Ofthe three primary phylogenetic domains Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes) -Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota {one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, 0. & Wheelis, M. L. (1990) Proc. Nadl. Acad. Sci. USA 87, 4576-4579J} have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also ofcrenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.Microbiologists have long understood the limitations of cultivation techniques in assessing the diversity of naturally occurring microbial communities. Commonly, only a small fraction of organisms observed microscopically can be cultivated using standard methods. Recently, sequence-based phylogenetic techniques have been used to alleviate the requirement for cultivation to identify microorganisms. Such studies have detected the presence of previously unknown organisms in each instance of their use (for review, see ref.1). These techniques sample microbial populations directly through isolating and sequencing specific genes from the environment. Phylogenetic comparative analysis of these sequences is then used to determine evolutionary relationships between members of the community and cultivated species. The results allow inference of some properties of otherwise unknown organisms in the environment, based on the properties of their studied relatives. In addition, the sequences can be used to design oligonucleotide probes for determination of morphotype and abundance of particular organisms and for assistance in cultivation efforts.We have employed molecular phylogenetic techniques to investigate the diversity of Archaea in a hot spring in Yellowstone National Park. Small-subunit rRNA genes were amplified by polymerase chain reaction (PCR) from DNA extracted directly from sediment, by using primers designed to amplify archaeal and eucaryal genes selectively. Amplification products were then cloned and the nucleotide sequences of the inserts were determined....
Allelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in HardyWeinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity. Through inference of this and other known attributes of closely related Candida species, such as sequence analysis of I S 1 and the ITS2 (internal transcribed spacer 2) region of the rDNA cistron, the deduced phylogeny suggests an evolutionarily recent origin for many frequently isolated strains. This finding will be of interest in the context of understanding pathogenicity and drug resistance in this human commensal yeast.
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