Nucleotide sequence corrections of the uidA open reading frame encoding β-glucuronidase

We report on the development of five missense mutants and one recombination substrate of the b-glucuronidase (GUS)-encoding gene of Escherichia coli and their use for detecting mutation and recombination events in transgenic Arabidopsis (Arabidopsis thaliana) plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:Gto-T:A transitions in a symmetrical sequence context and are in that respect complementary to previously published GUS point mutants. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. We show that spontaneous mutation and recombination events took place. Mutagenic treatment of the plants with ethyl methanesulfonate and ultraviolet-C increased the number of mutations, validating the use of these constructs to measure mutation and recombination frequencies in plants exposed to biotic or abiotic stress conditions, or in response to different genetic backgrounds. Plants were also subjected to heavy metals, methyl jasmonate, salicylic acid, and heat stress, for which no effect could be seen. Together with an ethyl methanesulfonate mutation induction level much higher than previously described, the need is illustrated for many available scoring systems in parallel. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli.

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“…M15182). The amino acids are identical to those shown for wild-type E. coli GUS (Schlaman et al, 1994; GenBank accession no. S69414).…”

Section: Discussionmentioning

confidence: 97%

“…c Positions and mutations relative to the wild-type E. coli sequence from Schlaman et al (1994;GenBank accession no. S69414).…”

Section: Discussionmentioning

confidence: 99%

Development and Application of Novel Constructs to Score C:G-to-T:A Transitions and Homologous Recombination in Arabidopsis

et al. 2007

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“…The oligonucleotides 5Ј-AGTCCCCCATGGTACGTC-CTGTCGACACCCC-3Ј and 5Ј-GGAAGATCTCCCCCATT-GCGAAGGC-3Ј anneal to positions þ75 to þ105 and þ1978 to þ1954 of E. coli gusA (Genbank accession no. S69414; Schlaman et al, 1994), respectively, and were used for cloning the gusA CDS from the E. coli K12 chromosome to the NcoI and Bgl II restriction sites within pIJ2920 (Janssen and Bibb, 1993) derivatives of pUL-AUG and pSD-AUG, replacing the lacZ CDS to make pUL-AUGgus and pSD-AUGgus. The start codons of pUL-AUGgus and pSDAUGgus were mutagenized by PCR using the oligonucleotide 5Ј-CAGAATTCTGGCACGACAGGTTTCCCGACTGG-3Ј which anneals to ¹130 to ¹98 of the lac transcriptional start site and the mutagenic oligonucleotide 5Ј-GGGGTGTCGA-CAGGACGTACCANGGACAC-3Ј or 5Ј-GGGGTGTCGACA-GGACGTACCANGGCTGTTTCC-3Ј (where N is an equal mix of G, T, and C) which anneals to þ61 to þ33 and þ23 to ¹10 of pSD-AUGgus and pUL-AUGgus gusA, respectively.…”

Section: Methodsmentioning

confidence: 99%

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Etten

Janssen

1998

Molecular Microbiology

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SummaryWe determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine-Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5Ј-AGGA-3Ј to 5Ј-UUUU-3Ј results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon-anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon-anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.

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“…The resulting PCR products were integrated into the plant pBI101u expression vector modified to harbor a USER cloning cassette upstream of GUS (uidA) (Schlaman et al, 1994). Constructs were confirmed by sequencing.…”

Section: Determination Of Tissue-specific Gene Expressionmentioning

confidence: 99%

Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool inArabidopsisFlowers

et al. 2013

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The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (2)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

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Nucleotide sequence corrections of the uidA open reading frame encoding β-glucuronidase

Cited by 26 publications

References 6 publications

Development and Application of Novel Constructs to Score C:G-to-T:A Transitions and Homologous Recombination in Arabidopsis

Development and Application of Novel Constructs to Score C:G-to-T:A Transitions and Homologous Recombination in Arabidopsis

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool inArabidopsisFlowers

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