Preliminary
sity (figs 2, 3), suggests several explanations. One is that the central more densely packed cells undergo fewer cell divisions than the cells in the more peripheral areas, as is true of undissociated clumps of limb bud mesenchyme [3]. More rapid cell division might hinder expression of the cartilage phenotype as it does for the expression of pigment in retinal pigment cells [6]. However, it is possible that the microenvironment of the closely aggregated cells may favor the expression of cartilage. To test this idea, small explants were cultured in small drops of medium in plastic dishes to favor the accumulation of metabolic products. These explants form cartilage, but no muscle (fig. 4). Similar small explants grown on lens paper in the organ culture assemblies differentiate into muscle, but not cartilage (fig. 5). Measurements revealed a greater level of DNA synthesis, and presumably cell division, in the small explants on lens paper that form muscle (table 3). It is possible that the accumulation of metabolic products in the small drops prevented myogenesis by reducing the incidence of cell division. Another explanation is that a lower degree of cell adhesion to the plastic substrate may have contributed to the failure of myogenesis in the small drop cultures. In any event, it is necessary to consider if the culture conditions actually cause determination resulting in undetermined cells becoming cartilage or muscle [5], or if the conditions merely selectively allow the differentiation of already determined cells [ 11.
notes
453
4. Hamburger, V & Hamilton, H L. J morphol X8 (1951) 49. 5. Searls, R L & Janners. M Y, J exp zoo1 170(1969) 365. 6. Whittaker, J R. J exp zool 169(1968) 143. Received February 6. 1976 Accepted February 12, 1976
Agglutination and labeling density of soybean agglutinin on young and old human red blood cells Y. MARIKOVSKY,’ R. LOTAN,’ H. LIS,’ N. SHARON2 and D. DANON,’ ‘Section of Biolokal Ultrastructure and the ‘Department of”Biophy&s, The Weizmann Institute of Science, Rehovot, Israel Summary. Young human red blood cells (RBCs) are agglutinated by soybean agglutinin (SBA) at a higher rate than old RBCs. Treatment of RBCs with trypsin or neuraminidase enhances the rate of agglutination of both populations, but abolishes the difference between them. The differential effect was most pronounced when agglutination was carried out with isolated SBA aggregates. The labeling density of SBA-ferritin on the surface of young RBCs is about twice as high as that of old RBCs. Treatment with neuraminidase increased significantly the labeling density of SBA-ferritin on the surface of RBCs of both age populations.
Soybean agglutinin (SBA) is known to agglutinate rabbit and human red blood cells (RBCs), and to bind to galactose-like saccharide sites on cell membranes [ 1,2]. This interaction can be used as a probe for studying the distribution of specific carbohydrate containing molecules on the surface of young and old human RBCs. We report the results of experiments on the rate of agglutination of separated human young and old RBCs with SBA. The influence of surface charge on the rate of agglutination was inferred from experiments with RBCs that had been treated with References neuraminidase or trypsin. The density and 1. Dienstman, S R, Biehl, J, Holtzer, S & Holtzer, H, distribution of SBA receptor sites on young Dev biol39 (1974) 83. and old separated human RBCs was stud2. Flickinger, R A, The role of RNA in reproduction and development (ed M C Niu & S Segal) p. 11. ied by electron microscopy using SBAElsevier, Amsterdam (1973). ferritin as a label. 3. Flickinger, R A, Devel biol41 (1974) 202. 30-761803
E.Ip C’rll H~.Y99 (I 976,
454
Preliminary
notes
Table 1. Density of ferritin
conjugated to SBA on membranes of young and old human RBCs, untreated and treated with neuraminidase and trypsina
Cell sample Young RBCs, untreated Old RBCs, untreated Young RBCs, neuraminidasetreated Old RBCs, neuraminidasetreated Young RBCs, trypsin-treated Old RBCs, trypsin-treated
Mean no. of ferritin particles/ pm
Range b
16 8
12-19 5-11
45
39-50
50 20 14
45-56 16-24 II-19
of separated young and old human RBCs, untreated or treated with neuraminidase or trypsin, was labeled with SBA-ferritin (1 mglml). In control experiments, SBA-ferritin was preincubated with o-galactose (0.3 M) for 15 min at room temperature and labeling was also done in the presence of the sugar. SBA-ferritin labeled cells were washed three times with Veronalbuffered saline to remove free, unbound SBA-ferritin, fixed with 2 % glutaraldehyde and post-fixed with 1% osmium tetroxide. The samples were embedded in Epon 812 and thin-sectioned with a Sorvall ultramicrotome. Electron micrographs were taken with a JEM-7 electron microscope at 80 kV. Counts of ferritin particles were performed on micrographs of perpendicularly sectioned RBC membranes, at a final magnification of 100000.
