Efficient site-directed mutagenesis by simultaneous use of two primers

Norris, Kjeld; Norris, Fanny; Christiansen, Lars; Fiil, Niels P.

doi:10.1093/nar/11.15.5103

Cited by 158 publications

(58 citation statements)

References 23 publications

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“…The test vector (Fig. 2) The clustered base changes in the T3 region were created by oligonucleotide-directed mutagenesis essentially as described by Norris et al (17). Mutant oligonucleotides extending from the BamHI site beyond the T3 box were used to prime the synthesis of a double-stranded DNA containing mismatches in the T3 region.…”

Section: Methodsmentioning

confidence: 99%

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

Labhart¹,

Reeder²

1987

Mol. Cell. Biol.

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rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.In Xenopus laevis oocytes, the entire ribosomal gene repeat unit is transcribed, except for about 200 base pairs (bp) upstream of the transcription start site (2, 10). As is diagrammed in Fig. 1, synthesis of a 7.8-kilobase-long precursor starts at the gene promoter, and its 3' end is formed by a type of RNA processing at a site we designated T2. Transcription continues beyond T2, but in oocytes at least, the RNA is very unstable. Synthesis of this unstable RNA continues across the intergenic spacer until site T3 is reached 215 bp upstream of the transcription initiation site of the next gene. Nuclear runoff assays clearly suggest that T3 is a termination site, since no transcription was detected between T3 and the adjacent transcription initiation site. Throughout this paper we refer to events at T3 as termination, although the precise mechanism of such events is not understood.Here we identify a 12-bp sequence element which is an essential part of the terminator. This 12-bp element contains a shorter 7-bp sequence (the T3 box) which has been conserved between different frog species (16) and which is also present at site T2 (10). This T3 box was required to specify the formation of correct 3' ends. We found that, when inserted between a promoter and site T2, the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself, both in steady-state and pulse-label experiments. In addition, our results indicate that, if taken out of its normal location, the region containing T3 did not appear to constitute a fully efficient terminator since it allowed a significant amount of readthrough transcripts to accumulate. MATERIALS AND METHODSPlasmid construction and mutagenesis. The test vector (Fig. 2) The clustered base changes in the T3 region were created by oligonucleotide-directed mutagenesis essentially as described by Norris et al. (17). Mutant oligonucleotides extending from the BamHI site beyond the T3 box were used to prime the synthesis of a double-stranded DNA cont...

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Section: Methodsmentioning

confidence: 99%

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

Labhart¹,

Reeder²

1987

Mol. Cell. Biol.

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“…This was done according to [29] using two synthetic oligonucleotide primers and single-stranded DNA from the recombinant phage mHElOlO as template. The first primer was HindIIIp, which introduces a six-base-pair insertion coding for a HindIII restriction site.…”

Section: Oligonucleotide-directed Mutagenesismentioning

confidence: 99%

Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

Hellinga

Evans

1985

European Journal of Biochemistry

135

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The gene for the major phosphofructokinase enzyme in Escherichia coli, pfk A, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA – tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate‐binding protein, sbp, and for a membrane‐bound enzyme, CDP‐diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA‐sbp‐cdh‐tpi. The pfkA gene has been subcloned into a high‐copy‐number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 × 103‐nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40–50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using ‘reverse genetics’.

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“…Hence the 5' end of the oligonucleotide is protected after extension and ligation. In the 'double priming' technique, a partial extension is made from a mutagenic oligonucleotide and a second oligonucleotide 5' to the mutagenic oligonucleotide to protect the mutation (Norris et al, 1983;Zoller & Smith, 1984).…”

Section: Mutagenesis Using Mismatched Oligonucleotidesmentioning

confidence: 99%