Worm Breeder's Gazette 10(1): 74
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Although C. elegans dauer larvae have been reported to lack translatable mRNA, the transcriptional activity of dauer larvae has not been assayed directly. Using the method of Dixon et al. (WBG 9:3, 73-74), we have isolated nuclei from staged cultures, including dauer larvae. For purposes of comparison among developmental stages, the amount of nuclear DNA was measured using a sensitive fluorometric assay. We have optimized conditions for run-on transcription by RNA polymerase II. Under these conditions, approximately 12,000 cpm or 6 pmol of [3H]-UTP were incorporated into total RNA by 10 l of an adult stage nuclear suspension containing 4ng of DNA. The table below shows the rate of nuclear transcription in a 10 min assay (20 C), using nuclei from various developmental stages. [See Figure 1] Relative to nuclei from other stages, dauer nuclei are certainly transcriptionally active, although less so than nuclei from late larval stages and adults. We will next compare transcription of specific mRNAs using appropriate probes. Using an immunological assay, we have measured the quantity of RpoII polypeptides (relative to total protein) in crude extracts from various developmental stages. Not surprisingly, the amounts of RpaII in extracts from all stages, including dauers, is closely similar. RpoII activity is also roughly similar in extracts from different stages. Thus, differences in transcriptional rates among different stages cannot be attributed to changes in enzyme level or activity in a nonspecific assay. In Western blots with anti-RpoII antibody, the polypeptide composition of RpoII from all stages is closely similar, with one exception. The amount of the 30 kd subunit detected by this probe is greatly reduced in whole cell and nuclear extracts of dauers, relative to other stages; this band reappears during dauer recovery. We are currently trying immunoprecipitation experiments to ask whether this change reflects loss or modification of the subunit. The use of nuclear extracts has led us to suspect modification of the large subunit of C. elegans RpoII, which has an Mr of 200,000. Crude extracts, but not purified enzyme, have small amounts of a polymerase-related polypeptide with Mr = 235,000. This is probably the worm equivalent of the IIo variant of the large subunit in other organisms, which is a phosphorylated form of IIA. In contrast to C. elegans whole-cell extracts, nuclear extracts have a high proportion of IIo (approximately 50% by antigenicity). This is useful information because IIo is the form believed to be active in in vitro transcription in other systems. In contrast to the situation with the 30 kDa subunit, modification of the large subunit appears to be similar in all stages, including dauers.