ISSN 0018-0661. Received February 9, 1976. HeLa cells were fused with protoplasts of carrot (Daucus carota) by the use of polyethylene glycol as fusion agent.
Hereditas 82: 121-124
(1976)
Fusion of human cells with carrot protoplasts induced by polyethylene glycol D. DUDITS,' I . RASKO,' GY. HADLACZKY' and A. LIMA-DE-FARIA*
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Biological Research Center, Institute of Genetics, Szeged, Hungary
' Institute of' Molecular Cytogenetics, University of Lund, Sweden
DUDITS,D., RASKO.I . , HADLACZKY, GY. and LIMA-DE-FARIA, A . 1976. Fusion of human cells with carrot protoplasts induced by polyethylene glycol. - Hereditas 82: 121-124. Lund, Sweden. ISSN 0018-0661. Received February 9, 1976 HeLa cells were fused with protoplasts of carrot (Daucus carota) by the use of polyethylene glycol as fusion agent. The two types of nuclei can be recognized in the heterokaryon by their differential staining ability and by the tritium labelling of HeLa cells. Seventy-two hours after fusion the cells appeared surrounded by cell walls and in some cases contained HeLa and carrot fused interphase nuclei. The percentage of fusion is estimated to be approximately 0.3 to 0.6%. A . Lima-de-Faria, Institute of Molecular Cytogenetics, S-223 63 Lund, Sweden
The regular distribution of various properties in the eukaryotic chromosome led to the formulation of the concept of the chromosome field, according to which the chromosome is considered to be an ordered system functioning as a whole. This concept was reinforced by the finding that the distribution of cistrons for 28s and 18s ribosomal RNA is so regular in both plants and animals that it can be predicted by means of an equation (LIMA-DE-FARIA 1954, 1972, 1973). Since the distribution of these genes and other properties is similar in lower plants and mammals (including man) this led to the postulate that eukaryotic chromosomes belonging to widely separated phyla are governed by the same essential type of organization mechanism (LIMA-DE-FARIA 1973, 1976). To test this postulate an experiment was designed to hybridize plant and human cells. Formation of interphylum heterokaryons has been reported for human and mosquito cells (ZEPP et al. 1971) and for hen erythrocytes and yeast protoplasts (AHKONGet al. 1975). UV inactivated Sendai virus and polyethylene glycol (PEG) were used as fusion agents in these experiments. PEG has been shown to induce fusion of protoplasts of higher plants (KAO and MICHAYLUK 1974; WALLINet al. 1974), 9
erythrocytes (AHKONGet al. 1975) and bacterium protoplasts (L. ALFOLDI,pers. comm.). We have succeeded in achieving the formation of heterokaryons between human cells (HeLa) and plant protoplasts by means of PEG. HeLa cells were grown as monolayers at 37°C in MEM (LOCKART and EAGLE1959) supplemented with 5% foetal calf serum (Biocult). In some experiments the HeLa cells were pre-incubated with 0.4 pCi/ml 3H thymidine (specific activity 19.6 Ci/mM) for 48 hours before fusion so that most nuclei could be labelled. The cells were used for fusion after trypsinisation. Plant protoplasts were obtained from cells of carrot (Daucus carota L. var. Nantaise Slenders) grown as suspension culture in B5 medium at 25°C (GAMBORG et al. 1968). The isolation of protoplasts was achieved by incubation of the cells with an enzyme solution, containing 4% Onozuka P 1500 (Yakult Bioche. Japan), 1% Driselase (Kyowa Hakko Kogyo, Japan), 2% Pectinase (Sigma), 1 % Rhozyme (Rhom and Haas, Canada) in a medium, described for Vicia protoplasts (KAOet al. 1974). After 5-6 hours incubation the protoplasts were washed by centrifugation and re-suspended in modified Hanks' solution (KCI was omitted and the
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FUSION OF HUMAN CELLS WITH CARROT PROTOPLASTS
