Acta histochem. (lena) 93, 298-306 (1992) Gustav Fischer Verlag lena' Stuttgart· New York
Max-Planck-Institut fur Biologie, Abteilung Mikrobiologie, Tiibingen, Germany, and Departamento de Biologia, Facultad de Ciencias, Universidad Aut6noma de Madrid, Spain
DNA cytochemistry in polytene chromosomes: Electron contrasting agents for the ultrastructural detection of chromatin DNA after alkaline hydrolysis/methylation-acetylation By JUAN CARLOS STOCKERT and CLAUS PELLING With 3 Figures (Received December 5, 1991) Key words: Electron microscopy, DNA cytochemistry, chromatin, alkaline hydrolysis, contrasting agents
Summary Salivary glands from Chironomus lemans larvae were fixed in glutaraldehyde and either subjected to alkaline hydrolysis followed by methylation-acetylation, or dehydrated without these treatments as controls. Ultrathin sections from Durcupan-embedded samples were contrasted by means of uranyl acetate, ruthenium red, indium trichloride, or the complex indium (ill)-hematoxylin. Electron microscopic observations revealed a general contrastiQg pattern in control sections, wljile after the hydrolytic and blocking procedure only chromatin from polytene chromosomes appeared selectively contrasted. The nucleolus, Balbiani ring granules and puff materials showed weak or no electron opacity. After toluidine blue staining of semithin sections, an orthochromatic blue colour was found in chromatin bands from treated samples.. ;These results indicate that alkaline hydrolysis/methylation-acetylation followed by contrasting with cationic heavy comPounds is a valuable procedure to visualize chromatin DNA in polytene chromosomes.
1. Introduction Simple salts of heavy elements are commonly applied to contrast chromatin DNA from tissue sections under the electron microscope (see ZOBEL and BEER 1965; HAYAT 1975; GAUTIER 1976; MOYNE 1980). For this purpose, one of the most widely used electron stains is uranyl acetate (see DERKSEN and MEEKES 1984; TANDLER 1990). Also heavy cations such as indium trichloride and ruthenium red have occasionally been employed (WATSON and ALDRIDGE 1961, 1964; STOCKERT and PANIAGUA 1980; GUTIERREZ-GONZALVEZ et al. 1984). Other electron dense stains (e.g., tungsten and molybdenum compounds), and procedures based on the use of silver or cationic dyes have also been reported (ADAMS et al. 1965; STOCKERT 1977a; EsQUIVEL et al. 1987; LAWTON 1990); however, their precise reaction mechanisms with chromatin components are not yet well understood. An important drawback of direct contrast of DNA by salts of heavy elements is that a nonspecific electron opacity of RNA and proteins is also produced (see LoMBARDI et al. 1971; TZAPHLinou et al. 1982; TATO et al. 1990). On the contrary, cytochemical methods selective for DNA based on acid hydrolysis are well known and some of them are the exclusive to offer the possibility to analyze the occurrence and localization of DNA at the ultrastructural level. Thus, a selective or almost specific contrast of DNA containing structures after HCl hydrolysis has been achieved by using silver methenamine (PETERS and GIESE 1970), the SCHIFF'S
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reagent and thallium ethylate (MOYNE 1973), platinum-pyrimidine complexes (AGAARWAL 1976), uranyl acetate (ERENPREISA 1981), and osmium ammine (DERENZINI et aI. 1982; OUNS et al. 1989). The selectivity of uranyl acetate for DNA is also improved after blocking procedures such as methylation-acetylation of proteins (TANDLER and SOLARI 1982). On the other hand, cytochemical studies of nucleic acids based on the alkaline hydrolysis of tissues are also known (SULKIN 1951; TANDLER 1959; GEYER and SCHEIBNER 1970; PETRAT et al. 1970). The alkaline treatment hydrolyses RNA and the phosphate group of phosphoproteins, while DNA remains unaffected (SCHMIDT and TANNHAUSER 1945). As an efficient blockage of carboxyl and amino groups from proteins is achieved by means of methylationacetylation (TANDLER and SOLARI 1982), the selectivity of electron dense stains for DNA can be increased. The combination of both treatments (alkaline hydrolysis and methylationacetylation) followed by contrasting with uranyl acetate has recently been used to visualize chromatin DNA in plant and mammalian tissues (RISUENO et al. 1990; TESTILLANO et aI. 1990; ROMERO et al. 1991). The present work deals with the application of some electron contrasting agents which allow a highly selective visualization of DNA in polytene chromosomes subjected to this hydrolytic and blocking procedure.
