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BCH 550
A.S Warsy
Topics to be covered
• Mutations
• Gene interaction: from genes to
phenotypes, diagnostic test for alleles,
interactions between the alleles of one
gene.
• DNA recombination, rearrangements:
Independent Assortment, CrossingOver, Linkage Maps, Mitotic CrossingOver, The Mechanism of Crossing-Over
, etc
The Central Dogma
The Human Chromosome
No. of Chromosomes in
Different Organisms
•
•
•
•
•
•
•
Bacteria
Fruit fly
Garden Pea
Yeast
Corn
Frog
Cat
1
8
14
16
26
26
38
•
•
•
•
•
•
•
Hydra
Fox
Mouse
Rat
Rabbit
Human
Chicken
34
34
40
42
44
46
78
HUMAN GENOME
23 pairs of chromosomes
3 x 109 bp
>90% non-coding
sequence
< 10% coding
sequence
Genes
2000-4000 genes/average chromosome
Gene per 30-60 kb
MITOCHONDRIAL DNA
16.6 kb double stranded circular
Encodes
37 mitochondrial genes
13 encode for polypeptides
involved in respiratory
chain
22 tRNA
24
2 rRNA
Genetic Material in the Living Cells
* Cells contain a nucleus surrounded
by a nuclear membrane in eukaryotic
cells, and a nuclear region in the
prokaryotic cells.
* In a non-dividing cell the nucleus is
filled with a thread-like material
known as "chromatin".
* Chromatin is made up of DNA and
proteins (mainly histones and some
non-histone acidic proteins).
Chromosomes
(Metaphase)
Chromatin
Cell Division
Cell Division
Mitosis:
Meiosis:
- Occurs in Somatic cells.
- Occur in cells of germ line.
- Division by which the body
- Only once in generation.
- Results in the formation of
haploid, reproductive cell
(gametes: ova and sperms).
- Chromosomes duplicates
followed by 2 cell divisions
resulting in cells with
half the number of
chromosomes (haploid).
grows, differentiates and repairs.
- Results in two identical daughter
Diploid cells with genes identical
to parent cells.
- Chromosomes are first doubled,
followed by cell division in which
the number in each cell is halved
(diploid).
Chromatin
Mitosis
G2
G1
S
Go
The Cell Cycle
Chromosomes
• Before
cell
division,
the
chromatin multiplies (replicate),
loses the relatively homogenous
appearances and condenses to
form rod like structures known
as "chromosomes“
(chroma:
colour; somes : bodies) as they
stain
deeply
with
certain
biological dyes.
• "Genes" are units of genetic
information present on the DNA
in
the
chromosomes
and
chromatin.
The Cell Cycle
G2
S
Mitosis
G1
Interphase and Metaphase
Chromosomes
Pair of identical
Interphase
Chromosomes
Centromere
DNA Replication
Metaphase
Chromosomes
Two setsone for each
daughter cell
Separation in Mitosis
The Normal Human Chromosomes
* Normal human cells contain 23
pairs
of
homologous
chromosomes:
- 22 pairs of autosomes
(numbered
as
1-22
in
decreasing order of size)
- 1 pair of sex chromosomes.
* Autosomes are the same in
males and females
* Sex chromosomes are:
- XX in females
- XY in males.
* Both X are homologous. Y is
much smaller than X and has
only a few genes.
p
q
Chromosomes
* One member of each chromosome pair is
derived from each parent.
* Somatic cells have diploid complement of
chromosomes i.e. 46.
* Germ cells (Gametes: sperm and ova) have
haploid complement i.e 23.
* The chromosomes of dividing cells are most
readily analyzed at the `metaphase' or
prometaphase stage of mitosis .
Chromosome Structure
Telomere
p
Centromere
q
Chromatids
* At the metaphase stage each
chromosome consists of two
chromatids joined at the
centromere or primary
constriction
• The centromere divides
chromosomes into short (p i.e.
petit) and long (q e.g. g=grand)
arms. The tip of each
chromosome is called telomere.
