298

12

DNA ORGAN IZATION IN CHROMOSOME S

[Figure 124(d)], two negative supercoils are introduced

spontaneously, reestablishing, in total, the original number
of turns in the helix. Use of the term negative refers to the fact
that, by denition, the supercoils are left-handed (whereas
the helix is right-handed). The end result is the formation of
a more compact structure with enhanced physical stability.
In most closed-circular DNA molecules in bacteria and
their phages, the DNA helix is slightly underwound [as in
Figure 124(c)]. For example, the virus SV40 contains 5200
base pairs. In energetically relaxed DNA, 10.4 base pairs occupy each complete turn of the helix, and the linking number
can be calculated as
L =

5200
= 500
10.4

However, analysis of circular SV40 DNA reveals that it is underwound by 25 turns, so L is equal to only 475. Predictably, 25
negative supercoils are observed in the molecule. In E. coli, an
even larger number of supercoils is observed, greatly facilitating chromosome condensation in the nucleoid region.
Two otherwise identical molecules that differ only in their
linking number are said to be topoisomers of one another. But
how can a molecule convert from one topoisomer to the other
if there are no free ends, as is the case in closed circles of DNA?
Biologically, this may be accomplished by any one of a group
of enzymes that cut one or both of the strands and wind or
unwind the helix before resealing the ends.
Appropriately, these enzymes are called topoisomerases.
First discovered by Martin Gellert and James Wang, these
catalytic molecules are known as either type I or type II, depending on whether they cleave one or both strands in the
helix, respectively. In E. coli, topoisomerase I serves to reduce
the number of negative supercoils in a closed-circular DNA
molecule. Topoisomerase II introduces negative supercoils
into DNA. This latter enzyme is thought to bind to DNA,
twist it, cleave both strands, and then pass them through
the loop that it has created. Once the phosphodiester bonds
are re-formed, the linking number is decreased and one or
more supercoils form spontaneously.
Supercoiled DNA and topoisomerases are also found
in eukaryotes. While the chromosomes in these organisms
are not usually circular, supercoils can occur when areas
of DNA are embedded in a lattice of proteins associated
with the chromatin bers. This association creates anchored ends, providing the stability for the maintenance
of supercoils once they are introduced by topoisomerases.
In both prokaryotes and eukaryotes, DNA replication and
transcription create supercoils downstream as the double
helix unwinds and becomes accessible to the appropriate
enzyme.
Topoisomerases may play still other genetic roles involving eukaryotic DNA conformational changes. Interestingly, these enzymes are involved in separating (decatenating)
the DNA of sister chromatids following replication.

12.3

Specialized Chromosomes Reveal

Variations in the Organization
of DNA
We now consider two cases of genetic organization that demonstrate specialized forms that eukaryotic chromosomes
can take. Both typespolytene chromosomes and lampbrush
chromosomesare so large that their organization was discerned using light microscopy long before we understood
how mitotic chromosomes form from interphase chromatin. The study of these chromosomes provided many of our
initial insights into the arrangement and function of the
genetic information. It is important to know that polytene
and lampbrush chromosomes are unusual and not typically
found in most eukaryotic cells, but the study of their structure has revealed many common themes of chromosome
organization.

Polytene Chromosomes
Giant polytene chromosomes are found in various tissues
(salivary, midgut, rectal, and malpighian excretory tubules)
in the larvae of some ies, as well as in several species of protozoans and plants. They were rst observed by E. G. Balbiani
in 1881. The large amount of information obtained from
studies of these genetic structures provided a model system for subsequent investigations of chromosomes. What is
particularly intriguing about polytene chromosomes is that
they can be seen in the nuclei of interphase cells.
Each polytene chromosome is 200 to 600 mm long, and
when they are observed under the light microscope, they
exhibit a linear series of alternating bands and interbands
(Figure 125 and 126). The banding pattern is distinctive
for each chromosome in any given species. Individual bands
are sometimes called chromomeres, a more generalized

FIGUR E 125
Polytene chromosomes derived from larval
salivary gland cells of Drosophila.