Results and Discussion
Young RBCs agglutinate with SBA at a higher rate than old RBCs (fig. 1A). The differential effect was most marked when agglutination was carried out with aggregated SBA (mol. wt3240000 [5, 9, lo] (fig. IB). Material and Methods After treatment with trypsin, the rate of agRed blood cells. Blood was collected from healthy glutination of both age populations was endonors (blood group B, Rh+) by venipuncture into heparinized test tubes. Young and old RBCs were hanced, but there was no longer any differseparated by the differential flotation method [3]. The separated cell fractions were washed twice in Verona1 ence between the young and old cells. An even larger increase in the rate of agglutinabuffered saline, pH 7.2. Agglutinins. SBA was purified by affinity chromatography [4] and stored in the lyophilized state. Ag- tion was observed after treatment of RBCs
a Ferritin particles were counted on 50 pm length of arbitrarily selected perpendicularly sectioned membranes. b Counts on 20 cells.
gregates formed in such preparations were separated from the unaggregated lectin by gel filtration [5]. Lectin preparations prior to gel filtration are designated as “unfractionated SBA”, and the isolated aggregates as “aggregated SBA”. Coupling of ferritin to SBA was carried out according to Avrameas [6], using equimolar concentrations of both proteins and in the presence of 1 M o-galactose. The ferritin-SBA complex was purified on an affinity column of Sepharose-N-E-aminocaproyl-@-D-galactopyranosylamine [4] at 4°C and separated from the residual uncoupled SBA by gel filtration on a column of Sephadex G-150 in phosphate-buffered saline [5]. The final product contained nearly equimolar proportions of SBA (estimated on the basis of its carbohydrate content, [7]) and ferritin. Poly-L-lysine, mol. wt 38000, was purchased from Miles-Yeda Ltd., Rehovoth, Israel. Enzymatic treatment. A 10% suspension of RBCs in Veronal-buffered saline were incubated with 50 U/ ml of protease-free neuraminidase from Vibrio cholerae (Behringwerke AG), or with 0.2 mg/ml trypsin (Worthington), at 37°C for 60 min. Enzyme-treated RBCs were washed twice with Veronal-buffered saline. Agglutination assay. The rate of agglutination at 24°C was automatically recorded for 12 min using a Fragiligraph, model D-2 (Elmedix Ltd., Tel Aviv, Israel) as previously described [8]. Electron microscopy. Two ml of a 10% suspension Exr, Cell Res 99 (1976)
I
1
’
’
’
‘I
Fig. 1. Abscissa: time (min); ordinate: light transmission (%). Rate of agglutination by SBA of erythrocytes. (A) Unfractionated SBA; (B) aggregated SBA, both at 0.4 mg/ml of cell suspension; (C) poly-L-lysine (mol. wt 38000,20 pg/ml suspension). 0, Young; 0, old untreated RBCs; young andiold I@,neuraminidasc-treated RBCs; A, trypsin-treated RBCs.
Preliminary notes
455
Fig. 2. Labeling with SBAferritin conjugate of human RBC membranes. (a) Young untreated human RBCs; (6) old untreated human RBCs; (c) young neuraminidasetreated human RBCs; (d) old neuraminidase-treated human RBCs. x 100000.
with neuraminidase. Here again, no difference between the two age populations was observed. With the positively charged poly-L-lysine, old RBCs were agglutinated at a higher rate than young RBCs (fig. 1C), reflecting surface charge differences between the young and the old cells [ 111. After treatment with trypsin, the rate of agglutination decreased for both age populations, while after treatment with neuraminidase, no agglutination by poly+lysine was observed, as previously noted [ 121.
