The Experts below are selected from a list of 117 Experts worldwide ranked by ideXlab platform
Daniel Schlieper - One of the best experts on this subject based on the ideXlab platform.
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a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
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A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
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A Lethal Mutant of the catabolite gene activator protein CAP of Escherichia coli.
Biological Chemistry, 1997Co-Authors: M. Lopata, Daniel Schlieper, B Von Wilcken-bergmann, Benno Müller-hillAbstract:: The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses its recognition helix to bind with each subunit the DNA sequence motif 5' G-7T-6G-5A-4 3'. It makes a direct amino acid-base contact with E181 and cytosine in position-5' on the reverse strand. While testing Mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is Lethal in high amounts for the E. coli strains we used for cloning. We cloned this CAP Mutant successfully in cya- strains, where CAP is inactive. Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity when present in low, non-Lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets. It binds well to CAP consensus with G or T in position-5, better to CAP consensus with A, C in position-5, quite well to lambda consensus operator with G in position-7 and rather weakly to lambda consensus.
Benno Mullerhill - One of the best experts on this subject based on the ideXlab platform.
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a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
Harald Sobek - One of the best experts on this subject based on the ideXlab platform.
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a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
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A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
Manfred Schmidt - One of the best experts on this subject based on the ideXlab platform.
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a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
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A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
Benno Müller-hill - One of the best experts on this subject based on the ideXlab platform.
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A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal Mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
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A Lethal Mutant of the catabolite gene activator protein CAP of Escherichia coli.
Biological Chemistry, 1997Co-Authors: M. Lopata, Daniel Schlieper, B Von Wilcken-bergmann, Benno Müller-hillAbstract:: The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses its recognition helix to bind with each subunit the DNA sequence motif 5' G-7T-6G-5A-4 3'. It makes a direct amino acid-base contact with E181 and cytosine in position-5' on the reverse strand. While testing Mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is Lethal in high amounts for the E. coli strains we used for cloning. We cloned this CAP Mutant successfully in cya- strains, where CAP is inactive. Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity when present in low, non-Lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets. It binds well to CAP consensus with G or T in position-5, better to CAP consensus with A, C in position-5, quite well to lambda consensus operator with G in position-7 and rather weakly to lambda consensus.