Sequencing Ribosomal RNA Genes

The nucleotide sequence of ribosomal RNA (rRNA) may be used to identify prokaryotes, particularly those that are difficult or currently impossible to grow in culture. The prokaryotic 70S ribosome, which plays an indispensable role in protein synthesis, is composed of protein and three different rRNAs (5S, 16S, and 23S) (figure 10.9). Because of their highly constrained and essential function, the nucleotide sequence changes that can occur in the rRNAs, yet still allow the ribosome to operate, are limited. This is why they have proved so useful in microbial classification and, more recently, identification. While earlier methods relied on determining the sequence of the rRNA molecule itself, newer techniques sequence rDNA, the DNA that encodes rRNA.

Of the different rRNAs, the 16S molecule has proved most useful in taxonomy because of its moderate size (approximately 1,500 nucleotides). The 5S molecule lacks the critical amount of information because of its small size (120 nucleotides), whereas

10.4 Characterizing Strain Differences 255

the larger size of the 23S molecule (approximately 3,000 nucleotides) has made it more difficult to sequence in the past.

Some regions of the 16S rRNA molecule are virtually the same in all prokaryotes, whereas others are quite variable. It is the variable regions that are used to identify an organism. Once the nucleotide sequence of that region has been determined, it can be compared with the 16S rDNA sequences of known organisms by searching extensive computerized databases.

Using rDNA to Identify Unculturable Organisms

In any environment, including soil, water, and the human body, a multitude of organisms exist that cannot yet be grown in culture. Some scientists estimate that every gram of fertile soil contains more than 4,000 different species of prokaryotes, the vast majority of which have not been identified. With current technologies that enable amplification of specific portions of DNA, followed by the cloning and then sequencing of those fragments, it is possible not only to detect such organisms, but also to obtain information about their identity.

One of the first examples of using molecular biology to identify an organism that had not been cultured was the characterization of the causative agent of a rare illness called Whipple's disease. This was done by using PCR to amplify bacterial 16S rDNA from intestinal tissue of patients who had symptoms of Whipple's disease. That DNA was then cloned and sequenced. The nucleotide sequence suggested that the causative agent was an actinomycete unrelated to any of those previously identified. It was given the name Tropheryma whippelii. Even though it had never been grown in culture, a specific probe was then developed that can detect it in intestinal tissue.

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