The possibility of intragenic heterogeneity between copies of the long intergenic (16S–23S rDNA) spacer region (LISR) was investigated by specific amplification of this region from 21 isolates. Three copies of the LISR ( and ) were demonstrated by hybridization of the LISR to genomic DNA cleaved with I-l and l When the LISR amplicon was digested with 5091, two known nucleotide substitutions were detected, one 4 nt upstream from the 5′ end of the tRNA gene (allele has the 509l site and and do not) and the other 22 nt downstream from the 3′ end of the tRNA gene ( has the 509l site). Sequence differences at these sites were detected at the allelic level (alleles and ) and different combinations of these alleles were designated Types. Using densitometry to analyse bands from electrophoresis gels, the intra-isolate ratios of the separate alleles () were determined in each Type: I (0:3:0), II (1:2:0), III (2:0:1), IV (3:0:0), V (2:1:0) and VI (1:1:1). Sequence variation between the three copies of the LISR was confirmed by the detection of at least five other intra-isolate nucleotide substitutions using heteroduplex analysis by conformation-sensitive gel electrophoresis (CSGE) that were not detected by 509I cleavage. Perpendicular denaturing gradient gel electrophoresis was capable of resolving homoduplexes; six to seven out of a possible nine curves were obtained in some isolates. In the isolate where seven curves were obtained one or more further nucleotide substitutions, not detected by 509l cleavage or CSGE, were detected. On the basis of LISR sequence heterogeneity, isolates were categorized into homogeneous (only one allele sequence present) and heterogeneous (two or three allele sequences present). The transition between homogeneous and heterogeneous LISRs may be useful in studying evolutionary mechanisms between isolates.


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