a quantitative recovery of variants was not obtained. Nevertheless, from the abundance of clones growing only at 33.5”C, after five rounds of selection, it was established that 2.5-5% of the surviving cells were both ts for growth and sufficiently viable to produce clones after the repeated 2&-day periods of growth arrest. Wild-type cells incubated at 33.5 or 39°C for 48 h with 10-j M BUdR and treated for the final 3 h with Hoechst 33258 and then near-visible light had a subsequent cloning efficiency at 33.5”C ~0.02% of that of cells cultured without the BUdR treatment. Similarly, ts cells treated in the same way with BUdR at 33.5”C had a relative cloning efficiency ~0.02%. By contrast, ts cells growth arrested by a preincubation of 18 h at 39°C were then fairly resistant to the 48 h BUdR treatment at 39°C. Such cells had a subsequent 33.5”C cloning efficiency 62.5 % that of ts cells cultured in the same way but without the BUdR treatment. These results suggest that BUdR incorporation into replicating cells was the basis for the selective action of the HAB reagent. Initial classi~cation. From three independent mutagenised cultures subjected to the HAB selection procedure, 2 500 clones were screened at the permissive and non-permissive temperatures. Of these, 26 clones, growing at 33.5”C but not at 39”C, were analysed further. On raising the incubation temperature of
442
Preliminary notes
these clones, several distinct phenotypes became evident. For the majority of the 26 clones, growth was arrested at the nonpermissive temperature of 39°C whereas the generation time at 33.5”C was 20 h like that of the wild-type cells (fig. 1). Twentyone of these clones grew in suspension whereas two clones grew as attached monolayers during routine passage (table 1 a). The suspension cells resembled wild-type cells in morphology and karyotype, having a modal number of 38-39 chromosomes including four biarmed chromosomes. The reversion frequency of representative mutants from each of the three independent selections varied from 1.0-3.5X 10P7. All of these clones were inducible with a variety of inducers at the permissive temperature, except for one clone which was noninducible and a second which synthesised hemoglobin constitutively (up to 45% cells benzidine-positive) over a 6 week period. Whether or not this clone contains a subpopulation of spontaneously inducing terminal cells has yet to be investigated. In order to determine the phase of the cell cycle in which the ts cells were arrested, the DNA content of cells cultured for 36 h at 39°C was established by flow cytofluorometry. About 60% cells accumulated in Gl , 30 % in S and 10 % in G2/M, whereas in permissive conditions about 25 % of cells were in Gl , 65 % in S and 10% in G2/M (fig. 2). Of the small proportion of cells in G2 at 39°C about 50 % were binucleate, presumably having a Gl content of DNA/nucleus. Essentially a similar result was obtained for each of the 21 suspension clones in which up to 75% cells were blocked in Gl after 48 h at 39°C (table 1 c). The incorporation of [3H]thymidine during a 1 h pulse by cells incubated 6, 12 and 40 h at 39°C was 27, 12 and 0.05% that of Eup Cr//Rr\126(1980)
wild-type cells in the same conditions, confirming that the arrested cell population does not enter DNA synthesis to any significant extent at 39°C. Viability of cells growth-arrested at 39°C was estimated from the growth curves of cells released from the Gl block and subsequently incubated at 33.5”C. After 24 or 48 h at 39°C the cells grew with initial generation times of about 24 or 40 h respectively (fig. 3). After 3 or 4 days at 39°C the poor initial recovery of cell growth suggested almost complete loss of viability. Contrasting the Gl-defective ts clones, three clones which were not strictly temperature sensitive for replication appear to be defective in accurate mitotic division. The cells had a generation time of 20 h at 33°C and were larger than wild-type cells. Karyotype analysis showed they were pseudotetraploid, having a modal chromosome number of 66-70 including 4-7 biarmed chromosomes (table 1 b). At 39°C these clones had a generation time of 48 h (table 1 d), and the cell population was then heterogeneous containing about 15 % giant multinucleate cells of up to 150 pm in diameter. The majority of cells had a chromosome complement between 50 and 65 with O-6 biarmed chromosomes; but about 17% of dividing cells had less than the mouse diploid complement whereas about 18 % had 100-150 chromosomes/cell. Of the latter multinucleate giant cells only a few of the nuclei were in mitosis at any time suggesting a lack of cell-cycle synchrony between the nuclei of a single cell. In order to investigate the viability of the heterogeneous population after 4 days of culture at 39°C the cells were cloned in methocel for 3 weeks at 335°C. Only 9 % of the cells formed colonies and these had modal chromosome complements varying between 60 and 71. Thus only cells with a
