Worm Breeder's Gazette 14(5): 71 (February 1, 1997)

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.

Transcriptionally-repressed germline blastomeres lack a specific subpopulation of phosphorylated RNA polymerase II

Geraldine Seydoux, Melanie A. Dunn

Dept. of Molecular Biology and Genetics. Johns Hopkins University School of Medicine 725 N. Wolfe St./515 PCTB Baltimore, MD 21205

        Embryonic germline blastomeres (P1, P2, P3 and P4) fail to
express many mRNAs expressed in somatic blastomeres (1).  In contrast,
rRNAs are expressed in both cell types (2).  In principle, this
difference could be due to the rapid degradation of newly transcribed
mRNAs in germline blastomeres, or to a block in mRNA transcription in
the germ lineage.  To begin to distinguish between these two
possibilities, we have characterized the distribution of phosphorylated
isoforms of the large subunit of RNA polymerase II (RNAP II LS) in
embryos.  For this purpose, we used 2 monoclonal antibodies, H5 and H14,
that recognize distinct phosphorylated epitopes on the carboxy terminal
domain (CTD) of RNAP II LS (3).  Phosphorylation of the CTD has been
correlated with transcriptional elongation; indeed, we find that H5 and
H14 start to stain somatic nuclei in the 4-cell stage, when these cells
are known to begin mRNA transcription.  However, no H5 staining, and
only weak H14 staining, were detected in germline blastomeres. In
contrast, an antibody (4) that recognizes both phosphorylated and
unphosphorylated RNAP II showed no difference in staining between
somatic and germline blastomeres. These results indicate that germline
blastomeres lack the subpopulation of phosphorylated RNAP II recognized
by H5, and have reduced levels of the subpopulation of phosphorylated
RNAP II recognized by H14.  These observations are consistent with a
block in RNAP II activity in the early germ lineage. 

        We have previously shown that the germline-specific factor PIE-1
is required to block mRNA production in the embryonic germ lineage (1). 
To test whether pie-1 is also required for the germline-specific
patterns of H5 and H14 staining described here, we stained embryos
derived from pie-1(zu154) mothers with H5 and H14.  We found that in the
absence of pie-1 activity, germ cells stain with H5 and H14 in a pattern
identical to that of somatic cells.  These results indicate that pie-1
activity is required for the soma-germline differences observed in H5
and H14 staining, and suggest that PIE-1 may function to repress,
directly or indirectly, RNAP II activity in the germ lineage.  

        We have also used the H5 and H14 antibodies to stain Drosophila
embryos. Remarkably, we find that pole cells (the Drosophila embryonic
germ cells) also fail to stain with H5, and stain only weakly with H14. 
These observations raise the possibility that a block in RNAP II
activity may be part of an evolutionarily conserved mechanism that
distinguishes germline from soma during early embryogenesis.

        We thank Miriam Golomb, Steve Warren and Jeff Corden for
providing antibodies, and Debbie Andrew for advice on Drosophila

1. Seydoux et al., 1996. Nature 382, 713-716.
2. Seydoux et al., 1996. East Coast Worm Meeting Abstracts, p 31.
3. Bregman et al., 1995. Journal of Cell Biology 129, 287-298; and M.
Patturajan and J. Corden, pers. comm. 
4. Sanford et al., 1985. Journal of Biological Chemistry 260, 8064-8069