58 BIOCHEMISTRY FUNDAMENTALS
such as cytochrome c 551 — electron transfer (et).^18 The authors created two
mutant azurins, one in which a glutamine residue was substituted for his35
(his35gln or H35Q mutant) and the other in which a leucine residue was sub-
stituted for his35 (his35leu or H35L mutant). An important fi nding resulting
from X - ray crystallographic structure of the H35Q mutant compared to the
wt azurin structure was that theβ barrel strand containing the mutation and
the unmutated strand to its left were shifted with respect to the confi guration
found in the wt azurin. The cleft created in the changed H35Q mutant exposes
the Q35 residue to solvent, a feature not found for the wt azurin in which his35
is shielded from solvent and buried within the protein structure. Figure 6 of
reference 18 shows the differences in tertiary structure in a stereo diagram.
(Stereo diagrams permit 3 - D visualization of a molecule on paper or a com-
puter screen. Helpful hints for stereoviewing are found in Section 4.6.1 .) The
technique for introducing point mutations using site - directed mutagenesis is
discussed in the following section.
2.3.5 Genes and Cloning,
Much of the information contained in this section and that following has been
obtained from the websites http://www.accessexcellence.org/RC/VL/GG/ ,
http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookDNAMOL
GEN.html , and references 1a , 2 , 7 , and 15. In eukaryotes, species whose cells
contain a nucleus and other intracellular structure, gene expression is more
complex than in prokaryotes, simpler life forms whose cells have no intracellu-
lar compartments. In eukaryotes, DNA is segmented into exons (regions that
encode for protein) and introns (regions that do not). When messenger RNA
(mRNA) is initially transcribed, both exon and intron regions are included.
Subsequently, the mRNA is spliced together at points so that only the exon
regions will be translated into protein in the ribosome. DNA molecules are
large and complex to work with in their native state. Usually, scientists wish to
work with only the DNA exons, and particularly the exons that code for the
specifi c protein(s) of interest. Recombinant DNA technology (also called
cloning and genetic engineering ) combines a number of different techniques
that allow scientists to manipulate and replicate DNA as well as study its struc-
ture and function. Recombinant DNA is DNA that has been created artifi cially
by techniques to be described in the following paragraphs. The recombinant
DNA molecule must be replicated many times to become useful for laboratory
research; this process is called cloning.
The fi rst step in the cloning process is to break the large DNA molecule
down into fragments. Sanger sequencing (also called the chain termination or
dideoxy method) uses an enzymatic procedure to produce DNA chains of
varying length in four different reactions, stopping DNA replication at posi-
tions occupied by one of the four bases and then determining the resulting
fragment lengths. This method uses naturally occurring enzymes called restric-
tion endonucleases. Restriction endonucleases are a class of enzymes, generally