1 2. 3

SPECIALIZED C HROMOS OME S RE VE AL VARIA TIONS IN THE ORG ANIZ A TION OF DNA

299

IB

IB

IB

Puff

FIGUR E 126
Photograph of a puff within a
polytene chromosome. The diagram depicts the uncoiling of strands within a band region (B) to produce
a puff (P) in a polytene chromosome. Each band (B)
represents a chromomere. Interband regions (IBs) are
also labeled.

term describing lateral condensations of material along the

axis of a chromosome.
Extensive study using electron microscopy and radioactive tracers led to an explanation for the unusual appearance
of these chromosomes. First, polytene chromosomes represent paired homologs. This in itself is highly unusual, since
they are present in somatic cells, where, in most organisms,
chromosomal material is normally dispersed as chromatin
and homologs are not paired. Second, their large size and
distinctive appearance result from their being composed of
large numbers of identical DNA strands. The DNA of these
paired homologs undergoes many rounds of replication, but
without strand separation or cytoplasmic division. As replication proceeds, chromosomes are created, having 1000 to
5000 DNA strands that remain in precise parallel alignment
with one another. Apparently, the parallel register of so
many DNA strands gives rise to the distinctive band pattern
along the axis of the chromosome.
The presence of bands on polytene chromosomes was
initially interpreted as the visible manifestation of individual
genes. The discovery that the strands present in bands undergo localized uncoiling during genetic activity further
strengthened this view. Each such uncoiling event results in
a bulge called a puff, so labeled because of its appearance
under the microscope (Figure 126). That puffs are visible
manifestations of a high level of gene activity (transcription
that produces RNA) is evidenced by their high rate of incorporation of radioactively labeled RNA precursors, as assayed
by autoradiography. Bands that are not extended into puffs
incorporate fewer radioactive precursors or none at all.
The study of bands during development in insects such as
Drosophila and the midge y Chironomus reveals differential
gene activity. A characteristic pattern of band formation that is
equated with gene activation is observed as development proceeds. Despite attempts to resolve the issue, it is not yet clear
how many genes are contained in each band. However, we
do know that in Drosophila, which contains about 15,000
genes, there are approximately 5000 bands. Interestingly,
a band may contain up to 107 base pairs of DNA, enough
DNA to encode 50 to 100 average-size genes.

122 After salivary gland cells from Drosophila are isolated and
cultured in the presence of radioactive thymidylic acid, autoradiography is performed, revealing polytene chromosomes.
Predict the distribution of the grains along the chromosomes.
H I N T : This problem involves an understanding of the organization
of DNA in polytene chromosomes. The key to its solution is to be aware
that 3H-thymidine, as a molecular tracer, will only be incorporated
into DNA during its replication.

Lampbrush Chromosomes
Another specialized chromosome that has given us insight
into chromosomal structure is the lampbrush chromosome,
so named because it resembles the brushes used to clean kerosene lamp chimneys in the nineteenth century. Lampbrush
chromosomes were rst discovered in 1882 by Walther Flemming in salamander oocytes, and then seen in 1892 by J. Ruckert in shark oocytes. They are now known to be characteristic
of most vertebrate oocytes, as well as the spermatocytes of
some insects. Therefore, they are meiotic chromosomes. Most
of the experimental work on them has been done with material taken from amphibian oocytes.
These unique chromosomes are easily isolated from oocytes in the diplotene stage of the rst prophase of meiosis,
where they are active in directing the metabolic activities of
the developing cell. The homologs are seen as synapsed pairs
held together by chiasmata. However, instead of condensing, as most meiotic chromosomes do, lampbrush chromosomes are often extended to lengths of 500 to 800 mm. Later,
in meiosis, they revert to their normal length of 15 to 20 m
m. Based on these observations, lampbrush chromosomes
are interpreted as being extended, uncoiled versions of the
normal meiotic chromosomes.
The two views of lampbrush chromosomes in Figure
127 provide signicant insights into their morphology.
Part (a) shows the meiotic conguration under the light microscope. The linear axis of each horizontal structure seen
in the gure contains a large number of condensed areas,