potassium phosphate buffer was substituted by HEPES, 0.01 M, at pH = 7.5). The fusion was carried out in 60 mm diameter plastic Petri dishes (Greiner), by the mixture of HeLa cells and protoplasts in modified Hanks solution in the ratios 1:1, 1.2, 2.1, respectively. Finally PEG solution (KAOand MICHAYLUK 1974) was added to the mixture. The addition of PEG resulted in intense clumping of HeLa cells and carrot protoplasts. After 40 min. incubation at room temperature with PEG, protoplast culture medium (KAOet al. 1974) was added and PEG was rinsed away. The cells were cultivated further in this medium at room temperature. The cell fusion was analysed by light microscopy after a differential staining described by VOSAand MARCHI(1972). A slight modification was applied, namely that the barium hydroxyde treatment was prolonged for 15-20 min. at room temperature. HeLa nuclei stained uniformly dark and the protoplast nuclei were pale, only small heterochromatic segments could be seen (Fig. 1). The highest fusion frequency was observed with a HeLa: protoplast ratio of 1.2 (approximately 0.3-0.6%, Fig. 2). After the fusion of labelled HeLa cells and carrot protoplasts, cell samples were taken at different times and preparations were made. The slides were immersed in Ilford K5 emulsion and processed for autoradiography. The autoradiographs also revealed that human-plant heterokaryons had been formed (Fig. 3, 4). The presence of labelled HeLa nuclei 48 hours after fusion in the heterokaryons means that they remained intact in the plant cytoplasm. Seventy-two hours after fusion the cells appeared to be surrounded by cell walls (Fig. 5, 6) and contained in some cases HeLa and carrot interphase nuclei which were fused (Fig. 5). Studies are in progress concerning the possibilities
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for the cultivation of the heterokaryons obtained, and the detection of biochemical activities of both partners. Literature cited AHKONG,Q. F., FISHER,D.. TAMPION, W. and LUCY,J. A. 1975. Mechanisms of cell fusion. - Nurure 253: 194-195 AHKONG,Q . F., HOWELL,J. I . and LUCY,J . A. 1975. Fusion of hen erythrocytes with yeast protoplasts induced by N a m e 255: 66-67 polyethylene glycol. GAMBORG, 0. L., MILLER,R. A. and OJIMA.K. 1968. Nutrient requirements of suspension cultures of soybean root cells. - Ezrp. Cell Res. 50: I51 158 KAO, K . N., CONSTABEL, F., MICHAYLUK, M. R . and GAMBORG, 0. L. 1974. Plant protoplast fusion and growth of intergeneric hybrid cells. Plania 120: 21 5-227 KAO,K. N . and MICHAYLUK, M . R. 1974. A method for highfrequency intergeneric fusion of plant protoplasts. - Plania 115: 355-367 LIMA-DE-FARIA. A. 1954. Chromosome gradient and chromosome field in Agaphanthus. - Chromosuma 6 : 330-370 LIMA-DE-FARIA, A. 1972. The specific location of ribosomal genes in the eukaryotic chromosome. - In Modern Aspects of Cjiugeneiics: Constitutive Hererochromaiin in Man. Symp. Med. Hoechsr 6 : 39--44 LIMA-DE-FARIA, A. 1973. Equations defining the position of ribosomal cistrons in the eukaryotic chromosome. - Naiure New Bid. 241: 136-139 LIMA-DE-FARIA, A. 1976. The chromosome field I . Prediction of the location of ribosomal cistrons. Hereditas 82 (in press) LOCKART,R. Z., JR and EAGLE,H. 1959. Requirements for growth of single human cells. - Science 129: 252-254 VOSA,C . G. and MARCHI,P. 1972. Quinacrine fluorescence and Giemsa staining in plants. - Naiure New, Biol. 237 191-192 WALLIN,A,, GLIMELIUS, K. and ERIKSSON,T. 1974. The induction of aggregation and fusion of Daucus curuia protoZ. Pjlanzenphysiol. 74: plasts by polyethylene glycol. 64-80 ZEPP, H. D., CONOVER, J. H., HIRSCHHORN, K. and HODES, H. L. 1971. Human-mosquito somatic cell hybrids induced by ultraviolet-inactivated Sendai virus. - Nature Neu' Biol. 229: 119-121 -
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Fig. 1-6. HeLa and Daucus curoia cells stained by a differential Giemsa staining procedure. HeLa cell nuclei are darkly stained, carrot cells appear pale with small chromocentres. As a control HeLa nuclei were labelled with tritiated thymidine in one of the experiments. -- Fig. I . Separate cells. - Fig. 2-6. Heterokaryons. - Fig. 2. Heterokaryon containing one human and three carrot nuclei in a common cytoplasm. - Fig. 3. Two or three carrot nuclei with one labelled HeLa nucleus. - Fig. 4. The same cell after cytoplasmic staining with carbolfuchsin to show more clearly the area occupied by the cytoplasm and the silver grains without nuclear staining. - Fig. 5. Two groups of nuclei within a common cytoplasm surrounded by a cell wall. The upper group shows interphase nuclear fusion between part of a HeLa nucleus and a carrot nucleus. - Fig. 6. Heterokaryon composed of two carrot nuclei and part of a HeLa nucleus in a cytoplasm surrounded by a cell wall. - Fig. 1, 3 , 4 and Fig. 2, 5. 6. 1 and 72 hours after fusion, respectively. x 1,300.
