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activity in isolated nuclei
372
Brain Research, 105 (I 976) 372-375 © Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands
Brain-specific effect of the S-100 protein on the RNA-polymerase I activity in isolated nuclei*
FABRIZIO MICHETTI, GABRIELLA DE RENZIS, ROSARIO DONATO AND NICOL0 MIANI Department of Anatomy, Universitd Cattolica, 00168 Rome ( ltaly)
(Accepted December 15th, 1975)
The brain-specific S-100 protein 19 is a component both of glial 2,5,21 and neuronal cytoplasm6,7As,z°,26; additional aliquots of the protein have also been extracted from particulate matterT,10, 2z or found in synapses 6,7,9. An interesting localization of the S-100 is in the nucleus, where the protein has been detected by immunohistological procedures13, 25 and has been studied in detail in subnuclear fractions by immunochemical assays 17. To gain additional information about the nuclear S-100, we demonstrated that the protein stimulates the RNA-polymerase I activity in isolated brain nuclei 16. Since S-100 is a brain-specific protein, an organ-specificity may be hypothesized for this action. The present study verifies this property of the protein. Nuclei from various sources were isolated according to the procedure described by Lovtrup-Rein and McEwen 14 with minor modifications, as indicated elsewherO 7. The RNA-polymerase I activity was measured according to the method described by Montanaro et al. 18. In a final volume of 0.5 ml the reaction mixture contained: 100 m M Tris.HCl buffer pH 8.0, 4 m M MgCI2, 14 m M 2-mercaptoethanol, 0.6 m M each of ATP, CTP and UTP, and 0.06 m M of [3H]GTP (sp. act. I00 Ci/mole). Nuclear suspensions were preincubated for l0 min at 37 °C in 100 mM Tris.HCI buffer, pH 8.0 containing 4 m M MgC12, in the presence or in the absence of S-100 protein. In the reaction tubes the DNA/S-100 ratio (2:1) was comparable to that which occurs in brain tissue of adult rat 16. The assay was started in the reaction mixture at 37 °C with 0.1 ml of preincubated suspension, and was stopped after 15 min with 5 ml of ice-cold perchloric acid (0.5 N) in 1 ~ of sodium pyrophosphate. One milligram of bovine serum albumin was added as a carrier. The precipitate was washed twice more with 6 ml of perchloric acid (0.2 N) in 1 o/,~of sodium pyrophosphate. The final precipitate was solubilized in 0.5 ml of NCS reagent (Nuclear Chicago Solubilizer), and 10 ml of toluene-based scintillation liquid were added for counting the radioactivity in a Nucclear Chicago scintillation spectrometer. Counting efliciencv~ was about 35 .... ,o. The activity was expressed in disint./min/mg DNA. D N A concentrations were determined * A preliminary account of these data was presented at the Fifth International Meeting of the International Society for Neurochemistry, Barcelona, Spain, September 1975.
373 TABLE I EFFECT OF THE S - 1 0 0 PROTEIN ON THE R N A - P O L Y M E R A S E NUCLEI OF CHICK EMBRYO
(llth
I ACTrvrrY IN
ISOLATED BRAIN A N D LIVER
DAY OF INCUBATION)
The enzyme activity (disint./min × 10 -4 incorporated/mg DNA) was determined as indicated in thetext. DNA per assay tube: about 120/~g. The values are the average of at least 4 experiments in duplicate, with a maximal variability of -1- 11%.
Control S-100 (100/tg/ml)
Brain
Liver
10.61 14.65
6.25 5.86
b y the m e t h o d o f B u r t o n 3. T h e S-100 p r o t e i n was p r e p a r e d a c c o r d i n g to the p r o c e dure o f MooreZL T a b l e I shows t h a t the s t i m u l a t i n g effect o f S-100 on the R N A - p o l y m e r a s e I activity in b r a i n nuclei i s o l a t e d f r o m chick e m b r y o is n o t elicited in liver nuclei. I n the light o f these p r e l i m i n a r y d a t a , a set o f e x p e r i m e n t s was e x t e n d e d to the n e w b o r n rat. Fig. 1 indicates t h a t the s t i m u l a t i n g effect is o b s e r v e d in nuclei isolated f r o m the b r a i n o f n e w b o r n rat, b u t n o t in liver o r k i d n e y o r heart, a n d very slightly in lung nuclei (0.1 > P > 0.05). I n the presence o f a c t i n o m y c i n D, n o i n c o r p o r a t i o n was observed, suggesting t h a t the s t i m u l a t o r y activity was d e p e n d e n t o n D N A t e m p l a t e activity a n d i n d e p e n d e n t o f h o m o p o l y m e r f o r m a t i o n . A s with b r a i n nuclei o f chick
Z
E
S-100 protein (200 /Jg/ml} [ ]
!
