Practical Methods in Molecular Biology

Schleif, Robert; Wensink, Pieter C.

doi:10.1007/978-1-4612-5956-5

Cited by 395 publications

(236 citation statements)

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“…Fragments of U urealyticum DNA extracted by the method according to Schleif and Wensingk [13] could not be amplified in the PCR performed.…”

Section: Suitability Of the Methods Investigated For The Detection Ofmentioning

confidence: 97%

“…Two methods were used for the extraction of the ureaplasma DNA. The first was a modification of the method described by Schleif and Wensingk [13]: 100 pl of liquid culture containing lo5 cfu of U urealyticum were mixed with 900 pl urine and subjected to microwave irradiation (Philips, Space Cube 50) for 20-30 s at 650 W. After centrifugation at 12000 g and 4°C for 30 min, the supernate was removed and mixed with 50 pl of a reaction buffer containing 0.1 M Tris, pH 7.8, 0.05 M EDTA, SDS 5% w/v and proteinase K (Boehringer, Mannheim, Germany) 500 pg and then incubated for I h at 56°C. After centrifugation at 12 000 g and 4°C for 15 min the DNA was separated by adding an equal volume of pheno1:chloroform:isoamyl alcohol (25:24: 1) to the supernate and shaking for 15 min at room temperature.…”

Section: Development Of a Protocol For The Detection Of U Urealyticummentioning

confidence: 99%

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Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction

Runge¹,

Rykena²,

Wildhagen³

et al. 1997

Journal of Medical Microbiology

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A method was developed to detect Ureaplasma urealyticum in urine by the polymerase chain reaction (PCR). A 457-bp fragment of the urease gene of U. urealyticum was amplified by PCR. Before PCR, components disturbing the amplification had to be reduced. This was possible by diluting the urine 1 in 10 with distilled water and by the extraction of the U. urealyticum DNA. Urine specimens from 41 patients with systemic lupus erythematosus (SLE) and 21 healthy individuals were treated by the dilution method and investigated by PCR for U. urealyticum DNA. The results were compared with those obtained by culture and the detection rates of PCR and culture were found to be identical. Also there was no difference in the detection rates of U. urealyticum from urine of SLE patients and healthy individuals; 10 (24.4%) of the 41 urine specimens from SLE patients and five (23.8%) of the 21 urine specimens from healthy individuals gave positive results for U. urealyticum. The results of this study do not indicate a decisive role for U. urealyticum in SLE.

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“…Fragments of U urealyticum DNA extracted by the method according to Schleif and Wensingk [13] could not be amplified in the PCR performed.…”

Section: Suitability Of the Methods Investigated For The Detection Ofmentioning

confidence: 97%

Section: Development Of a Protocol For The Detection Of U Urealyticummentioning

confidence: 99%

Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction

Runge¹,

Rykena²,

Wildhagen³

et al. 1997

Journal of Medical Microbiology

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“…Samples (10 ml) of cells were removed at several intervals for assays. The relative activity of permeabilized cells could then be calibrated by performing assays of total protein by the method of Lowry (34). We found that we are able to obtain values for specific activity of 3-galactosidase with an error margin of +20 to 30%.…”

mentioning

confidence: 97%

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

Cole¹,

Schild²,

Lovett³

et al. 1987

Mol. Cell. Biol.

133

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The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli I-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed ,I-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3-to 12-fold, in good agreeement with previous mRNA analyses. When MATa cells were arrested in Gl with a-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL) promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.The regulation of DNA repair and recombination in the procaryote Escherichia coli has been extensively studied. One result of these studies has been the elucidation of the inducible SOS repair pathway, a component of which, the recA gene produpt, appears to control a number of genes involved in both the repair of DNA lesions and general DNA recombination (for a review, see reference 38). The regulation of such activities in eucaryotic systems has not yet been thoroughly elucidated. In Saccharomyces cerevisiae, mutations in three separate epistasis groups have been demonstrated to cause sensitivity to DNA-damaging agents such as UV light, X-rays, and chemical mutagens (12,14). However, each of these groups has a different phenotypic response to different kinds of damage, leading to the hypothesis that each group is involved in a separate repair pathway (13,25). Some mutations in one of these epistasis groups, the RAD50-57 group of genes, have been shown to be not only sensitive to the effects of ionizing radiation, but also to block meiotic recombination and to reduce some types of mitotic recombination (16,27). For this reason, the repair mediated by the RAD50-57 pathway has been postulated to be recombinational repair. Mutations in RAD54 and RAD52, the genes whose regulation we describe here, have been shown to be blocked in double-strand break repair (6,20,28). Additionally, rad52 mutations block significant levels of recombination in meiosis and yield inviable spores (15, 16). Although rad54 mutations previously had been reported to have little if any effect on meiosis, deletion mutations of the RAD54 gene have recently been isolated and shown to decrease bot...

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“…DNA was labeled at the HindIII restriction site (+44) with y32P-ATP using bacterial alkaline phosphatase and polynucleotide kinase, digested with EcoRI and the 440 base-pair fragment isolated as described by Maxam & Gilbert (17). Sequences of mutants were first determined using DNA isolated by this rapid preparation, then the sequence of the entire relevant region was confirmed using CsCl purified DNA (14).…”

Section: Base Insertions and Deletionsmentioning

confidence: 99%

“…The proteins may interact directly (9,10), or the effects may be indirect, mediated through changes in the DNA structure over significant distances (11,12 All media, plates, and standard procedures including plasmid ligation, transformation and CsCl gradient plasmid isolation were as described in Schleif & Wensink (14).…”

Section: Introductionmentioning

confidence: 99%

Spacing mutations between theEscherichia coliPBAD RNA polymerase binding site and thearaC(I) induction site

1983

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Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function. INTRODUCTIONTranscription from the Escherichia coli L-arabinose operon promoter,

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Practical Methods in Molecular Biology

Cited by 395 publications

References 0 publications

Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction

Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

Spacing mutations between theEscherichia coliPBAD RNA polymerase binding site and thearaC(I) induction site

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