Chromosomes
1– general information
Genome Complexity
• Chromosomes are the structures that contain the
genetic material
– They are complexes of DNA and proteins
• The genome comprises all the genetic material
that an organism possesses
– In bacteria, it is typically a single circular
chromosome
– In eukaryotes, it refers to one complete set of
nuclear chromosomes
– Note:
• Eukaryotes possess a mitochondrial genome
• Plants also have a chloroplast genome
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-2
„
Bacteriophages may also contain a sheath, base plate and
tail fibers
Lipid bilayer
Picked up when
virus leaves host cell
Viral Genomes
• A viral genome is the genetic material of the virus
– Also termed the viral chromosome
• The genome can be
– DNA or RNA
– Single-stranded or double-stranded
– Circular or linear
• Viral genomes vary in size from a few thousand to
more than a hundred thousand nucleotides
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-7
BACTERIAL CHROMOSOMES
• The bacterial chromosome is found in a region
called the nucleoid
• The nucleoid is not membrane-bounded
– So the DNA is in direct contact with the cytoplasm
„
Bacteria may have one to four identical copies of
the same chromosome
„
The number depends on the species and growth
conditions
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
• Bacterial chromosomal DNA is usually a
circular molecule that is a few million
nucleotides in length
– Escherichia coli Æ ~ 4.6 million base pairs
– Haemophilus influenzae Æ ~ 1.8 million base
pairs
• A typical bacterial chromosome contains a few
thousand different genes
– Structural gene sequences (encoding
proteins) account for the majority of bacterial
DNA
– The nontranscribed DNA between adjacent
genes are termed intergenic regions
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
A few hundred
nucleotides in length
These play roles in DNA folding, DNA
replication, and gene expression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
• Eukaryotic genomes vary substantially in size
• In many cases, this variation is not related to
complexity of the species
– For example, there is a two fold difference in the size of
the genome in two closely related salamander species
– The difference in the size of the genome is not because
of extra genes
• Rather, the accumulation of repetitive DNA sequences
– These do not encode proteins
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-22
Has a genome that is more
than twice as large as that of
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-23
Heinrich Wilhelm Gottfried Waldeyer 1888
Chromosome
Chromo = colored in
response to dye
Some = body
Chromosome of Eukaryotes have been
the traditional subject for cytogenetic
analysis because they are large
enough to be examined with light
microscope
Cytogenetics = The study of chromosome
number, structure, function, and behavior
in relation to gene inheritance, organization
and expression
What is so special about chromosomes ?
1.They are huge:
One bp = 600 dalton, an average chromosome is 107 bp long = 109‐ 1010 dalton ! (for comparison a protein of 3x105 is considered very big.
What is so special about chromosomes ?
1.They are huge:
One bp = 600 dalton, an average chromosome is 107 bp long = 109‐ 1010 dalton ! (for comparison a protein of 3x105 is considered very big.
2. They contain a huge amount of non‐
redundant information (it is not just a big repetitive polymer but it has a unique sequence) .
What is so special about chromosomes ?
1.They are huge:
One bp = 600 dalton, an average chromosome is 107 bp long = 109‐ 1010 dalton ! (for comparison a protein of 3x105 is considered very big.
2. They contain a huge amount of non‐
redundant information (it is not just a big repetitive polymer but it has a unique sequence) .
