Preliminary
notes
413
The effects of environmental agents on human health is a subject of considerable interest at the present time. Of special concern to health scientists are the long-term effects of such agents and particularly their carcinogenic, mutagenic and teratogenic potentials which are believed to result, in part, from the ability of these agents to modify intracellular DNA. We have recently been investigating the DNA-modifying potential of a relatively new environmental agent which is widely used in pediatric therapeutics for the control of neonatal hyperbilirubinemia: high intensity illumination with visible light, i.e. phototherapy [l]. In the course of these studies we have found that (a) visible light (450 nm) was mutagenic for prokaryotes [2] and certain eukaryotes (yeasts) (unpublished results); (b) visible light (450 nm) in the presence of physiological photosensitizers was capable of altering the physical-chemical Received March 10, 1976 properties of isolated DNA [3-51; and (c) Accepted July 19, 1976 that visible light (450 nm) in the absence of added photosensitizers was capable of modifying the structure of intracellular DNA of human cells growing in tissue culImmunofluorescence for the detection of ture [6]. We have evidence that the aforephotochemical lesions in intracellular DNA mentioned DNA-modifying effects of visiB. GUTTER,’ Y. NISHIOKA, W. T. SPECK, H. S. ble light are dependent upon the generation ROSENKRANZ.’ BEVERLY LUBIT and B. F. ER- of singlet oxygen by intracellular photoLANGER, Departments of Microbiology and Pesensitizers with subsequent oxidation of the diatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA guanine moiety of DNA ([3, 51 and unpublished results). Because the effects were Summary. The ability of methylene blue to photooxidize the guanine moiety of intracellular DNA of seen with doses of visible light, only 5 % of living human cells was demonstrated by immuno- that received in a 24 h period by a newborn fluorescence using an antibody with a specificity for the unpaired cytosine residue of DNA. These findings infant undergoing phototheraphy in our which indicate that sufftcient amounts of the dye pene- nursery, we became interested in developtrate the mammalian cell to cause biological effects may present the investigator with the unique oppor- ing procedures to identify and quantitate in tunity of selectively altering the guanine residue in a rapid and reproducible fashion alterations DNA and thereby determining the genetic conin the genetic material of such infants. The sequences of this reaction. present communication describes a model system in which the guanine residues of the 1 Present address: Department of Microbiology, New DNA of human cells growing in culture are York Medical College, Valhalla, NY 10595,USA.
Caspersson, T, Zech, L, Modest, E J, Foley, Cl E, Wagh, U & Simonsson, E, Exp cell res 58 (1%9) 141. 15. Vosa, C G, Chromosoma 43 (1973) 269. 16. Bendich. A J & Bolton. E T. Plant LI ohvs 42 (1%7) ~ I 959. 17. Cullis, C A & Schweizer, D, Chromosoma 44 (1974)417. 18. Haitinger, M, Fluorescenz-Mikroskopie. Akademische Verlagsgesellschaft, Leipzig (1938). 19. Gellert, M, Smith, C E, Neville, D & Felsenfeld, G, J mol biol 11 (1965) 445. 20. Timmis, J N, Deumling, B &. Ingle, J, Nature 257 (1975) 152. 21. Ringer& N R & Bolund, L, Biochim biophys acta 174(1%9) 147. 22. La Cour, L F &Wells, B, J cell sci 14 (1974) 505. 23. Modest, E J & Sengupta, S K, Chromosome identification-techniaue and anulications in biology and medicine (ed’T Caspe&on & L Zech). Proc 23 Nobel symp. Stockholm (1972) p. 327. Nobel Foundation, Stockholm, and Academic Press, New York (1973). 24. Cionini, P G & Avanzi, S, Exp cell res 75 (1972) 154. 25. Schweizer, D, Chromosoma. In press. 26. Simola, K, Selander, R-K, de la Chapelle, A, Comeo, G & Ginelli, E, Chromosoma 51 (1975) 199. 27. Comings, D E, Chromosoma 52 (1975) 229. 28. Latt, S A & Wohlleb, J C, Chromosoma 52 (1975) 297. 14.
