12 Produce Degradation: Reaction Pathways and their Prevention
1.3.3 GENOMIC SUBTRACTION
In principle, genomic subtraction is identical to complete genome sequence com-
parison. The goal is to identify unique genomic information present in one strain
but absent in the other. The most common protocol for genomic subtraction is the
one from Straus and Ausubel (1990), who developed a technique for isolating the
DNA that was absent in deletion mutants. The method removes from wild-type DNA
the sequences that are present in both the wild-type and the deletion mutant genomes.
The process is achieved by allowing a mixture of denatured wild-type and biotiny-
lated mutant DNA to reassociate. After reassociation, the biotinylated sequences are
removed by binding to avidin-coated beads.
Early application of genomic subtraction was to develop detection probes.
Applying the genomic subtraction technique of Straus and Ausubel (1990), Darrasse
et al. (1994) isolated six DNA sequences from a strain of E. carotovora subsp.
atroseptica. One fragment was specific for typical E. carotovora subsp. atroseptica
strains, two hybridized with all E. carotovora subsp. atroseptica strains and with a
few E. carotovora subsp. carotovora strains, and two probes recognized only a subset
of E. carotovora subsp. atroseptica strains. The last probe was absent from the
genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the
genomes of many other strains, including those of other species and genera. Using
a nonradioactive system, Ward and De Boer (1994) also developed a DNA probe
that specifically detected Erwinia carotovora subsp. atroseptica.
Assuming a similar genetic background, as indicated by phenotypic similarity,
genes found in one soft rot erwinia but not in others may be involved in specific
pathogenesis or other plant-associated lifestyle characteristics. An example from an
early study with Listeria monocytogenes, a food-borne human pathogen (Chen et
al., 1993) can be referenced. Subtracter probe hybridization was used to screen a
partial genomic library of a clinical isolate of L. monocytogenes against the genome
DNA from L. innocua. L. monocytogenes and L. innocua are highly similar but the
latter is not pathogenic in humans. Three clones that hybridized with genomic DNA
from 174 strains of L. monocytogenes but not with genomic DNA from 32 strains
representing other Listeria species were recovered. Using the limited database avail-
able then, one of the clones was identified by BLAST to be related to inLAB, a
gene family associated with pathogenicity.
In most cases, the genetic nature of the probe DNA sequences is unknown.
Darrasse et al. (1994a) cloned and sequenced their DNA probe fragments. Probably
due to the limited sequence information available at the time, the genetic nature of
the probe sequences was not identified, with the exception of one. This probe is
homologous to the putP gene of Escherichia coli, which encodes a proline carrier.
To cope with the increase in sequence information a genomic analysis tool called
microarray has recently been developed. With microarray, thousands of genes or
DNA sequences can be analyzed in one hybridization experiment. In array technol-
ogy, the probe or known sequence is the arrayed material, whereas the unknown or
target sequence is labeled and hybridized to the array. The intensity of hybridized
sequences is quantified and the resulting data are subjected to detailed statistical or