PubMed Health⌕ Search

Biomedical subjects

J Timko

Publications and source records attributed to J Timko.

At least 19 recordsLinked to original sources
PubMed2006

Assay design and optimization of mutant-enriched PCR based method for detection of K-ras gene mutations in pancreatic carcinoma.

The aim of our work was to develop a fast, reliable and sensitive PCR method to detect K-ras mutations in various clinical samples. There is a need for an unimpeachable method for early diagnosis and/or screening of pancreatic cancer (PC). We optimized and subsequently analyzed four methods based on mutant-enriched PCR for the sensitivity, cost and time expense. Using the selected optimal method we examined codon 12 K- ras mutations in a study population of 59 patients with upper GIT malignancies. Reliability of the genotyping was confirmed by sequencing. By using the best of our modified mutant-enriched PCR methods we achieved sensitivity of 1:1 x 10(5). Further studies are necessary to determine the optimal biological material sampling in PC.

Adenocarcinoma↗
PubMed2005

Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20.

AIMS: Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251. METHODS AND RESULTS: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface. We observed strong adsorption ranging from ca 79 to 93% on the cells of B. flavum ATCC strains, but only ca 76% for B. flavum CCM 251. Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined. BFK 20 infection had no significant effect on growth and viability of C. glutamicum and B. lactofermentum, but significantly influenced growth and viability of B. flavum ATCC 21127, 21128 and 21474. Cell growth stopped in short time after infection but with no lysis. Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred. The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B. flavum CCM 251 and B. flavum ATCC strains after BFK 20 infection. Only weak hybridization signal was detected for DNA from infected cells of B. lactofermentum BLOB and no signal for C. glutamicum RM3. CONCLUSIONS: Based on the above results we suggest presence of a mechanism leading to abortive infection in B. flavum ATCC 21127, 21128 and 21474. In B. lactofermentum BLOB and C. glutamicum RM3 the adsorption barrier is more likely. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages.

Bacteriological Techniques↗
PubMed2004

DNA microarrays--techniques and applications in microbial systems.

Genome projects produce a huge amount of sequence information. As a result, the focus of genomics research is turning toward deduction of functional information about newly discovered genes. Thus structural genomics paves the way for a new discipline called functional genomics by providing the information required for microarray manufacture. Microarray technology is the result of automation and miniaturization in the detection of differential gene expression. By using this technology one can make a parallel analysis of RNA abundance and DNA homology for thousands of genes in a single experiment. Over the past several years, this unique technology has been used to explore hundreds transcriptional patterns and genome differences for a variety of microbial species. Applications of microarrays extend beyond the boundaries of basic biology into diagnostics, environmental monitoring, pharmacology, toxicology and biotechnology. We describe comprehensive nature of DNA microarray technology with emphasis on fabrication of DNA microarrays and application of this technology in biological environment with primary accent on microbial systems.

Bacteria↗
PubMed2001

An origin of DNA replication from streptomycete phage phi U1.

A DNA fragment from phage phi U1 containing an origin of DNA replication was identified. This fragment, designated ori, was able to support the maintenance in Streptomyces lividans of a plasmid lacking a functional Gram-positive ori. The sequence of the minimal ori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in this ori fragment indicates that host factors alone were sufficient to initiate replication at ori.

Bacteriophages↗
PubMed2001

Connection between foreign DNA replication and induced expression of the restriction-modification system in Streptomyces aureofaciens.

Tetracycline-producing strains of Streptomyces aureofaciens expressed SauLPI restriction-modification (R-M) system, which recognized specific DNA sequence 5'-GCCGGC-3' (isoschizomer Nael). The activation of the second R-M system SauLPII (5'-GAGCTC-3', isoschizomer of XhoI), which was silent during the growth cycle, after a foreign DNA transfer into this strain was observed. This phenomenon was tentatively explained as a response of the cells against the exogenous DNA entering the cells. The involvement of a SOS-like response in induction of R-M system genes in S. aureofaciens strains has been considered.

Bacteriophage mu↗
PubMed2001

Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum.

Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

Amino Acid Sequence↗
PubMed2000

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

We constructed new promoter-probe vectors for E. coli and corynebacteria based on the promoterless alpha-amylase gene originating from Bacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA. alpha-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of alpha-amylase activity.

