- Email: [email protected]
Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids
466
Preliminary notes
17. Lewis, P N, Bradbury. E M & Crane-Robinson, C, Biochemistry 14 (1975) 3391. 18. Riordan, J F& V&lee, B L, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 25 (ed C H W Hirs & S N Timasheff) pp. 494-499. Academic Press, New York (1972). 19. Ostrowski, K, Barnard, E A, Sawicki, W, Chorzelski, T, Langner, A & Mikulski, A, J histochem cytochem 18 (1970) 490. 20. Heinegard, D K & Hascall, V C, J biol them 254 (1979) 921. 21. Daniel, J W & Baldwin, H H, Methods in cell physiology (ed D M Prescott) vol. 1, pp. 9-41. Academic Press, New York (1964). 22. Guttes, E & Guttes, S, Methods in cell physiology (ed D M Prescott) vol. 1, pp. 43-54. Academic Press, New York (1964). 23. Mohberg, J & Rusch, H P, Exp cell res 66 (1971) 305. 24. Polman, B J J, Ph.D. thesis, University of Nijmegen, The Netherlands (1974). 25. Sures, I & Gallwitz, D, Biochemistry 19 (1980) 943. 2t5. Kervabon, -4, Mery, J & Parello. J, FEBS lett 106 11070) \”
,,,
a-4 ,d.
27. Burton, K, Biochem j 62 (1956) 315. 28. Mohberg, J. Babcock, K L, Haugli, F B & Rusch, H P, Dev biol34 (1973) 228. 29. Reeves. R & Candido. E P M. Nucleic acids res 8 (1980) i947. 30. Bradbury, E M, Inglis, R J, Matthews, H R & Sarner, N, Eurj biochem 33 (1973) 131. 31. Bradbury, E M, Inglis, R J & Matthews, H R, Nature 247 (1974) 257. 32. Matthews, H R, Recently discovered systems of enzyme regulation by reversible phosphorylation (ed P Cohen) pp. 235-254. Elsevierlh’orthHolland, Amsterdam (1980). Received October 15, 1981 Accepted December 15, 1981
Copyright .@ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0011-482:'81:04~66-04$0:.00,0
Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids CATHLEEN R. CARLIN’ and KATHLEEN W. RAO.? Biological Sciences Research Center. Genetics Curriculum, University of North Carolina, Chapel Hill, NC 27514, USA SumnzarJ. We have analysed metaphase chromosomes of man-mouse somatic cell hybrids using a sequential staining procedure involving the fluorescent DNAbinding stains, Hoechst 33258 and quinacrine musExp Cdl
Res 138 I 1982)
tard. This was found to be a simple and reliable means of differentiating the chromosomes of the two species and of identifying specific human chromosomes. In addition this method will permit the study of the segregation of human chromosome homologues that are discordant for quinacrine mustard fluorescent polymorphisms.
The preferential segregation of human chromosomes from man-mouse somatic cell hybrids is routinely exploited to associate the expression of human gene products with s,pecific human chromosomes. This method of gene mapping requires a chromosome staining procedure which will distinguish mouse from human chromosomes and then allow the identification of specific human chromosomes. The chromosomes of the two species can be distinguished from one another after staining with the benzimidazol compound Hoechst 33258 [l] or with alkaline Giemsa Lr21. 2 Human chromosomes are routinely identified by their characteristic Q-banding patterns after staining with quinacrine mustard [3] or by their G-banding patterns after staining with Giemsa [4]. Kozak et al. [5] have described a sequential staining procedure that combines G-banding and staining with Hoechst 33258 to facilitate the identification of human chromosomes in somatic cell hybrids. We now describe a sequential staining procedure that combines the differential centromere fluorescence produced by staining with Hoechst 33258 with the characteristic banding patterns produced by staining with quinacrine mustard. Materials and Methods The hybrid cell lines used in this study were derived from the polyethylene glycol-induced fusion [6] of cells from an HPRT-deficient mouse heteroploid
