246
Also, the amplification of heterologous DNAs can produce hybrid DNAs (chimeras) which
contain elements of two different templates (Meyerhaus et al., 1990).
At the present time, all of the above-described alternatives for isolating rRNA genes from
mixed populations have advantages and disadvantages (Table 1). The shotgun cloning
approach has been applied successfully to natural samples, and may avoid some complications
introduced by peR. However, this approach is very time consuming and labor intensive, even
for the analysis of just a single sample. Direct isolation of rRNA, and production and cloning
of ribosomal DNA is a tractable alternative, but full length rRNA clones appear to be rarely
recovered (Ward et al., 1990). Enzymatic amplification of rRNA genes using peR has also
been successfully applied, and is much more sensitive and rapid. peR may, however,
introduce artifacts due to the problems with Taq polymerase error described above. All the
techniques can potentially provide information about the phylogenetic diversity of mixed
populations, without requiring cultivation of the resident microbes. To demonstrate the utility
of these techniques, a brief review of some recent investigations, which shed light on the
phylogenetic identity of oceanic bacterioplankton, follows.
RECENT APPLICATIONS IN OCEAN SCIENCE
Giovanonni et al., (1990a) recently reported on the genetic diversity of Sargasso Sea
bacterioplankton. In these studies, large volumes of surface sea water were concentrated by
tangential flow filtration to obtain hundreds of milligrams wet weight of bacterioplankton cells
(Giovanonni et al., 1990b). DNA was extracted from this mixed cell population, and small
subunit rRNA genes were subsequently amplified from the mixture using the polymerase chain
reaction. The amplified rRNA genes were cloned, and phylogenetic analysis of small subunit
rRNA sequences performed. The sequences retrieved from the oligotrophic bacterioplankton
consortia fell into two main clusters: a cyanobacterial cluster, and a cluster most closely
affiliated with the alpha subdivision of the Proteobacteria (Stackebrandt et al., 1988). The
cyanobacterial sequences, not surprisingly, were very similar to those obtained from
well-characterized, motile, phycoerythrin containing Synechococcus cultivated from similar
habitats (Waterbury et al., 1985; Waterbury et al., 1986; D. Distel and J. Waterbury,
unpublished data). Although none of the cyanobacterial sequences retrieved from the Sargasso
Also, the amplification of heterologous DNAs can produce hybrid DNAs (chimeras) which
contain elements of two different templates (Meyerhaus et al., 1990).
At the present time, all of the above-described alternatives for isolating rRNA genes from
mixed populations have advantages and disadvantages (Table 1). The shotgun cloning
approach has been applied successfully to natural samples, and may avoid some complications
introduced by peR. However, this approach is very time consuming and labor intensive, even
for the analysis of just a single sample. Direct isolation of rRNA, and production and cloning
of ribosomal DNA is a tractable alternative, but full length rRNA clones appear to be rarely
recovered (Ward et al., 1990). Enzymatic amplification of rRNA genes using peR has also
been successfully applied, and is much more sensitive and rapid. peR may, however,
introduce artifacts due to the problems with Taq polymerase error described above. All the
techniques can potentially provide information about the phylogenetic diversity of mixed
populations, without requiring cultivation of the resident microbes. To demonstrate the utility
of these techniques, a brief review of some recent investigations, which shed light on the
phylogenetic identity of oceanic bacterioplankton, follows.
RECENT APPLICATIONS IN OCEAN SCIENCE
Giovanonni et al., (1990a) recently reported on the genetic diversity of Sargasso Sea
bacterioplankton. In these studies, large volumes of surface sea water were concentrated by
tangential flow filtration to obtain hundreds of milligrams wet weight of bacterioplankton cells
(Giovanonni et al., 1990b). DNA was extracted from this mixed cell population, and small
subunit rRNA genes were subsequently amplified from the mixture using the polymerase chain
reaction. The amplified rRNA genes were cloned, and phylogenetic analysis of small subunit
rRNA sequences performed. The sequences retrieved from the oligotrophic bacterioplankton
consortia fell into two main clusters: a cyanobacterial cluster, and a cluster most closely
affiliated with the alpha subdivision of the Proteobacteria (Stackebrandt et al., 1988). The
cyanobacterial sequences, not surprisingly, were very similar to those obtained from
well-characterized, motile, phycoerythrin containing Synechococcus cultivated from similar
habitats (Waterbury et al., 1985; Waterbury et al., 1986; D. Distel and J. Waterbury,
unpublished data). Although none of the cyanobacterial sequences retrieved from the Sargasso