Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain Reaction

Fromant, Michel; Blanquet, Sylvain; Plateau, Pierre

doi:10.1006/abio.1995.1050

Cited by 257 publications

(174 citation statements)

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“…Error-prone PCR with unbalanced dNTP concentration in the presence of MnCl 2 was used to generate random mutations in AP3(IKC) and PI(IKC) (Fromant et al, 1995;Vidal, 1997). PCR products were transformed together with the linearized yeast vectors into pJ69-4A a (for AP3 mutants) or pJ69-4A a (for PI mutants) to generate mutant libraries by homologous recombination (Vidal, 1997).…”

Section: Construction and Screening Of Ap3 And Pi Mutant Librariesmentioning

confidence: 99%

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

2003

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SummaryMADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic a-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.

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Section: Construction and Screening Of Ap3 And Pi Mutant Librariesmentioning

confidence: 99%

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

2003

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“…One primer was homologous to sequence within the ORF of the integrated LEU2 gene while the other to plo1 + sequences in the untransformed 3 region of the gene. The primers plo49 (5 -TCGTTTAAATTGCTCTACTTACC-3 ) and 3 UTRplo1 (5 -GACAGCATAGTAACTTAA-CGCC-3 ) were used in a mutagenic PCR reaction to amplify sequences from pura4plo1 + , according to the procedures of Fromant et al (1995), to generate a series of plo1 + :ura4 + fragments in which the mutation rate was roughly one change every 1-2 kb.…”

Section: Molecular Genetic Manipulationsmentioning

confidence: 99%

A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

MacIver

Glover

Hagan

2003

Yeast

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The completion of the Schizosaccharomyces pombe genome sequencing project has led to a dramatic acceleration of gene characterization in this system. Once a gene has been identified, the challenge then comes in using reverse genetics to generate a range of mutants in this gene of interest so that the powerful genetics and wealth of genetic backgrounds available in Sz. pombe can be exploited to study the function of the newly identified molecule. Beyond simple PCR-tagging approaches, the high frequency with which illegitimate recombination occurs in Sz. pombe has made the manipulation of some loci complex, time consuming and a process of trial and error. Here we describe a simple 'marker switch' approach that enables the rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. We use the generation of temperature-sensitive mutations in the plo1 + gene to validate this approach.

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“…The N-terminal 70 amino acid region of human JunD was ampli®ed by forced PCR (Fromant et al, 1995) using pACT2-JunD (1 ± 70) as template and Taq gold (Perkin Elmer, Foster City, CA, USA). Brie¯y, to decrease the ®delity of the Taq polymerase and induce base mismatches, a ®nal concentration of 9.5 mM MgCl 2 and 0.5 mM MnCl 2 were added to a 50 ml PCR reaction mix.…”

Section: Random Mutagenesis and Yeast Reverse Two-hybrid Library Scrementioning

confidence: 99%

Identification and characterization of JunD missense mutants that lack menin binding

Knapp¹,

Heppner²,

Hickman³

et al. 2000

Oncogene

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Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain Reaction

Cited by 257 publications

References 0 publications

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

Identification and characterization of JunD missense mutants that lack menin binding

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