Gene Cloning

226 Gene Cloning
The problem with trying to perform a pair-wise alignment, between
your query sequence and all of the sequences in one of the databases, is
that the databases are too big for it to be practical to perform such an
exhaustive search. However, a number of computational methods have
been developed which will perform a very good approximation to this type
of search. The two most commonly used programs are Basic Local
Alignment Search Tool (BLAST) and Fasta (it stands for FAST-All). Each of
these programs uses a short cut to allow it to produce a list of similar
sequences without performing an exhaustive alignment between the query
sequence and every entry in the database. There are numerous web-based
resources which allow you to run Fasta and BLAST searches. The addresses
for some of these can be found in the Further Reading section at the end of
this chapter.
A classical example, which demonstrates the power of this technique, is
the case of the gene responsible for cystic fibrosis. In this inherited disease,
thick mucus accumulates in the lungs and intestines, which results in frequent
lung infections and malnutrition. Patients also have high levels of
salt in their sweat and are often sterile. Intensive efforts to clone and
sequence the gene responsible culminated in 1989 in the publication, by a
large team led by Lap-Chee Tsui and John Riordan, of the sequence of the
CFTR gene; mutations in this gene give rise to cystic fibrosis. Performing a
similarity search with the CFTR gene today will yield a huge number of similar
sequences, as it belongs to a much studied family of membrane transporter
proteins. However, in the late 1980s the situation was very different
because there were far fewer known sequences. Homology searches at the
time showed that, somewhat surprisingly, the gene responsible for this
genetic disease in humans was similar to a family of proteins responsible
for resistance of bacteria to a wide range of antibacterial agents. These ABC
multidrug transporter proteins function by pumping the drugs out of the
bacteria. This was an important clue as to the function of the CFTR protein,
which is now known to be involved in pumping sodium and chloride ions
across membranes in mammalian cells.
8.10 Fasta
Typically, the output from a Fasta search produces a summary table such
as that shown in Figure 8.12a. The table lists the sequences from the database
which most closely match that of the query sequence. The first column
shows the name of the entry in the database, or database identifier,
followed by an extract from the description of the protein. Many web interfaces
for the Fasta program provide hyperlinks to the database entries so
that you have immediate access to a wealth of information about the proteins.
The other columns give statistical information, which enables you to
assess the match between your query and the database entry. In the example
shown you can see that most of the sequences are of a similar length to