A rapid and efficient one-tube PCR-based mutagenesis technique using<i>Pfu</i>DNA polymerase

Picard, Véronique; Ersdal-Badju, Eva; Lu, Aiqin; Bock, Susan

doi:10.1093/nar/22.13.2587

Cited by 225 publications

(179 citation statements)

References 8 publications

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“…To place the spoIIQ gene under the control of other promoters an XbaI restriction site was introduced by polymerase chain reaction (PCR) (Picard et al, 1994) 18 bp upstream of the spoIIQ translation initiation codon. Similarly, a NheI site was introduced downstream of the spoIIR promoter region, overlapping the spoIIR putative transcription start (Karow et al, 1995).…”

Section: Manipulating Expression Of Spoiiqmentioning

confidence: 99%

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

Fréhel

Stragier

1997

Molecular Microbiology

131

135

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SummaryA crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.

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Section: Manipulating Expression Of Spoiiqmentioning

confidence: 99%

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

Fréhel

Stragier

1997

Molecular Microbiology

131

135

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“…The N135A substitution eliminates binding heterogeneity associated with partial glycosylation of asparagine 135 (31) and directs production of a homogeneous ␤-ATIII-like parent molecule with high base-line affinity for heparin (37). Basic-to-Ala substitutions were introduced using a one-tube polymerase chain reaction mutagenesis method (38). Following sequence verification and subcloning, variants were expressed as described previously (37).…”

Section: Atiii Expression System and Site-directed Mutagenesis-mentioning

confidence: 99%

Identification of the Antithrombin III Heparin Binding Site

Ersdal-Badju

Zuo

et al. 1997

Journal of Biological Chemistry

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The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

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“…PCR-mediated in itro mutagenesis of the genes hmaL2 and humanL8 was performed in a one-tube megaprimer-PCR reaction [20]. The codon for histidine-199 of hmaL2 was replaced by the codons for arginine or glycine and the equivalent histidine-209 of humanL8 was exchanged with glycine or alanine codons.…”

Section: In Vitro Mutagenesismentioning

confidence: 99%

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Ühlein

Weglöhner

Urlaub

et al. 1998

Biochemical Journal

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The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In i o replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and

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A rapid and efficient one-tube PCR-based mutagenesis technique usingPfuDNA polymerase

Cited by 225 publications

References 8 publications

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

Identification of the Antithrombin III Heparin Binding Site

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

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