Gene Cloning

282 Gene Cloning
Third, we can make a translational fusion between the ORF for the protein,
and for another protein which is easier to detect, such as the green fluorescent
protein GFP. (See Box 9.1 for a discussion of translational fusions.)
This approach can make proteins which are present at low level more
“detectable”, and, as we shall see later, it can also be a powerful route to
determining the cellular location of the protein.
The construction of gene deletions
One of the first questions that is often asked of a newly identified gene is,
what is its role in the organism? This is a question which is being asked
more frequently in the age of large-scale sequencing projects, since often
these identify large numbers of genes which either do not resemble genes
in other organisms, or which are found in other organisms but for which no
function is known. To answer this question, we can use genetic means to
inactivate the gene, and then study the changes (if any) that result in the
organism. In this section we will consider some ways in which this can be
done, focusing on simple organisms such as bacteria and yeast. Gene deletions
or disruptions can also be made in complex organisms and may be
very informative; methods for doing this together with examples will be
discussed in Chapter 12. In the following section, we will look at methods
for reducing expression of genes without actually deleting the genes
themselves.
There are several approaches to disrupting the function of (often referred
to as “knocking out”) a gene. The most complete is to delete the whole gene
from the genome of the organism. Another is to insert a large piece of “junk”
DNA into the gene, which will prevent the mRNA being translated into the
normal protein, analogous to the way that a chunk of text pasted into a sentence
can disrupt the meaning of the sentence. We could also make a more
subtle change in the sequence of the gene, for example by introducing an
early stop codon into the gene, or by making a single base pair addition or
deletion so as to alter the reading frame. In general, the first two methods
are preferred, since they are unable to revert back to the wild-type.
Homologous recombination is always used to make either a targeted deletion
or disruption. For the simplest example of this, consider the situation
shown in Figure 10.1. Here, the regions on either side of the gene of interest
(which is shown in blue) have been cloned onto a plasmid which has then
been linearized and introduced back into the cell. A single homologous
recombination event between the linear DNA and the chromosome will produce
a broken chromosome, as shown. (If the chromosome itself is linear, this
will convert it into two linears; if it is circular, this will linearize it. In either
event, the chromosome will now be lost, either because it cannot replicate or
because it cannot successfully segregate to the daughter cells when the cell
divides.) However, a double recombination event as shown would essentially
remove the gene from the chromosome, and it would then be lost as it would