Abstract
A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allis CD, Glover CVC, Gorovsky MA (1979) Micronuclei of Tetrahymena contain two types of histone H3. Proc Natl Acad Sci; USA 76:4857–4861
Bonifaz EL, Plaut W (1974) The synthesis and distribution of DNA in the macronucleus of Euplotes eurystomus. Chromosoma 46:261–278
Brown M, Bollum FJ, Chung LMS (1981) Intracellular localization of DNA polymerase. Proc Natl Acad Sci 78:3049–3052
Butler AP, Laughlin TJ, Cadilla CL, Henry JM, Olins DE (1984) Physical structure of gene-sized chromatin from the protozoa Oxytricha. Nucleic Acids Res 12:3201–3217
Caro LG (1964) High-resolution autoradiography. In: Prescott DM (ed) Methods in cell physiology. Vol 1, Academic Press, New York, pp 327–363
Clavaguera A, Querol E, Coll D, Genesca J, Egozcue J (1983) Cytochemical studies on the nature of NOR (nucleolus organizer region) silver stainability. Cell Mol Biol 29:255–259
Faure-Fremiet EC, Rouiller C, Gauchery M (1957) La reorganisation macronucleaire chez les Euplotes. Exp Cell Res 12:135–144
Gall JG (1959) Macronuclear duplication in the ciliated protozoan Euplotes. J Biophys Biochem Cytol 5:295–308
Goodpasture C, Bloom SE (1975) Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37–50
Howell WM (1977) Visualization of ribosomal gene activity. Silver stains proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma 62:361–367
Isackson PJ, Fishback JL, Bidney DL, Reeck GR (1979) Preferential affinity of high molecular weight high mobility group nonhistone chromatin proteins for single-stranded DNA. J Biol Chem 254:5569–5572
Javaherian K, Liu LF, Wang JC (1978) Nonhistone proteins HMG1 and HMG2 change the DNA helical structure. Science 199:1345–1346
Johns EW, ed (1982) The HMG chromosomal proteins. Academic Press, Inc, New York
Kimball RF, Prescott (1962) Deoxyribonucleic acid synthesis and distribution during growth and amitosis of the macronucleus of Euplotes. J Protozool 9:88–92
Kluss BC (1962y7 Electron microscopy of the macronucleus of Euplotes eurystomus. J Cell Biol 13:462–465
Likovsky Z, Smetana K (1981) Further studies on the cytochemistry of the standardized silver staining of interphase nucleoli in smear preparations of Yoshida ascites sarcoma cells in rats. Histochemistry 72:301–313
Lischwe MA, Smetana K, Olson MOJ, Busch H (1979) Proteins C23 and B23 are the major nucleolar silver staining proteins. Life Sci 25:701–708
Mamrack MD, Olson MOJ, Busch H (1979) Amino acid sequence and sites of phosphorylation in a highly acidic region of nucleolar nonhistone protein C23. Biochemistry 18:3381–3386
Marushige Y, Marushige K (1978) Dispersion of mammalian sperm chromatin during fertilization: An in vitro study. Biochem Biophys Acta 16:570–575
McDonald BB (1958) Quantitative aspects of deoxyribuse nucleic acid (DNA) metabolism in an amicronucleate strain of tetrahymena. Biol. Bull. 114:71–94
Medina FJ, Risueno MC, Sanchez-Pina MA, Fernandez-Gomez (1983) A study on nucleolar silver staining in plant cells. The role of argyrophilic proteins in nucleolar physiology. Chromosoma 88:149–155
Olert J, Sawatzki G, Kling H, Gebauer J (1979) Cytological and histochemical studies on the mechanism of selective silver staining of nucleolus organizer regions (NORs). Histochemistry 60:91–99
Olins AL, Olins DE, Franke WW, Lipps HJ, Prescott DM (1981) Stereo-electron microscopy of nuclear structure and replication in ciliated protozoa (Hypotricha). Eur J Cell Biol 20:254–264
Olson MOJ, Ribers ZM, Thompson BA, Kao W-Y, Case ST (1983) Interaction of nucleolar phosphoprotein C23 with cloned segments of rat ribosomal deoxyribonucleic acid. Biochemistry 22:3345–3351
Perreault SD, Zirkin BR (1982) Sperm nuclear decondensation in mammals: Rule of sperm-associated proteinase in vivo. J Exp Zool 244:253–257
Prescott DM (1983) The C-value paradox and genes in ciliated protozoa. In: McIntosh JR (ed) Modern cell biology. Vol 2, Alan R Liss, Inc, New York, pp 329–352
Prescott DM, Kimball RF (1961) Relation between RNA, DNA and protein synthesis in the replicating nucleus of Euplotes. Proc Natl Acad Sci USA 47:686–693
Prescott DM, Rao MVN, Evenson DP, Stone GE, Thrasher JD (1966) Isolation of single nuclei and mass preparations of nuclei from several cell types. In: Prescott DM (ed) Methods in cell physiology. Vol 2, Academic Press, New York, pp 131–142
Ringertz NR, Ericsson JLE, Nilsson O (1967) Macronuclear chromatin structure in Euplotes. Exp Cell Res 48:97–117
Salvano P (1975) Comparaison du fonctionnement des bandes de reorganisation d'Euplotes crassus (Dujardin) et de Strombidium sulcatum claparede et linné apres analyse microspectrographique en UV. J Protozool 22:230–232
Schwarzacher HG, Mikelsaar AV, Schnedl W (1978) The nature of the Ag-staining of nucleolus organizer regions. Cytogenet Cell Genet 20:24–39
Sippel TO (1981) New fluorochromes for thiols: Maleimide and iodoacetamide deriatives of a 3-phenylcoumarin fluorophore. J Histochem Cytochem 29:314–316
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Allen, R.L., Olins, D.E. Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide. Chromosoma 91, 82–86 (1984). https://doi.org/10.1007/BF00286488
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00286488