scholarly journals Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

1988 ◽  
Vol 7 (8) ◽  
pp. 2579-2584 ◽  
Author(s):  
R. E. Doerig ◽  
B. Suter ◽  
M. Gray ◽  
E. Kubli
Keyword(s):  
Nonsense Mutation ◽  
Trna Gene ◽  
Suppressor Trna ◽  
Germline Transformation ◽  
Amber Suppressor

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

1989 ◽  
Vol 86 (17) ◽  
pp. 6696-6698 ◽  
Author(s):  
F A Laski ◽  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary ◽  
G M Rubin
Keyword(s):  
Drosophila Melanogaster ◽  
Trna Gene ◽  
P Element ◽  
Female Sterility ◽  
Suppressor Trna ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Male And Female Sterility ◽  
And Function

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr 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 amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.

scholarly journals A synthetic tyrosine suppressor tRNA gene with an altered promoter sequence. Its cloning and relative expression in vivo.

1979 ◽  
Vol 254 (21) ◽  
pp. 10803-10810
Author(s):  
M.J. Ryan ◽  
R. Belagaje ◽  
E.L. Brown ◽  
H.J. Fritz ◽  
H.G. Khorana
Keyword(s):  
Trna Gene ◽  
Promoter Sequence ◽  
Relative Expression ◽  
Suppressor Trna

scholarly journals Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

1985 ◽  
Vol 4 (1) ◽  
pp. 213-221 ◽  
Author(s):  
J.P. Capone ◽  
P.A. Sharp ◽  
U.L. RajBhandary
Keyword(s):  
Trna Gene ◽  
Trna Genes ◽  
Suppressor Trna

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.

scholarly journals Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR

2004 ◽  
Vol 186 (20) ◽  
pp. 6714-6720 ◽  
Author(s):  
Christopher D. Herring ◽  
Frederick R. Blattner
Keyword(s):  
Minimal Medium ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Oligonucleotide Microarrays ◽  
Wild Type ◽  
Suppressor Trna ◽  
Cellular Physiology ◽  
Putative Operon ◽  
Amber Suppressor ◽  
Reading Frames

ABSTRACT Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.

Transformation of Drosophila melanogaster with a suppressor tRNA gene from Schizosaccharomyces pombe

Genome ◽  
1988 ◽  
Vol 30 (2) ◽  
pp. 211-217 ◽  
Author(s):  
Charles M. Molnar ◽  
Tove Reece ◽  
James A. Williams ◽  
John B. Bell
Keyword(s):  
Drosophila Melanogaster ◽  
Salivary Gland ◽  
In Situ Hybridization ◽  
Schizosaccharomyces Pombe ◽  
Southern Hybridization ◽  
Trna Gene ◽  
P Element ◽  
Suppressor Trna ◽  
Good Agreement

P-element mediated transformation was utilized to introduce a suppressor tRNA gene [Formula: see text] from Schizosaccharomyces pombe into Drosophila melanogaster. Thirteen independently transformed lines were characterized as to the number and cytological locations of the transposons. It was ascertained that the suppressor tRNA gene of interest was introduced into each transformed strain. The helper P element used (pπ25.1) allows further transposition to occur, and it was determined that from one to seven copies of the heterologous [Formula: see text] gene per strain were present among the respective transformed strains. The number of transposons per transformed line was established by in situ hybridization to salivary gland chromosomes as well as by Southern hybridization analyses and there was good agreement in the totals determined by these two techniques.Key words: Drosophila, Schizosaccharomyces, tRNA suppressor, transformation, transposon.

scholarly journals Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

1996 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
Author(s):  
H J Drabkin ◽  
H J Park ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Trna Gene ◽  
Trna Synthetase ◽  
Developmentally Regulated ◽  
Coding Sequence ◽  
Suppressor Trna ◽  
Trna Synthetases ◽  
E Coli ◽  
Nonsense Codons

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

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