’ Current address: The Wistar Institute, 36th and Spruce Streets, Philadelphia, PA 19104, USA. 2 Current address: Department of Pediatrics. East Carolina University Medical School, Greenville, NC 27834, USA. Printed
in Susden
Preliminaq
notes
467
fibroblast line, A9 (Human Mutant Cell Repository; Camden, N.J.), and a normal human diploid skin fibroblast strain initiated in the laboratory of Dr Michael Swift, University of North Carolina: ChapeI Hill. N.C. Metaphase spreads for chromosome analvses were prepared in the following manner. Ceil division was arrested by treatment with 0.75 yg/ml of Colcemid (Calbiochem) for 3-4 h. Cells were swelled by incubation in 0.075 M KC1 hvnotonic solution for 20 miu at 37°C and tixed mith three changes of freshIy prepared Carnoy fixative. Cells resuspended in fixative were dropped onto cold (4°C) wet microscope slides and dried for approx. 1 min on a slide warmer at 60°C. Slides not stained immediately were stored at 4°C. Staining with Hoechst 33258 was performed by a modification of the method of Raposa & Natarajan [7]. A stock solution (10 pg/m! in Sorensen buffer, pH 5.5) of Hoechst 33258 (American Hoechst Corp.) can be stored for several months in a light-proof container at -2O’C. The working solution (0.05 wgfmi in Sorensen buffer, pH 5.5) was prepared week!y and stored in a light-proof container at CC. Shdes were soaked in Sorensen buffer, pH 5.5, for 2 min, stained in the working solution in the dark at 37%. and then rinsed twice in Sorensen buffer and once in deionized water. Staining for 12 min produced bright fhrorescence of mouse chromosome centromeres for the cei! line used in this study. Coverslips were mounted with sucrose dissolved in McIlvaine buffer, pH 7.0 (6 g sucrose/l0 ml buffer). Slides were examined within 24 h with a Zeiss Photomicroscope III equipped with the fohowing: A barrier filter that excludes wavelengths below 51OA; and, a narrow-band pass excitation filter with peak at 4356. Kodak Technical Pan 2415 film was used for all fluorescence photography: the film was developed with HCl 10 Deve!oper. Slides to be restained with quinacrine mustard (Polysciences) were soaked brieflv in deionized water to remove coverslips, air-dried? rinsed two or three times with xylene to remove immersion oil, and airdried again. Slides that were not restained with quin acrine mustard within 24 h were stored at 4Y. Q-banding was done by the method of Caspersson et al. [8]. The quinacrine mustard staining solution (50 &ml in McIlvaine buffer, pH 7.0) was prepared monthly and stored in a light-proof container at 4’C. Slides were first hydrated by sequential rinses of 2 min each in 100% ethanol, 95% ethanol. 50% ethanol. and deionized water. They were then soaked for 5 min in McIlvaine buffer, pH 7.0. followed by staining in quinacrine mustard in-the dark. Optimum staining 6me was 20 min on the average but varied with the batch of quinacrine mustard and the age of the working solution. Temperature was not critical in the quinacrine mustard staining protocol. After staining, the slides were soaked for 15 min in deionized water in the dark. I. (A) -4 partial hybrid metaphase spread that has been stained with Hoechst 33258. Arrows point to mouse chromosomes that do not display the expected bright centromere fluorescence. (B) The same hybrid metaphase spread restained with quinacrine mustard. Human chromosomes are labeled.
Fig.
468
Preliminary
notes
Fig. 2. (A) A partial hybrid metaphase spread that has been stained with Hoechst 33258. Arrows point to mouse chromosomes. (B) The same hybrid metaphase spread restained with quinacrine mustard showing a human chromosome 13 with the quinacrine mustard-bright centromere polymorphism [9].
Coverslips were mounted as before. Slides were analyzed and photographed with a Zeiss Photomicroscope III within a few hours.
