Detection of nucleic acid sequences from bacterial species with molecular genetic methods.

While blood products become more safe in terms of viral contamination, the risk of transfusion-related bacterial infection has re-emerged to one of the major hazards in transfusion medicine. In recent prospective studies the rate of contaminated platelets ranged from 0.04 to 0.

Differential display approach to quantitation of environmental stimuli on bacterial gene expression.

The differential display of the mRNA technique for eukaryotes is fruitful in identifying genes with altered transcription rates caused by exogenous or endogenous stimuli. Prokaryotic analogues of the method using arbitrary oligonucleotides may reach a complete statistical genome coverage. Thus a genome-wide mass screening for transcriptionally regulated sequences will be possible.

Effect of surface modifications of intraocular lenses on the adherence of Staphylococcus epidermidis.

The development of polymers with different surface properties and surface modifications of intraocular lenses (IOL) should reduce foreign body reactions after implantation by reducing the surface hydrophobicity of the lenses. It was examined how far such surface variations influenced the adhesiveness of bacteria. The most common organism isolated from cases of postoperative endophthalmitis is Staphylococcus epidermidis.

Localization of the iodomycin binding site in hamster P-glycoprotein.

P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. Plasma membrane vesicles isolated from multidrug-resistant Chinese hamster ovary B30 cells were photolabeled with [125I]iodomycin.

Primer design for a prokaryotic differential display RT-PCR.

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)].

Polymorphism of the pentanucleotide repeat d(AAAAT) within intron 1 of the human tumor suppressor gene p53 (17p13.1).

DyeDeoxy terminator cycle sequencing of allele-specific polymerase chain reaction products has shown that there is a highly polymorphic d(AAAAT) pentanucleotide repeat within the first intron of the human p53 gene. This provides a genetic marker for tumor suppressor p53 gene alterations.

[Theory and practice of macrorestriction analysis for the clonal analysis of pathogens].

The macrorestriction analysis of bacterial genomes is a method that is generally applicable to the typing of bacteria. The chromosome is cleaved with a restriction endonuclease that cuts infrequently and subsequently separated by pulsed-field gel electrophoresis. The fragment pattern defines the genotype of the strain.

Comparison of type IV-pilin genes of Pseudomonas aeruginosa of various habitats has uncovered a novel unusual sequence.

All known pilin sequences in Pseudomonas aeruginosa were amplified by a set of consensus primers located in the 5"-conserved region of pilA and the threonine-specific t-RNA following pilA. This also enabled the discovery of a novel pilin gene in a strain pair of clonal variants, which differs from known pilin genes in its increased GC-content. The mature protein has 173 amino acids making it the longest pilin known to date in P.

Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients.

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations.

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF).

Cystic fibrosis: the impact of analytical technology for genotype-phenotype studies.

The generalized exocrinopathy cystic fibrosis (CF) is the most common severe genetic disease in Caucasian populations. A panel of more than 700 chromosomes from German and Turkish CF patients was screened for disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch, single strand conformation polymorphism, restriction analysis and direct sequencing of genomic DNA amplified by polymerase chain reaction. Besides the major 3-bp deletion, delta F508 that was found on 73% of German CF chromosomes, more than 50 other missense, nonsense, frame-shift, and splice-site mutations have already been identified.