3. There is only one such molecule in each cell. (unlike any other molecule when lost it cannot be re‐synthesized from scratch or imported)
Organization of Eukaryotic
Chromosomes
A eukaryotic chromosome contains a long,
linear DNA molecule
• Three types of DNA sequences are required
for chromosomal replication and segregation
– Origins of replication
– Centromeres
– Telomeres
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-24
Heterochromatin vs Euchromatin
„
The compaction level of interphase chromosomes
is not completely uniform
„
Euchromatin
„
„
„
„
Less condensed regions of chromosomes
Transcriptionally active
Regions where 30 nm fiber forms radial loop domains
Heterochromatin
„
„
„
Tightly compacted regions of chromosomes
Transcriptionally inactive (in general)
Radial loop domains compacted even further
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-61
„
There are two types of heterochromatin
„
Constitutive heterochromatin
„
„
„
Regions that are always heterochromatic
Permanently inactive with regard to transcription
Facultative heterochromatin
„
„
Regions that can interconvert between euchromatin and
heterochromatin
Example: Barr body
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-62
Metaphase Chromosomes
„
As cells enter M phase, the level of compaction
changes dramatically
„
„
„
By the end of prophase, sister chromatids are entirely heterochromatic
Two parallel chromatids have an overall diameter of 1,400 nm
These highly condensed metaphase chromosomes
undergo little gene transcription
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-65
„
Two multiprotein complexes help to form and
organize metaphase chromosomes
„
Condensin
„
„
Cohesin
„
„
Plays a critical role in chromosome condensation
Plays a critical role in sister chromatid alignment
Both contain a category of proteins called SMC
proteins
„
„
Acronym = Structural maintenance of chromosomes
SMC proteins use energy from ATP and catalyze changes in chromosome
structure
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-67
The number of loops has not changed
However, the diameter of each loop is smaller
During interphase,
condensin is in the
cytoplasm
Condesin binds to
chromosomes and
compacts the
radial loops
Condesin travels
into the nucleus
The condensation of a metaphase
chromosome by condensin
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-68
Cohesins along
chromosome arms are
released
Cohesin at
centromer is
degraded
Cohesin
remains at
centromere
The alignment of sister chromatids via cohesin
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
10-69
Cytogenetic methods to detect chromosomal
abnormalities underlying human birth defects usually
involve analysis of mitotic chromosome
What tissues are appropriate for chromosome
study?
• Any tissue that can be stimulated to undergo
cell division in-vitro
………….the easiest way is to work on blood
cells,
of course carrying a nucleus as lymphocytes
Tissues and cultures for chromosomal analysis
Constitutional chromosomal anomalies
Peripheral blood sampling ⇒
⇒
Skin biopsy
Biopsies from other tissues ⇒
T lymphocytes
Fibroblasts
?
Amniocentesis
⇒
Amniocytes
Chorionic villi
⇒
Cytotrophoblast
Acquired chromosomal anomalies
Bone marrow, peripheral blood, lymphonodes, spleen, other
tissues containing ⇒
dysplastic or neoplastic cells
Biopsies from tumors
⇒
dysplastic or neoplastic cells
Blood sample ,
heparin
PHA stimulated cells
In vitro
cultures
Block mitosis,
Colchicine
Add hypotonic solution
Analysis
Lay cells on a slide, fix and stain
Amniotic fuid
Fetal cells
Placenta
Amniocentesis
Uterine wall
Centrifuge
Bioche mistry, Chromosomes
DNA
Fetal cells
Biochemistry,
DNA
Fetal cell cultures
Tjio and Levan, 1956
Chromosome Morphology
Telomere
Short
arm (p)
Centromere
Arm
Long
arm (q)
Telomere
Metacentric
Submetacentric
Acrocentric
Conferences and documents
Year
Denver Conference
1960
London Conference
1963
Chicago Conference
1966
Paris Conference
1971
Paris Conference (supplement)
1975
Stockholm-1977 ISCN
1978
Paris-1980 ISCN
1981
ISCN
1985
Cancer Supplement ISCN
1991
Memphis-1994 ISCN
1994
ISCN
2005
Staining (banding techniques)
Using intercalating agents each chromosome has its own
banding pattern:
Q Bands are obtained with Quinachrine mustard (fluorescent)
G Bands are obtained with Giemsa after various pre‐treatments R Bands are obtained with Giemsa o Acridine orange
Banding Techniques for specific chromosomal regions.