27-761809
Exp Cell Res 102 (1976)
414
Preliminary notes
I6 14
0,
-
Cells for physical
I2 IO864-
0
to react with KB cells for 1 h at 23°C (3x10’ cells/ 0.75 mg protein). The cells were removed by centtifugation and the clear supematant fluid was used for subsequent staining.
0.2
0.4
06
08
IO
fraction of total; ordinate: % of total radioactivity. 0, DNA extracted from cells exposed to methylene blue but kept in dark; 0, DNA from cells illuminated in the presence of methylene blue. Effect of illumination in the presence of methylene blue (1.5 h) on sedimentation behavior in alkaline sucrose gradients of cellular DNA. The direction of sedimentation is from left to right. Low molecular weight DNA is at the top of the gradient (lefr).
Fig. 1. Abscissa:
oxidized by singlet oxygen photogenerated from intracellular methylene blue. The modified DNA is then detectable by immunofluorescence using antibodies with a specificity for cytosine. In parallel it is demonstrated that the DNA isolated from such cells is smaller than that derived from untreated controls. Materials and Methods Methylene blue was purchased from Allied Chemical Co., and fluorescein isothiocyanate from the Sylvana Co. Preparation of anti C-antibody. The preparation of nucleoside conjugates and the procedure for immunization of rabbits has been described previously [7]. Conjugation of the fluorescein to the gamma globulin fraction of antibody was carried out by stirring an antibody solution (10 mg/ml) with fluorescein isothiocyanate (0.5 r&ml) overnight at 4°C in bicarbonate buffer (0.075 M pH 9.5). (Hsu, K. C., Personal communication (1972)). Conjugated and unconjugated fluorescein were separated by extensive dialysis against phosphate-buffered saline (PBS) in the cold (4°C). The precipitates that formed were subsequently removed by centrifugation. Prior to staining, the fluorescein-tagged antibody preparation was allowed
chemical
analysis of DNA.
Sus-
pensions of human (KB) cells growing in Eagle’s medium containing calf serum (10%) and glutamine (3~10~ cells/ml) were supplemented with [3H]TdR (6.0 Ci/mmole, final cont. 8.3 &i/ml). Five-ml portions of these cultures were distributed into 30 ml plastic tissue culture flasks and incubated for 24 h, whereupon the cel!s had settled to the plastic to form monolayers. The medium was then removed, the cells rinsed with PBS and fresh, non-radioactive growth medium was added. Some of the cultures received methylene blue (10 pg/ml of medium). Following an additional l-2 h period of incubation (in the dark), the monolayers were examined by light microscopy to be certain they were confluent. The cell sheets were then washed four times with 5 ml portion (PBS) and finally each flask received 5 ml of PBS. Illumination of cells. Cultures to be illuminated were exposed to a white light (Phototherapy Unit: DuraTest Vita Lite). The unit was protected from direct sunlight and air-cooled to maintain the cultures at 23°C. The sample distance from the light sources was adjusted to maintain a fluence rate (450 nm) of 141 pW/cm*. Photometric measurements were made with the IL600 A Photometer coupled to the IL600 Photodensitometer manufactured by International Light, Inc. Extraction and analysis of cellular DNA. Cells were harvested by scraping the surface of the plastic bottles, suspended in 1 ml of PBS, adjusted to 0.4 mg NapEDTA and 2% sodium laurylsulfate and supplemented with an equal volume of PBS-saturated phenol. After inverting gently 10 times, the aqueous phase was collected. Dilutions (0.1 ml volumes) of the aqueous phase were layered on top of an alkaline sucrose gradient (4.4 ml, 5-20 % sucrose in 0.1 M NaOH) containing a cushion of 50% sucrose in alkali. The specimens were spun for 2.5 h at 30000 rpm in the SW50.1 swinging buckets of the Spinco Model L-2 ultracentrifuge. Fractions were subsequently collected [8] and processed for determination of the radioactivity incorporated. Preparation of cells for immunofluorescence. KB cells were processed as described above except that
Materials.
Exp CellRes 102 (1976)
Table 1. Effect of duration of illumination on proportion offluorescent cells
Treatment
No. of fluorescing cells/Total no. of cells
%
Dark, 2 h White light, 1 h White light, 2 h
31214 %I241 1421246
1.5 40 58
Methylene blue cont. 10 pg/ml.
Preliminary notes
4 15
fluorescent microscope fitted with a HBO 200 W mercury lamp, a BG-12 exciter filter, a 530 nm barrier filter and a 100x planapochromatic objective.
RESULTS
Fig. 2. Immunofluorescence of KB cells illuminated in the presence of methylene blue. Cells were grown overnight in the dark, treated with methylene blue (10 w/ml) in the dark for 2 h and illuminated with white light for 1.5 h. Following the illumination cells were fixed and stained with fluorescein-labelled anti-C antibody. Fig. 3. Effect of illumination in the presence of methylene blue. The cells were treated as described in fig 2 except that the methylene blue concentration was 60 pglml.
they were grown in non-radioactive medium in 60 mm Petri plates containing two 22X22 mm coverslips (3 x lo6 cells/plate). Incubation was again in the dark in a CO,-incubator. After overnight incubation, the medium was removed and fresh medium containing methylene blue (10 or 60 pg/ml) was added. After additional incubation in the dark for l-2 h, the cells were washed thrice with PBS and illuminated in PBS for 1 or for 2 h. Staining procedure. Following illumination, the cells were washed with PBS, fixed with 95% ethanol for 1 min and airdried. The slides were then dipped into PBS and covered with fluorescein-labelled anti-C antibody (cytidine-specific antibody), diluted 1: 30 with PBS (final cont. 333 pg protein/ml) [9]. The cells were allowed to remain at room temperature for 30 min in a humid atmosphere, whereupon they were washed with 50 ml PBS and then placed in an inverted position with mounting solution of 1: 4 v/v glycerol : PBS. All slides were observed with the Zeiss
Analysis of DNA extracted from cells exposed to light in the presence of methylene blue revealed a sharp decrease in molecular weight when compared with the DNA of cells supplemented with methylene blue but incubated in the dark. Cells exposed to the same dose of light in the absence of methylene blue did not show this decrease in size (fig. 1). Examination of the cells by direct immunofluorescence with anti-C revealed fluorescence only in the cells illuminated in the presence of methylene blue (figs 2, 3). Illumination in the absence of the dye or cells exposed to methylene blue in the dark did not demonstrate immunofluorescence. The proportion of cells which fluoresced was dependent upon the duration of illumination in the presence of methylene blue (table 1) and on dye concentration (figs
273). Discussion Antinucleoside antibodies have been developed which are specific for the purine or pyrimidine group of the immunizing antigen. The specificities of the reactions have been shown by complement fixation, precipitation and radioimmunological techniques [lO-121. These antibodies react with singlestranded, denatured or partially denatured DNA [ 111.Because of their ability to combine with suitably modified metaphase chromosomes, they have also been used to study the structure of human chromosomes
[9,131. Certain dyes are capable, in the presence of oxygen and visible light, of selectively destroying the guanine moiety of DNA. Exp Cell Res 102 (1976)
416
Preliminary
notes
Simon & Van Vunakis [14] demonstrated that this property was exhibited by methylene blue (see also [15]). Garro et al. [ll] and Levine et al. [16] documented this selective photodegradation of guanine using cytosine-specific antinucleoside antibody, which reacted with the unpaired cytosine residue in the treated isolated DNA molecule. In the present study we have demonstrated, by a simple immunofluorescent procedure, that photo-oxidation of guanine residues in DNA by methylene blue can occur in living cells. On the assumption that bilirubin and riboflavin can also act as photosensitizers [3, 41, then the immunofluorescence technique described herein may be of value in the study of peripheral cells of newborn populations at the risk for similar structural changes in their DNA during phototherapy for neonatal jaundice. This study was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-l 1470 and AI-06860) and from George D. Smith Fund, Inc. One of the authors (H. S. R.) was a Research Career Development Awardee of the National Institute of General Medical Sciences (5 K3GM29,024).
