Genomes of higher organisms are larger than one typically expects. For example, the DNA of a single human cell is almost two meters long, the DNA in the human body covers the distance Earth-Sun approximately 140 times. This is often not considered in typical molecular biological approaches for DNA diagnostics, where usually only DNA of the length of a gene is investigated. Also, one basic aspect of sequencing the human genome is not really solved: the problem how to prepare the huge amounts of DNA required. Approaches from biomedical optics combined with new developments in single molecule biotechnology may at least contribute some parts of the puzzle. A large genome can be partitioned into portions comprising approximately 1% of the whole DNA using a laser microbeam. The single DNA fragment can be amplified by the polymerase chain reaction in order to obtain a sufficient amount of molecules for conventional DNA diagnostics or for analysis by octanucleotide hybridization. When not amplified by biotechnological processes, the individual DNA molecule can be visualized in the light microscope and can be manipulated and dissected with the laser microbeam trap. The DNA probes obtained by single molecule biotechnology can be employed for fluorescence in situ introduced into plant cells and subcellular structures even when other techniques fail. Since the laser microbeam trap allows to work in the interior of a cell without opening it, subcellular structures can be manipulated. For example, in algae, such structures can be moved out of their original position and used to study intracellular viscosities.
ABSTRACT We have isolated one sorbitol-nonfermenting (SNF) Escherichia coli O157:H7 isolate and five sorbitol-fermenting (SF) E. coli O157:H − isolates that do not contain Shiga toxin (Stx) genes ( stx ). Isolates originated from patients with diarrhea ( n = 4) and hemolytic-uremic syndrome (HUS) ( n = 2). All isolates harbored a chromosomal eae gene encoding gamma-intimin as well as the plasmid genes E- hly and etp . The E. coli O157:H7 isolate was katP and espP positive. Respective sera obtained from the patient with HUS contained antibodies to the O157 lipopolysaccharide antigen. The stx -negative E. coli O157:H7 isolate is genetically related to stx -positive SNF E. coli O157:H7. All stx -negative SF E. coli O157:H − isolates belong to the same genetic cluster and are closely related to stx -positive SF E. coli O157:H − isolates. Our data indicate that stx -negative E. coli O157:H7/H − variants may occur at a low frequency and cannot be recognized by diagnostic methods that target Stx.
ABSTRACT We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx 2f . This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx 2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.
Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx 1 , stx 2 and stx 2c ). The strains belonged to the serotypes O52:H19, O75:H− and O157:H− and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx 1 sequences, but stx 2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.
A cosmid library of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933 was constructed and clones carrying the stx2 gene were identified by colony blot hybridization with a stx2B specific probe. Nucleotide sequencing upstream of the stx2A gene revealed high sequence identities of 89.5% to the ileX tRNA gene found in E. coli. The ileX gene was located 260 bp from the translational start codon of stx2A. PCR analysis with primers specific for this analyzed region showed that in 11 Stx2-producing EHEC strains from patients with hemolytic uremic syndrome, all PCR-positive strains carried the ileX tRNA gene. However, PCR analysis of the respective region in 11 Stx1-producing EHEC strains detected no ileX genes. Although the role of ileX in Stx2-producing EHEC strains is not clear, its function in regard to the use of rare codons and as an integration site is discussed.
