We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species, and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structures, so that each DNA has its own structural fingerprint. Amplicons containing either a single 5-fluorescein or 5-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and-sensitive M. tuberculosis, the 5 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, Staphylococcus hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Band patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.

Floral induction in plants is a paradigm for signal perception, transduction, and physiological response. The introduction of root-inducing, left-hand transferred DNA (Ri T-DNA) into the genomes of several plants results in modifications of flowering (D Tepfer [1984] Cell 47: 959-967), including a delay in flowering in tobacco (Nicotiana tabacum). Conjugated polyamines are markers for flowering in numerous species of plants. In tobacco their accumulation is correlated with the onset of flowering (F Cabanne et al. [1981] Physiol Plant 53: 399-404). Using tobacco, we have explored the possibility of a correlation between the expression of Ri TL-DNA and changes in polyamine metabolism. We made use of two levels of phenotypic change, designated T and T', that retard flowering by 5 to 10 and 15 to 20 days, respectively. We show that delay in flowering is correlated with a reduction in polyamine accumulation and with a delay in appearance of conjugated polyamines, and we propose that genes carried by the Ri TL-DNA intervene either directly in polyamine metabolism or that polyamine metabolism is closely linked to direct effects of Ri T-DNA expression.

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all -dependent and -independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (␤N1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (␤R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for ␤N1072H. In contrast, ␤R352C and two adjacent substitutions (␤G351S and ␤S350F), but not ␤N1072H, formed open complexes of greatly reduced stability. The sequence in this region of ␤ modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in ␤, however, since the Rif r mutation rpoB2 in ␤ also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of ␤ region H in nucleoside triphosphate-binding and nucleotide addition and of ␤ region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.The two largest subunits of Escherichia coli RNA polymerase, ␤Ј (M r 155,063) and ␤ (M r 150,538) are homologous to the largest and second largest subunits, respectively, of all multisubunit RNA polymerases (Allison et al., 1985; Briggs et al., 1985;Falkenberg et al., 1987;Hudson et al., 1988;Leffers et al., 1989;Patel and Pickup, 1989). Most catalytic and regulatory functions of RNA polymerase appear to involve these subunits. Thus ␤ and ␤Ј are likely to form an enzymatic platform for RNA synthesis common to all forms of life, making elucidation of structure/function relationships in these subunits in a well studied prokaryote an ideal approach to understanding the mechanism of transcription. Sequence comparisons and recent crystallographic studies of single subunit DNA and RNA polymerases reveal broadly similar catalytic regions for both classes of enzyme (Steitz et al