References 1. Cramer, R G. Perrvman, P W & Richards. D H, Lancet i (1958) 1094. 2. Speck, W T & Rosenkranz, H S, Photochem photobio121 (1975) 369. 3. Sneck. W T. Chen. C C & Rosenkmnz. H S. Pkdiatres 9 (1975) 150. 4. Speck, W T & Rosenkranz, H S, Pediat res 9 (1975) 703. 5. Speck, W T, Rosenkranz, S & Rosenkranz, H S, Biochim biophys acta 435 (1976) 39. 6. Speck, W T & Rosenkranz, H S, Pediat res 10 (1976) 553. 7. Erlanger, B F & Beiser, S M, Proc natl acad sci US 52 (1964) 68. 8. Ellison, S A & Rosenkranz, H S, Analyt biochem 5 (1963) 263. 9. Schreck, R R, Warburton, D, Miller, 0 J & Beiser, S M, Proc natl acad sci US 70 (1973) 804. 10. Beiser, S M, Tanenbaum, S W & Erlanger, B F, Nature 203 (1964) 1381. 11. Garro, A J, Erlanger, B F & Beiser, S M, Nucleic acids in immunology (ed 0 J Plescia & W Btaun) pp 47-57. Springer-Verlag, New York (1968). Exp Cell Res 102 (1976)
12. Senitzer, D, Erlanger, B F & Beiser, S M, Immunochemistry 11 (1974) 321. 13. Schreck, R R,- Erlanger; B F & Miller, 0 J, Exp cell res 88 (1974) 3 1. 14. Simon, M I & Van Vunakis, H , J mol bio14 (1%2) 488. 15. Waskell, L A, Sastry, K S & Gordon, M P, Biochim biophys acta 129(1966) 49. 16. Levine, L, Seaman, E &Van Vunakis, H, Nucleic acids in immunology (ed 0 J Plescia & W Braun) pp 165-173. Springer-Verlag, New York (1968). Received April 22, 1976 Accepted June 17, 1976
Ribosomal bodies in early oogenetic stages of the lizard hcerta sicula Raf. C. TADDEI and S. FILOSA, Institute and Embryology,
University
of Histology of Naples, Naples, Italy
During hibernation, ribosomal bodies are present in the germ cells ofLacerta sicula, which have undergone meiotic prophase, but neither in oogonia nor in stroma cells. These bodies are similar in structure, although smaller in size, to those which have been described in the cytoplasm of growing oocytes; in the germ cells at zygopachytene, they are frequently associated with the nuclear envelope. Summary.
During hibernation complicated structural aggregates of ribosomes, named “ribosomal bodies”, have been observed in both previtellogenetic oocytes and follicle cells of the lizard Lacerta sicula Raf. [3, 61. These aggregates consist of stacks of crystalline sheets of ribosomes, similar to those described in the chick embryo after hypothermic treatment [I, 51, but regularly set between flat and parallel cistemae [6]. Experiments carried out both in vivo and in Fig. 1. Crystalline arrangement of the ribosomes in a ribosomal body (RB) in the cytoplasm of a germ cell at
zygopachytene. Synaptonemal complexes are present in the nucleus (arrow). x 18000. Fig. 2. A cap-like ribosomal body adjacent to a portion of the nuclear envelope, in a germ cell at zygopachytene. Note the dense material inserted between the sheets (crossed arrows) and the interruption of the cap at the nuclear pore level (arrow). x 16080. Fig. 3. A diverging crystalline sheet with interspersed dense material (arrow). ~26000. Fig. 4. Portion of a larger ribosomal body in the cytoplasm of an oocyte at early diplotene. X 12000.