C Bands
specific for constitutive heterochromatine (centromers)
CD Bands specific for proteins of the cinetochore
NOR Bands specific for NOR (acrocentric chromosomes )
Da‐DAPI
specific for heterochromatine of 1, 9, 15, 16, Yq
and more ………….
Q Bands (QFQ)
G Bands (GTG)
C Bands (CBG)
Visualizing Metaphase Chromosomes (Banding)
• Giemsa‐, reverse‐ or centromere‐stained metaphase chromosomes
G-Bands
R-Bands
C-Bands
NOR (Nucleolus Organizing Regions)
DA-Dapi (Distamicine A – Di Amino Phenil Indolo)
Banding
Banding
The analysis involves comparing chromosomes for their length, the placement of centromeres (areas where the two chromatids are joined), and the location and sizes of G‐bands.
International System for Cytogenetic
Nomenclature, (ISCN,1995)
• Short arm of the chromosome = p
• Long arm of the chromosome = q
• Bands are numbered independently on the short and long arms
• Centromeres = p10,q10
• Band numbers increase as move from the centromere to the telomere
Defining Chromosomal Location
Arm
Region
2
p
1
1
Band Subband
2
1
1
1
2
1
2
q
3
2
Chromosome 17
3
2
1
2
1
5
4
3
2
1
4
3
1
2
3
1
2, 3
4
1
2
3
17 q11.2
Karyotype
• International System for Human Cytogenetic Nomenclature (ISCN) – 46, XX – normal female
– 46, XY – normal male
• G‐banded chromosomes are identified by band pattern.
Chromosomes as seen at metaphase during cell division
Telomere
DNA and protein cap
Ensures replication to tip
Tether to nuclear membrane
Light bands
Replicate early in S phase
Less condensed chromatin
Transcriptionally active
Gene and GC rich
Centromere
Short arm
p (petit)
Joins sister chromatids
Essential for chromosome segregation at cell division
100s of kilobases of repetitive DNA: some non‐specific, some chromosome specific
Long arm
q
Telomere
Dark (G) bands
Replicate late
Contain condensed chromatin
AT rich
A pair of homologous chromosomes (number 1) as seen at metaphase
Locus (position of a gene or DNA marker)
Allele (alternative form of a gene/marker)
Metaphase chromosomes
Karyotyped chromosomes
Normal Female Karyotype (46, XX)
(G Banding)
Normal Female Karyotype
(High‐Resolution G Banding)
Banding patterns on human mitotic chomosomes
due to regions of condensed chomatin (darker - G
bands) and less condensed chromatin (lighter - R bands)
human chromosome 4 at varying resolutions due to exact mitotic
stage, (or degrees of spreading - squashing - stretching)
Human chromosome banding patterns seen on light microscopy
Chromosome 1
Different chromosome banding resolutions can resolve bands, sub-bands and sub-sub-bands
Chromosomes
Gene for cystic fibrosis (chromosome 7)
Gene for sickle cell disease (chromosome 11)
• Chromosomes are made of DNA. • Each contains genes in a linear order.
• Human body cells contain 46 chromosomes in 23 pairs – one of each pair inherited from each parent
• Chromosome pairs 1 – 22 are called autosomes.
• The 23rd pair are called sex chromosomes: XX is female, XY is male.
Total Genes On Chromosome: 723
373 genes in region marked red, 20 are shown FZD2
AKAP10
ITGB4
KRTHA8
WD1
SOST
Genes are arranged in linear order on chromosomes
MPP3
MLLT6
STAT3
BRCA1
GFAP
NRXN4
NSF
NGFR
Chromosome 17
source: Human Genome Project
CACNB1
HOXB9
HTLVR
ABCA5
CDC6
ITGB3
breast cancer 1, early onset
Hundreds of genes are encompassed within a single G‐band.
Therefore, most constitutional chromosome
abnormalities are associated with multiple
congenital anomalies.
Deletion of a single gene cannot be detected by G‐banding.