ones can be established using techniques of molecular phylogeny 

 (Olsen, 1988) . By establishing phylogenetic relationships, some 

 properties of an otherwise unknown organism then can be predicted 

 because representatives of particular phylogenetic groups are 

 expected to have common properties. The same accumulated mutations 

 that are the basis of the phylogenetic analysis provide sequence 

 variability that can be used to identify and quantify organisms in 

 the environment by hybridization with organism-specific probes. 



The phylogenetic identification of organisms can be determined 

 without cultivating them by extracting total nucleic acids from a 

 sample and analyzing rRNA sequences, as shown in Figure 1. The 

 four approaches outlined include (i) direct sequencing of purified 

 5S rRNAs; (ii) sequencing from a cDNA library of rDNAs following 

 reverse transcription of extracted RNAs; (iii) cloning and 

 sequencing of PCR amplified rRNAs; or (iv) cloning size- 

 fractionated DNA into phage lambda, and purifying and sequencing 

 cloned rDNAs. rRNA sequences are compared to a data base of known 

 rRNA sequences to identify phylogenetic relationships. 



Conserved and variable regions of rRNA sequences are useful as 

 hybridization sites for synthetic oligonucleotide probes to 

 identify or to distinguish organisms. The rRNAs are particularly 

 attractive hybridization targets, because there are as many as l(r 

 to 10 J ribosomes per cell. In situ hybridizations between 

 isotopically or fluorescently labeled probes and formaldehyde-fixed 

 cells permit the phylogenetic identification of individual cells 

 (Giovannoni et al., 1990; DeLong et al., 1989). One set of 

 fluorescent probes developed for single-cell analysis consists of 

 oligodeoxynucleotides that distinguish the three primary lines of 

 evolutionary descent: eubacteria, eukaryotes, and archaebacteria 

 (DeLong et al., 1989). Epifluorescence microscopy shows that a 

 eukaryotic-specif ic probe hybridizes selectively to cells of 

 Saccharomyces cerevisiae in a mixture of S. cerevisiae and Bacillus 

 megaterium , while a fluorescently labeled probe complementary to a 

 universally conserved region of the rRNA anneals to both. Similar 

 fluorescent probes also have been used to examine the microbial 

 ecology of mouse cecum samples (Amman et al., 1990). 



The combination of these techniques provides the potential to 

 identify microorganisms, visualize single cells, and quantitatively 

 assess the abundance of specific microorganisms, all without 



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