notes
413
The effects of environmental agents on human health is a subject of considerable interest at the present time. Of special concern to health scientists are the long-term effects of such agents and particularly their carcinogenic, mutagenic and teratogenic potentials which are believed to result, in part, from the ability of these agents to modify intracellular DNA. We have recently been investigating the DNA-modifying potential of a relatively new environmental agent which is widely used in pediatric therapeutics for the control of neonatal hyperbilirubinemia: high intensity illumination with visible light, i.e. phototherapy [l]. In the course of these studies we have found that (a) visible light (450 nm) was mutagenic for prokaryotes [2] and certain eukaryotes (yeasts) (unpublished results); (b) visible light (450 nm) in the presence of physiological photosensitizers was capable of altering the physical-chemical Received March 10, 1976 properties of isolated DNA [3-51; and (c) Accepted July 19, 1976 that visible light (450 nm) in the absence of added photosensitizers was capable of modifying the structure of intracellular DNA of human cells growing in tissue culImmunofluorescence for the detection of ture [6]. We have evidence that the aforephotochemical lesions in intracellular DNA mentioned DNA-modifying effects of visiB. GUTTER,’ Y. NISHIOKA, W. T. SPECK, H. S. ble light are dependent upon the generation ROSENKRANZ.’ BEVERLY LUBIT and B. F. ER- of singlet oxygen by intracellular photoLANGER, Departments of Microbiology and Pesensitizers with subsequent oxidation of the diatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA guanine moiety of DNA ([3, 51 and unpublished results). Because the effects were Summary. The ability of methylene blue to photooxidize the guanine moiety of intracellular DNA of seen with doses of visible light, only 5 % of living human cells was demonstrated by immuno- that received in a 24 h period by a newborn fluorescence using an antibody with a specificity for the unpaired cytosine residue of DNA. These findings infant undergoing phototheraphy in our which indicate that sufftcient amounts of the dye pene- nursery, we became interested in developtrate the mammalian cell to cause biological effects may present the investigator with the unique oppor- ing procedures to identify and quantitate in tunity of selectively altering the guanine residue in a rapid and reproducible fashion alterations DNA and thereby determining the genetic conin the genetic material of such infants. The sequences of this reaction. present communication describes a model system in which the guanine residues of the 1 Present address: Department of Microbiology, New DNA of human cells growing in culture are York Medical College, Valhalla, NY 10595,USA.
Caspersson, T, Zech, L, Modest, E J, Foley, Cl E, Wagh, U & Simonsson, E, Exp cell res 58 (1%9) 141. 15. Vosa, C G, Chromosoma 43 (1973) 269. 16. Bendich. A J & Bolton. E T. Plant LI ohvs 42 (1%7) ~ I 959. 17. Cullis, C A & Schweizer, D, Chromosoma 44 (1974)417. 18. Haitinger, M, Fluorescenz-Mikroskopie. Akademische Verlagsgesellschaft, Leipzig (1938). 19. Gellert, M, Smith, C E, Neville, D & Felsenfeld, G, J mol biol 11 (1965) 445. 20. Timmis, J N, Deumling, B &. Ingle, J, Nature 257 (1975) 152. 21. Ringer& N R & Bolund, L, Biochim biophys acta 174(1%9) 147. 22. La Cour, L F &Wells, B, J cell sci 14 (1974) 505. 23. Modest, E J & Sengupta, S K, Chromosome identification-techniaue and anulications in biology and medicine (ed’T Caspe&on & L Zech). Proc 23 Nobel symp. Stockholm (1972) p. 327. Nobel Foundation, Stockholm, and Academic Press, New York (1973). 24. Cionini, P G & Avanzi, S, Exp cell res 75 (1972) 154. 25. Schweizer, D, Chromosoma. In press. 26. Simola, K, Selander, R-K, de la Chapelle, A, Comeo, G & Ginelli, E, Chromosoma 51 (1975) 199. 27. Comings, D E, Chromosoma 52 (1975) 229. 28. Latt, S A & Wohlleb, J C, Chromosoma 52 (1975) 297. 14.
