Visualization of Nascent Transcripts on Drosophila Polytene Chromosomes Using BrUTP Incorporation

Polytene chromosomes from the salivary gland cells of Drosophila third instar larvae have been used extensively for cytological and genetic studies. Polytene chromosomes have also been used to study transcription because an actively transcribing gene is often manifested as a localized region of chromatin decondensation or “puff”. Several methods have been developed to detect sites of active transcription on polytenes, including direct autoradiography following tritiated nucleotide incorporation (7), hybridization of tritiated RNAs (6) and detection of RNA/DNA hybrids by immunofluorescence microscopy (8). Each of these methods has one or more drawbacks. Direct autoradiography is a fairly difficult technique and requires exposure times of up to several weeks. It is often difficult to precisely map the sites of transcription because of the migration of the decay emission. In addition, it is difficult to detect weakly transcribing sites because of high background. Analyzing RNA synthesis by in situ hybridization of labeled RNAs can be compromised if certain RNAs are lost during the RNA isolation procedure or if nonspecific hybridization or binding of the probe occurs (2). The detection of actively transcribing sites using antibodies that detect RNA/DNA hybrids has produced good results but is limited in that it is not suited for pulse-chase experiments and that commercial antibodies recognizing the hybrids are not readily available. Many of the disadvantages of studying nascent transcripts have been overcome through the use of nucleotide analogue incorporation followed by immunofluorescence detection. A comparison of biotin-11-UTP, digoxigenin11-UTP and bromo-UTP (BrUTP) indicated that of these three modified nucleotides, BrUTP was incorporated into transcripts the most efficiently in mammalian cells (5). While early studies using mammalian cells used permeabilization (5) or direct microinjection (9) to get BrUTP into cells, more recent methods have used the cationic lipid DOTAP (4) or the non-liposomal agent FuGENEÔ 6 (Roche Molecular Biochemicals, Laval, PQ, Canada) (3) to load BrUTP. All of these studies led us to investigate whether BrUTP could be used to label nascent transcripts on Drosophila polytene chromosomes. We have adapted the method described by Haukenes and co-workers (4) to label transcripts on polytene chromosomes. Salivary glands from wandering Drosophila melanogaster third instar larvae were first dissected out in a drop of modified TB1 buffer (15 mM HEPES, pH 6.8, 80 mM KCl, 16 mM NaCl, 5 mM MgCl2 and 1% PEG 6000) (1). All chemicals used in this procedure were from Sigma (St. Louis, MO, USA) unless otherwise noted. After dissection, isolated glands were allowed to sit for at least 1 h at room temperature in a depression slide containing 200 mL TB1 before labeling with BrUTP. The depression slide was kept in a humidified chamber to prevent evaporation of the buffer. In the transcription inhibition experiments, cells were incubated in TB1 for 1 h, then TB1 plus inhibitor [either actinomycin D (10 mg/mL) or a-amanitin (50 mg/mL)] for 1 h before labeling for 20 min in the presence of inhibitor. To make our standard 10 mM BrUTP labeling solution (50 mL), the following were combined in a 1.5-mL microcentrifuge tube: 35 mL TB1, 10 mL DOTAP (Roche Molecular Biochemicals) and 5 mL 100 mM BrUTP. The components were pipetted up and down gently to mix, and the mixture was incubated for 10 min at room temperature to allow the DOTAP to complex with the BrUTP. Early experiments also involved testing a lower concentration (1 mM) of BrUTP in the labeling mixture and mixtures containing different concentrations of BrUTP with and without DOTAP. In a typical experiment, one or two pairs of salivary glands were transferred to a 50-mL BrUTP labeling mixture in a depression slide using a siliconized pipet tip or forceps. After a specified labeling time at room temperature (typically 15 or 20 min), glands were transferred to a drop of fixative (50% acetic acid/3.7% formaldehyde) on a siliconized (SigmacoteÒ; Sigma) coverslip for 1–2 min and then squashed on a microscope slide. When “partial” squashes were performed (i.e., no pressure on the coverslip was applied to keep the cells intact), the fixation time was 2–3 min. Preparations were frozen in liquid nitrogen to facilitate the removal of the coverslip and then stored in 95% ethanol until immunostaining was performed. For the RNase-treated specimens, slides containing fixed squashed salivary gland cells that had incorporated Bromo-Uridine were washed briefly with 2 ́ SSC. Next, the slides were incubated for 2 h with 125 mL 1 mg/mL DNase-free RNase A (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in 2 ́ SSC. The slides were then rinsed with 2 ́ SSC and returned to a Coplin jar with 95% ethanol. Chromosome squash preparations were immunostained essentially following the method described by Westwood and co-workers (11). Squashes were first incubated with an anti-bromodeoxyuridine (anti-BrdU) mouse monoclonal antibody (Roche Molecular Biochemicals) diluted to a final concentration of 2 mg/mL in BTP (0.5% BSA, 0.1% TweenÒ 20 in PBS) for 1–2 h at room temperature. We have subsequently used anti-BrdU antibodies from Chemicon International (Temecula, CA, Benchmarks