Control
[]
ActinomycinD (2 pg/ml) I I m
o t-i L. e-,
:!: :::,
~2
::::
x
D-
Brain
Lung
Liver
Heart
Kidney
O.Ol~dn'O.O05
Fig. 1. Effect of the S-100 protein on the RNA-polymerase I activity in nuclei isolated from various organs of newborn rat. The enzyme activity was determined as indicated in the text. DNA per assay tube: about 200 #g. The values are the average of at least 3 experiments in duplicate.
374 e m b r y o , no stimulating effect was observed by bovine serum albumin, p o l y - L - a s p a r t a t e ( M . W . 4870) or poly-L-glutamate ( M . W . 19,700) e m p l o y e d as a c o n t r o l (not shown). D u t t o n a n d M a h l e r 8 f o u n d no S-100-dependent stimulating effect on the R N A p o l y m e r a s e activity in b r a i n nuclei isolated f r o m a d u l t rat. W e also f o u n d differences in the S-100 activity d e p e n d i n g on b r a i n m a t u r a t i o n 16. A t present it is unclear how m u c h these effects are representative in the general e c o n o m y o f S-100 in nervous function. A n u m b e r o f cellular localizations a n d functional i n t e r p r e t a t i o n s have been suggested for this protein4,9,11,12. F u r t h e r m o r e , the possibility t h a t the S-100 is in fact a class o f proteins has been proposed1,24, 27, so t h a t the a t t e m p t to identify a unique biological role m a y be w i t h o u t solution. Nevertheless, it is r e a s o n a b l e to hypothesize t h a t the p r o t e i n exerts an action in those c o m p a r t m e n t s where it is in a b o u n d form, n a m e l y in the nucleus as well as in m e m b r a n e s . Elsewhere a selective effect o f the p r o t e i n on the R N A p o l y m e r a s e I in i m m a t u r e nuclei has been i n d i c a t e d 16. Therefore, we have restricted o u r investigation to this field. The present d a t a show an a d d i t i o n a l selectivity in this in vitro system, as the effect a p p e a r s to be restricted to b r a i n nuclei. K e e p i n g in m i n d t h a t S-100 has been f o u n d in b r a i n nuclei a m o n g the nonhistone p r o t e i n s 17, the finding m a y be w o r t h consideration. In fact, the nuclear nonhistone proteins represent a class o f tissue-specific proteins which are believed to be involved in tissue-specific gene activation (for review see ref. 23). Nevertheless, the in vivo conditions include a great n u m b e r o f physiological p a r a m e t e r s which escape the present experimental system, so t h a t functional e x t r a p o l a t i o n s c a n n o t yet be m a d e . This investigation was p a r t i a l l y s u p p o r t e d by C . N . R . C o n t r a c t 74.00229.04. 1 ABE,K., TAKAHASH[,K., ANDANDO,T., Purification and properties of S-100 protein from porcine brain, J. Biochem., 75 (1973) 11-22. 2 BENDA, P., LIGHTBODY,J., SATO, G., LEVlNE, L., AND SWEET, W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 3 BURTON,K., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 4 CALISSANO,P., AND BANGHAM,A. D., Effect of two brain specific proteins (S-100 and 14.3.2) on cation diffusion across artificial membranes, Biochem. biophys. Res. Commun., 43 (1971) 504-509. 5 CICERO,T. J., COWAN,W. M., MOORE,B. W., AND SUNTZEFF,V., The cellular localization of the two brain specific proteins S-100 and 14.3.2, Brain Research, 18 (1970) 25-34. 