2. Material and methods Salivary glands from normal 4th instar larvae of Chironomus lenlans growing in standard culture were fixed with 2% glutaraldehyde in 0.066 moUI Sorensen' s phosphate buffer at pH = 7 for 2 h and washed in the buffer solution for 20 min. To avoid other sources of electron opacity as well as the known influence of osmium deposits on the affinity of tissue structures for subsequent staining processes, postfixation with OS04 was omitted. In this respect, it must be noted that OS04 reacts with single-stranded nucleic acids and also contrasts RNA containing structures (STOCKERT 1977b). Samples were subjected "in block" to an alkaline hydrolysis (TESTILLANO et al. 1990) by using 0.5 moUl NaOH in 4 % formaldehyde at room temperature (20°C) for 8 h. The material was washed first in I % acetic acid and then in distilled water for 30 min. After dehydration in methanol series, samples were subjected to the methylation-acetylation procedure (TANDLER and SOLARI 1982) using a freshly made and water-free methanolacetic anhydride solution (5: I = v/v) at room temperature for 8 h. Salivary glands were then washed in methanol for 45 min and embedded in Durcupan ACM (Fluka) as usual. Some samples not subjected to alkali/ methylation-acetylation were also dehydrated, embedded and used as controls. Semithin and ultrathin sections were obtained with an LKB Ultrolome /II and mounted on glass slides or copper grids without Formvar, respectively. To test the presence of nucleic acids, semithin sections from control and treated salivary glands were stained with 0.1 mg/ml toluidine blue 0 (Merck) in acetate buffer at pH = 5 for 2 h at 50°C (STOCKERT 1975 a). Preparations were washed in distilled water, air dried and directly observed under immersion oil and bright field illumination in a Zeiss photomicroscope III. Ultrathin sections were contrasted by using one of the following electron stains at room temperature for I h: a. A saturated and filtered solution of uranyl acetate (Merck) in distilled water (pH = 4); b. 0.5 mg/ml ruthenium red (Merck. batch No. 908K 10744419,99% pure) in borate buffer at pH = 9.2 (the product from Merck. M, = 551.22, appears formulated as RU2 (OHh CI 4 . 7NH 3 . 3H 20, in disagreement with the currently accepted chemical composition for ruthenium red, see HAYAT [1975], GUTIERREZ-GONZALVEZ et al. [1984]); c. 0.5 mg/ml indium trichloride (Merck) in I % acetic acid; d. A freshly made solution of the preformed complex indium (lII)-hematoxylin (GOMEZ et al. 1991), containing 0.1 % hematoxylin (Sc'hering; Kahlbaum. Berlin) and 0.3% indium trichloride (Merck). After staining. sections were briefly washed in distilled water and examined in a Zeiss 109 transmission electron microscope oper~ting at 60 kV.
3. Results Electron microscopic observations are summarized in Table I. Either untreated (control) or treated but unstained sections (Fig. I, A) revealed a very weak spontaneous contrast in
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Fig. I. Electron micrographs of C. tentans polytene chromosomes after alkaline hydrolysislmethylationacetylation. A: unstained (control) section, x 6,120; B: ruthenium red stained section, x 6,750. Note the weak (spontaneous) contrast of chromatin bands in A and the increased electron density in B.