Comparison of the rates of agglutination of young and old human RBCs, untreated or treated with neuraminidase or trypsin, by poly-L-lysine and SBA clearly shows that the agglutination by SBA is not due to the charge of the lectin. Both young and old RBCs were evenly but sparsely labeled with SBA-ferritin, the labeling density on the surface of the young cells being about twice as high as that of the old cells (fig. 2a, b ; table 1). Neuraminidase treatment of the cells caused a significant increase in labeling density-about 3 times Exp Cell Rcs Y9 (1976)
456
Preliminary
notes
as much for young cells, and about 6 times for old cells (fig. 2c, d; table 1). A slight increase in labeling density was observed on young and old RBCs treated with trypsin (table 1). In control experiments, where labeling was in the presence of n-galactose, no binding of SBA-ferritin was observed. The finding that SBA agglutinates young RBCs at a higher rate than old ones is somewhat surprising in view of the fact that the former contain 30 % more N-acetylneuraminic acid residues [12]. The increase in agglutinability can, however, be accounted for by the finding that young RBCs possess significantly more receptors for SBA (fig. 2a). Our results are thus in agreement with the report of Baxter & Beeley [13] that old RBCs contain 30 % less D-galactose residues on their surface than young cells. The ability of aggregated SBA [5, 93 to cause a pronounced differential agglutination of young versus old RBCs (fig. 1B) is probably the result of the increase in valency and in the size of the lectin. These properties of aggregated SBA allow it to form multiple cross-bridges between cells at distances larger than those across which the smaller unaggregated SBA can act. Therefore, the effect of electrostatic repulsion, which is higher between young RBCs as compared with old cells, is less pronounced with the aggregated SBA. The enhancement of agglutination by SBA of RBCs treated with neuraminidase can be explained by a reduction of the cell surface zeta-potential resulting from removal of most of the negatively charged Nacetylneuraminic residues, as well as to the exposure of new D-galactose sites [14]. Such treatment abolishes any differences in negative charge and number of SBA-binding sites originally present on old and young FU3Csand thus the rates of agglutination of both cell populations become the same. Exp Cell Res 99 (1976)
The authors are indebted to Mr Stanley Himmelhoch for his excellent technical help in thin sectioning and photography.
References I. Lis, H, Sela, B A, Sachs, L & Sharon, N, Biochim biophvs acta 21 I (1970) 582. 2. Gordon,‘JA, Sharon, iu &‘Lis, H, Biochim biophvs acta 264 (1972) 387. 3. banon, D & Marikovsky, Y, J lab clin med 64 (1964) 668. 4. Gordon, J A, Blumberg, S, Lis, H & Sharon, N, FEBS lett 24 (1972) 193. 5. Lotan, R, Lis, H & Sharon, N, Biochem biophys res commun 62 (1975) 144. Avrameas, S, Immunochemistrv 6 (l%9) 43. ? Lis, H, Sharon, N & Katchalski, E, J biol them 241 (1966) 684. 8. Danon, D, Marikovsky, Y & Kohn, A, Experientia 25 (1969) 104. 9. Lotan, R, Lis, H, Rosenwasser, A, Novogrodsky, A & Sharon, N, Biochem biophvs _ res commun 55 (1973) 1347. IO. - Ibid 56 (1974) 1100. II. Marikovsky, Y, Danon, D & Katchalsky, A, Biochim biophys acta 124 (1966) 154. 12. Marikovsky, Y & Danon, d, J cell biol 43 (1%9) I. 13. Baxter, A & Beeley, J G, Abstr paper presented at Symposium on glycoconjugates, Brighton (1975). 14. Nicolson, G L, J natl cancer inst 50 (1973) 1443. Received February 6, 1976 Accepted February 12, 1976
The solubility characteristics of SV 40 tumor antigen M. KELLERMAYER,‘* MARGARITA TALAS,z** CONNIE WONG,* H. BUSCH’ and JANET S. BUTEL,2 ‘Denartment of Pharmacology and 2Departmerit of Virology and Epidemiology, Baylor of Medicine, HOUSIOII, TX 77025, USA
College
The solubility characteristics of SV40 tumor (T) antigen were investigated. Cell nuclei were isolated from cover glass cultures of SVrlO-infected or -transformed cells by detergent treatment. When treated in a buffered-isotonic sucrose solution, the isolated nuclei contained tvpical SV40 T antigen bv immunofludrescence. If the-detergent was dissolved in an isotonic electrolyte solution containing 0.15 M monovalent cations (K+), the T antigen was extracted from the nuclei. Complement fixation tests demonstrated that the various solutions had no deleterious effects on the antigenicity of T antigen. Under identi-
Summary.
