Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

THE GLOBAL STRUCTURE OF CHROMOSOMES
209
DNA-binding
proteins
cross-link
formed
DNA probes used for PCR
TREAT
WITH
FORMALDEHYDE
CUT
WITH
RESTRICTION
NUCLEASE
DNA
LIGATION
REMOVE
CROSS-LINKS
BY HEAT TREATMENT
AND PROTEOLYSIS
TEST FOR JOINED
SEGMENTS BY
PCR
genome. Moreover, in this case, all the copies of each chromosome are aligned
side by side in exact register, like drinking straws in a box, to create giant polytene
chromosomes. These allow features to be detected that are thought to be shared
with ordinary interphase chromosomes, but are normally hard to see.
When polytene chromosomes from a fly’s salivary glands are viewed in the
light microscope, distinct alternating dark bands and light interbands are visible
(Figure 4–50), each formed from a thousand identical DNA sequences arranged
side by side in register. About 95% of the
MBoC6
DNA
m4.56/4.47
in polytene chromosomes is in
bands, and 5% is in interbands. A very thin band can contain 3000 nucleotide
pairs, while a thick band may contain 200,000 nucleotide pairs in each of its chromatin
strands. The chromatin in each band appears dark because the DNA is more
condensed than the DNA in interbands; it may also contain a higher concentration
of proteins (Figure 4–51). This banding pattern seems to reflect the same sort
of organization detected in the amphibian lampbrush chromosomes described
earlier.
There are approximately 3700 bands and 3700 interbands in the complete set
of Drosophila polytene chromosomes. The bands can be recognized by their different
thicknesses and spacings, and each one has been given a number to generate
a chromosome “map” that has been indexed to the finished genome sequence
of this fly.
The Drosophila polytene chromosomes provide a good starting point for examining
how chromatin is organized on a large scale. In the previous section, we
saw that there are many forms of chromatin, each of which contains nucleosomes
with a different combination of modified histones. Specific sets of non-histone
proteins assemble on these nucleosomes to affect biological function in different
ways. Recruitment of some of these non-histone proteins can spread for long
distances along the DNA, imparting a similar chromatin structure to broad tracts
DNA product is obtained
only if proteins hold the
two DNA sequences close
together in the cell
Figure 4–48 A method for determining
the position of loops in interphase
chromosomes. In this technique, known
as the chromosome conformation
capture (3C) method, cells are treated
with formaldehyde to create the indicated
covalent DNA–protein and DNA–DNA
cross-links. The DNA is then treated with
an enzyme (a restriction nuclease) that
chops the DNA into many pieces, cutting
at strictly defined nucleotide sequences
and forming sets of identical “cohesive
ends” (see Figure 8–28). The cohesive
ends can be made to join through their
complementary base-pairing. Importantly,
prior to the ligation step shown, the DNA
is diluted so that the fragments that have
been kept in close proximity to each other
(through cross-linking) are the ones most
likely to join. Finally, the cross-links are
reversed and the newly ligated fragments
of DNA are identified and quantified by
PCR (the polymerase chain reaction,
described in Chapter 8). From the results,
combined with DNA sequence information,
one can derive models for the interphase
conformation of chromosomes.
folded
chromatin
fiber
looped domain
high-level
expression
of genes
in loop
histonemodifying
enzymes
chromatin
remodeling complexes
RNA polymerase
proteins forming chromosome scaffold
Figure 4–49 A model for the organization of an interphase chromosome. A section of an interphase chromosome is shown folded into a series
of looped domains, each containing perhaps 50,000–200,000 or more nucleotide pairs of double-helical DNA condensed into a chromatin fiber.
The chromatin in each individual loop is further condensed through MBoC6 poorly m4.57/4.48 understood folding processes that are reversed when the cell requires
direct access to the DNA packaged in the loop. Neither the composition of the postulated chromosomal axis nor how the folded chromatin fiber is
anchored to it is clear. However, in mitotic chromosomes, the bases of the chromosomal loops are enriched both in condensins (discussed below)
and in DNA topoisomerase II enzymes (discussed in Chapter 5), two proteins that may form much of the axis at metaphase.