Добавлен: 13.02.2019
Просмотров: 6863
Скачиваний: 3
chromatin fiber that resembles a “beads on a string” appearance by electron mi-
croscopy. Figure 1-2 shows an electron micrograph of DNA that was isolated and
subjected to treatments to unfold DNA into a 10-nm diameter chromatin fiber. The
globular structure (“bead”; arrow 1) is the nucleosome. The linear structure
(“string”; arrow 2) is spacer DNA.
3.
The 10-nm diameter chromatin fiber is the first DNA structure that an endonucle-
ase attacks in an apoptotic cell.
4.
Histones are small proteins containing a high proportion of lysine and arginine
that impart a positive charge to the proteins that enhances its binding to negatively
charged DNA.
5.
Histone acetylation
reduces the affinity between histones and DNA. An increased
acetylation of histone proteins will make a DNA segment more likely to be tran-
scribed into RNA and hence any genes in that DNA segment will be expressed (i.e.,
c
acetylation of histones
expressed genes).
6.
Histone methylation
of lysine and arginine by methyltransferases also occurs.
C. 30-NM CHROMATIN FIBER
1.
The 10-nm nucleosome fiber is joined by H1 histone protein to form a 30-nm
chromatin fiber.
2.
During interphase of mitosis, chromosomes exist as 30-nm chromatin fibers or-
ganized in a primary loop pattern called extended chromatin (
300-nm diame-
ter). The extended chromatin can also be organized in a secondary loop pattern
as seen in condensed metaphase chromosomes. (Note: when the general term
“chromatin” is used, it refers specifically to the 30-nm chromatin fiber organized
as extended chromatin).
D. COMPACTION (Figure 1-3). During metaphase of mitosis, chromosomes can become
highly compacted. For example, human chromosome 1 contains about 260,000,000
bps. The distance between each base pair is 0.34 nm. So that the physical
length of the DNA comprising chromosome 1 is 88,000,000 nm or 88,000
m
(260,000,000
0.34 nm 88,000,000 nm).
During metaphase, all the chromosomes con-
dense such that the physical length of chro-
mosome 1 is about 10
m. Consequently, the
88,000
m of DNA comprising chromosome
1 is reduced to 10
m, resulting in a 8800-
fold compaction. Figure 1-3 shows double
helix DNA of chromosome 1 that is unrav-
eled and stretched out measuring 88,000
m
in length. When chromosome 1 condenses
as occurs during mitosis, the length is re-
duced to 10
m. This is a 8800-fold com-
paction.
3
CHROMOSOMAL DNA
B. NUCLEOSOME (Figure 1-2)
1.
The most fundamental unit of DNA pack-
aging is the nucleosome. A nucleosome
consists of a histone protein octamer (two
each of H2A, H2B, H3, and H4 histone
proteins) around which 146 bps of DNA
is coiled in 1.75 turns.
2.
The nucleosomes are connected by spacer
DNA, which results in 10-nm diameter
1
1
2
● Figure 1-2 Nucleosome.
Chromosome 1
● Figure 1-3 Chromosome Compaction.
LWBK771-c01_p1-8.qxd 9/29/10 6:48PM Page 3 aptara
A. A centromere is a specialized nucleotide DNA sequence that binds to the mitotic spin-
dle during cell division.
B. A major component of centromeric DNA is
-satellite DNA which consists of 171-bp
repeat unit.
-satellite DNA (a 68-bp repeat unit) and satellite 1 DNA (25–48-bp repeat
unit) are also components of centromeric DNA.
C. A centromere is also associated with a number of centromeric proteins, which include
CENP-A, CENP-B, CENP-C, and CENP-G.
D. All chromosomes have a single centromere which is observed microscopically as a pri-
mary constriction and which is the region where sister chromatids are joined.
E.
During prometaphase, a pair of protein complexes called kinetochores forms at the
centromere where one kinetochore is attached to each sister chromatid.
F.
Microtubules produced by the centrosome of the cell attached to the kinetochore
(called kinetochore microtubules) and pull the two sister chromatids toward opposite
poles of the mitotic cell.
A. Heterochromatin is condensed chromatin and
comprises
10% of the total chromatin.
B. Heterochromatin is transcriptionally inactive
and is electron dense (i.e., very black) in elec-
tron micrographs.
C. An example of heterochromatin is the Barr
body which is found in female cells and rep-
resents the inactive X chromosome.
D. Constitutive heterochromatin is always con-
densed (i.e., transcriptionally inactive) and consists of repetitive DNA found near the
centromere and other regions.
E.
Facultative heterochromatin can be either condensed (i.e., transcriptionally inactive)
or dispersed (i.e., transcriptionally active).
F.