* Department of Clinical Chemistry, Medical IJniversity of Pets, Pets, Hungary. ** Hungarian Academy of Sciences, Microbiological Research Group, Budapest 12, Hungary.
sity (figs 2, 3), suggests several explanations. One is that the central more densely packed cells undergo fewer cell divisions than the cells in the more peripheral areas, as is true of undissociated clumps of limb bud mesenchyme [3]. More rapid cell division might hinder expression of the cartilage phenotype as it does for the expression of pigment in retinal pigment cells [6]. However, it is possible that the microenvironment of the closely aggregated cells may favor the expression of cartilage. To test this idea, small explants were cultured in small drops of medium in plastic dishes to favor the accumulation of metabolic products. These explants form cartilage, but no muscle (fig. 4). Similar small explants grown on lens paper in the organ culture assemblies differentiate into muscle, but not cartilage (fig. 5). Measurements revealed a greater level of DNA synthesis, and presumably cell division, in the small explants on lens paper that form muscle (table 3). It is possible that the accumulation of metabolic products in the small drops prevented myogenesis by reducing the incidence of cell division. Another explanation is that a lower degree of cell adhesion to the plastic substrate may have contributed to the failure of myogenesis in the small drop cultures. In any event, it is necessary to consider if the culture conditions actually cause determination resulting in undetermined cells becoming cartilage or muscle [5], or if the conditions merely selectively allow the differentiation of already determined cells [ 11.
notes
453
4. Hamburger, V & Hamilton, H L. J morphol X8 (1951) 49. 5. Searls, R L & Janners. M Y, J exp zoo1 170(1969) 365. 6. Whittaker, J R. J exp zool 169(1968) 143. Received February 6. 1976 Accepted February 12, 1976
Agglutination and labeling density of soybean agglutinin on young and old human red blood cells Y. MARIKOVSKY,’ R. LOTAN,’ H. LIS,’ N. SHARON2 and D. DANON,’ ‘Section of Biolokal Ultrastructure and the ‘Department of”Biophy&s, The Weizmann Institute of Science, Rehovot, Israel Summary. Young human red blood cells (RBCs) are agglutinated by soybean agglutinin (SBA) at a higher rate than old RBCs. Treatment of RBCs with trypsin or neuraminidase enhances the rate of agglutination of both populations, but abolishes the difference between them. The differential effect was most pronounced when agglutination was carried out with isolated SBA aggregates. The labeling density of SBA-ferritin on the surface of young RBCs is about twice as high as that of old RBCs. Treatment with neuraminidase increased significantly the labeling density of SBA-ferritin on the surface of RBCs of both age populations.
Soybean agglutinin (SBA) is known to agglutinate rabbit and human red blood cells (RBCs), and to bind to galactose-like saccharide sites on cell membranes [ 1,2]. This interaction can be used as a probe for studying the distribution of specific carbohydrate containing molecules on the surface of young and old human RBCs. We report the results of experiments on the rate of agglutination of separated human young and old RBCs with SBA. The influence of surface charge on the rate of agglutination was inferred from experiments with RBCs that had been treated with References neuraminidase or trypsin. The density and 1. Dienstman, S R, Biehl, J, Holtzer, S & Holtzer, H, distribution of SBA receptor sites on young Dev biol39 (1974) 83. and old separated human RBCs was stud2. Flickinger, R A, The role of RNA in reproduction and development (ed M C Niu & S Segal) p. 11. ied by electron microscopy using SBAElsevier, Amsterdam (1973). ferritin as a label. 3. Flickinger, R A, Devel biol41 (1974) 202. 30-761803
E.Ip C’rll H~.Y99 (I 976,
454
Preliminary
notes
Table 1. Density of ferritin
conjugated to SBA on membranes of young and old human RBCs, untreated and treated with neuraminidase and trypsina
Cell sample Young RBCs, untreated Old RBCs, untreated Young RBCs, neuraminidasetreated Old RBCs, neuraminidasetreated Young RBCs, trypsin-treated Old RBCs, trypsin-treated
Mean no. of ferritin particles/ pm
Range b
16 8
12-19 5-11
45
39-50
50 20 14
45-56 16-24 II-19
of separated young and old human RBCs, untreated or treated with neuraminidase or trypsin, was labeled with SBA-ferritin (1 mglml). In control experiments, SBA-ferritin was preincubated with o-galactose (0.3 M) for 15 min at room temperature and labeling was also done in the presence of the sugar. SBA-ferritin labeled cells were washed three times with Veronalbuffered saline to remove free, unbound SBA-ferritin, fixed with 2 % glutaraldehyde and post-fixed with 1% osmium tetroxide. The samples were embedded in Epon 812 and thin-sectioned with a Sorvall ultramicrotome. Electron micrographs were taken with a JEM-7 electron microscope at 80 kV. Counts of ferritin particles were performed on micrographs of perpendicularly sectioned RBC membranes, at a final magnification of 100000.
Results and Discussion
Young RBCs agglutinate with SBA at a higher rate than old RBCs (fig. 1A). The differential effect was most marked when agglutination was carried out with aggregated SBA (mol. wt3240000 [5, 9, lo] (fig. IB). Material and Methods After treatment with trypsin, the rate of agRed blood cells. Blood was collected from healthy glutination of both age populations was endonors (blood group B, Rh+) by venipuncture into heparinized test tubes. Young and old RBCs were hanced, but there was no longer any differseparated by the differential flotation method [3]. The separated cell fractions were washed twice in Verona1 ence between the young and old cells. An even larger increase in the rate of agglutinabuffered saline, pH 7.2. Agglutinins. SBA was purified by affinity chromatography [4] and stored in the lyophilized state. Ag- tion was observed after treatment of RBCs
a Ferritin particles were counted on 50 pm length of arbitrarily selected perpendicularly sectioned membranes. b Counts on 20 cells.
gregates formed in such preparations were separated from the unaggregated lectin by gel filtration [5]. Lectin preparations prior to gel filtration are designated as “unfractionated SBA”, and the isolated aggregates as “aggregated SBA”. Coupling of ferritin to SBA was carried out according to Avrameas [6], using equimolar concentrations of both proteins and in the presence of 1 M o-galactose. The ferritin-SBA complex was purified on an affinity column of Sepharose-N-E-aminocaproyl-@-D-galactopyranosylamine [4] at 4°C and separated from the residual uncoupled SBA by gel filtration on a column of Sephadex G-150 in phosphate-buffered saline [5]. The final product contained nearly equimolar proportions of SBA (estimated on the basis of its carbohydrate content, [7]) and ferritin. Poly-L-lysine, mol. wt 38000, was purchased from Miles-Yeda Ltd., Rehovoth, Israel. Enzymatic treatment. A 10% suspension of RBCs in Veronal-buffered saline were incubated with 50 U/ ml of protease-free neuraminidase from Vibrio cholerae (Behringwerke AG), or with 0.2 mg/ml trypsin (Worthington), at 37°C for 60 min. Enzyme-treated RBCs were washed twice with Veronal-buffered saline. Agglutination assay. The rate of agglutination at 24°C was automatically recorded for 12 min using a Fragiligraph, model D-2 (Elmedix Ltd., Tel Aviv, Israel) as previously described [8]. Electron microscopy. Two ml of a 10% suspension Exr, Cell Res 99 (1976)
I
1
’
’
’
‘I
Fig. 1. Abscissa: time (min); ordinate: light transmission (%). Rate of agglutination by SBA of erythrocytes. (A) Unfractionated SBA; (B) aggregated SBA, both at 0.4 mg/ml of cell suspension; (C) poly-L-lysine (mol. wt 38000,20 pg/ml suspension). 0, Young; 0, old untreated RBCs; young andiold I@,neuraminidasc-treated RBCs; A, trypsin-treated RBCs.
Preliminary notes
455
Fig. 2. Labeling with SBAferritin conjugate of human RBC membranes. (a) Young untreated human RBCs; (6) old untreated human RBCs; (c) young neuraminidasetreated human RBCs; (d) old neuraminidase-treated human RBCs. x 100000.
with neuraminidase. Here again, no difference between the two age populations was observed. With the positively charged poly-L-lysine, old RBCs were agglutinated at a higher rate than young RBCs (fig. 1C), reflecting surface charge differences between the young and the old cells [ 111. After treatment with trypsin, the rate of agglutination decreased for both age populations, while after treatment with neuraminidase, no agglutination by poly+lysine was observed, as previously noted [ 121.
