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Nucleotide Sequence of the SUF2 Frameshift Suppressor Gene Of

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Proc. Nati Acad. Sci. USA Vol. 79, pp. 3565-3569, June 1982 Genetics Nucleotide sequence of the SUF2 frameshift suppressor gene of Saccharomyces cerevisiae (proline tRNA/branch-shift mutagenesis/nontriplet mRNA decoding/6 sequence) CLAUDIA M. CUMMINS*, THOMAS F. DONAHUEt, AND MICHAEL R. CULBERTSON* *Laboratones of Genetics and Molecular Biology, University ofWisconsin, Madison, Wisconsin 53706; and tSection of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 Communicated by Oliver E. Nelson, February 8, 1982 ABSTRACT To elucidate the molecular mechanism offrame- altered mRNAs produced in strains carrying these suppressible shift suppression by the SUF2 gene ofyeast, the sequences ofDNA mutations contain a 5'-CCCU-3' four-base sequence in place fragments carrying the SUF2-1 and suf2 + alleles of the gene and ofawild-type 5'-CCU-3' proline codon (9). Despite the fact that surrounding regions have been determined. Comparison of the these his4 mutants contain the same codon change at different suppressor and wild-type sequences indicates that the SUF2 gene positions in the his4 message, they are differentially suppressed product is a proline tRNA. Disregarding possible base modifica- by the six suppressors mentioned above. Two of the suppres- tions, we find that the wild-type suf2 + anticodon of the tRNA in- sors, SUF2 and SUF10, suppress both mutations whereas the ferred from the DNA sequence is 3'-GGA-5'. The SUF2-1 mu- other four suppressors, SUF7, SUF8, SUF9, and sufll, fail to tation represents the insertion of a G-C base pair at a position in suppress his4-712 (7). The molecular basis for this division of the gene that corresponds to the anticodon loop of the tRNA. Re- placement ofthe wild-type suf2 + anticodon bya 3'-GGGA-5' four- the suppressors is unknown. base anticodon enables the SUF2-1 tRNA to suppress the 5'- A general molecular cloning protocol based on complemen- CCCU-3' four-base codons generated as the result ofthe his4-712 tation in yeastfollowing transformation (10) has been specifically and hi&4-713 frameshift mutations. This nontriplet codon-anticodon adapted to clone DNA fragments carrying the yeast frameshift interaction restores the correct readingframe andallows synthesis suppressor genes and has been used to isolate a 10.7-kilobase of a functional his4 protein. fragment carrying the SUF2 suppressor gene (8). Three kinds of evidence support the conclusion that the cloned DNA frag- Frameshift ihutations result from addition or deletion of base ment carries the SUF2 gene. Plasmids carrying the fragment pairs in a gene encoding a protein product. Mutations of this confer suppression of SUF2-suppressible frameshift mutations type usually render the gene product nonfunctional due to in- when introduced into yeast by transformation. Appropriate ability of the translational apparatus to recognize the shift in plasmids carrying the fragment integrate by homologous re- readingframe. This results in an incorrect specification ofamino combination at the SUF2 locus. In addition, the fragment shares acids and in termination of translation at the first out-of-phase a homologous segment of DNA with yeast DNA contained in nonsense codon encountered. the plasmid pYe98F4T. This plasmid is known to carry the The correct reading frame can be restored in strains carrying CDC10 gene, which maps 1 centimorgan from the SUF2 locus frameshift mutations by various compensatory mechanisms. In near the chromosome III centromere (8, 11). some instances, suppressor mutations in genes external to that The genetic properties ofSUF2 and the implication ofaltered which carries the frameshift mutation have been shown to affect tRNAs in frameshift suppression led us to screen for the pres- the structures of tRNAs, tRNA base-modification enzymes, or ence of tRNA genes on the clone. Hybridization of restriction ribosomal proteins (1-3). Altered tRNAs containing an extra fragments from the SUF2 clone to 4S RNA revealed the pres- base in the anticodon have been implicated in suppression of ence ofat least two tRNA genes located on noncontiguous frag- + 1 frameshift mutations (1). Although such tRNAs may be ca- ments within the 10.7-kilobase segment. These two fragments pable ofreading four bases rather than the normal three bases, were subcloned. One ofthe two resulting plasmids was capable the exact mechanism governing four-base translocation on the of transforming an appropriate yeast recipient to a suppressor ribosome is uncertain (4). In the case of altered tRNA modifi- phenotype (8). It remained to be determined whether the tRNA cation enzymes and ribosomal proteins, the molecular mecha- gene carried on this restriction fragment was synonymous with nisms of frameshift suppression are unknown. the SUF2 gene. A complete molecular analysis of nontriplet decoding inter- In this communication we report the results of DNA