have also shown that interactions could occur with infecting viral sequences resulting in
transgene silencing. In transgene silencing, the transgene stops being expressed. For
many years there have been observations of gene silencing mediated through sequence
similarity between promoters introduced on transformation vectors and resident genes.
This area of research is relatively new, but information is rapidly accumulating on the
nature of the interactions that can modify expression and the orientation of gene elements
that will mediate targeted gene silencing. For example, inverted repeats within the DNA
sequence must be avoided (Fig. 9.1). Furthermore, vectors should be constructed so that
the promoters associated with the selectable markers are located at maximal distances
from promoters associated with the gene of interest or the borders with the plant DNA
(Fig. 9.6). The genome has an abundance of promoters that could interact with incoming
DNA on insertion. Indeed, this knowledge has been used for promoter discovery research
through the use of promoter or enhancer trap strategies (Fig. 9.5). Interestingly, a new strong
constitutive promoter, tCUP (Fig. 9.4), was discovered in this way and was found to be very
useful for driving the expression of selectable marker genes because it did not interact with
other promoters as extensively as the 35Spromoter (Fig. 9.6).
Nonselectable marker (reporter) genes have been used extensively to study the speci-
ficity and level of plant promoter activity; therefore, the promoters combined with reporter
genes have been much more diverse. It is common to have a selectable marker gene and a
Figure 9.6.Construction of plant transformation vectors to avoid interactions among promoters used
to drive selectable marker genes and genes of interest (GOI). Constitutive promoters, such as the 35S
promoter, are frequently used to drive expression of the selectable marker genes. However, the 35S
promoter will interact with other promoters (red arrows) within the transformation vector, particularly
if they are situated near each other (configuration 1). This can lead to aberrant or unpredictable
expression of the gene of interest (GOI). Similar interactions may occur with elements within the
plant DNA that become positioned close to promoters within the transferred DNA (configuration 2).
These interactions can be minimized by the design of the vector. The simplest approach is to use a
constitutive promoter that does not tend to interact with other promoters, for example, the tCUP pro-
moter (shown in configuration 3). The genes within the transferred DNA can also be positioned so that
their promoters are spaced as far away from each other as possible through their orientation relative to
each other (configuration 4) or by the insertion spacer DNA between them (configuration 5). These
manipulations will reduce the extent of the interactions (indicated by the broken arrows).
226 MARKER GENES AND PROMOTERS