6 DONATO, R., AND MICHETTI, F., S-100 protein in cerebral cortex synaptosomes, Experientia (Basel), 30 (1974) 511-512. 7 DONATO,R., MICHETTI,F., AND MIANI, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Research, in press. 8 DUTTON, G. R., AND MAULER,H. R., In vitro RNA synthesis by intact rat brain nuclei, J. Neurochem., 15 (1968) 765-780. 9 HAGLID,K. G., HAMBERGER,A., HANSSON,H.-A., HYDt~N, H., PERSSON,L., AND RONNB.~CK,L., S-100 protein in synapses of the central nervous system, Nature (Loud.J, (1974) 532-534. 10 HAGLID, K. G., AND STAVROU,O., Water soluble and pentanol extractable proteins in human brain normal tissue and human tumours, with special reference to S-100 protein, J. Neurochem., 20 (1973) 1523-1532. I 1 HYD~N, H., A calcium-dependent mechanism for synapse and nerve cell membrane modulation, Proc. nat. Acad. Sci. (Wash.), 8 (1974) 2965-2968. 12 HYD~N, H., AND LANGE,P. W., S-100 brain protein: correlation with behavior, Proc. nat. Acad. Sci. (Wash.}, 67 (1970) 1959-1966.
375 13 HYD~N, H., AND McEwEN, B. S., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 345-358. 14 LOVTRUP-REIN,H., AND McEwEN, B. S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405--415. 15 MIANI, N., DE RENZlS, G., MICHETTI, F., CORRER, S., OLIVIERI-SANGIACOMO,C., ANDCANIGLIA, A., Axonal transport of S-100 protein in mammalian nerve fibres, J. Neurochem., 19 (1972) 1387-1394. 16 MIANI,N., MICHETTI,F., DE RENZIS, G., ANDCANIGLIA,A., Effect of a brain-specific protein (S100 protein) on the nucleolar RNA-polymerase activity in isolated brain nuclei, Experientia (Basel), 29 (1973) 1499--1501. 17 MICHETTI,F., MIANI,N., DE RENZIS, G., CANIGLIA,A., AND CORRER,S., Nuclear localization of S-100 protein, J. Neurochem., 22 (1974) 239-244. 18 MONTANARO,N., NOVELLO, F., AND STIRPE, F., Effect of a-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning, Biochem. J., 125 (1971) 1087-1090. 19 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739-744. 20 PACKMAN, P. M., BLOMSTRAND,C., AND HAMBERGER, A., Disc electrophoretic separation of proteins in neuronal, glial, and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 479-487. 21 PEREZ, V. J., OLNEY, J. W.) CICERO,T. J., MOORE, B. W., AND BAHN,B. A., Wallerian degeneration in rabbit optic nerve: cellular localization in the central nervous system of the S-100 and 14.3.2 proteins, J. Neurochem., 17 (1970) 511-519. 22 RUSCA,G., CALISSANO,P., AND ALEM-~,S., Identification of a membrane-bound fraction of the S-100 protein, Brain Research, 49 (1972) 223-227. 23 STEIN, G. S., SPELSBERG,T. C., AND KLEINSMITH,L.J., Nonhistone chromosomal proteins and gene regulation, Science, 183 (1974) 817-824. 24 STEWART, J. A., Tissue specific S-100: a demonstration of multiple proteins, Biochim. biophys. Acta (Amst.), 263 (1972) 178-192. 25 SVIRIDOV,S. M., KOROCHKIN, L. I., IVANOV, V. N., MALETSKAYA,E. I., AND BAKHTINA,T. K., Immunohistochemical studies of S-100 protein during postnatal ontogenesis of the brain of two strains of rats, J. Neurochem., 19 (1972) 713-718. 26 TAaUCHI, K., AND KIRSCH, W. M., Immunocytochemical localization of S-100 protein in neurons and glia of hamster cerebellum, Brain Research, 92 (1975) 175-180. 27 UYEMURA,K., VINCENDON,G., GOMaOS, G., AND MANDEL,P., Purification and some properties of S-100 protein fractions from sheep and pig brains, J. Neurochem., 18 (1971) 429-438.
