Cell. RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope Rabbit Polyclonal to MBD3 for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ Quarfloxin (CX-3543) hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters Quarfloxin (CX-3543) of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to Quarfloxin (CX-3543) transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors. INTRODUCTION Previous results have demonstrated that spliceosome formation and/or splicing can be co-transcriptional (Beyer and Osheim, 1988 ; LeMaire and Thummel, 1990 ; Tennyson (1987) . Ultrathin sections cut on a Reichert Ultracut E Quarfloxin (CX-3543) ultramicrotome ((1997) was used. However, instead of two-step image acquisition of relocated cells, this modified protocol does not require cell relocation for the second fluorochrome. Briefly, whole RNA/DNA ISH using a mixture of biotin- and digoxigenin-labeled probes was performed; the probes were initially detected with anti-digoxigenin antibody and Cy2-conjugated secondary antibody. The cells were refixed with 4% paraformaldehyde in PBS for 5 min before RNase digestion of the targeted RNA. The probe hybridized to DNA was detected via ExtrAvidin-Cy3. This method allowed a more precise spatial discrimination between RNA and DNA using differentially labeled probes of the same sequence in a one-step hybridization protocol and in a one-step image acquisition. In this approach, the resolution of the signals is influenced by the optical system only and does not depend on the factor introduced by the investigator. When appropriate, the cells were counterstained for 5 min in 50 g/ml DAPI in PBS and mounted on glass slides in 2.3% (wt/vol) Mowiol 40C88 (Sigma), 42.5% glycerol, and 0.1 M Tris-HCl, pH 8.5, containing 134 mM 1,4-diazabicyclo[2.2.2]octane to reduce fading. Triple visualization of RNA, DNA, and protein in the same experiment required the consecutive labeling and refixation of the constituents in the order described above. Antibody against SC35 was detected using aminomethylcoumarin acetate (AMCA)-conjugated antibody ((1995) . Briefly, the spread cells were incubated with transcription mix at 37C in humidified chamber for 10C15 min. The transcription mix contained 600 M ATP, GTP, and UTP, 1 mM biotin-14-CTP (Life Technologies, Gaithersburg, MD), 37% buffer D (Dignam BX50 microscope) equipped with a universal plan-apochromat 100/1.35 numerical aperture (NA) objective lens. Fluoview was operated with excitation wavelengths of 488 nm (Cy2 fluorescence) and 568 nm (TRITC/Cy3 fluorescence) from an argon-krypton laser. Fluorescent signals of both fluorochromes were recorded simultaneously by two detectors at one scan. Fluorescence Microscopy.Samples were examined using an epifluorescence microscope (AX70 Provis; (1997) suggested complete colocalization of intron- and exon-specific probes over the full length of the RNA accumulations without Quarfloxin (CX-3543) any apparent loss in the intensity of the FISH signal along the track. This indicated that both introns and exons were situated along the entire RNA track. Similar results concerning exon- and intron-specific distribution along the RNA track were reported for the viral human cytomegalovirus immediate early antigen transcripts (Raap em et al. /em , 1991 ; Snaar em et al. /em , 1999 ). High levels of transcription can alter the apparent spatial relationship between genes and speckles, and the speckle proximity to the gene may thus be a result of dynamic interplay of gene activity and mass action of splicing factors (reviewed in Singer and Green, 1997 ; also see Xing em et al. /em , 1993 , 1995 ; Fakan, 1994 ; O’Keefe em et al. /em , 1994 ; Pombo em et.
