[4] Plasmid transformation of Escherichia coli and other bacteria

Hanahan, Douglas; Jessee, Joel; Bloom, Fredric R.

doi:10.1016/0076-6879(91)04006-a

Cited by 555 publications

(413 citation statements)

References 55 publications

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“…ElectroMax Escherichia coli strain DH10B (Life Technologies, Inc.) was used for transformation (19).…”

Section: Methodsmentioning

confidence: 99%

“…PCR reactions were performed as described above using the labeled oligonucleotides as primers and plasmids containing the appropriate ADE5,7 sequences as templates to amplify the region between Ϫ211 and Ϫ145 (relative to the ADE5,7 start codon). The templates were pR224, carrying the wild-type ADE5,7 fragment, or selected plasmids carrying mutated ADE5,7 promoter fragments, constructs numbered 5,7,10,11,13,14,19,20,27,29,34,35,39, and 43 as listed in Table IV. After PCR, a portion of each sample was separated by electrophoresis, and concentrations of the PCR products were estimated by ethidium bromide staining in comparison with known concentrations of duplex oligonucleotides of approximately the same length.…”

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

“…Fragments of the ADE5,7 promoter were generated by the PCR using plasmid pYEADE5,7(5.2R) (23) and the primers listed in Table I, as described in the relevant figure and table legends. PCR reactions were performed using Taq polymerase (Perkin-Elmer) under the following conditions: 30 cycles of denaturing at 95°C for 2 min, annealing at 55°C for 1 min, and chain extension at 72°C for 30 s. PCR fragments were digested with either XhoI or XhoI and BglII, separated from primers by electrophoresis, purified, and inserted into pLG699Z⌬XhoI or pCM81. The ligated products were transformed into competent E. coli by electroporation (19). Bacterial transformants were screened for insertions by colony hybridization (24), by restriction analysis (24), or by whole cell PCR (25).…”

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

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The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Rolfes

Zhang

Hinnebusch

1997

Journal of Biological Chemistry

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Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves downregulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

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“…ElectroMax Escherichia coli strain DH10B (Life Technologies, Inc.) was used for transformation (19).…”

Section: Methodsmentioning

confidence: 99%

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

Section: ј-Cgtcagatctgccccgnnngtagtgac-3јmentioning

confidence: 99%

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The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Rolfes

Zhang

Hinnebusch

1997

Journal of Biological Chemistry

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“…Digestion of template DNA was performed for 4 h using DpnI. Reaction mixtures (10 μL) were transformed into E. coli DH5α following the method of Hanahah et al (25).…”

Section: Molecularmentioning

confidence: 99%

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

et al. 2007

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Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteinefootprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.The mitogen-activated protein kinases (MAPKs) 1 are ubiquitous elements of eukaryotic signaling pathways that permeate nearly all aspects of signaling in multicellular organisms. In humans the loss of their regulation is associated with many diseases that encompass an expanding list of cancers as well as neurological and inflammatory diseases (1). Three main subgroups have been identified in humans, termed the ERKs (2), the JNKs (3,4), and the p38 MAPKs (5,6). Several newer members have recently been reviewed (7). Extracellular regulated protein kinase 2 (ERK2), the prototypical member of the MAP kinase family, catalyzes the † This research was supported in part by the Welch Foundation (F-1390) and the National Institutes of Health (GM59802). Mass spectra were acquired by Dr. Herng-Hsiang Lo in the CRED Analytical Instrumentation Facility Core supported by the NIEHS center grant ES07784. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transfer of the γ-phosphate of adenosine triphosphate to serine or threonine residues that generally lie in flexible regions of proteins. It is a remarkable enzyme whose ability to exhibit high specificity toward a discrete subset of structurally diverse proteins is poorly understood.Protein kinases generally recognize substrates through a consensus sequence that contains the phosphorylation site (8). This sequence is recognized, in part, by a region called the activation segment that encompasses residues 165 DFG to APE 195 of the catalytic domain of protein ki...

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“…Luria-Bertani (LB), tryptone broth (TB) and SM phage-dilution media were prepared as described by Silhavy et al (1984). The competent cells were grown in SOB medium (Hanahan et al, 1991). All media were supplemented with 100 mg ampicillin ml -1 (Amp) (Bristol-Myers Squibb), 50 mg kanamycin ml -1 (Km) (Roche Diagnostics), 50 mg chloramphenicol ml -1 (Cm) Phage lNK1316 (miniTn10 : : Km R cI 857 P am80 nin5 b522 att 2 ), neither replicates nor integrates into E. coli W3110 due to a mutation in gene P involved in replication, and by deletion of the site-specific recombination site (att 2 ) (Kleckner et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

et al. 2012

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We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage l as well. We propose that this mutation-andselection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

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[4] Plasmid transformation of Escherichia coli and other bacteria

Cited by 555 publications

References 55 publications

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

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