The ras genes are members of a highly conserved family of vertebrate genes first detected as the oncogenes of Harvey and Kirsten sarcoma viruses (1). They encode immunologically cross-reactive, guanine nucleotide binding proteins of -21,000 daltons (2). There are at least three human ras genes, H-, K-, and N-ras, which encode four distinct ras peptides of 188 or 189 amino acids, identical in sequence for the first 86 positions (3-5). Mutated ras genes have been detected in some human tumor cells by the ability of tumor cell DNA to induce tumorigenic transformation in NIH 3T3 cells upon DNA-mediated gene transfer. To date, many activating mutations of ras genes from human tumor cells have been analyzed and they all specify amino acid substitutions at position 12 or 61 of the encoded protein (3-11). To determine whether other mutations of the ras genes could also activate the transforming potential of the wild-type normal genes, we tested the transforming capacity of randomly mutagenized wild-type H-ras genes. MATERIALS AND METHODSCells, Transfections, and Immunoprecipitations. NIH 3T3 cells were cultured and transfected as described (12). Morphologically altered foci were scored 18 days after DNA transfer. Escherichia coli strains were MM294 (r-m+, rec+) for routine plasmid growths and BD1528 (ung-) (the gift of D. Shortle) was used only for transfection of DNA after bisulfite mutagenesis. E. coli transfections were performed as described (13). Cell labeling and immunoprecipitations were performed as described (14).Plasmid DNAs and Sequencing. The plasmid pT24 contained the transforming H-ras gene of T24 bladder carcinoma cells, encoding valine in position 12 (8, 15). The plasmid pP3 contained the wild-type human H-ras gene, encoding glycine in position 12, cloned initially from human placenta (8). The plasmid pTPT differs from pT24 only in containing the 242-base-pair (bp) Mst II/Xba I fragment of the wild-type H-ras gene contained in pP3. Its map is shown in Fig. 1. It therefore encodes glycine in position 12 and a normal H-ras protein. The plasmid EMS9 differs from pT24 in that it lacks any intron after the first coding exon. It was constructed by using the plasmids RC3 and RC6 containing cDNAs of the Hras transcript (15). The protein it encodes also contains valine in position 12. The plasmid EMSH3 differs from EMS9 in that it contains the 34-bp Hae II/Pvu II fragment of the viral Harvey ras gene (16), cloned into the corresponding Hae II/Pvu II site of plasmid EMS9. It therefore encodes arginine in position 12. Clones of bisulfite-mutagenized pTPT were initially grown in ung-BD1528 and then transfected into MM294 for large-scale growth. DNA restriction endonuclease fragments from the mutagenized regions were subcloned into the original pTPT plasmid and grown in MM294. Plasmid DNAs for DNA sequencing or transfection of animal cells was prepared by either of two methods. Large-scale individual plasmid DNAs were prepared from one-liter cultures of MM294 by the method of detergent lysis and CsCl/ethidium...

We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNATyr internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAPI (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap)-) mutant than in the wild type. The tap)-) mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap)-) mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts-growth defect, we cloned the wild-type TAP) gene. It is essential for yeast growth. We show in the accompanying report

Sequence data are presented for the Saccharomyces cerevisiae TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA(Tyr) allele SUP4A53T61. The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.

We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNA(Tyr) internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAP1 (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap1-1 mutant than in the wild type. The tap1-1 mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap1-1 mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts- growth defect, we cloned the wild-type TAP1 gene. It is essential for yeast growth. We show in the accompanying report (T. L. Aldrich, G. Di Segni, B. L. McConaughy, N. J. Keen, S. Whelen, and B. D. Hall, Mol. Cell. Biol. 13:3434-3444, 1993) that TAP1 is identical to RAT1, a yeast gene implicated in poly(A)+ RNA export and that the TAP1/RAT1 gene product has extensive sequence similarity to the protein encoded by another yeast gene (variously named DST2, KEM1, RAR5, SEP1, or XRN1) having exonuclease and DNA strand transfer activity (reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]).

Sequence data are presented for the Saccharomyces cerevisiae TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA(Tyr) allele SUP4A53T61. The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.