20
Chromosome, Microdissection and Microcloning
1
Chromosome Structure and Organization
DNA is coiled in eukaryotic chromosomes
in a hierarchical fashion with several
levels, to achieve the highest degree of
condensation. Chromosome organization
is described in the following orders of
DNA folding (from lowest to highest):
naked double-stranded DNA, DNA coiled
around core nucleosomes, the 30 nm chro-
matin Fber, the 250 nm chromatin Fber
containing DNA loops, helical coiling of
the 250 nm chromatin Fber, chromosome
bands, and Fnally chromosome regions.
At the Frst level of DNA folding, linear
double-stranded DNA is coiled around a
histone core octamer (two copies each of
H2A, H2B, H3, and H4). The DNA under-
goes two left-handed superhelical turns
around the core histone octamer to form
a
nucleosome.
Adjacent nucleosomes are
connected by ‘‘linker’’ DNA, which may
be 8 to 114 bp long in different eukaryotic
species. Nucleosomes are folded in the
form of a simple solenoid structure that
generates a 30 nm
chromatin fber
.The
re
are six nucleosomes per turn (approxi-
mately 1.2 kb DNA). In both the interphase
and metaphase nucleus, the 30 nm Fber is
folded in a loop structure. The chromatin
loops contain between 5 and 100 kb of
DNA. One model predicts radial arrays of
ten30kbloopsy
ie
ld
inganarrayofabou
t
300 kb – that is, approximately 250 nm in
width. The chromatin loops are anchored
to a nonhistone protein structure that is
referred to as the
nuclear matrix
or the
chro-
mosome scaFFold
. The metaphase chromo-
some scaffold is positioned along the cen-
tral axis of the chromatid.
Topoisomerase II,
a major component of the scaffold struc-
ture, is localized along the length of the
chromatid. Topoisomerase II appears to
behave as a ‘‘loop fastener’’ in addition
to its catalytic function during replication
and transcription. DNA segments that in-
teract with the scaffold (scaffold-associated
regions: SAR) or with the nuclear ma-
trix (matrix-associated regions: MAR) are
0.6 to 1.0 kb in length and are presumed
to form the base of the loop structure.
The metaphase scaffol can be visualized
microscopically by using treatments that
partially deplete chromosomes of histones.
Under these conditions, the scaffold has
been observed to undergo a helical coiling
resulting in a further ninefold compaction
of the chromosome in metaphase. This
structural feature accounts for the zigzag
appearance of chromatids and probably
represents partially uncoiled structures.
During interphase, the extended, un-
coiled
chromosome
scaffold
structure
yields chromatids approximately 250 nm
in width, whereas the condensed, coiled
scaffold in metaphase yields a width of
700 nm. After metaphase, there is se-
lective decondensation (uncoiling) of eu-
chromatic chromosome regions. SpeciFc
mammalian chromosomes can be iden-
tiFed by the pattern of transverse bands
produced by a fluorescent dye or by Giemsa
stain. Chromosome bands have become
topographical landmarks used to map
genes, inherited traits, and chromosome
structural abnormalities, and as reference
points for large-scale genomic sequencing
projects. Chromosome bands represent
not only structural but also functional com-
partmentalization of the genome.
The
number
of
visible
bands
(e.g.
Giemsa dark bands or ‘‘dark G-bands’’)
depends on the exact stage in the cell
cycle at which chromosomes are exam-
ined. Chromosomes in midprophase will
yield approximately 2000 bands in a hu-
man karyotype. Later, in early metaphase,
when chromosomes are condensed, only
450 to 800 G-bands will be visible. An
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