Results
When metaphase spreads from the mouse parental cell line were stained with Hoechst 33258, three to four of the mouse chromosomes did not display the expected bright centromere fluorescence (arrows, fig. 1A). However, these chromosomes were distinguishable from human chromosomes after staining with quinacrine mustard because of their unique Q-banding patterns. By comparing fig. 1A and B, it can be seen that all Hoechst 3325Sinduced fluorescence of mouse chromosome centromeres was extinguished by hydration of chromosome preparations prior to restaining with quinacrine mustard. None of the human chromosome centromeres were brightly fluorescent after staining with Hoechst 33258, although the centromere of human chromosome 3 displayed slight fluorescence. However, this particular human chromosome retained centromere fluorescence after restaining with quinacrine mustard, while all mouse centroExp CeNRes 138 11982)
mere fluorescence was extinguished (fig. 1B). Pretreatment of human chromosomes with Hoechst 33258 did not alter the characteristic Q-banding patterns of the human chromosomes produced by staining with quinacrine mustard. Quinacrine mustardbright polymorphisms [9] also retained their brilliance after pretreatment with Hoechst 33258 (fig. 2), suggesting that this technique can be used to follow the segregation of homologues that are discordant for quinacrine mustard fluorescent polymorphisms. Adequate results could be obtained from this procedure with slides that had been stored at 4°C for as long as 5-6 months. The best results, however, were obtained with preparations that were less than 1 month old. Q-banding was optimal if preparations were restained with quinacrine mustard within l-2 days after staining with Hoechst 33258. Discussion
Raposa & Natarajan [7] have reported that human metaphase chromosomes that have been stained with Hoechst 33258 under the appropriate conditions can be identified by their characteristic fluorescent banding patterns. The Hoechst 33258 staining conditions that produce optimal banding patterns, however, are not the same as those that result in optimal mouse chromosome centromere fluorescence. For this reason, we found that restaining with quinacrine mustard was necessary for the identification of human chromosomes that were not adequately banded after staining with Hoechst 33258. The sequential staining protocol described in this paper offers a method for the rapid screening of man-mouse somatic cell hybrid clones. Although it does not permit the identification of interspecific translocations as precisely as the alkaline Giemsa
Preliminary izoles technique [L?], clones which are suspected of carrying such rearrangements can be further analysed with alkaline Giemsa. In many cases, adequate cytogenetic characterization of a large number of hybrid clones can be achieved by using a combination of the two techniques: Rapid screening of ail clones with the Hoechst 3325Squinacrine mustard sequential technique, followed by further delineation of discordant clones with alkaline Giemsa. The sequential staining of metaphase spreads with Hoechst 33258 and quinacrine mustard as a method of identifying human chromosomes in man-mouse somatic cell hybrids offers several advantages. First, human and mouse chromosomes are readily distinguished, either by their differential centromere fluorescence after staining with Hoechst 33258 or by their unique Q-banding patterns. Second, hydration of chromosome preparations prior to restaining with quinacrine mustard extinguishes the centromere fluorescence of all mouse chromosomes. Third, the quality of Q-banding is sufficient to allow for the identification of specific human chromosomes. Fourth, the use of Q-banding rather than G-banding has the advantage of allowing us to follow the segregation of specific homologues in the case of human chromosome pairs discordant for quinacrine mustard polymorphisms. This staining procedure, therefore, can be used to study the expression of dominant traits in human cells heterozygous for the trait in the case that the gene coding for the trait and a QM-bright polymorphism are linked. Finally, the procedure is easy to reproduce once optimal staining times have been determined. We are indebted to Drs Michael Swift and Philip Buchanan for making their laboratory facilities available for this work and for their critical evaluations of the manuscript. We also wish to thank Drs Henry N. Kirkman and Charles Timmons for their reading
469
of the manuscript and Dorothea Baker for preparation of the manuscript. This work was supported by NIH grants NIGMST32, GM07092, and HD03110.
References 1. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. 2. Bobrow. M&Gross. J. Nature 251 (1974) 77, 3. Caspersson, T: Lomakka. G & Zech, L,‘Hereditas 67 (1970) 89. 4. Sumner, A T, Evans, H J Br Buckland. R A$ Nature 232 (1971) 31. 5. Kozak, C A. Lawrence, J B B Ruddte. % H. Exp cell res 105 (1977) 109. 6. Davidson. R L & Gerald, P S, Somatic ceii genet 2 (1976) 165. 7. Raposa, T & Natarajan, A ‘I, Humangenetik 21 (1974) 221. 8. Caspersson. T, Zech, L B Johansson. C. Bxp ceil res 60 (1970) 315. 9. Hamerton. J L, Jacobs, P A & Khnger, H P. Cytogenetics !l (1972) 313. Received October 19, 1981 Accepted December 11. 1981
Repair DNA synthesis in heterokaryons during reactivation of chick erythroeytes fused with human diploid fibroblasts or HeLa cells E. VAN DER VEER and D. BOOTSMA, Depm:merit of Cell Biology and Genetics. sity Rotterdam, 3000 DR Rotterdam.
Erasmus UniterThe ,%‘etizerlands
Suppression of unscheduled DNA synthe
Summay.
