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Correspondence to: Marc Thiry, Laboratory of Cell and Tissue Biology, Univ. of Liège, Rue de Pitteurs, 20, B-4020 Liège, Belgium.
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Summary |
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Recently, we developed the terminal deoxynucleotidyl transferase (TdT)immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evaluated using the giant polytene chromosomes from Chironomus tentans salivary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and interbands of the polytene chromosomes but also over the BR structures. In the BRs, gold particles were present over segments of active transcription units, each with a central chromatin axis and a number of growing RNP products attached to the axis. One third of the transversely sectioned transcription units showed labeling in the central parts, i.e., where the unfolded chromatin axis is located, whereas the growing RNP fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA antibodies readily decorated the RNP fibers. The nuclear sap and cytoplasm displayed no significant label. These results clearly indicate that the TdTimmunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability to efficiently label a single DNA molecule demonstrates the method's very high sensitivity. (J Histochem Cytochem 46:345351, 1998)
Key Words: DNA, Balbiani ring genes, immunogold labeling, technique
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Introduction |
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A WIDE VARIETY of cytological techniques are now available for studying nucleic acids in situ at the ultrastructural level in biological materials. The most commonly used techniques include the Feulgen-like reaction with osmium ammine (
To further elucidate the question of specificity and sensitivity, we chose to investigate the Balbiani rings (BRs) of the giant polytene chromosomes of Chironomus tentans salivary glands. The BRs are sites at which large mRNA molecules are actively produced (
In this study we used the in situ terminal deoxynucleotidyl transferase (TdT)immunogold method to investigate the precise location of DNA in this model. This recent technique combines a molecular biology procedure with an immunocytological method (
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Materials and Methods |
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EM Preparations
Fourth instar larvae were decapitated and the salivary glands were isolated. Small pieces of various Chironomus tentans salivary glands were fixed for 2 hr at 4C in 2% glutaraldehyde dissolved in 0.1 M sodium cacodylate-HCl buffer (pH 7.2). The pieces were then rinsed four times with the same buffer for 15 min. Next, they were dehydrated in a graded ethanol series and soaked for 15 min at room temperature (RT) in ethanol:Agar 100 resin (3:1), for another 15 min in ethanol:Agar 100 resin (2:1), overnight in ethanol:Agar 100 resin (1:1), then finally embedded in Agar 100 resin (Agar Scientific; Essex, UK) and polymerized first at 45C, then at 60C. The glands were sectioned with a diamond knife in a Reichert Ultracut ultramicrotome. Optimal BRs were located by light microscopy on thick sections stained with toluidine blue. Subsequently, ultrathin sections were collected in platinum rings (4-mm diameter) formed by a platinum wire (0.1-mm in diameter; SA Johnson Matthey, Bruxelles, Belgium) and stored in distilled water until used.
TdTImmunogold method
Ultrathin sections were first floated for 60 min at RT on a saturated solution of sodium metaperiodate, then incubated for 10 min at 37C at the surface of the following medium: 20 µM 5 bromo-2-deoxyuridine (BUdR) triphosphate (Sigma; St. Louis, MO), 100 mM sodium cacodylate (pH 7), 2 mM MnCl2, 10 mM ß-mercaptoethanol, 50 µg/ml BSA, 125 U/ml calf thymus TdT (Boehringer Mannheim; Mannheim, Germany). (
Several kinds of control experiments were carried out. First, TdT or labeled nucleotides were omitted from the TdT medium. In a second control, BUdR triphosphate was replaced by BUdR monophosphate. Third, the grids were incubated with antibody-free particles. In the fourth control, the primary antibody was omitted. Finally, some cell sections were preincubated at 37C for 120 min with 1 mg/ml DNase I (Sigma; Type DN-Ep) in PBS (0.14 M NaCl, 6 mM Na2 HPO4, 4 mM KM2 PO4, pH 6.8) containing 7 mM MgCl2. Other sections were preincubated at 56C for 120 min with 1 or 10 mg/ml RNase A (Boehringer Mannheim) in 10 mM Tris-HCl (pH 7.4) containing 15 mM NaCl.
Immunocytological Technique for RNA
The procedure, involving two mouse monoclonal anti-RNA antibodies (D444, BWR5), has been described (
Control experiments were carried out as previously described (
Finally, the ultrathin sections were mounted on nickel grids and stained with uranyl acetate and lead citrate before examination in a JEOL CX 100 electron microscope at 60 kV.
