Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C.

Kudo, Ichiro; Leineweber, Michael; RajBhandary, Uttam L.

doi:10.1073/pnas.78.8.4753

Cited by 49 publications

(14 citation statements)

References 38 publications

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Section: Methodsmentioning

confidence: 99%

“…A single-base change (see Fig. 1A) was introduced in the Drosophila tRNAT~, gene (13) contained in the 0.9-kilobase EcoRI-Sal I fragment of the plasmid p22F91Rs (gift of Michael Hoffman, Harvard University) by site-directed mutagenesis (14)(15)(16) using the ohigonucleotide 5'-GGTGGACTCTAGATTGG-3'. The EcoRISal I fragment containing the wild-type or mutated tRNA gene was cloned between the EcoRI site (at position 1601 of the P-element sequence) and the Sal I site (at position 2110 of the P-element sequence) of the P element Pc[ry] (17) Proc.…”

Section: Methodsmentioning

confidence: 99%

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Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Laski

Ganguly²,

Sharp³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATY gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of ambersuppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.Nonsense suppressors are useful tools for the genetic analysis of organisms. In Escherichia coli (1), yeast (1), and Caenorhabditis elegans (2) nonsense suppressors were isolated as second-site mutations able to suppress mutations in many other genes. However, no nonsense suppressors have been identified in mammals or Drosophila. Suppressor tRNA genes constructed in vitro and transformed into mammalian tissue culture cells were shown to have nonsense-suppressing activity (3-11). In this paper we describe the activity of a Drosophila amber-suppressor tRNATIr gene after introduction into flies by P-element-mediated transformation. METHODSConstruction of P[iy, DtT(Su+)]. A single-base change (see Fig. 1A) was introduced in the Drosophila tRNAT~, gene (13) contained in the 0.9-kilobase EcoRI-Sal I fragment of the plasmid p22F91Rs (gift of Michael Hoffman, Harvard University) by site-directed mutagenesis (14)(15)(16)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Laski

Ganguly²,

Sharp³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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“…The ability to introduce site-specific mutations into tRNA genes (1)(2)(3)(4) and to study the properties of mutant tRNAs in vitro and in vivo has led to major advances in identification of sequence elements important in recognition of tRNAs by the corresponding aminoacyl-tRNA synthetases. These studies (reviewed in refs.…”

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confidence: 99%

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

Lee

RajBhandary²

1991

Proc. Natl. Acad. Sci. U.S.A.

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We recently described mutants ofEschenchia coU initiator tRNA that suppress amber termination codons (UAG) in E. coUl. These mutants have changes in the anticodon sequence (CAU --CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevsiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification ofthe C1 G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified the generation and characterization of mutants that suppress amber codons in E. coli (16,23). One of these mutants has a ClG72 base pair, at the end of the acceptor stem. This feature is otherwise found only in tyrosine tRNA of eukaryotic cells and archaebacteria and in proline tRNAs of eubacteria and bacterial viruses (24). We therefore investigated the possibility that this mutant might function as an amber suppressor in the yeast Saccharomyces cerevisiae. We show here that it does and that it is aminoacylated with tyrosine by yeast extracts. Results ofaminoacylation assays on several of the mutants suggest an absolute requirement for the C1lG72 base pair for aminoacylation with tyrosine. Comparison of kinetic parameters in aminoacylation suggests that one of the mutant E. coli initiator tRNAs is as good a substrate for yeast TyrRS as a yeast tyrosine-inserting amber suppressor tRNA studied by Bare and Uhlenbeck (21). We discuss the implications of these results for recognition of tRNAs by yeast TyrRS.The ability to introduce site-specific mutations into tRNA genes (1-4) and to study the properties of mutant tRNAs in vitro and in vivo has led to major advances in identification of sequence elements important in recognition of tRNAs by the corresponding aminoacyl-tRNA synthetases. These studies (reviewed in refs. 5-7) show that, in most cases, sequences important for specificity in recognition are located primarily in the anticodon sequence (8-10), the acceptor stem (11, 12), or both (13-16). However, there are several examples in which nucleotides important for recognition are more widely dispersed, including a combination of anticodon sequence, acceptor stem, dihydrouridine stem, dihydrouridine loop, variable loop, etc. (4, 17-19). Details of the nature of interactions between aminoacyl-tRNA synthetases and tRNAs are beginning to emerge from parallel work on x-ray crystallographic structure of the enzyme-tRNA complexes (13,15,20). In the two known structures of such complexes (15,20), the enzyme makes extensive contact with the tRNA substrate and there is substantial change in the local structure of the tRNA in the anticodon loop and...

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“…pRSVcat DNA was digested with HindlIl and BamHI, and the small fragment, which contains the cat gene, was purified and cloned into similarly digested bacteriophage vector M13mp8 replicative-form (RF) DNA. Single-stranded virion DNA, designated M13cat and containing the transcribed strand of the cat gene, was prepared and purified by alkaline sucrose gradient centrifugation (18). This DNA was used as a template for site-directed mutagenesis to introduce amber and ochre nonsense mutations into the cat gene.…”

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Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

Capone¹,

Sedivy²,

Sharp³

et al. 1986

Mol. Cell. Biol.

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We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse N1113T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.Nonsense mutations, which cause premature termination of protein synthesis, can be suppressed by tRNAs which recognize termination codons. The availability of a wide variety of nonsense suppressor tRNA genes has played a crucial role in the isolation and study of nonsense mutations in procaryotes and lower eucaryotes (7). In mammalian cells, it has so far not been possible to isolate cell lines carrying nonsense suppressors by using classic genetic selections; consequently, nonsense mutations have been identified in only a few nonviral genes (8,21).Recently, we demonstrated that both a Xenopus laevis tRNATYr gene and a human tRNAser gene could be altered by oligonucleotide-directed site-specific mutagenesis to recognize and suppress amber (UAG) and ochre (UAA) codons in vivo (5,19,20). An opal (UGA) suppressor tRNA gene derived from a human tRNAser gene was also constructed (5). However, since the opal suppressor tRNA gene could not be propagated in mammalian cells as part of a replicating simian virus 40 (SV40) recombinant, it was not possible to demonstrate its functional expression. In addition, the X.laevis tRNATYr amber and ochre suppressors were also used to establish permanent Su' mammalian cell lines (16,19), although the level of nonsense suppression in these cell lines was low (36). It is likely that many other suppressor tRNA genes which are active in mammalian cells will be constructed and used to isolate mammalian cell lines containing functional suppressors. A knowledge of the levels of suppression in these various cell lines will aid significantly in their eventual use for the isolation and characterization of nonsense mutations in cellular and viral genes. It is therefore important to have a rapid, quantitative, and sensitive method to assay for the * Corresponding author. level of suppression of nonsense mutations in different cell types. This paper repo...

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Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C.

Abstract: We have cloned the Escherichia coli tyrosine-inserting amber suppressor tRNA gene into the recombinant singlestrand phage M13mp3. By using the M13mp3Sum' recombinant phage DNA as template and an oligonucleotide bearing a mismatch as primer, we have synthesized in vitro an M13mp3SuIgl

Cited by 49 publications

References 38 publications

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

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