Brain Research, 105 (I 976) 372-375 © Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands
Brain-specific effect of the S-100 protein on the RNA-polymerase I activity in isolated nuclei*
FABRIZIO MICHETTI, GABRIELLA DE RENZIS, ROSARIO DONATO AND NICOL0 MIANI Department of Anatomy, Universitd Cattolica, 00168 Rome ( ltaly)
(Accepted December 15th, 1975)
The brain-specific S-100 protein 19 is a component both of glial 2,5,21 and neuronal cytoplasm6,7As,z°,26; additional aliquots of the protein have also been extracted from particulate matterT,10, 2z or found in synapses 6,7,9. An interesting localization of the S-100 is in the nucleus, where the protein has been detected by immunohistological procedures13, 25 and has been studied in detail in subnuclear fractions by immunochemical assays 17. To gain additional information about the nuclear S-100, we demonstrated that the protein stimulates the RNA-polymerase I activity in isolated brain nuclei 16. Since S-100 is a brain-specific protein, an organ-specificity may be hypothesized for this action. The present study verifies this property of the protein. Nuclei from various sources were isolated according to the procedure described by Lovtrup-Rein and McEwen 14 with minor modifications, as indicated elsewherO 7. The RNA-polymerase I activity was measured according to the method described by Montanaro et al. 18. In a final volume of 0.5 ml the reaction mixture contained: 100 m M Tris.HCl buffer pH 8.0, 4 m M MgCI2, 14 m M 2-mercaptoethanol, 0.6 m M each of ATP, CTP and UTP, and 0.06 m M of [3H]GTP (sp. act. I00 Ci/mole). Nuclear suspensions were preincubated for l0 min at 37 °C in 100 mM Tris.HCI buffer, pH 8.0 containing 4 m M MgC12, in the presence or in the absence of S-100 protein. In the reaction tubes the DNA/S-100 ratio (2:1) was comparable to that which occurs in brain tissue of adult rat 16. The assay was started in the reaction mixture at 37 °C with 0.1 ml of preincubated suspension, and was stopped after 15 min with 5 ml of ice-cold perchloric acid (0.5 N) in 1 ~ of sodium pyrophosphate. One milligram of bovine serum albumin was added as a carrier. The precipitate was washed twice more with 6 ml of perchloric acid (0.2 N) in 1 o/,~of sodium pyrophosphate. The final precipitate was solubilized in 0.5 ml of NCS reagent (Nuclear Chicago Solubilizer), and 10 ml of toluene-based scintillation liquid were added for counting the radioactivity in a Nucclear Chicago scintillation spectrometer. Counting efliciencv~ was about 35 .... ,o. The activity was expressed in disint./min/mg DNA. D N A concentrations were determined * A preliminary account of these data was presented at the Fifth International Meeting of the International Society for Neurochemistry, Barcelona, Spain, September 1975.
373 TABLE I EFFECT OF THE S - 1 0 0 PROTEIN ON THE R N A - P O L Y M E R A S E NUCLEI OF CHICK EMBRYO
(llth
I ACTrvrrY IN
ISOLATED BRAIN A N D LIVER
DAY OF INCUBATION)
The enzyme activity (disint./min × 10 -4 incorporated/mg DNA) was determined as indicated in thetext. DNA per assay tube: about 120/~g. The values are the average of at least 4 experiments in duplicate, with a maximal variability of -1- 11%.
Control S-100 (100/tg/ml)
Brain
Liver
10.61 14.65
6.25 5.86
b y the m e t h o d o f B u r t o n 3. T h e S-100 p r o t e i n was p r e p a r e d a c c o r d i n g to the p r o c e dure o f MooreZL T a b l e I shows t h a t the s t i m u l a t i n g effect o f S-100 on the R N A - p o l y m e r a s e I activity in b r a i n nuclei i s o l a t e d f r o m chick e m b r y o is n o t elicited in liver nuclei. I n the light o f these p r e l i m i n a r y d a t a , a set o f e x p e r i m e n t s was e x t e n d e d to the n e w b o r n rat. Fig. 1 indicates t h a t the s t i m u l a t i n g effect is o b s e r v e d in nuclei isolated f r o m the b r a i n o f n e w b o r n rat, b u t n o t in liver o r k i d n e y o r heart, a n d very slightly in lung nuclei (0.1 > P > 0.05). I n the presence o f a c t i n o m y c i n D, n o i n c o r p o r a t i o n was observed, suggesting t h a t the s t i m u l a t o r y activity was d e p e n d e n t o n D N A t e m p l a t e activity a n d i n d e p e n d e n t o f h o m o p o l y m e r f o r m a t i o n . A s with b r a i n nuclei o f chick
Z
E
S-100 protein (200 /Jg/ml} [ ]
!
