Chromosome, Microdissection and Microcloning
21
average dark G-band in prophase contains
approximately 1.5 Mb of DNA, whereas an
average-sized light G-band in metaphase
contains 10 to 30 Mb. Therefore, con-
densed metaphase bands are more hetero-
geneous (i.e. composed of multiple band
types) than those found in midprophase.
Each metaphase G-band contains about 15
to 50 fg of DNA. Chromosome bands can
be visualized using other fluorochromes.
Quinacrine mustard and its derivatives
have speciFcity for chromatin regions that
are rich in deoxyadenine (dA) and de-
oxythymidine (dT). Quinacrine staining
produces a banding pattern (Q-bands) that
is different from that due to fluorochromes
with afFnity for deoxycytidine (dC) and
deoxyguanidine (dG) residues (e.g. chro-
momycin A3 and mitramycin). Treatment
of Fxed chromosomes with trypsin fol-
lowed by staining with Giemsa (GTG
banding) yields dark (G) and light (reverse
or R) bands. R-bands usually correlate
with bright bands detected with quinacrine
(Q-bands). Giemsa staining of human
chromosomes after heat treatment results
in preferential staining of centromeric and
paracentromeric regions, as well as regions
of chromosomes 1, 9, and 16 and the telom-
ere of the Y chromosome; these patterns
are referred to as
C-bands
. C-bands are re-
gions of constitutive heterochromatin and
remain condensed throughout the cell cy-
cle, except for a short period of time during
DNA replication.
The structural compartmentalization of
chromosomes into transverse bands ob-
tained with Giemsa and fluorescent dyes
can also be seen by treating chromosome
spreads with the restriction endonuclease
Hae
III, as well as with
in situ
hybridization
using repeat-sequence DNA probes. Long
interspersed element (LINE) repeats are
found predominantly in G-bands, whereas
short interspersed element (SINE) repeats
or
Alu
sequences are found in R-bands.
The differing base composition of these
two repeat DNA groups accounts for the
higher GC content of R-bands in contrast
to higher AT content of G-bands. C bands
do not contain SINEs or LINEs but are
made of highly repetitive sequences called
satellite DNA sequences
.
The structural organization of chromo-
some bands has profound implications for
the regulation of gene expression. Most
housekeeping genes and the majority of
tissue-speciFc genes are found in R-bands.
In this regard, clustering of nonmethy-
lated CpG dinucleotides (HT± islands),
which is usually found 5
0
to ‘‘housekeep-
ing’’ genes, appears to be restricted to
R-bands. The staining techniques likely
to generate bands for microdissection are
summarized in a number of student cy-
togenetic manuals. Other chromosomal
structural regions such as the centromeres
and telomeres are also very amenable to
microdissection. The primary constriction
in mammalian chromosomes is called the
centromere
, and its location is useful in
the preliminary grouping of human chro-
mosomes. The centromere is composed
of constitutively condensed chromatin,
α
-satellite DNA, composed of multiple
copies of highly diverging basic repeat
units of approximately 171 bp. Tandem
repeats of
α
-satellite DNA are further
organized into micro repeat units rang-
ing in length from 0.5 to 10 Mb. Hu-
man chromosomes can be distinguished
from one another by their divergent
α
-
satellite sequences.
The chromosome ends, or
telomeres
,are
specialized structures that are essential for
the maintenance of chromosome integrity.
Telomeres are likely to be important in the
spatial positioning of the chromosomes in
the nucleus as well. The primary structure
of telomeres is a conserved G/C-rich repeat
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