Looking at mRNA Splicing and Transport in Situ

This chapter describes protocols for in situ analysis of the organization of intact salivary gland polytene nuclei with particular attention to pre-messenger RNA (mRNA) metabolism. The chapter discusses the construction of chimeric genes with high expression levels in polytene salivary gland nuclei. A crucial feature of this experimental system is the capability to engineer and reintroduce genes heavily transcribed in salivary gland nuclei and specifically designed for analysis. The Drosophila salivary gland is enclosed in a basement membrane-like structure that must be breached to permit efficient access of nucleic acid and antibody probes. This is done in two ways. The first is to generate “thick” cryosections. These cut a salivary gland lobe in roughly five serial sections with complete and nearly complete nuclei in essentially every section. The second strategy is to generate “smear” preparations. The basement membrane is ruptured by gentle squashing that leaves most nuclear envelopes intact. The resulting nuclei are somewhat “flattened;” however, many nuclear structures are preserved and much information can be gained from such preparations.