• The exact function of the
centromere is not clear, but it is
known to be responsible for the
movement of the chromosomes
at cell division.
Schematic Presentation of Eucaryotic
Chromosome Structure
Chromosome Arm
‘p’
Replication origins
Telomere
Chromosome Arm
‘q’
Centromere
Replication origins
Telomere
Classification Of Chromosomes
* Morphologically chromosomes are classified
according to the position of the centromere as
(i) metacentric,
(ii) sub-metacentric,
(iii) acrocentric,
(iv) telocentric (with centromere at one end.
This occurs in other species, but not in man).
Metacentric
Chromosomes
p
q
Sub-Metacentric
Chromosomes
Centromere
Telomeres
* Acrocentric chromosomes (13, 14, 15, 21 and
22) have a small mass of chromatin known
as satellite attached to their short arm by
narrow stalks (secondary constrict).
* The stakes contain genes for 18S and 28S
rRNA.
Satellite
Stalk
Staining methods for cytogenetic
analysis of chromosomes
• Several staining methods for cytogenetic
analysis of chromosomes.
• Each stain produces specific banding
patterns known as "Chromosome Banding"
- G banding,
- Q banding,
R banding,
C banding.
• The pattern is specific for each chromosome,
and is the characteristic utilized to identify
each chromosome.
Staining Methods for Cytogenetic
Analysis
G Banding:
Treat with trypsin and then with Geimsa Stain.
R Banding:
Heat and then treat with Geimsa Stain.
Q Banding:
Treat with Quinicrine dye. Fluorescent bands.
C Banding:
Staining of the Centromere.
The G-Banding
Pattern of
Chromosomes
DNA CONTENT OF HUMAN CHROMOSOME
Chromosome
% of total
Amount
length
(Mb) 1
8.3
Amount of
DNA (Mb)
250
Chromosome
% of total
13
length
3.6
of DNA
110
2
7.9
240
14
3.5
105
3
6.4
190
15
3.3
100
4
6.1
180
16
2.8
85
5
5.8
175
17
2.7
80
6
5.5
165
18
2.5
75
7
5.1
155
19
2.3
70
8
4.5
135
20
2.1
65
9
4.4
130
21
1.8
55
10
4.4
130
22
1.9
60
11
4.4
130
X
4.7
140
12
4.1
120
Y
2.0
60
Each species has a characteristic gene
map i.e. the chromosomal location of
the genes, and it is the same in all
normal individuals of each species
Centromere and Telomere structure
(contd)
• Relative position of centromere is
characteristics of each chromosome.
• Centromere is an A-T regions of about 130 bp
& binds some proteins with high affinity
(kinetochrome). It provides anchor for mitotic
spindle formation.
• The ends of chromosomes are telomeres.
– contain short, repeat TG-rich element e.g.
human telomere
5’ – TTAGGG-3’ extending for several
kilobases
SCHEMATIC STRUCTURE OF A HUMAN METAPHASE
CHROMOSOME
Telomere
(TTAGGG)
T
T
A
.
.
A
A
T
.
.
Centromere
(AT rich region)
.
Telomere
TELOMERASE
• Telomerase is an enzyme responsible for
telomere synthesis and maintaining length of
the telomeres.
• Telomere shortening is associated with:
– Malignant transformation.
– Aging
Composition of Chromosome
DNA
Histones
(Major proteins)
Non-histone
(Small amounts)
DNA packing in the
Chromosomes
Composition of Nucleosomes
DNA
Histones
Nucleosome (10 nm diameter):
8 histones in bead and 1 outside.
Each bead: is surrounded by 140 bp
DNA and there are 60 bp in the
linker region.
Space between beads is about 14 nm.
Summary of Chromosome
Folding
Level of folding
Consists of
Base pair/turn
DNA double helix
Nucleotides
10
Nucleosomes
200 bp each
100
30 Nanometer
fiber
6 Nucleosomes
/turn
1,200
Loops
50 Solenoids/loop
60,000
Miniband
18 loops
1,080,000
Chromatid
1,000,000
minibands
Structure of Nucleic Acids
Nucleotide
DNA
Phosphate
Nitrogenous Bases
Polynucleotides
Sugar
RNA
Nucleoside
Nucleotides
De-oxyriboguanosine
triphosphate
Riboguanosine
triphosphate
OH
OH
OH
Polynucleotide
formation
5’
Phosphodiester
bond
OH OH
3’
OH OH
DNA Double strand
ssDNA
dsDNA
Nitrogenous bases in DNA
Purines
Pyrimidines
DNA Double Helical Structure
(Watson and Crick)
2.0 nm
Major
groove
Pitch
3.6 nm
10 bp
Minor groove
Major Groove
Minor Groove
Complementary bases:
A-T
G-C
Chargaff’s Ratio
A = T; G =C
A/T = 1; G/C = 1
A+G = T+C
A+G+C+T = 1
This makes it possible to calculate the composition of DNA
if the concentration of one of the nucleotides is known.
e.g if G= 20 %.
then C= 20%
and T=30%.
and A=30%.
Different Structural forms of Nuclear DNA
A
B
Z
B form= Rt handed;Most stable form; 10 bp/turn; Pitch= 3.4 nm; Normal form
A form= Rt handed;Formed in dehydrated state; 11 bp/turn; Pitch= 2.3nm
Z form= Left handed; 12 bp/turn; Pitch= 3.8 nm; Some nucleotide sequences
form Z form; Short Z sequences may occur in cells. May be involved
in regulation of gene expression.
Histones
•
•
•
•
Basic proteins---rich in Arg and Lys.
Positively charged.
Five main types: H1, H2a, H2b, H3 and H4
Two molecules each of H2a, H2b, H3 and
H4 are present in nucleosomes. H1 is
present outside the nucleosome. H1 is Lys
rich.
• Salt bridges between positively charged
histones and negatively charges DNA play
a major role in stabilizing DNA-histone
complex.
Characteristics of Histones
Name
Mol.Wt
H1
Lys/Arg
ratio
20
Copies /
H2a
1.2
14,500
1 (not in
bead)
2 (in bead)
H2b
2.5
13,700
2 (in bead)
H3
0.7
15,300
2 (in bead)
H4
0.8
11,300
2 (in bead)
nucleosome
21,000
Types of Histone Modification
Acetylation
Methylation
Phosphorylation
Covalent linkage
ADP-Ribosylation
to Ubiquitin
(a nuclear protein)
POSSIBLE ROLES OF MODIFIED
HISTONES
• Acetylation of histone H3 and H4 is associated
with activation or inactivation of gene
transcription.
• Acetylation of core histones is associated with
chromosomal assembly during DNA replication.
• Phosphorylation of histone H1 is associated with
condensation of chromosome during the
replication cycle.
• ADP-ribosylation of histones is associated with
DNA repair.
ROLE OF NUCLEOPLASMIN IN
NUCLEOSOME FORMATION
• As histones are strong cations and DNA is a
strong anion, they can bind by salt bridges.
This non-specific interaction would present
nucleosome formation.
• Nucleoplasmin is an anionic pentameric
protein binds to histone octamer, preventing
histones to adhere non-specificity to DNA
surface.
– maintain environment conducive to
assembly of nucleosomes.
• As nucleosomes assemble, nucleoplasmin is
released from histones.
• Exact mechanism not known.
Mitochondrial DNA
Several thousand mitochondria/ cell, each has its own mitochondrial DNA (mtDNA).
mtDNA is circular.mtDNA genome is very compact with very little repetitive sequences.
mtDNA structure is the same as that of nuclear DNA, except the ends are joined.
Genetic code is slightly different then nuclear DNA.
Mitochondria of fertilized zygote are inherited from oocytes.
mtDNA has higher rate of mutations then nuclear DNA.
Accumulation of mutations in mtDNA may be responsible for the somatic effects seen with
ageing.
Normally all mtDNA from different mitochondria in a cell are alike- homoplasmy, but if mutation
occurs in some, then a state of heteroplasmy
exists.
Mt DNA
Mitochondrial
Inheritance
Variation in presentation of Mt
diseases