Worm Breeder's Gazette 10(2): 66
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.
My laboratory is interested in isolating maternal and early embryonic mRNAs which segregate with, or are produced in, the two germ line precursor cells of the early nematode embryo. I propose a final assay for putative messages to be in situ hybridization to Ascaris embryos. Because the vitelline membrane of Ascaris is highly impermeable, it has been necessary to use frozen 10 m sections of eggs (sliced eggs!). This has worked well for the probes tested to date. A 1.0 kb subclone from the 26 S subunit of an Ascaris ribosomal gene ( Bach, et al., (1984) NAR 12-3:1313.) has been put into the SP6 transcription vector in both orientations. With the antisense transcript all cells of the paraformaldehyde-fixed embryos hybridize strongly to the hydrolyzed [35S] RNA probe, while the sense strand shows no hybridization above background. In order to determine the resolution of our assay and see if the two germ cells can be selectively hybridized, I have also used an RNA probe from the Ascaris satellite sequence which is lost from all the somatic lineages and retained in the germ line during chromatic diminution (Muller, et al., (1982) NAR 10-23:7493.). With 60 cell embryos (after diminution), the satellite probe shows high grain accumulation in the two primordial germ cells. The pattern seen in Ascaris looks much like the antibody staining with the anti-P granule antibodies in C. elegans, the two germ cells being covered with silver grains in this case. The sensitivity of the hybridization has yet to be tested, as both the ribosomal and satellite probes are abundant in the RNA and germ line genomic DNA, respectively. Helpful, current resource material on in situ hybridization techniques includes: Jorgensen, E. M. and R. L. Garber. Pro Mega Notes 10:2-5 (1987). Angerer, L. M., et al. Methods in Enzymology 152:649-661 (1987).