By Norman R. Pace, David A. Stahl, David J. Lane (auth.), K. C. Marshall (eds.)
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Failure to accomplish this results in confusion of evolution within the gene family with the evolution of the organisms. Of the rRNAs, only the 5S rRNA appears to present this difficulty; as noted above, significantly different sequences can be found in a single organism. Minor heterogeneity in the high-molecular-weight rRNAs commonly occurs, but the number of sequence position changes does not significantly disturb phylogenetic calculations. However, as the rRNAs from additional organisms are characterized, one must remain aware of the possibility of this complication.
Hybridization of an organism-specific sequence to bulk DNA derived from the natural sample then is a measure of the abundance of that gene in the original population. Hybridization of one of the above-mentioned oligonucleotide primers, complementary to all 16S genes so far as we know, provides an estimate of total rDNA present. The specific/total rDNA ratio really is only an approximate estimate of relative cell numbers, since rRNA gene copy numbers vary in different organisms. The inspected archaebacteria, for instance, have single copies of the rRNA genes; eubacteria commonly have 5-10 rDNA copies; and eucaryotes may have hundreds.
An examination ofthis is shown in Fig. 8. Here, base-fragmented and labeled 16S rRNAs from the aforementioned kingdom representatives are hybridized individually, under conditions oflow stringency, to cloned DNA containing the smallsubunit rRNA genes of Paramecium, Trypanosoma, Rattus, Dictyostelium, Escherichia coli, and Sulfolobus solfataricus. Conditions -for lowstringency hybridization have been optimized to reduce background hybridization to nonribosomal DNA; this is represented by the combined "plus strand" DNA target.
Advances in Microbial Ecology by Norman R. Pace, David A. Stahl, David J. Lane (auth.), K. C. Marshall (eds.)