In previous studies on repair DNA synthesis (unscheduled DNA synthesis, UDS)
Preliminary notes
17. Lewis, P N, Bradbury. E M & Crane-Robinson, C, Biochemistry 14 (1975) 3391. 18. Riordan, J F& V&lee, B L, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 25 (ed C H W Hirs & S N Timasheff) pp. 494-499. Academic Press, New York (1972). 19. Ostrowski, K, Barnard, E A, Sawicki, W, Chorzelski, T, Langner, A & Mikulski, A, J histochem cytochem 18 (1970) 490. 20. Heinegard, D K & Hascall, V C, J biol them 254 (1979) 921. 21. Daniel, J W & Baldwin, H H, Methods in cell physiology (ed D M Prescott) vol. 1, pp. 9-41. Academic Press, New York (1964). 22. Guttes, E & Guttes, S, Methods in cell physiology (ed D M Prescott) vol. 1, pp. 43-54. Academic Press, New York (1964). 23. Mohberg, J & Rusch, H P, Exp cell res 66 (1971) 305. 24. Polman, B J J, Ph.D. thesis, University of Nijmegen, The Netherlands (1974). 25. Sures, I & Gallwitz, D, Biochemistry 19 (1980) 943. 2t5. Kervabon, -4, Mery, J & Parello. J, FEBS lett 106 11070) \”
,,,
a-4 ,d.
27. Burton, K, Biochem j 62 (1956) 315. 28. Mohberg, J. Babcock, K L, Haugli, F B & Rusch, H P, Dev biol34 (1973) 228. 29. Reeves. R & Candido. E P M. Nucleic acids res 8 (1980) i947. 30. Bradbury, E M, Inglis, R J, Matthews, H R & Sarner, N, Eurj biochem 33 (1973) 131. 31. Bradbury, E M, Inglis, R J & Matthews, H R, Nature 247 (1974) 257. 32. Matthews, H R, Recently discovered systems of enzyme regulation by reversible phosphorylation (ed P Cohen) pp. 235-254. Elsevierlh’orthHolland, Amsterdam (1980). Received October 15, 1981 Accepted December 15, 1981
Copyright .@ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0011-482:'81:04~66-04$0:.00,0
Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids CATHLEEN R. CARLIN’ and KATHLEEN W. RAO.? Biological Sciences Research Center. Genetics Curriculum, University of North Carolina, Chapel Hill, NC 27514, USA SumnzarJ. We have analysed metaphase chromosomes of man-mouse somatic cell hybrids using a sequential staining procedure involving the fluorescent DNAbinding stains, Hoechst 33258 and quinacrine musExp Cdl
Res 138 I 1982)
tard. This was found to be a simple and reliable means of differentiating the chromosomes of the two species and of identifying specific human chromosomes. In addition this method will permit the study of the segregation of human chromosome homologues that are discordant for quinacrine mustard fluorescent polymorphisms.
The preferential segregation of human chromosomes from man-mouse somatic cell hybrids is routinely exploited to associate the expression of human gene products with s,pecific human chromosomes. This method of gene mapping requires a chromosome staining procedure which will distinguish mouse from human chromosomes and then allow the identification of specific human chromosomes. The chromosomes of the two species can be distinguished from one another after staining with the benzimidazol compound Hoechst 33258 [l] or with alkaline Giemsa Lr21. 2 Human chromosomes are routinely identified by their characteristic Q-banding patterns after staining with quinacrine mustard [3] or by their G-banding patterns after staining with Giemsa [4]. Kozak et al. [5] have described a sequential staining procedure that combines G-banding and staining with Hoechst 33258 to facilitate the identification of human chromosomes in somatic cell hybrids. We now describe a sequential staining procedure that combines the differential centromere fluorescence produced by staining with Hoechst 33258 with the characteristic banding patterns produced by staining with quinacrine mustard. Materials and Methods The hybrid cell lines used in this study were derived from the polyethylene glycol-induced fusion [6] of cells from an HPRT-deficient mouse heteroploid