27-761809
Exp Cell Res 102 (1976)
414
Preliminary notes
I6 14
0,
-
Cells for physical
I2 IO864-
0
to react with KB cells for 1 h at 23°C (3x10’ cells/ 0.75 mg protein). The cells were removed by centtifugation and the clear supematant fluid was used for subsequent staining.
0.2
0.4
06
08
IO
fraction of total; ordinate: % of total radioactivity. 0, DNA extracted from cells exposed to methylene blue but kept in dark; 0, DNA from cells illuminated in the presence of methylene blue. Effect of illumination in the presence of methylene blue (1.5 h) on sedimentation behavior in alkaline sucrose gradients of cellular DNA. The direction of sedimentation is from left to right. Low molecular weight DNA is at the top of the gradient (lefr).
Fig. 1. Abscissa:
oxidized by singlet oxygen photogenerated from intracellular methylene blue. The modified DNA is then detectable by immunofluorescence using antibodies with a specificity for cytosine. In parallel it is demonstrated that the DNA isolated from such cells is smaller than that derived from untreated controls. Materials and Methods Methylene blue was purchased from Allied Chemical Co., and fluorescein isothiocyanate from the Sylvana Co. Preparation of anti C-antibody. The preparation of nucleoside conjugates and the procedure for immunization of rabbits has been described previously [7]. Conjugation of the fluorescein to the gamma globulin fraction of antibody was carried out by stirring an antibody solution (10 mg/ml) with fluorescein isothiocyanate (0.5 r&ml) overnight at 4°C in bicarbonate buffer (0.075 M pH 9.5). (Hsu, K. C., Personal communication (1972)). Conjugated and unconjugated fluorescein were separated by extensive dialysis against phosphate-buffered saline (PBS) in the cold (4°C). The precipitates that formed were subsequently removed by centrifugation. Prior to staining, the fluorescein-tagged antibody preparation was allowed
chemical
analysis of DNA.
Sus-
pensions of human (KB) cells growing in Eagle’s medium containing calf serum (10%) and glutamine (3~10~ cells/ml) were supplemented with [3H]TdR (6.0 Ci/mmole, final cont. 8.3 &i/ml). Five-ml portions of these cultures were distributed into 30 ml plastic tissue culture flasks and incubated for 24 h, whereupon the cel!s had settled to the plastic to form monolayers. The medium was then removed, the cells rinsed with PBS and fresh, non-radioactive growth medium was added. Some of the cultures received methylene blue (10 pg/ml of medium). Following an additional l-2 h period of incubation (in the dark), the monolayers were examined by light microscopy to be certain they were confluent. The cell sheets were then washed four times with 5 ml portion (PBS) and finally each flask received 5 ml of PBS. Illumination of cells. Cultures to be illuminated were exposed to a white light (Phototherapy Unit: DuraTest Vita Lite). The unit was protected from direct sunlight and air-cooled to maintain the cultures at 23°C. The sample distance from the light sources was adjusted to maintain a fluence rate (450 nm) of 141 pW/cm*. Photometric measurements were made with the IL600 A Photometer coupled to the IL600 Photodensitometer manufactured by International Light, Inc. Extraction and analysis of cellular DNA. Cells were harvested by scraping the surface of the plastic bottles, suspended in 1 ml of PBS, adjusted to 0.4 mg NapEDTA and 2% sodium laurylsulfate and supplemented with an equal volume of PBS-saturated phenol. After inverting gently 10 times, the aqueous phase was collected. Dilutions (0.1 ml volumes) of the aqueous phase were layered on top of an alkaline sucrose gradient (4.4 ml, 5-20 % sucrose in 0.1 M NaOH) containing a cushion of 50% sucrose in alkali. The specimens were spun for 2.5 h at 30000 rpm in the SW50.1 swinging buckets of the Spinco Model L-2 ultracentrifuge. Fractions were subsequently collected [8] and processed for determination of the radioactivity incorporated. Preparation of cells for immunofluorescence. KB cells were processed as described above except that
Materials.
