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.

Are Dauer Larvae Transcriptionally Active?

B. Dalley and M. Golomb

Figure 1

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.

Figure 1