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Table I. Contrasting patterns in Chironomus tentans salivary gland cells after glutaraldehyde fixation, alkaline hydrolysis/methylation-acetylation (AH/MA), and treatment of thin sections with uranyl acetate (VA), ruthenium red (RR) , indium trichloride (In), and indium (lII)-hematoxylin (In-H). - indicates no contrast; + to + + + + indicates increasing electron contrast. Contrasting intensity
Method
Glutaraldehyde
AHIMA
Chromatin
Nucleoli
Balbiani rings and puffs
Ribosomes
UA RR In In-H
+ ++++ ++++ +++ +++
+ +++ +++ +++ +++
++ ++ ++ ++
++ ++ ++ ++
UA RR In In-H
+ ++++ ++++ +++ +++
+ + + + +
{ {
chromatin bands from polytene chromosomes and nucleoli. Balbiani rings, puffs, ribosomes, and other cytoplasm organelles appeared practically uncontrasted and they only showed diffuse outlines. When sections from untreated salivary glands were stained with electron dense agents, a general contrasting pattern was observed in most cell structures. Chromatin bands and ribonucleoprotein containing components (nucleoli, ribosomes, Balbiani ring granules, and fibrillo-granular materials in large puffs) presented considerable electron opacity. The heavy compounds used in this work showed similar contrasting properties, uranyl acetate and ruthenium red giving a somewhat higher electron density than the other agents. On the contrary, a clearly different pattern of contrast was observed after application of the electron dense stains on sections of salivary glands previously subjected to alkaline hydrolysis/ methylation-acetylation, allowing the selective visualization of chromatin DNA. Bands of condensed chromatin as well as chromatin material from interband regions were the cell components which showed the highest electron contrast (Fig. I, B). RNA containing structures such as nucleoli, ribosomes, Balbiani ring granules, and puffed chromosome regions revealed very scarce or no contrast (Fig. 2). Only the spontaneous electron density of the nucleolus was observed after this procedure, and no differences between its fibrillar and granular regions were apparent. Balbiani ring granules and puff materials could be not detected and as in the case of nucleoli, only a network of branched chromatin fibres were found selectively contrasted within Balbiani rings and puffed chromosomes regions (Fig. 3). Toluidine blue staining of semithin sections from control salivary glands showed orthochromatic chromosome bands (blue) and a metachromatic reaction in nucleoli, Balbiani rings and basophilic cytoplasm (violet), indicating the presence of DNA and RNA, respectively, in the corresponding structures (see STOCKERT 1975a; COLMAN and STOCKERT 1983). After alkaline hydrolysis/methylation-acetylation, chromatin DNA in toluidine blue stained sections appeared blue, while RNA containing components did not stain.
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B Fig. 2.
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and C.
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p
Fig. 3. Electron micrograph of a large chromosome puff after the hydroIY'jc-blocking procedure and uranyl acetate contrasting. Ribonucleoproteins in the puffed region (P) appear uncontrasted and only chromatin branches (arrowheads) show electron opacity; x 6,750.