se- actions, typified by suppression offrameshift mutations, can be quence analyses of restriction fragments carrying the SUF2-1 expected to provide a more detailed view of the normal in vivo suppressor mutation and the wild-type suf2 + allele ofthe gene. decoding mechanism and of the mechanisms for translational The DNA sequences show that the SUF2-1 suppressor phe- control of protein synthesis. To examine this problem in the notype results from insertion of a G-C base pair within a gene lower eukaryote Saccharomyces cerevisiae, mutationally in- encoding a proline tRNA. The implications for tRNA-mRNA duced nontriplet reading systems have been developed in our interactions resulting in suppression of frameshift mutations laboratory by the isolation ofexternal suppressors offrameshift are discussed. mutations at the his4 locus. Suppressor mutations mapping at 25 different loci have been identified (5-8). MATERIALS AND METHODS Six of these suppressors have been shown to suppress the Enzymes and Chemicals. [a-32P]dNTPs (840 Ci/mmol; 1 Ci +1 G-C insertion mutations his4-712 and his4-713 (7, 9). The = 3.7 x 10'° becquerels) were purchased from New England Nuclear. Restriction enzymes and T4 DNA ligase were products The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations:bp, basepair(s);ICR-170, 2-methoxy-6-chloro-9[3-(ethyl- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2-chloroethylamino)propylamino]acridine. 3565 Downloaded by guest on September 29, 2021 3566 Genetics: Cummins et aL Proc. Natl. Acad. Sci. USA 79 (1982) of Bethesda Research Laboratories. DNA polymerase I was The presence of these restriction sites was confirmed by sub- purchased from Sigma, and DNase I was from Worthington sequent DNA sequence analysis. In addition, a third Xba I site Biochemicals. DEAE-cellulose (DE-52) was purchased from was revealed in close proximity to the terminal Pst I site. The Whatman. Avian myeloblastosis virus RNA-dependent DNA strategy for analysis of the DNA sequence from the most left- nucleotidyltransferase (reverse transcriptase) was obtainedfrom ward Xba I site to the rightward Xho I site is shown in Fig. 1. the Division ofCancer Cause and Prevention, National Cancer The sequence was determined for both strands of the 867-bp Institute. Xba I/Xho I fragment. Plasmids. Plasmids pCC5 and pCC6 are subclones of pre- The SUF2 Gene Encodes a Proline tRNA. The DNA se- viously described plasmids, pCC1 and pCC4 (8). To construct quence of the suf2' allele on pCC6 is shown in Fig. 2. When pCC5, the Pst I/HindIII fragment ofYIp33 (12) was removed this sequence was compared with the corresponding sequence and replaced with a Pst I/HindIII fragment from pCC1 that ofthe Xba I/Xho I fragment derived from pCC5, which carries carries the SUF2-1 allele. Similarly, pCC6 was generated by the SUF2-1 allele, the two sequences were found to differ at a replacing the Pst I/HindIII fragment of YIp33 with a Pst I/ single position. An autoradiogram of a portion of a gel encom- HindIII fragment from pCC4 that carries the suf2' allele. passing the site ofthe mutational difference is shown in Fig. 3. DNA Preparation. Plasmid DNA was prepared according to The evidence provided by this gel supports the conclusion that the method of Hicks and Fink (13). [a-3zP]dATP was incorpo- the SUF2-1 suppressor phenotype results from insertion of a rated into plasmid DNA by nick-translation with DNA poly- single G-C base pair in the DNA. This resultwas not unexpected merase I (14). DNA was transferred from agarose gels to nitro- considering that the SUF2-1 allele was induced by using the cellulose filters by using the method of Southern (15). mutagen 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethylamino)- DNA Sequence Analysis. DNA fragments to be analyzed propylamino]acridine dihydrochloride (ICR-170), which is were labeled at their 3' ends by using reverse transcriptase and known to cause G-C base-pair insertions in the DNA of both [a-32P]dNTPs as described by Smith and Calvo (16). The se- prokaryotes (20) and eukaryotes (9). quence of the DNA was determined by the method of Maxam By examining the DNA sequence in the regions surrounding and Gilbert (17). In some cases, 15 ,ul ofglacial acetic acid was the site ofthe + 1 G-C base-pair insertion, we found that a RNA substituted for 5 M NaCl in the cytosine-specific reactions (18). sequence complementary to one ofthe two DNA strands could be drawn that has features characteristic of tRNAs. In partic- ular, the RNA sequence deduced from the DNA sequence can RESULTS be folded into the clover-leaf secondary structure typical ofall Subeloning, Restriction Mapping, and Strategy for Analysis tRNAs whose sequences have thus far been determined (Fig. of the DNA Sequence. A restriction map of the 10.7-kilobase 4). In addition, various constant regions found among different BamHI fragment originally found to carry the SUF2-1 allele of tRNAs are present at their appropriate positions in this RNA the SUF2 gene is shown in Fig.
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