The electron micrograph in Figure 1-4 shows a nucleus containing predominately euchro-
matin (E), peripherally located heterochromatin (H), and a centrally located nucleolus (NL).
A. EUCHROMATIN is dispersed chromatin and comprises
90% of the total chromatin.
B. Ten percent of euchromatin is transcriptionally active and 80% is transcriptionally
inactive.
C. When chromatin is transcriptionally active, there is weak binding to the H1 his-
tone protein and acetylation of the H2A, H2B, H3, and H4 histone proteins occurs.
Studying Human Chromosomes (Figure 1-5)
A. MITOTIC CHROMOSOMES are fairly easy to study because they can be observed in
any cell undergoing mitosis.
● Figure 1-4 Heterochromatin and Euchro-
matin.
4
CHAPTER 1
LWBK771-c01_p1-8.qxd 9/29/10 6:48PM Page 4 aptara
5
CHROMOSOMAL DNA
B. MEIOTIC CHROMOSOMES are much more
difficult to study because they can be ob-
served only in ovarian or testicular samples.
In the female, meiosis is especially difficult
because meiosis occurs during fetal develop-
ment. In the male, meiotic chromosomes can
be studied only in a testicular biopsy of an
adult male.
C. Blood is the most convenient source of hu-
man cells for karyotype analysis. Blood cells are
cultured and a mitogen is added to the culture
media to stimulate the mitosis of lymphocytes.
Subsequently, colchicine is added to the media which arrests the lymphocytes in
metaphase. It is often preferable to use prometaphase chromosomes because they are
less condensed and therefore show more detail. The lymphocytes are then concentrated
and treated with a hypotonic solution to lyse the lymphocytes and aid in spreading the
chromosomes. The cell preparation is then spread on a microscope slide, fixed, and stained
by a variety of methods (see section VII: Staining of Chromosomes). The separated
metaphase chromosomes are then identified and photographed. The photos of all the chro-
mosomes are then cut out and arranged in a standard pattern called the karyotype.
Figure 1-5 shows the G-banding pattern of metaphase chromosomes arranged in a
karyotype.
Metaphase or prometaphase chromosomes are prepared
for karyotype analysis (see section VI: Studying Human Chromosomes).
A. CHROMOSOME BANDING. The chromosome-banding technique is based on denat-
uration and/or enzymatic digestion of DNA followed by incorporation of a DNA-
binding dye. This results in chromosomes staining as a series of dark and light bands.
1.
G Banding
a.
G banding uses the Giemsa dye and is now the standard analytical method in
cytogenetics.
b.
Giemsa staining produces a unique pattern of dark bands (Giemsa positive;
G bands) which consist of heterochromatin, replicate in the late S phase, are
rich in A-T bases, and contain few genes.
c.
Giemsa staining also produces a unique pattern of light bands (Giemsa nega-
tive; R bands) which consist of euchromatin, replicate in the early S phase,
rich in G–C bases, and contain many genes.
2.
R Banding
a.
R banding uses the Giemsa dye (as above) to visualize light bands (Giemsa
negative; R bands) which are essentially the reverse of the G-banding pat-
tern.
b.
R banding can also be visualized by G–C specific dyes (e.g., chromomycin A
3
,
oligomycin, or mithramycin).
3.
Q Banding.
Q banding uses the fluorochrome quinacrine (binds preferentially to
A–T bases) to visualize Q bands which are essentially the same as G bands.
4.
T Banding.
T banding uses severe heat denaturation prior to Giemsa staining or a
combination of dyes and fluorochromes to visualize T bands which are a subset of
R bands located at the telomeres.
5.
C Banding.
C banding uses barium hydroxide denaturation prior to Giemsa stain-
ing to visualize C bands which are constitutive heterochromatin located mainly at
the centromere.
•
Figure 1-5 Human Karyotype.
LWBK771-c01_p1-8.qxd 9/29/10 6:48PM Page 5 aptara
6
CHAPTER 1
B. FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
1.
The FISH technique is based on the ability of single-stranded DNA (i.e., a DNA
probe) to hybridize (bind or anneal) to its complementary target sequence on a
unique DNA sequence that one is interested in localizing on the chromosome.
2.
Once this unique DNA sequence is known, a fluorescent DNA probe can be con-
structed.
3.
The fluorescent DNA probe is allowed to hybridize with chromosomes prepared
for karyotype analysis and thereby visualizes the unique DNA sequence on spe-
cific chromosomes.
C. CHROMOSOME PAINTING
1.
The chromosome painting technique is based on the construction of fluorescent
DNA probes to a wide variety of different DNA fragments from a single chromo-
some.
2.
The fluorescent DNA probes are allowed to hybridize with chromosomes prepared
for karyotype analysis and thereby visualize many different loci spanning one
whole chromosome (i.e., chromosome paint). Essentially, one whole particular
chromosome will fluoresce.
