Gene Cloning

Gene Cloning in the Functional Analysis of Proteins 309
individual bases in this region can now be mutated to any other desired
base using site-directed mutagenesis, and the mutated DNA checked for
the extent to which protein will still bind to it.
Site-directed mutagenesis is perhaps most widely used, however, to
probe protein function. By changing the DNA sequence of the gene that
encodes a protein, we can change any amino acid to any other amino acid,
and this is used to test hypotheses about the roles of particular amino acids
in proteins. These hypotheses are often generated by examining the structure
of a protein, either on its own or in a complex with other proteins or
ligands. For example, it is known that prior to infection of human cells by
the HIV virus, the virus has to bind to the surface of the cells. This binding
is due to an interaction between the viral coat protein gp120 and a cell surface
protein present on some human cells (all parts of the immune system)
called CD4. Early structural studies on CD4 identified a number of amino
acids which were good candidates for being important in the interaction
with gp120, including an exposed hydrophobic residue, phenylalanine, as
position 43 in the chain. This residue was mutated to alanine, and it was
shown that when this was done the affinity of CD4 for gp120 dropped by
about 500-fold, confirming the importance of this residue for gp120 binding.
When a structure was obtained (some 5 years later) of the two proteins
in a complex together, it was confirmed that the side chain of this phenylalanine
protruded into a pocket on the gp120 protein, explaining in terms
of structure why this residue is so important in binding.
Similar experiments have been done to probe the importance of individual
amino acid residues in enzyme mechanisms, protein structure,
interactions with other proteins and DNA, post-translational modification
of proteins, localization of proteins, and many more issues.
Questions and Answers
Q10.1. DNA in different organisms varies in the relative proportion of A+T
vs. G+C base pairs it contains. Some organisms are referred to as being
“GC-rich”, which simply means that G and C base pairs are found more frequently
in their genomes than would be expected by random chance;
others are “AT-rich” for the converse reason. If you were to compare the
genomes of an AT-rich and a GC-rich organism, which would you predict
would contain the higher number of long ORFs that do not encode proteins
but have simply arisen by chance, and why?
A10.1. GC-rich DNA has a higher frequency of long ORFs. This is because the
stop codons (TAA, TAG, and TGA) are relatively AT-rich – only two of the
nine bases are not A or T. On a purely random basis, then, these stop codons
will be relatively rarer in DNA that has fewer As and Ts, and so there will be
more longer ORFs in this DNA.