The transcription elongation factor S-II, also designated TFIIS, stimulates the nascent transcript cleavage activity intrinsic to RNA polymerase II. Rpb9, a small subunit of RNA polymerase II, enhances the cleavage stimulation activity of S-II. Here, we investigated the role of nascent transcript cleavage stimulation activity on the maintenance of transcriptional fidelity in yeast. In yeast, S-II is encoded by the DST1 gene. Disruption of the DST1 gene decreased transcriptional fidelity in cells. Mutations in the DST1 gene that reduce the S-II cleavage stimulation activity led to decreased transcriptional fidelity in cells. A disruption mutant of the RPB9 gene also had decreased transcriptional fidelity. Expression of mutant Rpb9 proteins that are unable to enhance the S-II cleavage stimulation activity failed to restore the phenotype. These results suggest that both S-II and Rpb9 maintain transcriptional fidelity by stimulating the cleavage activity intrinsic to RNA polymerase II. Also, a DST1 and RPB9 double mutant had more severe transcriptional fidelity defect compared with the DST1 gene deletion mutant, suggesting that Rpb9 maintains transcriptional fidelity via two mechanisms, enhancement of S-II dependent cleavage stimulation and S-II independent function(s).

The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.

These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation.

SDT1(suppressor of disruption of TFIIS 1, YGL224c, also known as SSM1, suppressor of S-II null mutant 1) is Saccharomyces cerevisiae gene identified as a multicopy suppressor of 6-azauracil sensitivity in a null mutant of the transcription elongation factor S-II. We found that overproduction of SDT1 caused hyposensitivity to not only 6-azauracil but also 5-fluorouracil and 5-fluorocytosine. This hyposensitivity was limited to pyrimidine derivatives, and no effect was observed for non-pyrimidine drugs including such clinically used anti-fungal drugs as amphotericin B and fluconazole. Purified recombinant SDT1 protein specifically dephosphorylated 5-UMP and 5-CMP. These results suggested that SDT1 conferred pyrimidine-specific hyposensitivity by dephosphorylating active metabolites of 6-or 5-modified pyrimidines, i.e. 6-or 5-modified UMP. This is the first description of a highly specific pyrimidine 5-nucleotidase in S. cerevisiae.A Saccharomyces cerevisiae null mutant of the transcription elongation factor S-II is hypersensitive to 6-AU 1 (1) and MPA, and now a number of transcription elongation-related factors and RNA polymerase II mutants are also known to be 6-AUhypersensitive (2-6). In yeast cells, 6-AU is transformed to 6-azaUMP, which inhibits both IMP dehydrogenase and orotidylic acid decarboxylase, key enzymes of the purine and pyrimidine nucleotide synthesis pathways, respectively, thereby lowering intracellular GTP and UTP levels and inhibiting transcription elongation (7). MPA specifically inhibits IMP dehydrogenase and lowers the intracellular GTP level (7). The transcriptional stimulation and arrest relief activity of S-II are necessary to support cell proliferation in the presence of 6-AU (8). We have identified SDT1 (9) (also known as SSM1 (10)) as a multicopy suppressor of the 6-AU hypersensitivity of an S-II null mutant (10). The SDT1 null mutant is hypersensitive to 6-AU but not to MPA, and SDT1 overexpression confers hyposensitivity to 6-AU but not to MPA (this study and Ref. 10). Thus, although SDT1 was identified by the screening for a functional substitute for S-II, it can support growth in the absence of S-II only partially if at all. The deduced amino acid sequence of SDT1 has 30 -50% identity with S. cerevisiae YER037w, Schizosaccharomyces pombe SPAC24B11, and a putative sugar starvation-induced protein of Arabidopsis thaliana (GenBank TM accession number AC006223). These genes share a haloacid dehalogenase-like hydrolase consensus sequence, but their biological functions are unknown. To elucidate the mechanism of 6-AU hypersensitivity suppression by SDT1 overexpression, we first tested the drug sensitivity of a SDT1 overproducer. The result suggested that drug resistance by SDT1 overexpression is specific to pyrimidine derivatives. Because SDT1 has a hydrolase consensus sequence, we assumed that SDT1 is a metabolic enzyme of pyrimidines and found that the UMPase activity in a yeast cell extract was proportional to the SDT1 gene dosage. We then expressed recombinant SDT1 protein in Escher...

In order to examine a role of carcinoembryonic antigen (CEA) in metastasis, cDNA encoding CEA was introduced into a clone of human colorectal carcinoma SW1222 cells. Western blot analysis revealed that all transfectants express CEA of 180 kDa while the parent clone does not. In the transfectants, the level of CEA expression in clone 3 was higher than that of clone 1. Clone 3 formed aggregates rapidly after suspended by trypsinization while clone 1 did not. In experimental metastasis assay where tumor cells were injected intrasplenically, clone 3 exhibited a higher liver-metastatic activity than clone 1. Fab fragment of anti-CEA antibody significantly inhibited both the cell aggregation and the liver metastases caused by clone 3. These findings suggested that CEA expressed on the cell surface may play an important role in hepatic metastasis from colorectal carcinoma, possibly through its cell adhesion activity.