Rapid phylogenetic identification of single microbial cells was achieved with a new staining method. Formaldehyde-fixed, intact cells were hybridized with fluorescently labeled oligodeoxynucleotides complementary to 16S ribosomal RNA (rRNA) and viewed by fluorescence microscopy. Because of the abundance of rRNA in cells, the binding of the fluorescent probes to individual cells is readily visualized. Phylogenetic identification is achieved by the use of oligonucleotides (length 17 to 34 nucleotides) that are complementary to phylogenetic group-specific 16S rRNA sequences. Appropriate probes can be composed of oligonucleotide sequences that distinguish between the primary kingdoms (eukaryotes, eubacteria, archaebacteria) and between closely related organisms. The simultaneous use of multiple probes, labeled with different fluorescent dyes, allows the identification of different cell types in the same microscopic field. Quantitative microfluorimetry shows that the amount of an rRNA-specific probe that binds to Escherichia coli varies with the ribosome content and therefore reflects growth rate.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.

Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The selfcleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysishas not yet been proven. A minimal natural ribozyme sequence with proficient in viiro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (Pl-P4).The two pairings that define the 5' and 3' boundaries of the ribozyme, PI and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of PI, is in close proximity to three singlestranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.

The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88°C outflow from Octopus Spring, Yellowstone National Park, was examined. Three phylogenetic types ("phylotypes"), designated EM 3, EM 17, and EM 19, were identified by cloning and sequencing the small subunit rRNA genes (16S rDNA) obtained by PCR amplification of mixed-population DNA. All three phylotypes diverge deeply within the phylogenetic domain Bacteria sensu Woese (C. R. Woese, 0. Kandler, and M. L. Wheelis, Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). No members of the Archaea or Eucarya were detected. EM 3 comprises a unique lineage within the Thermotogales group, and EM 17 and EM 19 are affiliated with the Aquificales. A total of 35 clones were examined, of which the majority (26 clones) were of a single sequence type (EM 17) closely related to Aquifex pyrophilus. In situ hybridization with clone-specific probes attributes the majority sequence, EM 17, to the pink filaments.