Comparison of the rates of agglutination of young and old human RBCs, untreated or treated with neuraminidase or trypsin, by poly-L-lysine and SBA clearly shows that the agglutination by SBA is not due to the charge of the lectin. Both young and old RBCs were evenly but sparsely labeled with SBA-ferritin, the labeling density on the surface of the young cells being about twice as high as that of the old cells (fig. 2a, b ; table 1). Neuraminidase treatment of the cells caused a significant increase in labeling density-about 3 times Exp Cell Rcs Y9 (1976)
456
Preliminary
notes
as much for young cells, and about 6 times for old cells (fig. 2c, d; table 1). A slight increase in labeling density was observed on young and old RBCs treated with trypsin (table 1). In control experiments, where labeling was in the presence of n-galactose, no binding of SBA-ferritin was observed. The finding that SBA agglutinates young RBCs at a higher rate than old ones is somewhat surprising in view of the fact that the former contain 30 % more N-acetylneuraminic acid residues [12]. The increase in agglutinability can, however, be accounted for by the finding that young RBCs possess significantly more receptors for SBA (fig. 2a). Our results are thus in agreement with the report of Baxter & Beeley [13] that old RBCs contain 30 % less D-galactose residues on their surface than young cells. The ability of aggregated SBA [5, 93 to cause a pronounced differential agglutination of young versus old RBCs (fig. 1B) is probably the result of the increase in valency and in the size of the lectin. These properties of aggregated SBA allow it to form multiple cross-bridges between cells at distances larger than those across which the smaller unaggregated SBA can act. Therefore, the effect of electrostatic repulsion, which is higher between young RBCs as compared with old cells, is less pronounced with the aggregated SBA. The enhancement of agglutination by SBA of RBCs treated with neuraminidase can be explained by a reduction of the cell surface zeta-potential resulting from removal of most of the negatively charged Nacetylneuraminic residues, as well as to the exposure of new D-galactose sites [14]. Such treatment abolishes any differences in negative charge and number of SBA-binding sites originally present on old and young FU3Csand thus the rates of agglutination of both cell populations become the same. Exp Cell Res 99 (1976)
The authors are indebted to Mr Stanley Himmelhoch for his excellent technical help in thin sectioning and photography.
References I. Lis, H, Sela, B A, Sachs, L & Sharon, N, Biochim biophvs acta 21 I (1970) 582. 2. Gordon,‘JA, Sharon, iu &‘Lis, H, Biochim biophvs acta 264 (1972) 387. 3. banon, D & Marikovsky, Y, J lab clin med 64 (1964) 668. 4. Gordon, J A, Blumberg, S, Lis, H & Sharon, N, FEBS lett 24 (1972) 193. 5. Lotan, R, Lis, H & Sharon, N, Biochem biophys res commun 62 (1975) 144. Avrameas, S, Immunochemistrv 6 (l%9) 43. ? Lis, H, Sharon, N & Katchalski, E, J biol them 241 (1966) 684. 8. Danon, D, Marikovsky, Y & Kohn, A, Experientia 25 (1969) 104. 9. Lotan, R, Lis, H, Rosenwasser, A, Novogrodsky, A & Sharon, N, Biochem biophvs _ res commun 55 (1973) 1347. IO. - Ibid 56 (1974) 1100. II. Marikovsky, Y, Danon, D & Katchalsky, A, Biochim biophys acta 124 (1966) 154. 12. Marikovsky, Y & Danon, d, J cell biol 43 (1%9) I. 13. Baxter, A & Beeley, J G, Abstr paper presented at Symposium on glycoconjugates, Brighton (1975). 14. Nicolson, G L, J natl cancer inst 50 (1973) 1443. Received February 6, 1976 Accepted February 12, 1976
The solubility characteristics of SV 40 tumor antigen M. KELLERMAYER,‘* MARGARITA TALAS,z** CONNIE WONG,* H. BUSCH’ and JANET S. BUTEL,2 ‘Denartment of Pharmacology and 2Departmerit of Virology and Epidemiology, Baylor of Medicine, HOUSIOII, TX 77025, USA
College
The solubility characteristics of SV40 tumor (T) antigen were investigated. Cell nuclei were isolated from cover glass cultures of SVrlO-infected or -transformed cells by detergent treatment. When treated in a buffered-isotonic sucrose solution, the isolated nuclei contained tvpical SV40 T antigen bv immunofludrescence. If the-detergent was dissolved in an isotonic electrolyte solution containing 0.15 M monovalent cations (K+), the T antigen was extracted from the nuclei. Complement fixation tests demonstrated that the various solutions had no deleterious effects on the antigenicity of T antigen. Under identi-
Summary.
* Department of Clinical Chemistry, Medical IJniversity of Pets, Pets, Hungary. ** Hungarian Academy of Sciences, Microbiological Research Group, Budapest 12, Hungary.
