Worm Breeder's Gazette 10(1): 76

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.

Synchronous Young Ascaris Embryos Are Transcribing

B. Dalley, M. Golomb and K. Bennett

Ascaris embryos offer several advantages with regard to studying 
early nematode development, including the capability of obtaining 
large populations of synchronous embryos.  The fertilized, but 
uncleaved eggs remain arrested and viable at 4 C for months.  With the 
intention of producing an oocyte and an embryonic cDNA library to 
screen with our P granule antibodies, Dr.  Bennett's new laboratory 
has recently isolated polyA+ RNAs from Ascaris oocytes and from 
Ascaris embryos at two stages, 4-8 cells and 16-32 cells.  These RNAs 
have been tested with various probes, including our Ascaris actin gene.
All contain actin message, but it had yet to be established whether 
young Ascaris embryos carry out new zygotic transcription, or rely 
entirely on maternal messages.  We wanted to establish that the 
embryonic library will not be redundant.
To address this question, we have isolated nuclei from Ascaris 
embryos using the protocol of Dixon et al.(WBG 9:3,73-74).  The 
embryos used were slightly older than those used in the RNA 
preparations, in the 5-6th cleavage divisions, our reasoning being 
with more nuclei we'd have a better chance of success with the first 
isolations.  The nuclei are intact, as assayed by 
diamidinophenylindole (DAPI) staining.  We have used this preparation, 
along with nuclei from a mixed population of Caenorhabditis eggs, in 
run-on transcription assays, essentially as described by McKnight and 
Palmiter (JBC 1979,  254:9050).
These Ascaris nuclei incorporate significant amounts of label (~35 
fold over background).  Preliminary results of testing the alpha-
amanitin sensitivity of the run-on transcripts show these young 
Ascaris nuclei have a striking profile, with the transcripts being 
over 80% sensitive to 1 microgram/ml alpha-amanitin, implying high 
levels of Polymerase II transcription.  After assuring ourselves that 
we are optimizing conditions for Ascaris (we're currently using those 
worked out in the Golomb lab for Caenorhabditis), we will assay 
transcription with specific probes, including actin.  We plan to look 
at earlier embryos; the synchronous Ascaris populations offer the 
opportunity to profile transcription throughout nematode embryogenesis.
And it's on for the embryonic