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J Kur

Publications and source records attributed to J Kur.

At least 37 records · Page 2Linked to original sources
PubMed1998

In vitro interaction of the IHF-like proteins Acinetobacter junii and Proteus vulgaris with ihf sites.

The ability of the IHF-like proteins of Acinetobacter junii and Proteus vulgaris to interact with the H' attP and pR' ihf sites of lambda DNA was investigated. IHF from A. junii and P. vulgaris was found to bind the examined ihf sites in a way similar to IHF from Escherichia coli as shown by gel mobility shift DNA binding assays and footprinting analysis. The three IHF proteins bound to the pR' ihf site that overlaps the-35 region of that promoter and in vitro repression of transcription by each IHF was observed. These results confirm that IHF-like proteins from Gram-negative bacteria can recognize the same specific DNA sequences and appear to be important in regulation of transcription.

Acinetobacter↗
PubMed1998

The construction and use of a PCR internal control.

An example of the application and contruction of a polymerase chain reaction (PCR) internal control is presented. The internal control is synthesized in one PCR reaction. The primers used in this reaction possess 5' over-hanging ends which are identical to the primers used in the diagnostic reaction, whereas their 3' ends are complementary to a predetermined DNA sequence (pUC19 in this case) of defined length and sequence. As the sequence of the control except for primer sites, is not homologous to the PCR signal product, the formation of heteroduplexes and non-specific PCR products should not occur. Neither is there a risk that the target DNA will contaminate the internal control. However, the simultaneous amplification of two different DNA fragments flanked by the same primer sites resulted in either inhibition or enhancement of one or both products depending on the molar ratio of those DNA fragments. The presented method may be applied to construction of internal controls for quantitative PCR. The internal control was developed and tested for use in a PCR detection system for Agrobacterium tumefaciens.

Agrobacterium tumefaciens↗
PubMed1998

Effect of integration host factor of RNA II synthesis in replication of plasmid containing orip 15A.

The synthesis rates of the replication control RNAs of plasmid orip15A. RNA I, an inhibitor of replication, and RNA II, the primer, have been determined using lacZ fusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) in Escherichia coli integration host factor-positive (IHF+) and -negative (IHF-) strains containing pACYC184 plasmid (orip15A). In the absence of IHF (E. coli IHF-), expression of the lacZ gene from the PRNAII promoter increased by a factor of 4 compared with the E. coli wild type (IHF+). Also, the increase in expression was more pronounced when the IHF protein was mutated in the ihfB gene than in the ihfA gene. For the PRNAII promoter of oripMB1 (pBR322), no significant differences were found in expression of the lacZ gene in he E. coli strains examined. The level of beta-galactosidase expression from the PRNA promoter of orip 15A shows that the absence of functional IHF in the transformed strains has no effect on expression of the lacZ gene. The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 for E. coli IHF+ and 4.4 for E. coli IHF-. Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I in E. coli IHF+ and IHF-, are equal, but the relative level of RNA II in E. coli IHF is about four times higher than in E. coli IHF+. These results indicate that the IHF protein inhibits transcription from the PRNAII promoter of orip15A plasmid.

Artificial Gene Fusion↗
PubMed1998

Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei.

Complete PCR-derived DNA fragments containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector. The clones expressing thermostable His-tagged DNA polymerases were selected. The cloned fragments were sequenced. The DNA sequences were verified to be authentic by sequencing several clones. The nucleotide (nt) sequence revealed that DNA polymerase of P. woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566. It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P. furiosus (Pfu DNA polymerase). The results confirm that nucleotide sequences of both archaeons (P. furiosus and P. woesei) are highly similar. The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-affinity chromatography. We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG. However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition. The proteins were purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni2+-Sepharose columns. The enzymes were characterized and displayed high DNA polymerase activity and thermostability. This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases.

Amino Acid Sequence↗
PubMed1998

Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment.

The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.

Amino Acid Sequence↗
PubMed1998

[Use of PCR methods for identification of Listeria monocytogenes in milk].

The aim of this work was to estimate the limit of Listeria monocytogenes cfu in polymerase chain reaction (PCR) for a DNA fragment of listeriolysine O (hly A) gene. The PCR method, with used primers selected in areas of the listeriolysin O gene, allows to differentiate L. monocytogenes strains from other Listeria species. The amplified fragment (456 bp) of hly A gene was obtained for all strains L. monocytogenes and no other Listeria species. The PCR method with the selected primers allowed to detect 50-500 cfu L. monocytogenes/ml suspended in water or milk. Among 20 samples of raw milk from cows, 10 samples contained > 50 cfu L. monocytogenes/ml. Obtained results indicate that the PCR assay of L. monocytogenes identification is technically simple and may be conduct with minimal time. So, it could be recommended as quick diagnostic method in identification L. monocytogenes in milk.

Animals↗
PubMed1998

Recombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase.

The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli. The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography. The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus. The applied overexpression system was very efficient giving 700,000 u of DNA polymerase activity from 1 liter of induced culture. The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase.

Amino Acid Sequence↗
PubMed1998

Recombinant His-tagged DNA polymerase. II. Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment).