Preliminary notes chromosomal constitution similar to that of the stable chromosome complement at 33.X were able to produce clones, suggesting giant cells and cells with less than about 60 chromosomes were non-viable. Alternatively, during the 3 week cloning period at permissive temperature, giant cells may have undergone reversal of a defect in cytokinesis to produce viable cells having the stable karyotype. Discussion This report is as far as we know the first to contain a description of Friend cells temperature-sensitive for (1) entry into the DNA synthesis phase of the cell cycle, and (2) accurate mitotic division, including cytokinesis. Initially, uncloned ts lines were derived by repeated selection at 39°C in 10 pCi/ml [3H]thymidine. However, growth of these lines was only transiently temperaturesensitive, the ts phenotype being lost within two passages following selection. Furthermore, the cultures were then HAT-sensitive, suggesting overgrowth by TK- cells no longer ts for growth. TK- cells arise in Friend cell populations at a rate of 2.1 x 10P6/cell/generation (Ivor Hickey, personal communication), whereas in L5178Y populations the rate is 5 x IO-“/cell/generation [8]. This may explain why [3H]thymidine, or BUdR alone, previously employed successfully in a number of isolations (reviewed by Basilic0 [9]) could not readily be used in the isolation of ts Friend cells. Therefore the basis for the selection system devised was 2-fold. In addition to selecting against replicating wild-type cells hypersensitive to light because of BUdR incorporation, aminopterin was used to select against TK- cells. Furthermore, in the presence of aminopterin the wild-type cells
443
were under pressure to incorporate the thymidine analogue in the absence of the de novo synthetic pathway. Since BUdR acts as a mutagen in mammalian cells [lo] this may have complemented the effects of MNNG. For the majority of the ts Friend cell clones, the cells survived the selection procedure by avoiding incorporation of BUdR, the lethal constituent of HAB. After about 40 h at the non-permissive temperature the cell population acquired a Gl DNA content and failed to incorporate [3H]thymidine while retaining viability, suggesting that these clones have a primary ts defect in Gl progression. However, the results do not distinguish between a ts defect throughout Gl and a function defective at a specific point in Gl as described by Basilic0 [9], Naha et al. [ll] and Floros et al. [12]. Regardless of this incomplete characterisation, Gl-arrested Friend cells should be useful when studying whether differentiation in response to inducers is cell-cycle dependent. Other mutants have been isolated in which a lengthened generation time of 48 h, equivalent to the time of exposure to BUdR during selection may be the basis for surviva1 . At the non-permissive temperature, these cells appeared to have aberrations of the mitotic process resulting in heterogeneity in cell size and DNA content. In addition to small cells, giant multinucleate cells were observed at 39°C representing about 15% of the population. A similar mutant, isolated by Hatzfeld & Buttin [ 131 from CHO cells, had a majority (up to 63%) of multinucleate cells after 48 h at 39°C together with a significant fraction of cells less than onethird the volume of control cells. Another ts cell defective in cytokinesis produced mainly binucleate cells [14]. Contrasting the
444
Preliminary
notes
mutant isolated by Hatzfeld & Buttin [13], the nuclei of the Friend giant cells were not synchronised with respect to the cell cycle. Multinucleate cells could not be cloned at 33 .YC suggesting that they were inviable or that the defect in cytokinesis could be reversed to yield viable cells with lower chromosome numbers. In these multinucleate cells it would be of interest to measure the activity of ornithine decarboxylase, the enzyme regulating polyamine synthesis, since polyamines may have a role in cell division and cytokinesis [ 151. In addition, these cells may be useful in studies aimed at assigning to specific chromosomes the role of controlling accurate mitotic division. This work has been supported by grants from the Medical Research Council and the Cancer Research Campaign. We gratefully acknowledge the excellent technical assistance of Janis Fleming, Rory Sillars and David Tallach.