Exp CellRes 102 (1976)
Table 1. Effect of duration of illumination on proportion offluorescent cells
Treatment
No. of fluorescing cells/Total no. of cells
%
Dark, 2 h White light, 1 h White light, 2 h
31214 %I241 1421246
1.5 40 58
Methylene blue cont. 10 pg/ml.
Preliminary notes
4 15
fluorescent microscope fitted with a HBO 200 W mercury lamp, a BG-12 exciter filter, a 530 nm barrier filter and a 100x planapochromatic objective.
RESULTS
Fig. 2. Immunofluorescence of KB cells illuminated in the presence of methylene blue. Cells were grown overnight in the dark, treated with methylene blue (10 w/ml) in the dark for 2 h and illuminated with white light for 1.5 h. Following the illumination cells were fixed and stained with fluorescein-labelled anti-C antibody. Fig. 3. Effect of illumination in the presence of methylene blue. The cells were treated as described in fig 2 except that the methylene blue concentration was 60 pglml.
they were grown in non-radioactive medium in 60 mm Petri plates containing two 22X22 mm coverslips (3 x lo6 cells/plate). Incubation was again in the dark in a CO,-incubator. After overnight incubation, the medium was removed and fresh medium containing methylene blue (10 or 60 pg/ml) was added. After additional incubation in the dark for l-2 h, the cells were washed thrice with PBS and illuminated in PBS for 1 or for 2 h. Staining procedure. Following illumination, the cells were washed with PBS, fixed with 95% ethanol for 1 min and airdried. The slides were then dipped into PBS and covered with fluorescein-labelled anti-C antibody (cytidine-specific antibody), diluted 1: 30 with PBS (final cont. 333 pg protein/ml) [9]. The cells were allowed to remain at room temperature for 30 min in a humid atmosphere, whereupon they were washed with 50 ml PBS and then placed in an inverted position with mounting solution of 1: 4 v/v glycerol : PBS. All slides were observed with the Zeiss
Analysis of DNA extracted from cells exposed to light in the presence of methylene blue revealed a sharp decrease in molecular weight when compared with the DNA of cells supplemented with methylene blue but incubated in the dark. Cells exposed to the same dose of light in the absence of methylene blue did not show this decrease in size (fig. 1). Examination of the cells by direct immunofluorescence with anti-C revealed fluorescence only in the cells illuminated in the presence of methylene blue (figs 2, 3). Illumination in the absence of the dye or cells exposed to methylene blue in the dark did not demonstrate immunofluorescence. The proportion of cells which fluoresced was dependent upon the duration of illumination in the presence of methylene blue (table 1) and on dye concentration (figs
273). Discussion Antinucleoside antibodies have been developed which are specific for the purine or pyrimidine group of the immunizing antigen. The specificities of the reactions have been shown by complement fixation, precipitation and radioimmunological techniques [lO-121. These antibodies react with singlestranded, denatured or partially denatured DNA [ 111.Because of their ability to combine with suitably modified metaphase chromosomes, they have also been used to study the structure of human chromosomes
[9,131. Certain dyes are capable, in the presence of oxygen and visible light, of selectively destroying the guanine moiety of DNA. Exp Cell Res 102 (1976)
416
Preliminary
notes
Simon & Van Vunakis [14] demonstrated that this property was exhibited by methylene blue (see also [15]). Garro et al. [ll] and Levine et al. [16] documented this selective photodegradation of guanine using cytosine-specific antinucleoside antibody, which reacted with the unpaired cytosine residue in the treated isolated DNA molecule. In the present study we have demonstrated, by a simple immunofluorescent procedure, that photo-oxidation of guanine residues in DNA by methylene blue can occur in living cells. On the assumption that bilirubin and riboflavin can also act as photosensitizers [3, 41, then the immunofluorescence technique described herein may be of value in the study of peripheral cells of newborn populations at the risk for similar structural changes in their DNA during phototherapy for neonatal jaundice. This study was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-l 1470 and AI-06860) and from George D. Smith Fund, Inc. One of the authors (H. S. R.) was a Research Career Development Awardee of the National Institute of General Medical Sciences (5 K3GM29,024).