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12

DNA ORGAN IZATION IN CHROMOSOME S

highly condensed structures during mitosis. However,

after chromosome separation and cell division, cells
enter the interphase stage of the cell cycle, at which
time the components of the chromosome uncoil
and decondense into a form referred to as chromatin. While in interphase, the chromatin is dispersed
Chiasma
throughout the nucleus. As the cell cycle progresses,
cells may replicate their DNA and reenter mitosis,
whereupon chromatin coils and condenses back into
visible chromosomes once again. This condensation
represents a length contraction of some 10,000 times
for each chromatin ber.
The organization of DNA during the transitions
(b)
just described is much more intricate and complex
than in viruses or bacteria, which never exhibit a
complex process similar to mitosis. This is due to
the greater amount of DNA per chromosome in eukaryotes, as well as the presence of a large number
Loops
of proteins associated with eukaryotic DNA. For example, while DNA in the E. coli chromosome is 1200 mm
long, the DNA in each human chromosome ranges
Central axis
from 19,000 to 73,000 mm in length. In a single huwith
chromoman nucleus, all 46 chromosomes contain sufcient
meres
DNA to extend almost 2 meters. This genetic material, along with its associated proteins, is contained
within a nucleus that usually measures about 5 to 10
FIG U R E 12 7
Lampbrush chromosomes derived from amphibian
mm in diameter.
oocytes. (a) A photomicrograph. (b) A scanning electron micrograph.
Such intricacy parallels the structural and biochemical
diversity
of the many types of cells present in a
which, as with polytene chromosomes, are referred to as
multicellular
eukaryotic
organism. Different cells assume
chromomeres. Emanating from each chromomere is a pair of
specic functions based on highly specic biochemical aclateral loops, giving the chromosome its distinctive appeartivity. Although all cells carry a full genetic complement,
ance. In part (b), the scanning electron micrograph (SEM)
different cells activate different sets of genes. Clearly, then,
shows many adjacent pairs of loops in detail along one of
a highly ordered regulatory system must exist to govern the
the axes. As with bands in polytene chromosomes, much
use of the genetic information. Such a system must in some
more DNA is present in each loop than is needed to enway be imposed on or related to the molecular structure of
code a single gene. Such an SEM provides a clear view of the
the genetic material.
chromomeres and the chromosomal bers emanating from
(a)

them. Each chromosomal loop is thought to be composed of

one DNA double helix, while the central axis is made up of
two DNA helices. This hypothesis is consistent with the belief that each meiotic chromosome is composed of a pair of
sister chromatids. Studies using radioactive RNA precursors
reveal that the loops are active in the synthesis of RNA. The
lampbrush loops, in a manner similar to puffs in polytene
chromosomes, represent DNA that has been reeled out from
the central chromomere axis during transcription.
12.4

DNA Is Organized into Chromatin

in Eukaryotes
We now turn our attention to the way DNA is organized in
eukaryotic chromosomes, which are most clearly visible as

Chromatin Structure and Nucleosomes

As we have seen, the genetic material of viruses and bacteria consists of strands of DNA or RNA relatively devoid of
proteins. In contrast, eukaryotic chromatin has a substantial
amount of protein associated with the chromosomal DNA
in all phases of the cell cycle. The associated proteins can
be categorized as either positively charged histones or less
positively charged nonhistone proteins. Of these two groups,
the histones play the most essential structural role. Histones
contain large amounts of the positively charged amino
acids lysine and arginine, making it possible for them to
bond electrostatically to the negatively charged phosphate
groups of nucleotides. Recall that a similar interaction has
been proposed for several bacterial proteins. The ve main
types of histones are shown in Table 12.2.