[21] Recovery of plasmids from yeast into Escherichia coli: Shuttle vectors

Strathern, Jeffrey N.; Higgins, David R.

doi:10.1016/0076-6879(91)94024-7

Cited by 142 publications

(62 citation statements)

References 13 publications

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“…The yeast-impregnated filters were lifted from the plates and treated with X-Gal (5-bromo-4-chloro-3-indolyl ␤-D-galactoside) to assay colonies for ␤-galactosidase activity (15). Plasmid DNA from transformants that formed blue colonies on X-Gal-treated filters was recovered (16), propagated in E. coli DH5␣, and transformed back into the yeast reporter strains to confirm activity.…”

Section: Methodsmentioning

confidence: 99%

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Stockinger

Gilmour

Thomashow

1997

Proc. Natl. Acad. Sci. U.S.A.

1,574

1,203

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Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat͞dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat͞DRE binding factor, CBF1 (C-repeat͞DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat͞DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat͞DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat͞DRE DNA regulatory element and, thus, is likely to have a role in cold-and dehydrationregulated gene expression in Arabidopsis.

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Section: Methodsmentioning

confidence: 99%

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Stockinger

Gilmour

Thomashow

1997

Proc. Natl. Acad. Sci. U.S.A.

1,574

1,203

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“…The transformants were tested for terbinafine sensitivity on YPD plates containing 10 g/ml of terbinafine. Plasmid DNA was isolated from S. cerevisiae KLN1 transformants that were unable to grow on terbinafine-containing plates (34), amplified in E. coli XL1, and characterized by DNA sequence analysis. Recombinant plasmids pG 30 S, pL 37 P, and pR 269 G, which carry different erg1 alleles, were isolated by this method.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

Ruckenstuhl

Lang

Poschenel

et al. 2007

Antimicrob Agents Chemother

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Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G 25 S, D 335 X (W, F, P), and G 210 A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G 30 S and L 37 P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R 269 G variant exhibited moderately reduced SE activity and a 5-to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.

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“…Plasmids were released from yeast minicultures by lysis with SDS and glass beads (19). The cDNA inserts were amplified by PCR using vector-specific primers flanking the cloning site.…”

Section: Methodsmentioning

confidence: 99%

A voltage-gated ion channel expressed specifically in spermatozoa

Quill

Ren

Clapham

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

279

276

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Calcium ions play a primary role in the regulation of sperm cell behavior. We report finding a voltage-gated ion channel (CatSper2) that is expressed in male germ cells but not in other cells. The putative channel contains 6 transmembrane segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore domain sequence resembles that of a Ca v channel. The mRNA is expressed during the meiotic or postmeiotic stages of spermatogenesis, and the protein is localized to the sperm flagellum, suggesting a role in the regulation of sperm motility. The mRNA for the channel is present in mouse, rat, and human sperm cells, and the gene is found on chromosome 2 E5-F1 in the mouse and 15q13 in the human. Recently, another voltagegated channel (CatSper) that has features similar to the one reported here was discovered. It also is expressed within the flagellum and is required for normal fertility of mice. However, expression of CatSper2 alone or coexpression with CatSper in cultured cells, or attempts to coimmunoprecipitate the two proteins from germ cells failed to demonstrate that these two unique but similar ␣-like subunits form either a homo-or heterotetramer. It is possible, therefore, that two independent ␣ subunits, different from other known voltage-gated channels, regulate sperm motility.

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[21] Recovery of plasmids from yeast into Escherichia coli: Shuttle vectors

Cited by 142 publications

References 13 publications

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants

A voltage-gated ion channel expressed specifically in spermatozoa

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