Quantitative Evaluations
To evaluate the labeling density, we first determined the area (Sa) of each compartment morphometrically by the point-counting method (
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Results |
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BR Structures Contain DNA-positive Sites
To pinpoint the location of DNA within the polytene chromosomes of Chironomus tentans salivary glands, and notably within the BRs, we applied the TdTimmunogold procedure to ultrathin sections. In this procedure, free DNA ends generated by sectioning are elongated by the enzyme TdT in the presence of bromodeoxyuridine triphosphates. The thymidine analogue is visualized by immunogold electron microscopy. The micrographs were examined visually and evaluated quantitatively. Under our experimental conditions, intense label was always found over both the bands and the interbands of the giant polytene chromosomes (Figure 1). The bands were more intensely labeled than the interbands. Evident but less intense label was consistently found over the BR structures (Figure 2), whereas the extrachromosomal areas of salivary gland cell interphase nuclei appeared completely gold-free. Cytoplasmic areas (mitochondria excepted) were devoid of label.
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Table 1 summarizes the numerical data on gold particle distribution over the various compartments. These data confirm our subjective observations and further demonstrate the high labeling specificity. Over the ribosome-rich cytoplasmic areas, the labeling density is insignificant; over the BR structures, it is significant but lower than over areas known for their high DNA content, such as the bands and interbands of chromosomes.
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The specificity of the TdT reaction was tested in several ways. When TdT or labeled nucleotides were omitted from the TdT medium, the ultrathin sections were devoid of label. Likewise, no labeling was observed when BUdR triphosphate was replaced by BUdR monophosphate. When cell sections were incubated with RNase, the labeling persisted. It was almost completely abolished, however, when DNA was specifically digested with DNase I. The immunolabeling specificity was also tested. When the primary antibody was omitted, virtually no labeling occurred. Gold lacking the antibody tag did not bind to the sections.
Detection of DNA in the Axis of a BR Gene
Careful examination of the BR structures shows a "preference" of the gold particles for active transcription unit segments with growing RNP products (Figure 2). Because of intertwining of the segments, it was usually difficult to attribute label to any particular substructure of the loop segments. To achieve this, we had to examine the precise location of label over segments whose axis was separate from and not touching other active genes. A segment's typical labeling pattern was found to depend on the angle at which it was sectioned (Figure 3). It should be recalled that the method detects only DNA ends present at the surface of sections (
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By comparison, the presence of RNA in BR structures was investigated using a postembedding and immunogold labeling with two RNA-specific monoclonal antibodies. Except for the chromosome bands, all the compartments labeled by the TdT procedure were also labeled by this technique. Label was evident over the BR structures (Figure 4). Gold particles were found over almost all active transcription unit segments with growing RNP products, irrespective of the sectioning angle. In particular, in transversely sectioned transcription units, label was revealed over lateral RNP fibers (Figure 4). Label was as abundant over the growing RNP fibers of the proximal segment as over the stalked granules of the distal segment.
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Discussion |
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This study provides considerable information about the potential value of the TdTimmunogold technique in pinpointing the location of DNA in biological materials.
Our results show that the procedures used here can detect DNA in transcriptionally very active genes, such as the 75S RNA genes present in the BRs of C. tentans salivary glands.
The in situ TdTimmunogold technique labels specifically and with high resolution all known DNA-containing structures. In the polytene chromosomes it reveals not only the condensed chromatin of the chromosome bands but also the relatively decondensed chromatin of the interbands. Previous reports have shown the technique to detect both the condensed and dispersed chromatin of interphase nuclei from various tissues and cultured cell lines (
Although the restriction of labeling to the surface of the sections is limiting (
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Acknowledgments |
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Supported by the Fonds de la Recherche Scientifique Médicale (grant no. 3.4516.96). M. Thiry is a research associate of the National Fund for Scientific Research (Belgium).
We are grateful to Prof G. Goessens (University of Liège, Belgium) for encouraging discussions and for critical reading of the manuscript. We also acknowledge the skillful technical and secretarial assistance provided by Ms F. Skivée, Mr D. Bourguignon, and Ms S. Bodson.
Received for publication June 18, 1997; accepted October 13, 1997.
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Literature Cited |
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