244
relationships of resident microbes, even those which resist cultivation. This information may
be used to infer the likely physiological or biochemical properties of population members, and
design experiments to test these hypotheses. In addition, the sequence information can be used
to construct organism-specific nucleic acid hybridization probes, for monitoring the abundance
and distribution of individual microbial species.
Isolation of small subunit rRNA genes from complex populations provides a way to identify
members of the original consortia, without requiring isolation of individual organisms in pure
culture. The approach hinges on the construction of recombinant DNA libraries, from which
rRNA genes can be identified, sorted and sequenced. Several strategies are now available for
preparation of recombinant DNA libraries from mixed population nucleic acids (Pace et al. ,
1986a; Weller and Ward, 1989; DeLong et al., 1989b; Giovanonni et al., 1990a). The first
to be proposed (Pace et al., 1986a) involves the initial isolation of fairly large quantities
(> 100 p.g) of high molecular weight, total population DNA. This mixed population DNA is
partially cleaved with a restriction endonuclease. DNA fragments in the 10-20 kilobase size
class are then purified, and inserted into a well characterized cloning vector (eg. bacteriophage
lambda). These cloning vectors allow the stable propagation and in vivo amplification of the
isolated DNA fragment. The end product is a recombinant DNA library, consisting of
hundreds of thousands of bacteriophage, each of which contain a DNA fragment originating
from the mixed population DNA. Individual clones containing rRNA genes can be identified
using radioactively labeled, broad-spectrum rRNA probes (Pace et al., 1986a). These probes,
by virtue of Watson-Crick base pairing, bind specifically to recombinant DNA clones which
contain rRNA genes. Individual clones which contain rRNA gene inserts are next isolated and
purified. The rRNA sequences of cloned rRNA genes are then determined, and compared to
a data base of sequences from well-characterized organisms. In this fashion, the phylogenetic
identity of individual population members can be ascertained (Pace et al., 1986a; DeLong
et al., 1989b; Schmidt et al., submitted).
Alternative methods for retrieving small subunit rRNA genes from natural populations have
recently been developed. Weller and Ward (1989) proposed the initial isolation of mixed
population rRNA, rather than DNA. Using the mixed population rRNA as a template,
ribosomal DNA (rDNA) can be generated enzymatically using the enzyme reverse
relationships of resident microbes, even those which resist cultivation. This information may
be used to infer the likely physiological or biochemical properties of population members, and
design experiments to test these hypotheses. In addition, the sequence information can be used
to construct organism-specific nucleic acid hybridization probes, for monitoring the abundance
and distribution of individual microbial species.
Isolation of small subunit rRNA genes from complex populations provides a way to identify
members of the original consortia, without requiring isolation of individual organisms in pure
culture. The approach hinges on the construction of recombinant DNA libraries, from which
rRNA genes can be identified, sorted and sequenced. Several strategies are now available for
preparation of recombinant DNA libraries from mixed population nucleic acids (Pace et al. ,
1986a; Weller and Ward, 1989; DeLong et al., 1989b; Giovanonni et al., 1990a). The first
to be proposed (Pace et al., 1986a) involves the initial isolation of fairly large quantities
(> 100 p.g) of high molecular weight, total population DNA. This mixed population DNA is
partially cleaved with a restriction endonuclease. DNA fragments in the 10-20 kilobase size
class are then purified, and inserted into a well characterized cloning vector (eg. bacteriophage
lambda). These cloning vectors allow the stable propagation and in vivo amplification of the
isolated DNA fragment. The end product is a recombinant DNA library, consisting of
hundreds of thousands of bacteriophage, each of which contain a DNA fragment originating
from the mixed population DNA. Individual clones containing rRNA genes can be identified
using radioactively labeled, broad-spectrum rRNA probes (Pace et al., 1986a). These probes,
by virtue of Watson-Crick base pairing, bind specifically to recombinant DNA clones which
contain rRNA genes. Individual clones which contain rRNA gene inserts are next isolated and
purified. The rRNA sequences of cloned rRNA genes are then determined, and compared to
a data base of sequences from well-characterized organisms. In this fashion, the phylogenetic
identity of individual population members can be ascertained (Pace et al., 1986a; DeLong
et al., 1989b; Schmidt et al., submitted).
Alternative methods for retrieving small subunit rRNA genes from natural populations have
recently been developed. Weller and Ward (1989) proposed the initial isolation of mixed
population rRNA, rather than DNA. Using the mixed population rRNA as a template,
ribosomal DNA (rDNA) can be generated enzymatically using the enzyme reverse