Chapter 79 Enrichment of mRNA Encoding Flagellar Proteins

Abstract

Publisher Summary In many cases, growth of cilia or flagella is accompanied by large increases in levels of translatable mRNAs encoding flagellar proteins and correspondingly large increases in synthesis of flagellar polypeptides. Thus, the process of cilium/flagellum formation provides the opportunity to study controls over the expression of a specific large set of structurally diverse yet functionally related genes encoding ciliary or flagellar proteins. It also provides an opportunity to isolate mRNA enriched in transcripts encoding flagellar proteins, thus permitting construction of cDNA libraries enriched for these sequences. This chapter discusses a basic protocol for stimulating wild-type Chlamydornonas cells by pH shock, collecting samples of fixed cells for flagellar length measurements, and collecting RNA samples enriched for flagellar mRNAs before and 15, 45, 90, and 120 minutes after stimulation. RNA samples prepared in this manner have been used for cDNA library preparation, Northern, and S1 nuclease protection analysis, and in vitro translation. At the end of this description, variations in the basic method are discussed. As some cells lose their flagella during concentration, allow cells to recover from concentration by gently stirring with a magnetic stirrer under illumination for 120 minutes. After recovery, fix a 100-μl aliquot of cells by pipetting them into a tube containing an equal volume of glutaraldehyde, for the later measurement of flagellar lengths in nonstimulated cells.