A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.

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A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups:chordates, echinoderms, arthropods, and eucoelomate protostomes.

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were partially sequenced by a dideoxynucleotide-terminated, primer extension method. A least-squares distance matrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green algae, and higher plants only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C. paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.The cyanobacteria are one of the most morphologically diverse and conspicuously successful procaryotic groups. It is generally believed that the cyanobacteria were the first major group of phototrophs to arise with a two-stage photosynthetic pathway capable of oxidizing water to produce molecular oxygen. Geochemical and fossil evidence indicates that in the Precambrian Era they caused the transition in the Earth's atmosphere from its primordial, anaerobic state to its current, aerobic condition (19,36,43). Moreover, molecular phylogenetic analysis of c-type cytochrome and rRNA sequences have established a relationship between cyanobacteria and the green (euglenoids, green algae, and higher plants) and red (rhodophyte) chloroplasts, thus supporting the procaryotic origins of chloroplasts.Because of their ubiquity, rRNA sequences are particularly useful for establishing evolutionary relationships among diverse organisms. Woese and colleagues, using partial (RNase Tl-generated oligonucleotide catalogs) and complete 16S rRNA sequences, have defined about 10 major divisions (phyla) of eubacteria (45). The cyanobacteria are one of these phyla. However, too few strains (eight) of cyanobacteria had been investigated to develop a comprehensive overview of the diversity of the group.We have used a method for directly sequencing 16S rRNA to explore the evolutionary relationships among 30 representatives of the diverse cyanobacterial groups, including the photosynthetic organelle of the phytoflagellate Cyanophora paradoxa. The results shed new light on the relative ages of * Corresponding author. t Present address: