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THE USE OF FLUORESCENTIN SITU HYBRIDIZATIONFOR TIIE ANALYSIS OF NUCLEAR ARCH/TECTUREBY CONFOCALMICROSCOPY
R. Huispas and J.G.J. Bauman Dept. Molecular Pathology, ITRI-TNO, P.O. Box 5815, 2280 HV Rijswijk, NL
INTRODUCTION
The DNA of an eukaryotic cell, containing all the genetic information, is present within the nucleus. During interphase, heterogeneous nuclear-mRNA (hn-mRNA) or pre-mRNA, is produced by transcription of genes. This pre-mRNA is processed within the nucleus to mature mRNA, which is transported to the cytoplasm where it can be translated into the amino acid sequence. With respect to DNA the interphase can be divided in three stages: Gl-stage (transcription), S-stage (duplication) and G2-stage (cell contains double amount of DNA). The interphase is followed by mitosis, during which there is no gene transcription. The degree of accuracy by which these processes are performed is startling, especially when one takes into account the enormous amount of DNA that is present in a nucleus. An average diploid mammalian nucleus has a diameter of + ? pm and contains nearly 2 m DNA (~ = 20/~). The need for a proper structural organization is obvious. The DNA is packaged into nucleosomes that are organized in solenoids. The solenoids are further organized into loop domains, which are attached to nuclear matrix components (Nelson et al., 1986; Manuelidis and Chen, 1990). The sequences responsible for attachment are still under investigation. When the nuclear matrix is considered as a nuclear framework, the anchoring of DNA could mean that a specific chromatin or chromosome organization exists (Hilliker and Appels, 1989). It has been proposed that the heterochromatin (condensed, inactive form) is mainly located at the periphery of the nucleus, whereas the euchromatin (less condensed, active form) is mostly found in the interior of the cell (Bodnar, 1988). Blobel (1985) proposed that a gene changes of nuclear position in order to be transcribed. Next, the mRNA is processed and transported from this transcriptionally active site through the nuclear pores to the cytoplasm (Schr6der et al., 1987). This makes the position of the gene within the interphase nucleus an important feature of gene expression and gene control. Chromatin domains can be investigated by means of classical cytochemical staining techniques. These techniques are based upon general features of chromatin, such as nucleic acid type and charge (acridine-orange staining), local chromosome folding (propidium iodide, DAPI, Giemsa), protein composition, GC content or protein-RNA interactions with chromatin (Giemsa). However, to study the individual chromosome or gene distribution these features cannot be used. The fluorescent in situ hybridization (FISH) technique is very suitable to visualize the DNA of individual chromosomes. With FISH it is also possible to detect RNA species. The potential shnultaneous detection of DNA and RNA of individual genes makes FISH a powerful technique in the study of the nuclear architecture. 0309-16511921080739-9/$03.00/0
© 1992 Academic Press Ltd
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SOME RECENT HNDINGS D N A d i s t r i b u t i o n - Studies on the distribution of DNase I sensitive regions are done in relation to the distribution of active genes. Compartments of DNase I sensitive regions were found preferential at the periphery of interphase nuclei by De Graaf et al. (1990). It should be mentioned that not all DNase I sensitive regions represent active chromatin (Bodnar, 1988). By comparison of DNA extracts from nuclear matrices and DNA extracts from whole nuclei of rat fihroblasts, it was concluded that an active gene is preferentially attached to nuclear matrix components, whereas an inactive gene is not (Ogata, 1990). However, the nuclear position of the gene can not be determined by this technique. The first indication that chromosome position may be involved in the functional status of a tissue was published by Borden and Manuelidis (1988). In normal human cortex cells they found the chromosome X centromere region located close to the nuclear envelope, whereas in epileptic human cortex cells this centromere region was located in the interior of the nucleus. Using FISH on suspended