NUCLEIC ACIDS 57
resulting in an incorrect series of amino acids following the frame shift. An
example is shown in Figure 2.18.
Organisms have elaborate proofreading and repair mechanisms that recog-
nize false base - pairings and other types of DNA damage, and they repair these.
Genetic mutations that occur in spite of proofreading and repair mechanisms
cause genetic diseases. Recent advances in genetic manipulation have allowed
the source of genetic diseases to be discovered and in some cases treated.
Publication of the human genome sequence in 2001, as discussed below, con-
stitutes a large step forward in the information available for scientists studying
genetic disease and treatment.
Scientists use mutations to study proteins by introducing amino acid sub-
stitutions in crucial locations that determine protein structure or catalytic,
binding, and regulatory functions. In order to carry out these mutations a
process calledsite - directed mutagenesis is invoked, during which the coding
gene must be isolated, cloned, sequenced, and then changed in a specifi c
manner. These processes of DNA isolation and cloning, called recombinant
DNA techniques, will be discussed further in the next section. Site - directed
mutagenesis enables the scientist to change any residue in any protein to any
other residue by changing the DNA code and then transcribing and translating
the mutated DNA. The essential process involves extracting the plasmid con-
taining the gene of interest from bacterial cells (usuallyEscherichia coli cells)
and then using a series of enzymatic reactions to switch codons. The plasmid
is then reintroduced to theE. coli cells for expression — transcription and
translation — into the mutated protein. The techniques are described in more
detail in the following section. The three - dimensional structure of the mutated
protein is studied by many methods, one of the most popular being X - ray
crystallography. One such study has been carried out on the copper - containing
electron transfer metalloenzyme azurin. In studies of the electron transfer
properties of this protein, it was found that the his35 residue (the histidine
residue found 53 residues along from the N - terminal end of the protein) was
part of a hydrophobic region or patch possibly involved in electron transfer
between azurin and itself — electron self - exchange (ese) — or a redox partner
Figure 2.18 Frame shift mutation caused by insertion of one base, G.CTT CCT CAC CGC AGT
leucine proline histidine arginine serineCTT CCT GCA CCG CAG Tinsert Gleucine proline alanine prolineglutamine