scholarly journals Nonsense suppression in archaea

2015 ◽  
Vol 112 (19) ◽  
pp. 6015-6020 ◽  
Author(s):  
Arpita Bhattacharya ◽  
Caroline Köhrer ◽  
Debabrata Mandal ◽  
Uttam L. RajBhandary
Keyword(s):  
Nonsense Suppression ◽  
Trna Genes ◽  
Bacterial Strains ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Viral Genetics ◽  
Stop Codons ◽  
Amber Suppressor ◽  
Nonsense Suppressor

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeonHaloferax volcaniiare active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons inH. volcanii. Additionally, transformation of aΔpyrE2 H. volcaniistrain with plasmids carrying the genes for apyrE2amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in thepyrE2gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.

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

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

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 NIH3T3 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.

scholarly journals 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.

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067 ◽  
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

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 NIH3T3 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.

scholarly journals Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

1998 ◽  
Vol 18 (8) ◽  
pp. 4418-4425 ◽  
Author(s):  
Ho-Jin Park ◽  
Uttam L. RajBhandary
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
E Coli ◽  
Tetracycline Transactivator ◽  
Hela Cell Lines ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

Cell ◽  
1982 ◽  
Vol 31 (1) ◽  
pp. 137-146 ◽  
Author(s):  
Robert M. Hudziak ◽  
Frank A. Laski ◽  
Uttam L. Rajbhandary ◽  
Phillip A. Sharp ◽  
Mario R. Capecchi
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Trna Genes ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Mammalian Cell Lines

scholarly journals Mutations in Eukaryotic Release Factors 1 and 3 Act as General Nonsense Suppressors in Drosophila

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 601-612
Author(s):  
Anna T Chao ◽  
Herman A Dierick ◽  
Tracie M Addy ◽  
Amy Bejsovec
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Nonsense Suppression ◽  
Release Factor ◽  
Nonsense Mutations ◽  
Stop Codons ◽  
Larval Stages ◽  
Mutant Phenotypes ◽  
Polypeptide Chains ◽  
Nonsense Suppressors

Abstract In a screen for suppressors of the Drosophila winglessPE4 nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens.

scholarly journals Suppression of Nonsense Mutations in Cell Culture and Mice by Multimerized Suppressor tRNA Genes

2000 ◽  
Vol 20 (9) ◽  
pp. 3116-3124 ◽  
Author(s):  
Massimo Buvoli ◽  
Ada Buvoli ◽  
Leslie A. Leinwand
Keyword(s):  
Skeletal Muscle ◽  
Trna Gene ◽  
Premature Termination Codon ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Intact Mouse ◽  
Suppressor Trna ◽  
Expression Plasmids

ABSTRACT We demonstrate here the first experimental suppression of a premature termination codon in vivo by using an ochre suppressor tRNA acting in an intact mouse. Multicopy tRNA expression plasmids were directly injected into skeletal muscle and into the hearts of transgenic mice carrying a reporter gene with an ochre mutation. A strategy for modulation of suppressor efficiency, applicable to diverse systems and based on tandem multimerization of the tRNA gene, is developed. The product of suppression (chloramphenicol acetyltransferase) accumulates linearly with increases in suppressor tRNA concentration to the point where the ochre-suppressing tRNASer is in four- to fivefold excess over the endogenous tRNASer. The subsequent suppressor activity plateau seems to be attributable to accumulation of unmodified tRNAs. These results define many salient variables for suppression in vivo, for example, for tRNA suppression employed as gene therapy for nonsense defects.

Construction of Escherichia coli amber suppressor tRNA genes

1990 ◽  
Vol 213 (4) ◽  
pp. 705-717 ◽  
Author(s):  
Lynn G. Kleina ◽  
Jean-Michel Masson ◽  
Jennifer Normanly ◽  
John Abelson ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Trna Genes ◽  
Suppressor Trna ◽  
Amber Suppressor

Construction of Escherichia coli amber suppressor tRNA genes

1990 ◽  
Vol 213 (4) ◽  
pp. 719-726 ◽  
Author(s):  
Jennifer Normanly ◽  
Lynn G. Kleina ◽  
Jean-Michel Masson ◽  
John Abelson ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Trna Genes ◽  
Suppressor Trna ◽  
Amber Suppressor

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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