nuclei and colffocal laser scanning microscopy, the position of pcricentromeric DNA of chromosome I in human peripheral lymphocytes and in HL-60 interphase nuclei has been studied. The majority of the centromere 1 regions was detected at the periphery of the nucleus. The spatial angle between the two centromere 1 homoloques and the center of mass of the nucleus was also measured. Statistical analysis revealed that the distribution of the two homoloques near the surface of the nuclei could not be distinguished from a random distribution (Van Dekken et al., 1990b). In contrast, Amoldus et al. (1989, 1991) reported non-random distributions of chromosomes 1 and 17, using FISH on sections of human normal brain tissue. Whether these homoloques were located at the nuclear periphery has not been reported. With FISH and immunofluorescence Masumoto et al. (1989) demonstrated the association of kinetochores with centromeric DNA in HeLa cells throughout the cell cycle. This indicated that the distribution of all centromeric DNA can also be studied by immunofluorescence, as had been done by Hadlaczky et al. (1986). They found arrangement in pairs of kinetochores in some G 1-nuclei of rat-kangaroo ovary tumour cells and male Indian muntjac ceils, using immunofluorescence. Some rat-kangaroo nuclei showed clustering of kinetochores as proposed in the Rabl configuration (Rabl, 1985). The fact that these different arrangements were not seen in all nuclei, indicates that other parameters must be involved. Haaf et al. (1990) found a correlation between the distribution of kinetochores and transcriptional activity of ribosomal genes ha murine Sertoli cells. After chromosome-staining with DAPI the chromosome arrangement in D r o s o p h i l a embryo cells was followed from interphase to mitosis relative to the surface of the embryo (Hiraoka et aI., 1990). These studies indicate that positioning of chromosomes is not only related to cell cycle stage, but also to a biological reference point outside the nucleus. FISH with probes from chromosome specific DNA libraries can be used to paint specific chromosomes in interphase cells to analyse the organization of chromosomal domains in 3D (Pinkel et al., 1986; Lichter et al., 1988). Comparison between small and large sized chromosomes showed that small chromosomes were preferentially found in the interior of the nucleus (Cremer et al., 1991).
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R N A distribution - T h e distribution of ribosomal RNA and poly-(A)+ RNA in mouse bone marrow and HL-60 cells was studied using FISH and confocal microscopy on cells in suspension (Banman et al., 1990). Ribosomal RNA was detected in the cytoplasm and in the nucleoli and the distribution of poly-(A)+ RNA within the nucleus was non-homogeneous. The major components of the splicing apparatus are found in loci (Carmo-Fonesca et al., 1991; Zamore and Green, 1991). The precise distribution of these loci within the individual cell nuclei however, has still to be studied. Spector (1990) presented a model of the 3D organization of the marrunalian nucleus. In this model the small nuclear ribonucleoproteins (snRNPs) are distributed in a reticular network around which the bulk DNA is located. Using FISH, Lawrence et al. (1989) showed that it is possible to detect specific mRNA 'tracks' in an Epstein-Barr virus transfected cell line. These tracks probably represent specific parts of the network in which the pre-mRNA is processed and transported to the nuclear pores. Using FISH and confocal microscopy we have analized the position of the track in 3D (Bauman et al., 1991). FISH With FISH it is possible to detect specific base sequences in chromosomes, cells and tissue sections. Detection of the hybridized probe is done by means of a fluorochrome, which can be visualized by fluorescent microscopy. Two types of FISH can be distinguished: the indirect and the direct method. In the direct method the fluorochrome is directly bound to the probe. In the indirect method the probe contains an element (e,g. biotin) with a specific affinity to another element which is fluorochrome conjugated (e.g. avidin-F1TC). The first successful FISH has been reported by Rudkin and Stollar (1977) and involved the indirect method. Nowadays, many FISH systems are commercially