Bacillus subtilis↗
PubMed1995

The properties of the multicopy suppressor of the ogd1 mutation in yeast.

The 8.1 kb chromosomal fragment partially suppressing the ogd1 mutation in Saccharomyces cerevisiae has been cloned. The molecular analysis revealed that its suppressor gene codes for a natural glutamine tRNA(CAG) and maps on chromosome XIII in the upstream region of the URA10 gene. The multicopy plasmids containing this tRNA gene also suppressed the standard trp1-1 amber mutation and conferred the sensitivity of yeast cells to paromomycin and increased temperature.

Chromosome Mapping↗
PubMed1994

Purification and some characteristics of the acetylxylan esterase from Schizophyllum commune.

Acetylxylan esterase from Schizophyllum commune was purified using ion-exchange and hydrophobic chromatography. The enzyme has a molecular mass of 31 kDa, as determined by SDS/PAGE, or 18 kDa, according to gel filtration. Glycosylation of the enzyme was not detected. Acetylxylan esterase is relatively stable under laboratory conditions; it retains full activity at pH 6.2-8.5 upon incubation at 25 degrees C for 7 h, but loses nearly the whole activity upon incubation at 60 degrees C for 30 min. The pH optimum of the enzyme activity is 7.7 and its temperature optimum lies between 30 and 45 degrees C. Ca2+ and Co2+ inhibit markedly the activity of acetylxylan esterase at a concentration of 10 mM, as do Mn2+, Zn2+, Fe2+ and Cu2+ at a concentration of 1 mM.

Acetylesterase↗
PubMed1994

Characterization and sequence analysis of the F2 promoter from corynephage BFK20.

F2 promoter from corynephage BFK20 was isolated and characterized. It was functional in Escherichia coli and Corynebacterium glutamicum. Cloning of the F2 promoter into the pJUP05 promoter probe vector caused an increase of the neomycin phosphotransferase II specific activity. According to the Northern blot hybridization the nptII gene was expressed from the cloned F2 promoter. The apparent transcription start point in E. coli and C. glutamicum was determined. The -35 region of F2 promoter showed high similarity to that of E. coli promoter consensus sequence, but its -10 region was G+C rich and had no significant homology to that.

Bacteriophages↗
PubMed1992

Characterization of bacteriophage BFK20 from Brevibacterium flavum.

Bacteriophage BFK20 was isolated from a Brevibacterium flavum strain that had become contaminated during industrial fermentation. BFK20 has a polyhedral head 50 nm wide and a non-contractile tail 200 nm long and 10 nm in diameter. The genome of this bacteriophage consists of a linear double stranded DNA molecule of 44-45 kb with cohesive ends. The capsid of phage BFK20 contains nine polypeptides with molecular masses from 22.0-108.0 kDa. BFK20 DNA was used as a donor for fragments carrying promoters and transcription-terminators.

Bacteriophages↗
PubMed1990

Construction of a promoter-probe shuttle vector for Escherichia coli and brevibacteria.

We constructed a promoter-probe vector, pJUP05, for brevibacteria and Escherichia coli based on the promoterless neomycin-resistance (neoR) gene from Tn5. This gene confers resistance to the aminoglycosides, kanamycin and neomycin. The promoter of the neoR gene was deleted and replaced by a suitable multiple cloning site. There are translation stop codons in all three reading frames upstream from the neoR gene. The plasmid contains functional origins of DNA replication for both brevibacteria and E. coli, and permits selection for chloramphenicol- and/or ampicillin-resistance markers.

Base Sequence↗
PubMed1981

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2.

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and ara C mutant strains of S. typhimurium. The DNA sequence was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence for the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.

Arabinose↗
PubMed1981

Characterization of a site-specific restriction endonuclease from Streptomyces aureofaciens.

A new type II sequence-specific restriction endonuclease, SauI, was isolated from Streptomyces aureofaciens IKA18/4. The purified enzyme was free of contaminating exonuclease and phosphatase activities. SauI cleaved lambda DNA at two sites, but did not cleave pBR322, simian virus 40, or phi X174 DNA. SauI recognized the septanucleotide sequence 5'-CCTNAGG-3' and cleaved at the position indicated by the arrow, producing a trinucleotide 5'-terminal extension.

Base Sequence↗