’ Current address: The Wistar Institute, 36th and Spruce Streets, Philadelphia, PA 19104, USA. 2 Current address: Department of Pediatrics. East Carolina University Medical School, Greenville, NC 27834, USA. Printed
in Susden
Preliminaq
notes
467
fibroblast line, A9 (Human Mutant Cell Repository; Camden, N.J.), and a normal human diploid skin fibroblast strain initiated in the laboratory of Dr Michael Swift, University of North Carolina: ChapeI Hill. N.C. Metaphase spreads for chromosome analvses were prepared in the following manner. Ceil division was arrested by treatment with 0.75 yg/ml of Colcemid (Calbiochem) for 3-4 h. Cells were swelled by incubation in 0.075 M KC1 hvnotonic solution for 20 miu at 37°C and tixed mith three changes of freshIy prepared Carnoy fixative. Cells resuspended in fixative were dropped onto cold (4°C) wet microscope slides and dried for approx. 1 min on a slide warmer at 60°C. Slides not stained immediately were stored at 4°C. Staining with Hoechst 33258 was performed by a modification of the method of Raposa & Natarajan [7]. A stock solution (10 pg/m! in Sorensen buffer, pH 5.5) of Hoechst 33258 (American Hoechst Corp.) can be stored for several months in a light-proof container at -2O’C. The working solution (0.05 wgfmi in Sorensen buffer, pH 5.5) was prepared week!y and stored in a light-proof container at CC. Shdes were soaked in Sorensen buffer, pH 5.5, for 2 min, stained in the working solution in the dark at 37%. and then rinsed twice in Sorensen buffer and once in deionized water. Staining for 12 min produced bright fhrorescence of mouse chromosome centromeres for the cei! line used in this study. Coverslips were mounted with sucrose dissolved in McIlvaine buffer, pH 7.0 (6 g sucrose/l0 ml buffer). Slides were examined within 24 h with a Zeiss Photomicroscope III equipped with the fohowing: A barrier filter that excludes wavelengths below 51OA; and, a narrow-band pass excitation filter with peak at 4356. Kodak Technical Pan 2415 film was used for all fluorescence photography: the film was developed with HCl 10 Deve!oper. Slides to be restained with quinacrine mustard (Polysciences) were soaked brieflv in deionized water to remove coverslips, air-dried? rinsed two or three times with xylene to remove immersion oil, and airdried again. Slides that were not restained with quin acrine mustard within 24 h were stored at 4Y. Q-banding was done by the method of Caspersson et al. [8]. The quinacrine mustard staining solution (50 &ml in McIlvaine buffer, pH 7.0) was prepared monthly and stored in a light-proof container at 4’C. Slides were first hydrated by sequential rinses of 2 min each in 100% ethanol, 95% ethanol. 50% ethanol. and deionized water. They were then soaked for 5 min in McIlvaine buffer, pH 7.0. followed by staining in quinacrine mustard in-the dark. Optimum staining 6me was 20 min on the average but varied with the batch of quinacrine mustard and the age of the working solution. Temperature was not critical in the quinacrine mustard staining protocol. After staining, the slides were soaked for 15 min in deionized water in the dark. I. (A) -4 partial hybrid metaphase spread that has been stained with Hoechst 33258. Arrows point to mouse chromosomes that do not display the expected bright centromere fluorescence. (B) The same hybrid metaphase spread restained with quinacrine mustard. Human chromosomes are labeled.
Fig.
468
Preliminary
notes
Fig. 2. (A) A partial hybrid metaphase spread that has been stained with Hoechst 33258. Arrows point to mouse chromosomes. (B) The same hybrid metaphase spread restained with quinacrine mustard showing a human chromosome 13 with the quinacrine mustard-bright centromere polymorphism [9].
Coverslips were mounted as before. Slides were analyzed and photographed with a Zeiss Photomicroscope III within a few hours.