available. The most frequently used system is that of the biotin-modified nucleotides, visualized with a fluorochrome conjugated avidin. Another very useful system is the digoxigenin/anti-digoxigenin system. We developed a procedure to directly label RNA probes with a fluorochrome (Bauman et .al., 1980). This allows visualization of the formed hybrids immediately after the FISH procedure. Recently, fluorochrome-labeUed nucleotides to make directly labelled DNA probes were introduced by Boehringer and applied in FISH (Wiegant et al., 1991). Combinations of different probe systems and fluorochromes permits to perform multiple FISH experiments to detect various base sequences simultaneously (Hopman et al., 1985; Van Dekken et al., 1990a; Nederlof et al., 1990). FISH and immuno-fluorescence experiments can be combined to visualize nucleotide sequences and pro-teins at the same time (Masumotto et al., 1989; Mielke et al., 1990; Singer et al., 1989). T e c h n i c a l a s p e c t s o f F I S H - When using FISH the cells or tissue have to be prepared such that the probe will have access to the target, and will not bind to other material besides the target. In case of the indirect method the same criteria hold for the fluorochrome-conjugated element. Many problems in FISH concern the accessibility of the target. The accessibility is influenced by the way the biological material is fixed, the denaturation method (only for DNA targets) and the size of the probe. Optimal fixation procedures have to be determined experimentally because of the great variety of biological materials and applications. For example, metaphase chromosome spreads are routinely fixed with methanol/acetic acid, and cells or tissue -
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sections with (para)formaldehyde. Usually, permeabilization treatments of the fixed material is necessary to enhance the accessibility. The accessibility is also influenced by the denaturation procedure. When FISH is performed on double stranded DNA targets, the two strands have to be seperated in order to render it accessible for hybridization. This denaturation can be done by heating the target DNA in a formamide containing solution. This method can result in destruction of protein structures (Masumotto, 1989). An alternative is to treat the DNA with 0.07 N NaOH for 2-5 minutes at room temperature. Van Dekken et al. (1988) showed that single stranded target D N A can also be produced by a mild enzymatic treatment. Finally, the size of the probe plays a very important role in accessibility. Small probes can easily penetrate through ceils or tissue sections. The way the material is fixed influences the penetration of the probes. This explains why a permeabilization treatment is sometimes necessary. In general, a probe size of about 200 bp is sufficient for most applications. Applying FISH on paraformaldehyde fixed cells in suspension, we found out that a probe with an average size of 1000 bp (range 400-2000 bp) did not penetrate (completely) through the nuclear membrane. Fluorescent signals were seen only at the periphery of the nuclear membrane. Using an average probe size of 250 bp (range 150-350 bp), we observed fluorescent signals throughout the nucleus (Fig. 1). Probably, large probes are trapped near the nuclear membrane so that forming of concatemeres happens in the cytoplasm. On the other hand, an average probe size of 160 bp (range 150-175 bp) showed a general fluorescent glare in the cytoplasm and very low specific signals in the nucleus (data not shown). Small probe concatemers can easily be trapped by cytoplasmic structures. Only a small amount of probe will reach the target, which explains the low signal in the nucleus.
Fig. 1. Visualization by confocal microscopy of a centromeric DNA specific probe (p82H; Mitchell et al., 1985) after FISH on HL-60 ceils in suspension (essentially as described by Van Dekken et al., 1990b). The nucleus is stained with propidium iodide. Only one optical x-y section out of 20 from a z-serie is shown. A: Average probe size 800 bp, range 400-2000 bp the fluorescent spots are found at the periphery of the nuclear membrane. Bar represents 5 pm. B: Average probe size 250 bp, range 150-350 bp the signal is found throughout the nucleus. Bar represents 5 grn.