Control
[]
ActinomycinD (2 pg/ml) I I m
o t-i L. e-,
:!: :::,
~2
::::
x
D-
Brain
Lung
Liver
Heart
Kidney
O.Ol~dn'O.O05
Fig. 1. Effect of the S-100 protein on the RNA-polymerase I activity in nuclei isolated from various organs of newborn rat. The enzyme activity was determined as indicated in the text. DNA per assay tube: about 200 #g. The values are the average of at least 3 experiments in duplicate.
374 e m b r y o , no stimulating effect was observed by bovine serum albumin, p o l y - L - a s p a r t a t e ( M . W . 4870) or poly-L-glutamate ( M . W . 19,700) e m p l o y e d as a c o n t r o l (not shown). D u t t o n a n d M a h l e r 8 f o u n d no S-100-dependent stimulating effect on the R N A p o l y m e r a s e activity in b r a i n nuclei isolated f r o m a d u l t rat. W e also f o u n d differences in the S-100 activity d e p e n d i n g on b r a i n m a t u r a t i o n 16. A t present it is unclear how m u c h these effects are representative in the general e c o n o m y o f S-100 in nervous function. A n u m b e r o f cellular localizations a n d functional i n t e r p r e t a t i o n s have been suggested for this protein4,9,11,12. F u r t h e r m o r e , the possibility t h a t the S-100 is in fact a class o f proteins has been proposed1,24, 27, so t h a t the a t t e m p t to identify a unique biological role m a y be w i t h o u t solution. Nevertheless, it is r e a s o n a b l e to hypothesize t h a t the p r o t e i n exerts an action in those c o m p a r t m e n t s where it is in a b o u n d form, n a m e l y in the nucleus as well as in m e m b r a n e s . Elsewhere a selective effect o f the p r o t e i n on the R N A p o l y m e r a s e I in i m m a t u r e nuclei has been i n d i c a t e d 16. Therefore, we have restricted o u r investigation to this field. The present d a t a show an a d d i t i o n a l selectivity in this in vitro system, as the effect a p p e a r s to be restricted to b r a i n nuclei. K e e p i n g in m i n d t h a t S-100 has been f o u n d in b r a i n nuclei a m o n g the nonhistone p r o t e i n s 17, the finding m a y be w o r t h consideration. In fact, the nuclear nonhistone proteins represent a class o f tissue-specific proteins which are believed to be involved in tissue-specific gene activation (for review see ref. 23). Nevertheless, the in vivo conditions include a great n u m b e r o f physiological p a r a m e t e r s which escape the present experimental system, so t h a t functional e x t r a p o l a t i o n s c a n n o t yet be m a d e . This investigation was p a r t i a l l y s u p p o r t e d by C . N . R . C o n t r a c t 74.00229.04. 1 ABE,K., TAKAHASH[,K., ANDANDO,T., Purification and properties of S-100 protein from porcine brain, J. Biochem., 75 (1973) 11-22. 2 BENDA, P., LIGHTBODY,J., SATO, G., LEVlNE, L., AND SWEET, W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 3 BURTON,K., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 4 CALISSANO,P., AND BANGHAM,A. D., Effect of two brain specific proteins (S-100 and 14.3.2) on cation diffusion across artificial membranes, Biochem. biophys. Res. Commun., 43 (1971) 504-509. 5 CICERO,T. J., COWAN,W. M., MOORE,B. W., AND SUNTZEFF,V., The cellular localization of the two brain specific proteins S-100 and 14.3.2, Brain Research, 18 (1970) 25-34. 