The Stoffel DNA fragment, shortened by 12 bp from 5' end, coding for Stoffel DNA polymerase (missing 4 amino acids at N-terminus of Stoffel amino-acids sequence) from the thermophilic Thermus aquaticus (strain YT-1) was amplified, cloned and expressed in Escherichia coli. The recombinant Stoffel fragment contained a polyhistidine tag at the N-terminus (21 additional amino acids) that allowed its single-step isolation by Ni2+ affinity chromatography. The enzyme was characterized and displayed high DNA polymerase activity and thermostability evidently higher than the native Taq DNA polymerase.

Amino Acid Sequence↗
PubMed1998

Thermal profile with alternately raised and lowered annealing temperature improves the PCR amplification using highly degenerate primers.

The proposed here PCR thermal profile improves the specificity and efficiency of PCR using highly degenerate primers, especially in the case of larger PCR products (around 2000 bp and more). The improvement is achieved by the use of a specific annealing temperature in the beginning cycles and the alternate lowering and raising of the annealing temperature in the subsequent cycles.

DNA Primers↗
PubMed1997

Effect of Escherichia coli IHF mutations on plasmid p15A copy number.

Four isogenic strains (himAhimD double mutant, himA and himD single mutants, and their wild type counterpart) harboring orip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number of orip15A plasmid increases about four times in the himAhimD double (65-70 copies per cell) and himD single mutant cells (50-56 copies per cell) and was almost the same in himA mutant (17-18 copies per cell) and wild type cells (14-16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation of orip15A plasmid copy number. Complementation experiments of himAhimD double mutant cells using plasmid carrying himA and himD genes (pPLhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number of orip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (using himAhimD double mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.

Bacterial Proteins↗
PubMed1997

Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates.

In this study we have developed a modified CLARK and DIAMOND (1992, 1993) method using the polymerase chain reaction to amplify amoebic ribosomal RNA genes that allow either specific detection of Entamoeba histolytica s. str. or amoeba species identification. DNA was isolated from cultured protozoa or from stool samples (cysts and/or trophozoites). Cultures of xenic or axenic material (also stool samples) were treated with Easy Genomic DNA Prep or Genomic DNA Prep Plus (A&A Biotechnology, Poland) for DNA isolation. With these new procedures, DNA can be obtained effectively and with high degree of purity. 13 amoeba strains were investigated. Three strains produced an 876 bp fragment with Psp5 and Psp3 primers (specific product for E. histolytica s. str). Three strains gave a specific 876 bp product for E. dispar with NPsp5 and NPsp3 primers. Two strains were E. moshkovskii as identified by KpnI digestion of PCR products obtained with RD5 and RD3 primers. Four strains (one of them was cultured in two laboratories) were E. invadens as identified by Hinfl or HhaI digestion of PCR products obtained with RD5 and RD3 primers. Characteristic RFLP patterns with these enzymes was also obtained for E. terrapinae and Blastocystis hominis. This RFLP analysis show significant promise as a medical diagnostic tool particularly useful for species identification.

Animals↗
PubMed1997

A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.

The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future.

Agrobacterium tumefaciens↗
PubMed1996

A proposal for using modified site-specific recombination systems for making insertions into a chosen chromosomal site in vivo based on the analysis of lambda phage integration.

A proposal is presented here to use modified mobile genetic elements for making chromosomal insertions in vivo, into any desired chromosomal site. The idea lies in replacing the sequences which direct specificity of integration of those elements with other DNA sequences, homologous to the desired target chromosomal site. The idea is analysed on the basis of a lambda phage integration system. A design for a universal system for making chromosomal insertions into any site of choice is proposed.

Bacteriophages↗
PubMed1996

Differentiation of seventeen genospecies of Acinetobacter by multiplex polymerase chain reaction and restriction fragment length polymorphism analysis.

The 17 described genomic species (DNA groups) of the genus Acinetobacter, including the type strains of the seven named species, were studied by using a multiplex PCR. The multiplex PCR assay combined two primer sets (rA1 and rA2 for recA gene target; rib1 and rib2 for 16S rDNA sequence) in a single reaction. Restriction analysis with two enzymes (Mbol and Hinfl) of the enzymatically amplified products allowed identification of all genospecies. This technique proved to be a rapid and reliable method for the identification of the Acinetobacter genomic species including the closely related DNA groups (1, 2, 3, 13). The results of this study suggest that the proposed method can be used for the identification of Acinetobacter spp. and as such may help to elucidate the ecology and clinical significance of the different species of this genus.

Acinetobacter↗
PubMed1996

Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR.

The presence or absence of the mecA gene, the determinant of resistance to all beta-lactam antibiotics, was examined in clinical isolates of Staphylococcus aureus by multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bp nuc- based PCR fragment (amplification product of the nuc gene encoding specific Staphylococcus aureus nuclease) and a 533-bp mecA-based PCR fragment (amplification product of the mecA gene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.

Genes, Bacterial↗
PubMed1996

Purification of IHF-like protein from gram-negative bacteria in one chromatographic step.

We describe a fast and very efficient method of purification which yields highly purified integration host factor-like proteins in one chromatographic step. IHF-like proteins from Acinetobacter junii or Proteus vulgaris are each an alpha beta heterodimer (subunits of 10 and 11 kDa) similar to the IHF of Escherichia coli when analyzed by polyacrylamide gel electrophoresis. The purified IHF are able to bind to the same ihf sites as IHF of E. coli. The results presented confirm that IHF is conserved during evolution in gram-negative bacteria.

Acinetobacter↗