Results
When metaphase spreads from the mouse parental cell line were stained with Hoechst 33258, three to four of the mouse chromosomes did not display the expected bright centromere fluorescence (arrows, fig. 1A). However, these chromosomes were distinguishable from human chromosomes after staining with quinacrine mustard because of their unique Q-banding patterns. By comparing fig. 1A and B, it can be seen that all Hoechst 3325Sinduced fluorescence of mouse chromosome centromeres was extinguished by hydration of chromosome preparations prior to restaining with quinacrine mustard. None of the human chromosome centromeres were brightly fluorescent after staining with Hoechst 33258, although the centromere of human chromosome 3 displayed slight fluorescence. However, this particular human chromosome retained centromere fluorescence after restaining with quinacrine mustard, while all mouse centroExp CeNRes 138 11982)
mere fluorescence was extinguished (fig. 1B). Pretreatment of human chromosomes with Hoechst 33258 did not alter the characteristic Q-banding patterns of the human chromosomes produced by staining with quinacrine mustard. Quinacrine mustardbright polymorphisms [9] also retained their brilliance after pretreatment with Hoechst 33258 (fig. 2), suggesting that this technique can be used to follow the segregation of homologues that are discordant for quinacrine mustard fluorescent polymorphisms. Adequate results could be obtained from this procedure with slides that had been stored at 4°C for as long as 5-6 months. The best results, however, were obtained with preparations that were less than 1 month old. Q-banding was optimal if preparations were restained with quinacrine mustard within l-2 days after staining with Hoechst 33258. Discussion
Raposa & Natarajan [7] have reported that human metaphase chromosomes that have been stained with Hoechst 33258 under the appropriate conditions can be identified by their characteristic fluorescent banding patterns. The Hoechst 33258 staining conditions that produce optimal banding patterns, however, are not the same as those that result in optimal mouse chromosome centromere fluorescence. For this reason, we found that restaining with quinacrine mustard was necessary for the identification of human chromosomes that were not adequately banded after staining with Hoechst 33258. The sequential staining protocol described in this paper offers a method for the rapid screening of man-mouse somatic cell hybrid clones. Although it does not permit the identification of interspecific translocations as precisely as the alkaline Giemsa
Preliminary izoles technique [L?], clones which are suspected of carrying such rearrangements can be further analysed with alkaline Giemsa. In many cases, adequate cytogenetic characterization of a large number of hybrid clones can be achieved by using a combination of the two techniques: Rapid screening of ail clones with the Hoechst 3325Squinacrine mustard sequential technique, followed by further delineation of discordant clones with alkaline Giemsa. The sequential staining of metaphase spreads with Hoechst 33258 and quinacrine mustard as a method of identifying human chromosomes in man-mouse somatic cell hybrids offers several advantages. First, human and mouse chromosomes are readily distinguished, either by their differential centromere fluorescence after staining with Hoechst 33258 or by their unique Q-banding patterns. Second, hydration of chromosome preparations prior to restaining with quinacrine mustard extinguishes the centromere fluorescence of all mouse chromosomes. Third, the quality of Q-banding is sufficient to allow for the identification of specific human chromosomes. Fourth, the use of Q-banding rather than G-banding has the advantage of allowing us to follow the segregation of specific homologues in the case of human chromosome pairs discordant for quinacrine mustard polymorphisms. This staining procedure, therefore, can be used to study the expression of dominant traits in human cells heterozygous for the trait in the case that the gene coding for the trait and a QM-bright polymorphism are linked. Finally, the procedure is easy to reproduce once optimal staining times have been determined. We are indebted to Drs Michael Swift and Philip Buchanan for making their laboratory facilities available for this work and for their critical evaluations of the manuscript. We also wish to thank Drs Henry N. Kirkman and Charles Timmons for their reading
469
of the manuscript and Dorothea Baker for preparation of the manuscript. This work was supported by NIH grants NIGMST32, GM07092, and HD03110.
References 1. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. 2. Bobrow. M&Gross. J. Nature 251 (1974) 77, 3. Caspersson, T: Lomakka. G & Zech, L,‘Hereditas 67 (1970) 89. 4. Sumner, A T, Evans, H J Br Buckland. R A$ Nature 232 (1971) 31. 5. Kozak, C A. Lawrence, J B B Ruddte. % H. Exp cell res 105 (1977) 109. 6. Davidson. R L & Gerald, P S, Somatic ceii genet 2 (1976) 165. 7. Raposa, T & Natarajan, A ‘I, Humangenetik 21 (1974) 221. 8. Caspersson. T, Zech, L B Johansson. C. Bxp ceil res 60 (1970) 315. 9. Hamerton. J L, Jacobs, P A & Khnger, H P. Cytogenetics !l (1972) 313. Received October 19, 1981 Accepted December 11. 1981
Repair DNA synthesis in heterokaryons during reactivation of chick erythroeytes fused with human diploid fibroblasts or HeLa cells E. VAN DER VEER and D. BOOTSMA, Depm:merit of Cell Biology and Genetics. sity Rotterdam, 3000 DR Rotterdam.
Erasmus UniterThe ,%‘etizerlands
Suppression of unscheduled DNA synthe
Summay.
In previous studies on repair DNA synthesis (unscheduled DNA synthesis, UDS)