Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

Busby, Stephen; Irani, M; Crombrugghe, Benoít de

doi:10.1016/0022-2836(82)90060-2

Cited by 77 publications

(43 citation statements)

References 13 publications

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“…This is in contrast to hydroxylamine treatment of the ga promoter region, where only -20% of the mutations were subsequently found within the promoter/RBS (48).…”

Section: Dna Segregation Analysismentioning

confidence: 64%

“…The major differences here are that a mutagen, nitrous acid, is reacted with the promoter/RBS, and the indicator gene is cloned in phage M13 (29,33,48). This approach to local mutagenesis is also similar to that of Hirose et.…”

Section: Dna Segregation Analysismentioning

confidence: 99%

“…With the techniques of local DNA mutagenesis described here and elsewhere (48,50,51) multiple mutations can now be induced in a single binding site or any regulatory region of DNA. Hypotheses, such as those involving secondary structure models, can then be tested by introducing specific single nucleotide changes with the powerful technique of oligonucleotide-directed mutagenesis (27,51).…”

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confidence: 99%

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Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E.coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased a-galactosidase synthesis, were selected for DNA sequencing. One clone has a G--A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C--T and T--C) between the Shine-Dalgarno sequence and initiation codon which raise expression of a-galactosidase two-fold.A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present.These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

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“…This is in contrast to hydroxylamine treatment of the ga promoter region, where only -20% of the mutations were subsequently found within the promoter/RBS (48).…”

Section: Dna Segregation Analysismentioning

confidence: 64%

Section: Dna Segregation Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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“…pYS-GAL104 (10 g of DNA/500-l reaction) was mutagenized by incubation with 1 M hydroxylamine (pH 5.5-6.5) at 75°C for 45-90 min (Busby et al, 1982). After phenol-chloroform purification, 1/100 of each preparation was transformed into KC8 bacteria (Table 1), and colonies were replica plated to ϩ/Ϫ uracil plates to assess the mutagenesis frequency in the URA3 marker.…”

Section: Mutagenesis Screenmentioning

confidence: 99%

Dominant Gain-of-Function Mutations in Hsp104p Reveal Crucial Roles for the Middle Region

Schirmer

Homann

Kowal

et al. 2004

MBoC

108

111

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Heat-shock protein 104 (Hsp104p) is a protein-remodeling factor that promotes survival after extreme stress by disassembling aggregated proteins and can either promote or prevent the propagation of prions (protein-based genetic elements). Hsp104p can be greatly overexpressed without slowing growth, suggesting tight control of its powerful protein-remodeling activities. We isolated point mutations in Hsp104p that interfere with this control and block cell growth. Each mutant contained alterations in the middle region (MR). Each of the three MR point mutations analyzed in detail had distinct phenotypes. In combination with nucleotide binding site mutations, Hsp104p T499I altered bud morphology and caused septin mislocalization, colocalizing with the misplaced septins. Point mutations in the septin Cdc12p suppressed this phenotype, suggesting that it is due to direct Hsp104p-septin interactions. Hsp104p A503V did not perturb morphology but stopped cell growth. Remarkably, when expressed transiently, the mutant protein promoted survival after extreme stress as effectively as did wild-type Hsp104p. Hsp104p A509D had no deleterious effects on growth or morphology but had a greatly reduced ability to promote thermotolerance. That mutations in an 11-amino acid stretch of the MR have such profound and diverse effects suggests the MR plays a central role in regulating Hsp104p function. INTRODUCTIONThe AAA ϩ proteins are ATPases associated with various cellular activities. They are important proteins with a great diversity of functions, including protein folding, membrane trafficking, organelle biogenesis, proteolysis, intracellular motility, and DNA replication (Neuwald et al., 1999;Vale, 2000;Ogura and Wilkinson, 2001). Little is known about how most AAA ϩ proteins recognize substrates and use ATP binding and hydrolysis to remodel them. The intrinsic complexity and multiplicity of conformational states of the AAA ϩ proteins make them difficult to study. The Clp/ HSP100 proteins are members of the AAA ϩ superfamily and have been the subject of intense biochemical analysis in vitro and genetic analysis in vivo (Wickner et al., 1999;Glover and Tkach, 2001). The functional unit of the yeast HSP100 heatshock protein 104 (Hsp104p) is composed of six monomers, each with two ATP binding sites (nucleotide binding domains one and two; NBD1 and NBD2) flanked by aminoterminal, middle, and carboxy-terminal regions (Figure 1).Hsp104p has remarkable functions, one of which is to allow survival after extreme stress. For example, yeast cells expressing Hsp104p are 1000 times more viable after exposure to temperatures Ն50°C or to an ethanol concentration of 20% than cells carrying deletions of HSP104 (Sanchez and Lindquist, 1990;Sanchez et al., 1992). This survival capacity is directly attributable to Hsp104p's ability to resolubilize protein aggregates and, together with Hsp70p and Hsp40p, return them to their folded and active states (Parsell et al., 1994;Glover and Lindquist, 1998;Goloubinoff et al., 1999). This is in contrast to other...

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“…The resulting plasmid, named pDH6, was subjected to hydroxylamine mutagenesis as described (Busby et al, 1982). Progress of the mutagenesis with time was monitored by the drop in transformation frequency of DH5␣, a 90% reduction in frequency being considered optimum.…”

Section: Materials-chemicalsmentioning

confidence: 99%

Casein Kinase II Is Required for Cell Cycle Progression during G1 and G2/M in Saccharomyces cerevisiae

Hanna

Rethinaswamy

Glover

1995

Journal of Biological Chemistry

208

198

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The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2 ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% largebudded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G 1 . Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G 2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G 1 , at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G 2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.Casein kinase II (CKII) 1 is a serine/threonine protein kinase which is ubiquitous among eukaryotic organisms (for review, see Issinger, 1993;Pinna, 1990;Tuazon and Traugh, 1991). The enzyme is composed of a catalytic ␣ and regulatory ␤ subunit that combine to form a native ␣ 2 ␤ 2 holoenzyme which is constitutively active in vitro. How (and indeed whether) the enzyme is regulated in vivo is unknown, though regulation via allosteric effectors (e.g. polyamines), covalent modification, cellular redistribution, and substrate-directed effects have all been proposed. CKII recognizes a Ser of Thr residue followed by a series of acidic residues and phosphorylates a broad and intriguing spectrum of both nuclear and cytoplasmic substrates.Although the physiological role of CKII is not known, several lines of evidence suggest a role for the enzyme in cell prolifer-

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Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

Cited by 77 publications

References 13 publications

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Dominant Gain-of-Function Mutations in Hsp104p Reveal Crucial Roles for the Middle Region

Casein Kinase II Is Required for Cell Cycle Progression during G1 and G2/M in Saccharomyces cerevisiae

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