D. SPECTRAL KARYOTYPING OR 24 COLOR CHROMOSOME PAINTING
1.
The spectral karyotyping technique is based on chromosome painting whereby
DNA probes for all 24 chromosomes are labeled with five different fluo-
rochromes so that each of the 24 chromosomes will have a different ratio of flu-
orochromes.
2.
The different fluorochrome ratios cannot be detected by the naked eye, but com-
puter software can analyze the different ratios and assign a pseudocolor for each
ratio.
3.
This allows all 24 chromosomes to be painted with a different color. Essentially,
all 24 chromosomes will be painted a different color.
E.
COMPARATIVE GENOME HYBRIDIZATION (CGH)
1.
The CGH technique is based on the competitive hybridization of two fluores-
cent DNA probes: one DNA probe from a normal cell labeled with a red fluo-
rochrome and the other DNA probe from a tumor cell labeled with a green flu-
orochrome.
2.
The fluorescent DNA probes are mixed together and allowed to hybridize with
chromosomes prepared for karyotype analysis.
3.
The ratio of red to green signal is plotted along the length of each chromosome as
a distribution line. The red/green ratio should be 1:1.
a.
The tumor DNA is missing some of the chromosomal regions present in
normal DNA (more red fluorochrome and the distribution line shifts to the
left).
b.
The tumor DNA has more of some chromosomal regions than present in nor-
mal DNA (more green fluorochrome and the distribution line shifts to the
right).
A. GENERAL FEATURES
1.
The appearance of chromosomal DNA can vary considerably in a normal resting
cell (e.g., degree of packaging, euchromatin, and heterochromatin) and a dividing
cell (e.g., mitosis and meiosis).
2.
The pictures of chromosomes seen in karyotype analysis are chromosomal DNA
at a particular point in time, that is, arrested at metaphase (or prometaphase)
of mitosis. Early metaphase karyograms showed chromosomes as X shaped
LWBK771-c01_p1-8.qxd 9/29/10 6:48PM Page 6 aptara
7
CHROMOSOMAL DNA
because the chromosomes were at a point in mitosis when the protein cohesin
no longer bound the sister chromatids together, but the centromeres had not
yet separated.
3.
Modern metaphase karyograms show chromosome as I shaped because the chro-
mosomes are at a point in mitosis when the protein cohesin still binds the sister
chromatids together and the centromeres are not separated. In addition, many
modern karyograms are prometaphase karyograms where the chromosomes are I
shaped.
B. CHROMOSOME NOMENCLATURE (Figure 1-6)
1.
A chromosome consists of two characteristic
parts called arms. The short arm is called the
p (petit) arm and the long arm is called the q
(queue) arm.
2.
The arms can be subdivided into regions
(counting outward from the centromere), sub-
regions (bands), subbands (noted by the ad-
dition of a decimal point), and sub-subbands.
3.
For example, 6p21.34 is read as the short arm
of chromosome 6, region 2, and subregion
(band) 1, subband 3, and sub-subband 4. This
is NOT read as the short arm of chromosome
6, twenty-one point thirty-four.
4.
In addition, locations on an arm can be
referred to in anatomical terms: proximal
is closer to the centromere and distal is
farther from the centromere.
5.
Figure 1-6 shows the G-banding pattern of
a metaphase chromosome along with the centromere, p arm, and q arm.
DNA Melting Curve (Figure 1-7)
A. The denaturation of double-stranded DNA to
single-stranded DNA can be achieved by heat-
ing a solution of DNA to a temperature high
enough to break the hydrogen bonds holding
the two complementary strands together.
B. The denaturation of DNA can be followed by
measuring the optical density of the DNA at a
wavelength of 260 nm (ultraviolet light)
which is called the optical density at 260 nm
(OD
260
).
C. A measure of double-stranded DNA stability is the melting temperature (T
M
) which is
the temperature where 50% of the double-stranded DNA has been converted to single-
stranded DNA.
D. Denaturation of DNA is dependent on
1.
Base composition
a.
DNA with a high guanine and cytosine content will have a high T
M
because
guanine and cytosine are connected by three hydrogen bonds (c GC content
c
T
M
).
b.
DNA with a high adenine and thymine content will have a low T
M
because ade-
nine and thymine are connected by two hydrogen bonds (c AT content
T T
M
).
● Figure 1-6 G-banding Pattern of a
Metaphase Chromosome.
Temperature (°C)
% 260 nm Absorbance
10
30
50
70
90
110
130
100
50
0
T
m
AT
pH
urea
GC
Na
Natural
mammilian
DNA
● Figure 1-7 DNA melting curves.
LWBK771-c01_p1-8.qxd 9/29/10 6:48PM Page 7 aptara