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The specificity of probe binding is determined by the melting temperature (T,,) of the hybrid. The T= of the hybrid depends on its chemical environment and the percentage of mismatched base pairs. The hybridization specificity (% mismatched base pairs) can be regulated by manipulating the temperature, the Na÷-concentration and/or the percentage of formamide (for details see Bauman et al., 1989). Binding of the probe or the conjugate to non-nucleic acid components, can be prevented by competition reactions. Single stranded herring or salmon sperm D N A or E. coli tRNA is mostly used to avoid non-specific binding of the probe. Bovine serum albumin, non-fat dry milk, Tween-20 or normal goat serum have been used to avoid non-specific binding of the conjugate. Using FISH on tissue sections and on ceils in suspension to study the nuclear architecture at the light microscopical level, DNA and RNA sequence can be visualized and examined directly in their 'natural' position. The availability of antibodies specific to nuclear components and the possibility to combine the FISH technique with immtmofluorescence, may complement data obtained by other techniques. Analyzing F I S H experiments - The specific advantage of hybridizing cells in suspension, is that the fluorescent signals of individual cells can be quantitated by flow cytometry and localized by confocal microscopy. Large quantities of cells can be analized by flow cytometry (Bauman and Bentvelzen, 1988; Trask et al., 1985; Van Dekken et al., 1990a). Multiple parameters of each cell (e.g. size, light absorption, multiple fluorescence signals) can be rapidly measured shnultaneously. The ability to sort cells on the basis of one or more parameters enables one to analyse the nuclear architecture in relation to specific cell types or cell cycle stages. Originally, the confocal microscope was designed to improve resolution with respect to the fluorescence microscope, using visible light. An improvement of the resolution by a factor +2 was established, which gives the confocal microscope a (theoretic) limit resolution of 0.14 /am (on the x-y-plane). However, the exclusion of the out-of-focus light by the confocal principle, became a major advantage. This makes it possible to exmnine various sections of an object without physically making sections of it (Pawley, 1990). Various software approaches are available to build 3D images of the obtained data (Brakenhoff et al., 1988; Peeples and Goldstein, 1989; Van der Voort et al., 1989). In cytometric instruments the light source, excitation falters, dichroic mirrors and emission filters play an ilnportant role in the interpretation of the results. However, the autofluorescence of the objects to be analized can still obscure the results. Although much of the autofluorescence will not be detected when using optimal filter sets (e.g. double bandpass filters), there is always some autofluorescence of the same wavelength as the fluorochrome of interest. Another inevitable phenomenon is bleaching of fluorescence. Especially, when very weak signals have to be detected, these are major problems. Depending on the kind of cells and how the cells are fixed and treated, the autofluorescence is strong or weak. For example, we observed in formaldehyde fixed mouse bone marrow cells, an increase in autofluorescence related to time of hybridization (Fig. 2A). To detect weak signals one can amplify the signal or use anti-bleaching (e.g. Dabco) or autofluorescence-reducing agents (e.g. DTF, see Fig. 2B).
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Fig. 2. Autofluorescence induction at 520 um as a function of hybridization time (A) and prevention by addition of DTF (B). Murine bone marrow ceils formaldehyde fixed and hybridized in 50% formamide, 2XSSC at 37°C. Fluorescence measured by flow cytometry and expressed in arbitrary units (a.u.) as described (Bauman and Bentvelzen, 1988). I : fluorescence after 0 h. &: fluorescence after 20 h. PERSPECTIVES In our study on distribution of specific DNA sequences in the nucleus, we use FISH on parafonnaldehyde fixed cells in suspension. For the analysis of the distribution of centromeric DNA, a general biological marker that can serve the purpose of a topological point of reference is needed. A useful point of reference is the nucleolus. Visualization of the nucleolus can be done by chromatin staining dyes, like propidium iodide and DAPI, or by FISH with ribosomal RNA probes. In our studies we chose the microtubule organizing center (MTOC) as a point of reference. During mitosis the microtubules, originating from the MTOC's (or centrosomes), play an important role in chromosome movement. Both the position of the MTOC and the distribution of the chromosomes are related to the cell cycle stage. We have developed a technique to combine FISH and immunofluorescence for cells in suspension to visualize centromeres and centrosomes simultaneously. After FISH, cells are sorted with respect to DNA content and subsequently analyzed by means of confocal microscopy. Using this approach, we are able to study the localization of DNA sequences relative to the MTOC in specific stages of the cell cycle (publication in progress). Results of this study will increase the knowledge of the nuclear architecture, which will lead to an improved understanding of nuclear processes, such as replication, transcription gene and regulation. REFERENCES Amoldus E.P.J., Peters A.C.B., Bots G.T.A.M., Raap A.K. and Van der Ploeg M. (1989) Somatic pairing of chromosome 1 centromeres in interphase nuclei of human cerebellum. Hum. Genet. 83: 231-234. Amoldus E.P.J., Noordermeer I.A., Peters A.C.B., Raap A.K. and Van der Ploeg M. (1991) Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal human brain tissue, but no trisomy 7 or sex-chromosome loss. Cytogenet. Cell Genet. 56: 214-216.
