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.
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 library!