Chimpanzee Genome
557
of closely related species than the compar-
ison of proteins. First, the molecular clock
of DNA sequences ticks faster than that of
protein sequences and, therefore, provides
a better resolution in time. This is be-
cause in coding sequences of genes, three
types of positions can be distinguished:
nondegenerated sites where any of the
three possible nucleotide substitutions is
nonsynonymous, that is, it alters the en-
coded amino acid. Twofold-degenerated
sites where only two of the three possible
nucleotide substitutions are nonsynony-
m
ou
s
.And±n
a
l
l
y
,f
ou
r
f
o
ld
-d
e
g
en
e
r
a
t
ed
sites where no nucleotide substitution
changes the encoded amino acid. Thus,
only a subset of DNA sequence changes
translates into changes of the respective
protein. Furthermore, a change of the pro-
tein sequence generally interferes with
protein function and is therefore com-
monly removed by purifying selection.
Therefore, the accumulation of nonsyn-
onymous DNA sequence differences and
the consequent accumulation of amino
acid sequence differences in proteins is
considerably slow. In contrast, nucleotide
sequence changes that leave the encoded
protein unaltered are less likely subject
to purifying selection and thus, accumu-
late differences at a substantially higher
rate. Second, comparative molecular anal-
yses can be extended to those regions in
thegenometha
ta
reno
tp
ro
te
in
-
cod
ing
,
and in the optimal case, not functional at
all. The latter sequences should be most
informative for phylogenetic analyses of
closely related species since they are likely
to evolve free of selective constraint.
1.2.1
Comparative DNA–DNA
Hybridization
DNA
occurs
in
nature
usually
as
a
double helix where the two complementary
strands are connected by hydrogen bonds
formed between guanine and cytosine, and
adenine and thymine respectively. Heating
DNAduplexesbreaksthehydrogenbonds
and the two strands are separated – the
DNA is ‘‘denatured’’ or ‘‘melted’’. The
progress of this process can be quanti±ed
as it gradually increases the UV absorption
at 260 nm of the DNA solution. Upon
cooling, complementary DNA strands will
rehybridize to duplex DNA. Performing
the rehybridization in the presence of DNA
from a second species, heteroduplexes can
be formed where the two complementary
strands stem from different species.
The temperature required to separate
50% of the DNA duplexes, also called
the
melting temperature
(
T
m
), increases, de-
pending on a number of factors: (1) The
guanine and cytosine content (GC con-
tent) of the DNA: guanine and cytosine
pair with three hydrogen bonds whereas
adenine and thymine pair only with two
hydrogen bonds; (2) the length of the
analyzed fragments; (3) the degree of se-
quence similarity between the two strands
forming the duplex. Accordingly, control-
ling for base composition and duplex
length, the difference between the melt-
ing temperature of DNA heteroduplexes
and that of the corresponding homodu-
plexes – both strands stem from the same
species – (
1
T
m
)se
rve
sa
samea
su
refo
r
the genetic difference between species.
For closely related species, the correla-
tion between
1
T
m
and the number of
mismatches between the complementary
strands of a DNA duplex is approxi-
mately linear.
An application of this method to the
problem of hominoid phylogeny con-
±rmed
the
conclusions
from
protein
comparisons that gibbons, followed by
orangutans, are the most diverged species.
Furthermore, additional support was given
to the view that humans and chimpanzees
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