The Eukaryotic Chromosome: An Organelle for Packaging and Managing DNA

1
The Eukaryotic ChromosomeAn Organelle for
Packaging and Managing DNA
2

• 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.

3
(No Transcript)
4
(No Transcript)
5

• 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.

6

• 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.

7

• 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).

8

• 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
  primers 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
  bind two types of proteins (i) a protective
  protein which binds to the single-stranded TTAGGG
  sequence for shielding and protection and (ii)
  attract telomerase, a ribonucleoprotein which
  extends broken telomeres.

9
(No Transcript)
10
(No Transcript)
11
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. (i) They hold sister chromatids
together and (ii) ensure proper segregation of
chromosome (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.
12
(No Transcript)
13
Chromosome structure Gene Expression - Gene
expression occurs in the interphase.
Decompaction of higher folding precedes
transcription. - Non-histone proteins unwind
the nucleosomes allowing RNA polymerase access
which proceeds in transcription 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
14
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

15
Studies using DNase identify decompacted regions
Fig. 12.12 a
16
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

17
Heterochromatin versus euchromatin

• Heterochromatin is darkly stained
• Euchromatin is lightly stained
• C-banding techniques stains constitutive
  heterochromatin near centromere

Fig. 12.13
18
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
19
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
20
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

21
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
22

• Darkly stained highly condensed bands
  interspersed with lightly stained less condensed
  bands

Fig. 12.15 b
23
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
24
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
25

• Newly transcribed rRNAs appear as short, wispy
  strands emanating from the DNA in this electron
  micrograph of nucleolus chromatin

Fig. 12.16 b