References 1. Cramer, R G. Perrvman, P W & Richards. D H, Lancet i (1958) 1094. 2. Speck, W T & Rosenkranz, H S, Photochem photobio121 (1975) 369. 3. Sneck. W T. Chen. C C & Rosenkmnz. H S. Pkdiatres 9 (1975) 150. 4. Speck, W T & Rosenkranz, H S, Pediat res 9 (1975) 703. 5. Speck, W T, Rosenkranz, S & Rosenkranz, H S, Biochim biophys acta 435 (1976) 39. 6. Speck, W T & Rosenkranz, H S, Pediat res 10 (1976) 553. 7. Erlanger, B F & Beiser, S M, Proc natl acad sci US 52 (1964) 68. 8. Ellison, S A & Rosenkranz, H S, Analyt biochem 5 (1963) 263. 9. Schreck, R R, Warburton, D, Miller, 0 J & Beiser, S M, Proc natl acad sci US 70 (1973) 804. 10. Beiser, S M, Tanenbaum, S W & Erlanger, B F, Nature 203 (1964) 1381. 11. Garro, A J, Erlanger, B F & Beiser, S M, Nucleic acids in immunology (ed 0 J Plescia & W Btaun) pp 47-57. Springer-Verlag, New York (1968). Exp Cell Res 102 (1976)
12. Senitzer, D, Erlanger, B F & Beiser, S M, Immunochemistry 11 (1974) 321. 13. Schreck, R R,- Erlanger; B F & Miller, 0 J, Exp cell res 88 (1974) 3 1. 14. Simon, M I & Van Vunakis, H , J mol bio14 (1%2) 488. 15. Waskell, L A, Sastry, K S & Gordon, M P, Biochim biophys acta 129(1966) 49. 16. Levine, L, Seaman, E &Van Vunakis, H, Nucleic acids in immunology (ed 0 J Plescia & W Braun) pp 165-173. Springer-Verlag, New York (1968). Received April 22, 1976 Accepted June 17, 1976
Ribosomal bodies in early oogenetic stages of the lizard hcerta sicula Raf. C. TADDEI and S. FILOSA, Institute and Embryology,
University
of Histology of Naples, Naples, Italy
During hibernation, ribosomal bodies are present in the germ cells ofLacerta sicula, which have undergone meiotic prophase, but neither in oogonia nor in stroma cells. These bodies are similar in structure, although smaller in size, to those which have been described in the cytoplasm of growing oocytes; in the germ cells at zygopachytene, they are frequently associated with the nuclear envelope. Summary.
During hibernation complicated structural aggregates of ribosomes, named “ribosomal bodies”, have been observed in both previtellogenetic oocytes and follicle cells of the lizard Lacerta sicula Raf. [3, 61. These aggregates consist of stacks of crystalline sheets of ribosomes, similar to those described in the chick embryo after hypothermic treatment [I, 51, but regularly set between flat and parallel cistemae [6]. Experiments carried out both in vivo and in Fig. 1. Crystalline arrangement of the ribosomes in a ribosomal body (RB) in the cytoplasm of a germ cell at
zygopachytene. Synaptonemal complexes are present in the nucleus (arrow). x 18000. Fig. 2. A cap-like ribosomal body adjacent to a portion of the nuclear envelope, in a germ cell at zygopachytene. Note the dense material inserted between the sheets (crossed arrows) and the interruption of the cap at the nuclear pore level (arrow). x 16080. Fig. 3. A diverging crystalline sheet with interspersed dense material (arrow). ~26000. Fig. 4. Portion of a larger ribosomal body in the cytoplasm of an oocyte at early diplotene. X 12000.