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Bauman J.G.J., Wiegant J., Borst P. and Van Duijn P. (1980) A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labeled RNA. Exp. Cell Res. 138: 485-490. Bauman J.G.J. and Bentvelzen P. (1988) Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization. Cytometry 9: 517-524. Bauman J.G.J., Pinkel D., Trask B. and Van der Ploeg M. (1989) In: Flow Cytogenetics. Gray, J.W. ed., pp. 276-284, Academic Press, London. Bauman J.G.J., Bayer J.A. and Van Dekken H. (1990) Fluorescent in situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy. J. Microsc. 157: 73-81. Bauman J.G.J., Fogarty K., Bowman D., Fay F. and Lawrence J.B. (1991) The 3D analysis of the EBV RNA tracks in interphase nuclei of transformed cell lines. Cytometry Suppl. 5: 26. Blobel G. (1985) Gene gating: A hypothesis. Proc. Natl. Acad. Sci. USA 82: 8527-8529. Bodnar J,W. (1988) A domain model for eukaryotic DNA organization: A molecular basis for cell differentiation and chromosome evolution. J. Theor. Biol. 132: 479-507. Borden J. and Manuelidis L. (1988) Movement of the X chromosome in epilepsy. Science 242: 1687-1691. Brakenhoff G.J., Van der Voort H.T., Baarslag M.W., Marls B., Oud J.L., Zwart R. and Van Driel R. (1988) Visualization and analysis techniques for three dimensional information acquired by colffocal microscopy. Scan. Microsc. 2: 1831-1838. De Graaf A., Van Hemert F., Linnemans W.A.M., Brakenhoff G.J., De Jong L., Van Renswoude J. and Van Driel R. (1990) Three-dimensional distribution of DNase I-sensitiv.e chromatin regions in interphase nuclei of embryonal carcinoma cells. Eur. J. Cell Biol. 52: 135-141. Carmo-Fonseca M., Toilervey D., Pepperkok R., Barabino S.M.L., Merdes A., Brenner C., Zamore P.D., Green M.R., Hurt E. and Lamond A.I. (1991) Mmnmalian nuclei contain loci which are highly enriched in components of the pre-lnRNA splicing machinery. EMBO J. 10: 195-206. Cremer T., Remm B., Kharboush I., Jauch A., Wienberg J., Stelzer E. and Cremer C. (1991) Non-isotopic in situ hybridization and digital image analysis of chromosomes in mitotic and interphase cells. RBM 13: 50-54. Haaf T., Steinlein C. and Schmid M. (1990) Nucleolar transcriptional activity in mouse Sertoli cells is dependent on centromere arrangement. Exp. Cell Res. 191: 157-160. Hadlaczky G., Went M. and Ringertz N.R. (1986) Direct evidence for the non-random localization of mammalian chromosomes in the interphase nucleus. Exp. Cell Res. 167: 1-15. Hilliker A.J. and Appels R. (1989) The arrangement of interphase chromosomes: Structural and functional aspects. Exp. Cell Res. 185: 297-318. Hiraoka Y., Agard D.A. and Sedat J.W. (1990) Temporal and spatial coordination of
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chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila metanogaster embryos. J. Cell Biol. 111: 2815-2828. Hopman A.H.N., Wiegant J., Raap A.K., Landegent J.E., Van der Ploeg M. and Van Duijn P. (1985) Bi-color detection of two target DNAs by non-radioactive in situ hybridization. Histochemistry 85: 1-4. Lawrence J.B., Singer R.H. and Marselle L.M. (1989) Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57: 493-502. Lichter P., Cremer T., Borden J., Manuelidis L. and Ward D.C. (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-234. Manuelidis L. and Chen T.L. (1990) A unified model of eukaryotic chromosomes. Cytometry 11: 8-25. Masumotto H., Sugimoto K. and Okazaki T. (1989) Alphoid satelite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle. Exp. Cell Res. 181: 181-196. Mielke V., Bauman J.G.J., Sticherling M., Ibs T., Zomershoe A.G., Seligrnann K., Henneicke H., SchriSder J., Sterry W. and Christophers E. (1990) Detection of neutrophil activating peptide NAP/IL-8 and NAP/IL-8 rrtRNA in human recombinant IL-la- and human recombinant tumor necrosis factor-a- stimulated human dermal fibroblasts. J. Immunol. 