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Background: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. Methods: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 L plasma in 1 batch. Results: The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1-or HCV-positive plasma samples. The method linearity (R 2 ) was >0.99 for the viruses evaluated. Conclusions: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel

In eubacteria, the tRNA transglycosylase (Tgt) in specific tRNAs exchanges a guanine in the anticodon for 7-aminomethyl-7-deazaguanine, which is finally converted to queuosine. The tgt gene of Escherichia coli has been mapped at 9 min on the genome, and mutant pairs containing an intact or mutated tgt allele were obtained after transduction of the tgt locus by P1 bacteriophages into a genetically defined E. coli strain (S. Noguchi, Y. Nishimura, Y. Hirota, and S. Nishimura, J. Biol. Chem. 257:6544 6550, 1982). These tgt mutants grew anerobicafly with fumarate as an electron acceptor, while nitrate or trimethylamine N-oxide could not be reduced. Furthermore, molybdate reductase activity was almost lacking and the characteristic absorption maxima, corresponding to cytochrome a, and the cytochrome d complex, were not detectable in lowtemperature reduced-minus-oxidized difference spectra in anaerobically grown cells. Transduction of the mutated tgt locus into another E. coli recipient resulted in tgt mutants without anaerobic defects. Transformation of the original tgt mutants with anfnr gene-containing plasmid reversed the anaerobic defects. Clearly, the original tgt mutants harbor a second mutation, affecting the anaerobic regulator protein Fnr. The results suggest that fnr is involved in anaerobic control of components of the cytochrome d complex and of the redox system that transfers electrons to molybdate. F' plasmids containing a fused lacI-lacZ gene with the nonsense codon UAG at different positions in the lacl part were transferred to E. coli strains with a mutated or nonmutated tgt locus but intact infnr. A twofold increase in the frequency of incorrect readthrough of the UAG codon, dependent on the codon context, was observed in the tgt mutant and is suggested to be caused by a tRNATYr with G in place of queuosine.Eseherichia coli mutants with a defined genetic background were isolated, containing or lacking the tRNA guanine transglycosylase (Tgt), and consequently containing or lacking queuosine (Q), 7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)-methyl)-7-deazaguanosine, in tRNA (31).The transglycosylase enzyme catalyzes (in tRNAsGUN specific for Asn, Asp, His, and Tyr) the exchange of the guanine residue in the first position of the anticodon for 7-aminomethyl-7-deazaguanine, a precursor of Q (32, 33). The cyclopentenediol moiety of Q is then synthesized at the level of tRNA and involves epoxy-Q which is finally converted to Q by a cobamide-dependent enzyme system (7).Noguchi et al. (31) isolated tgt mutants by random screening from a collection of Escherichia coli K-12 mutants obtained by treatment with N-methyl-N'-nitrosoguanidine. tRNA was isolated from about 400 strains, and three mutants were found with respective tRNAs containing the anticodon GUN, where N is one of the canonical nucleosides. The defective gene, named tgt, was mapped at about 9 min on the E. coli chromosome, and the gene order was shown to be phoB tgt tsx. The tgt locus was transferred into E. coli ANLO5 (see Table 1) by P1 ba...

The tyrT locus in Escherichia coli codes for two gene copies of tRNAT yr. Both genes are organized in one operon, which has a unique structure. The two tRNA genes are separated by a spacer segment highly homologous to a part of a unit which is repeated three times in the distal portion of the locus. This operon also contains coding capacity for a small basic protein. A genomic deletion of this locus was constructed and marked by a kanamycin resistance cassette. Deletion mutants exhibited a characteristic phenotype when cells were shifted from rich medium to minimal medium. The cells entered a transient lag phase, apparently resulting from specific glycine starvation. This phenotype involved stringent response and was therefore not observed in relA derivatives. The genomic deletion was complemented in trans by a plasmid-borne tyrT locus. From deletion mapping, it can be concluded that a product of the tyrT operon is responsible for complementation. However, neither the tRNATYr nor the proposed basic protein is the complementation-competent entity.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)