6 DONATO, R., AND MICHETTI, F., S-100 protein in cerebral cortex synaptosomes, Experientia (Basel), 30 (1974) 511-512. 7 DONATO,R., MICHETTI,F., AND MIANI, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Research, in press. 8 DUTTON, G. R., AND MAULER,H. R., In vitro RNA synthesis by intact rat brain nuclei, J. Neurochem., 15 (1968) 765-780. 9 HAGLID,K. G., HAMBERGER,A., HANSSON,H.-A., HYDt~N, H., PERSSON,L., AND RONNB.~CK,L., S-100 protein in synapses of the central nervous system, Nature (Loud.J, (1974) 532-534. 10 HAGLID, K. G., AND STAVROU,O., Water soluble and pentanol extractable proteins in human brain normal tissue and human tumours, with special reference to S-100 protein, J. Neurochem., 20 (1973) 1523-1532. I 1 HYD~N, H., A calcium-dependent mechanism for synapse and nerve cell membrane modulation, Proc. nat. Acad. Sci. (Wash.), 8 (1974) 2965-2968. 12 HYD~N, H., AND LANGE,P. W., S-100 brain protein: correlation with behavior, Proc. nat. Acad. Sci. (Wash.}, 67 (1970) 1959-1966.
375 13 HYD~N, H., AND McEwEN, B. S., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 345-358. 14 LOVTRUP-REIN,H., AND McEwEN, B. S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405--415. 15 MIANI, N., DE RENZlS, G., MICHETTI, F., CORRER, S., OLIVIERI-SANGIACOMO,C., ANDCANIGLIA, A., Axonal transport of S-100 protein in mammalian nerve fibres, J. Neurochem., 19 (1972) 1387-1394. 16 MIANI,N., MICHETTI,F., DE RENZIS, G., ANDCANIGLIA,A., Effect of a brain-specific protein (S100 protein) on the nucleolar RNA-polymerase activity in isolated brain nuclei, Experientia (Basel), 29 (1973) 1499--1501. 17 MICHETTI,F., MIANI,N., DE RENZIS, G., CANIGLIA,A., AND CORRER,S., Nuclear localization of S-100 protein, J. Neurochem., 22 (1974) 239-244. 18 MONTANARO,N., NOVELLO, F., AND STIRPE, F., Effect of a-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning, Biochem. J., 125 (1971) 1087-1090. 19 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739-744. 20 PACKMAN, P. M., BLOMSTRAND,C., AND HAMBERGER, A., Disc electrophoretic separation of proteins in neuronal, glial, and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 479-487. 21 PEREZ, V. J., OLNEY, J. W.) CICERO,T. J., MOORE, B. W., AND BAHN,B. A., Wallerian degeneration in rabbit optic nerve: cellular localization in the central nervous system of the S-100 and 14.3.2 proteins, J. Neurochem., 17 (1970) 511-519. 22 RUSCA,G., CALISSANO,P., AND ALEM-~,S., Identification of a membrane-bound fraction of the S-100 protein, Brain Research, 49 (1972) 223-227. 23 STEIN, G. S., SPELSBERG,T. C., AND KLEINSMITH,L.J., Nonhistone chromosomal proteins and gene regulation, Science, 183 (1974) 817-824. 24 STEWART, J. A., Tissue specific S-100: a demonstration of multiple proteins, Biochim. biophys. Acta (Amst.), 263 (1972) 178-192. 25 SVIRIDOV,S. M., KOROCHKIN, L. I., IVANOV, V. N., MALETSKAYA,E. I., AND BAKHTINA,T. K., Immunohistochemical studies of S-100 protein during postnatal ontogenesis of the brain of two strains of rats, J. Neurochem., 19 (1972) 713-718. 26 TAaUCHI, K., AND KIRSCH, W. M., Immunocytochemical localization of S-100 protein in neurons and glia of hamster cerebellum, Brain Research, 92 (1975) 175-180. 27 UYEMURA,K., VINCENDON,G., GOMaOS, G., AND MANDEL,P., Purification and some properties of S-100 protein fractions from sheep and pig brains, J. Neurochem., 18 (1971) 429-438.