A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

Vandeyar, Mark A.; Weiner, Michael P.; Hutton, Carolyn J.; Batt, Carl A.

doi:10.1016/0378-1119(88)90425-8

Cited by 293 publications

(157 citation statements)

References 5 publications

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“…[34] Mutations were introduced by PCR using the QuikChange TM site-directed mutagenesis kit from Stratagene according to the manufacturer's protocol. [43] High level protein expression was achieved using a slightly modified version of the protocol already published. [34] By incubating the starter culture for only 4 h, using terrific broth (TB)-medium for expression and prolonging cultivation time after induction to 16 h at 25°C, 800 to 1200 nmol (96 to 144 mg) of soluble CYP102A1 per litre of cell culture were obtained.…”

Section: Design Of Nadh-dependent Reductase Mutantsmentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP + -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD + -dependent FDH was used.Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.

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Section: Design Of Nadh-dependent Reductase Mutantsmentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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“…Construction of point mutations in the core of attI by site-directed mutagenesis Directed point mutations in attI were introduced by the hemimethylation protection method described by Vandeyar et al (1988) in the integron fragment of plasmid 2273 cloned in M13mp18 or M13mp19, using synthetic degenerate mutagenesis primers according to Table 5. Three mutants out of each experiment were selected after sequencing 24 clones.…”

Section: Construction Of Deletion Mutationsmentioning

confidence: 99%

Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets

Hansson

Sköld

Sundström

1997

Molecular Microbiology

108

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SummaryGenes borne on cassettes are mobile owing to sitespecific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R-plasmids. In these processes, the palindromic site, attC (59-base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross-overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross-over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5Ј of the cross-over point. A region 27-36 bp 5Ј of attI influenced recombination with attC sites only, whereas a sequence 9-14 bp 5Ј of the cross-over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site-specific recombinase to new sequences.

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“…JM101 [A(lac-proAB)supE thi F'(traD36 proA +B+ lacIVZAM15)] (10) was used for cloning and propagating M13 derivatives. SDM [hsdRl7 mcrAB recAl supE44 Tetr A(lac-proAB) F'(traD36 proA+B+ lacIVZAM15)] was used to grow M13 derivatives which had undergone mutagenesis as described by Vandeyar et al (22). JP3923 (thr-1 leuB6 thi-1 lacZAM15 lacYl gal-351 supE44 tonA21 hsdR4 gyrA379 rpsL743 recA56 srl-1300::TnJO aroL513) was used for all f-galactosidase assays.…”

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confidence: 99%

Mutations affecting pseudoknot control of the replication of B group plasmids

1993

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The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure.Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major elfects on the translation of repA.Plasmid replication in prokaryotes is, in many cases, dependent on a replication initiation protein (Rep), whose expression determines a plasmid's copy number and stability. In the case of pT181 and IncFII plasmids, the regulators of Rep synthesis are small countertranscript RNAs which inhibit Rep expression by binding with their target RNAs. In pT181, binding of the countertranscript RNA to the mRNA for the Rep protein is proposed to cause premature transcriptional termination by altering the folding of the RNA (9, 13). The mechanism by which the countertranscript RNAs of the IncFII plasmids Rl and NR1 regulate Rep expression is not clear. However, it is thought that they indirectly regulate Rep expression by sterically inhibiting the translation of a leader peptide. Translation of the Rep protein is believed to be dependent on the translation of the leader peptide, and the two genes are said to be translationally coupled (4, 23).The replication frequency of the B group miniplasmid pMU720 is thought to be dependent on the expression of the repA gene, which is negatively regulated primarily at the posttranscriptional level by a small countertranscript RNA, RNAI (14,15). RNAI is transcribed from the opposite strand of, and is complementary to, the leader region of the mRNA coding for repA (RNAII) (see Fig. 1). Computer analysis of the folding of RNAII indicates that the translational initiation region (TIR) of repA is sequestered within a secondary structure designated stem-loop III (see Fig. 1 and Fig. 2). It is postulated that stem-loop III inhibits ribosome access to the repA TIR. Previous studies have revealed that for repA to be expressed, stem-loop III must be disrupted by the translation and termination of a small leader peptide RepB, and a pseudoknot has to form (15). Pseudoknot formation is essential for the translation of repA and involves pairing between complementary sequences in RNAII. One of these sequences lies in the loop of a large structure called stem-loop I (proximal pseudoknot sequence), which is complementary to RNAI (see Fig. 2), and the other involves bases adjacent to the ShineDalgarno (SD) sequence of repA (distal pseudoknot sequence). RNAI is thought to...

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A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

Cited by 293 publications

References 5 publications

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets

Mutations affecting pseudoknot control of the replication of B group plasmids

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