References P R. Conkie. D. Rutherford. T & Yeoh. (BSCB G, Stem cells and tissue homeostasis svmn. 2) (ed B T Lord. C S Potten & R J Cole) p. 241. Cambridge University Press (1978). 2. Paul, J, Cell and tissue culture, 5th edn. Churchill Livingstone (1975). 3. Paul, j & Conkie, D, Exp cell res 77 (1973) 105. 4. Fried. J. Commuters and biomed res 9 (1976) 263. 5. Puck; T T &-Kao, F, Proc natl acad sci ‘US 58 (1967) 1227. Naha, PM, Nature 223 (1969) 1380. 67: Stetten, G, Davidson, R L & Latt, S A, Exp cell res 108 (1977) 447. 8. Clive, D, Flam, W G, Machesko, M R & Bernheim, N J, Mutat res 16 (1972) 77. 9. Basilica, C, Adv cancer res 24 (1977) 223. 10. Chu, E H Y, Sun, N C & Chang, C C, Proc natl acad sci US 69 (1972) 3459. 11. Naha, P M, Meyer, A L & Hewitt, K, Nature 258 (1975) 49. 12. Floras. J. Ashihara. T & Baseraa. R. Cell biol int repts 2(1978) 259. 13. Hatzfeld, J & Buttin, G. Cell 5 (1975) 123. 14. Smith, R J & Wigglesworth, N M, J cell physiol80 (1972) 253. 15. Sunkara, P S, Rao, P N, Nishioka, K & Brinkley, B R, Exp cell res 119 (1976) 63. 1. Harrison,
Received April 19, 1979 Revised version received November Accepted November 22, 1979 Exp Cell Res 126(19801
13, 1979
Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form rererved 0014.4R27/80/04044405$02.00/0
Distribution
of repetitive DNA sequences in
Vi& faba chromosomes R. E. ROWLAND,l Botuny Depurtment, Victoria University of Wellington, Wellington, Neal Zealand Summury. In situ hybridization of labelled complementary RNA transcribed from whole DNA to metaphase chromosomes indicates the presence of repetitive DNA in both euchromatin and heterochromatin of the Viciafaba genome.
The development of in situ hybridization has demonstrated that certain heterochromatic sites are rich in repetitive DNA sequences [l-3]. In situ hybridization studies in Viciafaba have shown that satellite DNA is distributed diffusely throughout all the chromosomes [4]. It is unusual to find such non-localization of a satellite DNA component on the chromosomes of higher organisms. Scilla sibirica (Liliaceae) satellite DNA, for example, is strongly localized on the chromosomes [4]. The present investigation was undertaken to establish the relationship between the localization of heterochromatin and repetitive DNA in Vicia faba chromosomes by hybridizing labelled cRNA transcribed from whole DNA. The conditions were such as to allow hybridization of reiterated sequences only. Materials and Methods Isolation of DNA. Whole DNA was extracted from bean seedlings according to the method of Marmur [5]. For final puhfication the DNA was pelleted by preparative centrifugation for 4 h at 200000 g. The pellet was dissolved in O:l X SSC. Beckman Model-E uhracentrifuge analysis of the DNA was performed in CsCl using P&dot&as aeruginosa DNA (1.726 g/cm”) as a marker. Calculation of buoyant density was made according to Vinograd & Hearst [6]. DNA transcription. The method of transcribing hiahlv radioactive RNA complementarv to DNA was essentially that described by Pardue et al. [7]. Approx.
’ Current address: Botany Department, University of Nairobi, P.O. Box 30197, Nairobi, Kenya. Printed
in Sweden