144: 153-161. Mitchell A.R., Gosden J.R. and Miller D.A. (1985) A cloned sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes. Chromosoma 92: 369-377. Nederlof P.M., Van der Flier S., Wiegant J., Raap A.K., Tanke H.J., Ploem J.S. and Van der Ploeg M. (1990) Multiple fluorescence in situ hybridization. Cytometry 11: 126-131. Nelson W.G., Pienta K.J., Barrack E.R. and Coffey D.S. (1986) The role of the nuclear matrix in the organization and function of DNA. Ann. Rev. Biophys. Chem. 15: 457-475. Ogata N. (1990) Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian virus-40-pBR322 recombinant plasmid. Biochem. J. 267: 385-390. Pawley J.B. (ed.) (1990) Handbook of biological confocal microscopy. Plenum Press, New York. Peeples W.J. and Goldstein P. (1989) BioGraf 3D: an algorithm for optimization and graphical representation of nuclear structure spatial distributions. Cytobios. 58: 109-123. Pinkel D., Gray J.W., Trask B., Van den Engh G., Fuscoe J. and Van Dekken H. (1986) Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic probes. Cold Spring Harbor Symp. Quant. Biol. LI: 151-157. Rabl C. (1885) In: "Uber Zelltheilung. Morphologisches Jahrbuch. 10: 214-330. Rudkin G.T. and Stollar B.D. (1977) High resolution detection of DNA-RNA hybrids
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in situ by indirect immunofluorescence. Nature 265: 472-473. Schrtider H.C., Bachmann M., Diehl-Seifert B. and Miiller W.E. (1987) Transport of mRNA from nucleus to cytoplasm. Prog. Nucl. Ac. Res. Mol. Biol. 34: 89-142. Singer R.H., Langevin G.L. and Lawrence J.B. (1989) Ultrastructural visualization of cytoskeletal mRNA and their associated proteins using double-label in situ hybridization. J. Cell Biol. 108: 2343-2353. Spector D.L. (1990) Higher order nuclear organization: Three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc. Natl. Acad. Sci. USA 87: 147-151. Trask B., Van den Engh G., Landegent J., Jansen in de Wal N. and Van der Ploeg M. (1985) Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230: 1401-1403. Van Dekken H., Pinkel D., MuUikin J. and Gray J.W. (1988) Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization. Chromosoma 97: 1-5. Van Dekken H., Arkesteijn G.J.A., Visser J.W.M. and Bauman J.G.J. (1990a) Flow cytometric quantitation of chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to human lymphocyte interphase nuclei. Cytometry 11: 153-164. Van Dekken H., Van Rotterdam A., Jonker R., Van der Voort H.T.M., Brakenhoff G.J. and Bauman J.G.J. (1990b) Spatial topography of a pericentromeric region (1Q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy. Cytometry 11: 570-578. Van der Voort H.T., Brakenhoff G.J. and Baarslag M.W. (1989) Three-dimensional visualization methods for confocal microscopy. J. Microsc. 153: 123-132. Wiegant J., Ried T., Nederlof P.M., Van der Ploeg M., Tanke H.J. and Raap A.K. (1991 ) In situ hybridization with fluoresceinated DNA. Nucl. Acid Res., in press. Zamore P.D.. and Green M.R. (1991) Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 10:207-214