A rapid and highly efficient method for PCR-based site-directed mutagenesis using only one new primer

Boles, Eckhard; Miosga, Thomas

doi:10.1007/bf00315788

Cited by 27 publications

(22 citation statements)

References 5 publications

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“…The plasmids carrying the E4orf6 and E1B 55-kDa protein genes were described previously (23,45). Most E4orf6 variants were created by site-directed mutagenesis using PCR (7,49). Briefly, arginine codons were changed by performing PCR with a 5Ј oligonucleotide primer containing the altered E4orf6 sequences and a 3Ј primer corresponding to sequences beyond the 3Ј end of the E4orf6 coding region.…”

Section: Methodsmentioning

confidence: 99%

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

Orlando

Ornelles

2002

J Virol

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The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic ␣-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic ␣-helix were analyzed. Two of the six arginine residues within the ␣-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic ␣-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

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Section: Methodsmentioning

confidence: 99%

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

Orlando

Ornelles

2002

J Virol

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“…To measure the influence of two putative Gcn4p binding sites and of a putative Leu3p binding site found in the BAP2 promoter, the binding sites were inactivated by PCR-based site-directed mutagenesis by the method described by Boles and Miosga (2). Plasmid pTD17 is a derivative of pTD14, with only a 683-bp BAP2 promoter fragment fused to lacZ instead of the 1.7-kb promoter fragment in pTD14.…”

Section: Methodsmentioning

confidence: 99%

Amino acids induce expression of BAP2, a branched-chain amino acid permease gene in Saccharomyces cerevisiae

et al. 1996

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Branched-chain amino acid uptake in Saccharomyces cerevisiae is mediated by at least three transport systems: the general amino acid permease Gap1p, the branched-chain amino acid permease Bap2p, and one or more so far unknown permeases. Regulation of the transcription of BAP2 is mainly subject to the presence of certain amino acids in the medium. The level of transcription is low during growth on a minimal medium with proline as the sole nitrogen source. As assayed with a lacZ fusion, the level of transcription is slightly higher (3-fold) on a minimal medium with ammonium ions as a nitrogen source, and transcription is induced about 20-fold by addition of leucine (0.2 mM). As little as 10 M leucine causes a fivefold induction. Addition of L-leucine to minimal proline medium, on the other hand, has no effect on BAP2 transcription. The two known permeases for transport of branched-chain amino acids, Gap1p and Bap2p, are thus not active at the same time. The BAP2 promoter contains one or two putative Gcn4p binding sites and one putative Leu3p binding site. None of the three is needed for induction by leucine. Induction of BAP2 transcription by leucine is accompanied by an increase in branched-chain amino acid uptake. This elevation is interpreted to be partly the result of an increased level of the Bap2p permease in the plasma membrane, because deletion of BAP2 slightly decreases the induction of uptake. There is still a leucine-inducible increase in branched-chain amino acid uptake in a ⌬gap1 ⌬bap2 strain, indicating that BAP2 shares leucine induction with at least one remaining branched-chain amino acid-transporting permease.

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“…The fragments were purified, cut, and ligated into pUC19 (Vieira and Messing, 1991). Activated alleles of all Rho-GTPases were constructed using the method by Boles and Miosga (1995) with primers named AgRHOx and AgCDC42 plus the corresponding nucleotide exchange. DNA of wild-type and mutant alleles was cloned into pGBT9 (Bartel et al, 1993) using again EcoRI and BamHI.…”

Section: Plasmids and Constructsmentioning

confidence: 99%

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

Schmitz

Kaufmann

Köhli

et al. 2006

MBoC

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Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

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A rapid and highly efficient method for PCR-based site-directed mutagenesis using only one new primer

Cited by 27 publications

References 5 publications

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

Amino acids induce expression of BAP2, a branched-chain amino acid permease gene in Saccharomyces cerevisiae

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

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