16S rDNAs amplified by PCR from 22 hyperthermophilic methanococci isolated from deep-sea hydrothermal vents were compared with those of the six type strains of the genus Methanococcus by RFLP analysis. Restriction fragments obtained with Haelll enabled four of the type species to be distinguished. Restrictions with Hhal, Bstul and Mspl were necessary to differentiate Methanococcus jannaschii and Methanococcus fervens. The results indicate that the 16S rDNA PCR-RFLP method provides a rapid and reliable tool for the identification of newly isolated hyperthermophilic Methanococcus spp.
The family Chlamydiaceae contains two genera and nine species. Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations. Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from >50 chlamydial strains representing all nin species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial species were reliably distinguished in the tests. The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria. The result was that a PCR method for species identification of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created. This method can be used to rapidly identify known species of the family Chlamydiaceae.
Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.