Download Chromosome structure & Gene Expression

The Eukaryotic Chromosome:
An Organelle for Packaging and
Managing DNA
Eukaryotic Chromosomes:
• A chromosome consists of a single double-helix DNA molecule
starting at one end of the chromosome going through the centromere
and ending at the other end of the chromosome.
• Chromatin consists of 1/3 DNA, 1/3 histones and 1/3 non-histones
• Histones are five types, H1, H2A, H2B, H3 and H4. They are the
same in all cell types of an organism and in all different eukaryotic
organisms.
• Histones are highly conserved basic proteins that form nucleosomes,
a spool-like structure upon which 160 base pairs of DNA is wound.
Linker DNA between nucleosomes is 40 base pairs long.
• Non-histones are all other types of proteins (enzymes included) that
are responsible for DNA replication, expression and also cell division.
These are very heterogeneous group of proteins.
Karyotypes: represent the metaphase chromosomes of a cell that are
fully condensed then stained with Giemsa stain. This staining forms
G bands which are interchangeable dark and light bands along the
chromosome. These bands are identical and characteristic for each
pair of homologous chromosomes but differ between different
chromosomes. At low resolution, human chromosomes have 300
dark G bands and light interbands. At high resolution there are 2000
of such bands.
• Banding pattern of G bands is species specific.
• Bands are used to locate and map genes, especially useful when
mapping disease-causing genes. For example the the X-linked gene
for color blindness resides at q27-qter.
• Evolutionary relationships can be explained by G banding patterns.
Chromosome 1 of great apes have are very similar G bands as those
of chromosome 1 in humans. Chromosome 2 of humans appears to
have resulted from the fusion of acrocentric chromosomes of apes.
• G bands of human chromosomes have been used to identify genetic
diseases. Missing of a light band in X chromosome was linked to the
appearance of four X-linked diseases in a single individual.
Chromosome structure ensures accurate replication &
segregation:
1. Origins of replication:
• 10,000 in mammalian cells scattered throughout the chromosomes
and are separated by 30-300 kb of DNA.
• At any origin of replication, the replication occurs at both ends of
the replication bubble (replication fork) in opposite directions. The
DNA between two origins of replications is called a replicon or a
replication unit.
• Origins of replication consist of an AT-rich sequence (consensus) that
is adjacent to special flanking sequences.
• In yeast, DNA sequences containing origins of replication are
isolated by their ability to replicate plasmids when incorporated into
their DNA. Hence they are called autonomous replicating sequences
(ARS).
• Origins of replication sequences are not associated with
nucleosomes and are accessible to enzymes.
2. Telomeres ensure that chromosomes do not lose their termini at
each round of replication:
• DNA polymerase is unable to fill in an RNA primer’s length of
nucleotides at the 5’ end of a new strand at chromosome tips.
• This results in shortening the ends of a chromosome, with all the
relative genes it carries, a bit at a time with every round of DNA
replication.
• Telomeres are 250-1500 repeats of the sequence TTAGGG at the
ends of chromosomes. Such repeats have 2 functions: (i) form a
hairpin loop by unusual G-G hydrogen bonding. This provides a
free 3’ end for finishing the replication of the 5’ end of newly
synthesized DNA strands. (ii) attract telomerase, a
ribonucleoprotein which extends broken telomeres.
Centromeres:
- are primary constriction in chromosomes which contain blocks of
repetitive, noncoding DNA sequences known as satellite DNA
- satellite DNA consist of short tandem repeats (5-300 base pairs long).
In humans, a 171 bp satellite DNA is present in tandem repeats at the
centromere region.
- Centromeres have two functions. They hold sister chromatids
together and ensure proper segregation of chromosome segregation
(separation and distribution) through their kinetochore region with
motor proteins that specifically bind to microtubules of the spindle
apparatus.
- In yeast, centromeres consist of two highly conserved sequences each
10-15 bp separated by 90 bp of AT-rich DNA. Higher eukaryotes have
larger and more complex centromeres. Yeast artificial chromosomes
(YAC) demonstrate the important elements for chromosome function.
Chromosome structure & Gene Expression:
- Gene expression occurs in the interphase. Decompaction of higher
folding precedes transcription.
- RNA polymerase unwinds the nucleosome and proceeds in the
direction 5’ to 3’. DNA left behind the polymerase during
transcription rewinds again around histones to form nucleosomes.
- DNase I treatment experiments showed that DNase Hypersisitive
sites (DH) that contain few nucleosomes are found at the 5’ end of
genes.
- Extreme condensation causes the formation of heterochromatin
which could be constitutive or facultative.
- Position effect in Drosophila is an example of facultative
heterochromatin and Barr bodies in humans are constitutive
heterochromatin
How chromosomal packaging
influences gene activity
• Decompaction precedes gene expression
– Boundary elements delimit areas of
decompaction
– Nucleosomes in the decompacted area unwind
to allow initiation of transcription
• Transcription factors (nonhistone proteins) unwind
nucleosomes and dislodge histones at 5’ end of
genes
• Unwound portion is open to interaction with RNA
polymerase which can recognize promotor and
initiate gene expression
Studies using DNase identify
decompacted regions
Fig. 12.12 a
Extreme condensation silences
expression
• Heterochromatin
–
–
–
–
Darkly stained region of chromosome
Highly compacted even during interphase
Usually found in regions near centromere
Constitutive heterochromatin remains condensed most
of time in all cells (e.g., Y chromosomes in flies and
humans)
• Euchromatin
– Lightly stained regions of chromosomes
– Contains most genes
Heterochromatin versus euchromatin
• Heterochromatin is
darkly stained
• Euchromatin is
lightly stained
• C-banding
techniques stains
constitutive
heterochromatin
near centromere
Fig. 12.13
Position effect variegation in Drosophila: moving a
gene near heterochromatin prevents it expression
• Facultative
heterochromatin
– Moving a gene
near
heterochromatin
silences its
activity in some
cells and not
others
Fig. 12.14 a
Position effect variegation in Drosophila: moving a
gene near heterochromatin prevents it expression
• A model for
position-effect
variagation
– Heterochromatin
can spread
different
distances in
different cells
Fig. 12.14 b
Barr bodies: example of heterchromatin
decreasing gene activity
• Barr bodies – inactivation of one X
chromosome to control for dosage
compensation in female mammals
– One X chromosome appears in interphase cells
as a darkly stained heterochromatin mass
Unusual chromosome structures clarify the correlation
between chromosome packaging and gene function
• Polytene chromosomes
magnify patterns of gene
expression and tie them to
gene expression
– Drosophila larvae salivary gland
cells – chromosomes replicate
10 rounds without mitosis
– 210 = 1024 sister chromatids
plus homolougous chromosome
tightly wound together – 2048
double helices of DNA
– Chromosomal puff of gene
activity
Fig. 12.15 a
• Darkly stained highly condensed bands
interspersed with lightly stained less
condensed bands
Fig. 12.15 b
Polytene chromosomes are an
invaluable tool for geneticists
• in situ hybridization
of white gene to a
single band (3C2)
near the tip of the
Drosophila X
chromosome
Fig. 12.15 c
Decondensed chromatin in the nucleolus of
interphase cells contains rRNA genes actively
undergoing transcription
• Nucleolus contains hundreds of copies
of rRNA genes
Fig. 12.16 a
 Newly transcribed rRNAs appear as
short, wispy strands emanating from the
DNA in this electron micrograph of
nucleolus chromatin
Fig. 12.16 b