In an attempt to identify temperature sensitive alleles that prevent transcription at the restrictive temperature, we have identified two ts alleles of the TFIID initiation factor subunit, taf-6.2. These alleles were isolated by M. Wallenfang in the Seydoux lab in a screen for temperature sensitive embryonic lethals (Golden et al., 2000). The strains JH873 (ax701) and JH686 (ax514) were thought to have ts defective transcription based on the fact that they 1) did not express an early zygotic reporter, and 2) when adults were shifted to 25°C the embryos arrested at a point similar to ama-1(RNAi) (Wallenfang et al., 2002; Powell-Coffman et al., 1996).

We first mapped the ax701 mutation in JH873 to chromosome IV, and positioned it between dpy-13 and unc-17 (8/9 Dpy non-Unc; 9/53 Unc non-Dpy). A candidate gene in this region that matched the phenotypes observed for ax701 was taf-6.2 (Y37E11AL.8). Sequencing of taf-6.2 in JH873 (ax701) identified 2 missense mutations (R144H, G459A) in the TAF6 domain (AA105-475) of the taf-6.2 coding region. During the course of mapping and sequencing ax701 it was found that ax514 did not complement ax701, suggesting that ax514 was also located in taf-6.2. Sequencing of taf-6.2 in JH686 identified a missense mutation (G421E) which was also in the TAF6 domain of taf-6.2. These mutations were not found in either an N2 strain or the parent strain mutagenized in the original screen, JH150. In addition, no mutations were found in non-coding regions of the taf-6.2 ORF of either strain. The strains were outcrossed six times each, and the lesions we identified following the ts embryonic lethal (Emb) phenotype in the outcross.

Finally, the Emb phenotypes can be rescued with a WT copy of taf-6.2. A construct containing the taf-6.2 ORF plus 200 bp upstream and downstream of the ORF (pBAB1) was injected into the outcrossed ax701 strain with PD118.20 (myo-3:GFP). Nine independent GFP+ rescued lines that could grow at the restrictive temperature (25°C) were obtained. The taf-6.2 transgene also rescued JH686 (ax514) to viability at 25°C.

In addition to the phenotypes mentioned above, further lines of evidence suggest that ax701 prevents transcription at the restrictive temperature. Embryos raised at the restrictive temperature failed to gastrulate, as is observed in ama-1(RNAi) (Powell-Coffman et al., 1996). In addition, shifted ax701 unc-17 embryos showed a lack of a transcriptional elongation marker, phosphorylation of Ser2 on the CTD repeat peptide (H5 antibody).

In summary, we have identified two temperature sensitive alleles of the transcription initiation factor, taf-6.2. While we have confirmed that mutations in taf-6.2 are responsible for embryonic arrest phenotypes that suggest that RNA Polymerase II transcription is shut down or severely compromised in these strains, we have not yet verified the transcription defect using high resolution or biochemical assays. Overall, we think these alleles will be important to the worm community as a way to modulate transcription in a time- and temperature-sensitive manner. We plan to make the unc-17 taf-6.2 outcrossed strains available to the community through the CGC [KW1973: taf-6.2(ax701) unc-17(e113) IV and KW1975: taf-6.2(ax514) unc-17(e113) IV].