4. Discussion The heavy compounds used in this study have found wide use as contrasting agents in electron microscopy (ZOBEL and BEER 1965; HAYAT 1975; TATO et al. 1990), and the strong affinity of some of them for nucleic acids is a well documented feature. Uranyl acetate is one of the most popular contrasting agents and its use as a selective electron stain for DNA and RNA is already known (HUXLEY and ZUBAY 1961; STOECKENIUS 1961; ZOBEL and BEER 1961, 1965). Although the large amount of phosphate groups in DNA seems to correspond to obvious binding sites for the uranyl cation (ZoBEL and BEER 1961), it has recently been reported that a selective and strong binding mode may occur in the minor groove of DNA at the level of adenine-thymine sequences (NIELSEN et al. 1990). In addition, uranyl ions can stain proteins, glycoproteins, and ribonucleoproteins (HUXLEY and ZUBAY 1961; STERNBERGER 1961; STOCKERT 1975b; DERKSEN and MEEKES 1984), mainly by complexing to carboxyl and amino groups (ZOBEL and BEER 1961, 1965; LOMBARDI et al. 1971; TZAPHLIDOU et al. 1982). This unspecific binding limits the cytochemical value of direct contrasting by uranyl acetate. Other heavy compounds also show similar limitations. Ruthenium red has a strong affinity for doublestranded nucleic acids (KARPEL et al. 1981), but in addition to chromatin DNA (STOCKERT and PANIAGUA
Fig. 2. Electron micrographs of sections from C. tentans salivary glands showing part of nuclei. Alkaline hydrolysis/methylation-acetylation. A: contrasted by ruthenium red, x 4,620; B: contrasted by indium trichloride, x 7,200. The nucleolus (NU) and Balbiani ring (BR) appear with scarce or no electron opacity, while bands and branched chromatin fibres from polytene chromosomes show considerable contrast.
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1980), it binds and stains other cell and tissue components such as acidic proteins and glycosaminoglycans (HANKE and NORTHCOTE 1975; GUTIERREZ-GONZALVEZ et al. 1984; TATO et al. 1990). Nucleic acid containing structures are well contrasted by using both indium trichloride (WATSON and ALDRIDGE 1961, 1964; ALDRIDGE and COLEMAN 1966) and the complex indium (lII)-hematoxylin (GOMEZ et al. 1991), but other cell substrates also show considerable electron opacity. Since heavy cations which contrast DNA also bind to RNA and proteins, selective extraction or blocking methods must be applied in order to bring about a more specific contrast of chromatin DNA. The alkaline treatment derived from the SCHMIDT-THANNHAUSER'S (1945) analytical method for nucleic acids removes RNA (by hydrolysis of phosphodiester linkages) and the phosphate group from phosphoproteins. The large difference in lability to dilute alkali between DNA and RNA has been utilized to effect the separation and estimation of nucleic acids (FONO 1947; MARKHAM and SMITH 1952; SCOTT et al. 1956). Some cytochemical studies have also been carried out after alkaline hydrolysis (SULKIN 1951; AVERS 1963; GEYER and SCHEIBNER 1970; PETRAT et al. 1970), and as DNA remains unaffected it can be easy and selectively demonstrated in light microscopy (TANDLER 1959). The possibility of interaction between carboxyl groups of proteins and heavy cations used as electron stains may reduce the cytochemical value of alkaline hydrolysis for DNA localization. It has been shown that a mixture of methanol and acetic anhydride at room temperature produces methylation of carboxyl groups and acetylation of amino groups in proteins (BLACKBURN and PHILIPS 1944). More recently, this procedure has been applied as an efficient blocking method in electron microscopic cytochemistry (TANDLER and SOLARI 1982; TESTILLANO etal. 1990; RISUENO et al. 1990). Soluble phosphate compounds (e.g., inorganic phosphate, free nucleotides) are easily lost and without OS04 fixation, most phospholipids are removed mainly during dehydration and embedding. DNA remains as the only polyanionic macromolecule which conserves a strong affinity for cationic electron stains, thus allowing the highly selective contrast of chromatin. After this hydrolytic and blocking technique, toluidine blue staining of semithin sections indicates that RNA is entirely removed and chromatin DNA appears preserved in a native-like condition. Although the knowledge of the precise binding mechanism between DNA and the used electron stains requires further investigation, they are very suitable for the selective contrasting of DNA in ultrastructural studies.
Acknowledgements We are greatly indebted to J. W. BRAND and C. J. TANDLER for valuable collaboration. This work was supported by a grant from the Cooperation Program between Max-Planck-Gesellschaft (Germany) and Consejo Superior de Investigaciones Cientfficas (Spain).
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