Transcript bioc801a

MOLECULAR GENETICS
CLASS SESSIONS:
1. DNA, Genes, Chromatin
2. DNA Replication, Mutation, Repair
3. RNA Structure and Transcription
4. Eukaryotic Transcriptional Regulation
5. CLASS DISCUSSION – GENETIC DISEASES
6. RNA Processing
7. Protein Synthesis and the Genetic Code
8. Protein Synthesis and Protein Processing
9. CLASS DISCUSSION – GENETIC DISEASES
10. DNA Cloning and Isolating Genes
THE FLOW OF GENETIC INFORMATION
2
DNA
3
RNA
PROTEIN
1
DNA
1. REPLICATION
(DNA SYNTHESIS)
2. TRANSCRIPTION (RNA SYNTHESIS)
3. TRANSLATION
(PROTEIN SYNTHESIS)
DNA Structure and Chemistry
a). Evidence that DNA is the genetic information
i). DNA transformation – know this term
ii). Transgenic experiments – know this process
iii). Mutation alters phenotype – be able to define
genotype and phenotype
b). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
ii). Structure of the DNA double helix
iii). Complementarity of the DNA strands
c). Chemistry of DNA
i). Forces contributing to the stability of the double helix
ii). Denaturation of DNA
Structures of the bases
Purines
Pyrimidines
Adenine (A)
Thymine (T)
5-Methylcytosine (5mC)
Guanine (G)
Cytosine (C)
Nucleoside
[structure of deoxyadenosine]
Nucleotide
Nomenclature
Base
Nucleoside
+deoxyribose
Purines
adenine
guanine
adenosine
guanosine
hypoxanthine
inosine
Pyrimidines
thymine
cytosine
thymidine
cytidine
+ribose
uracil
Nucleotide
+phosphate
uridine
ii). Structure
the
Structure
of theofDNA
DNA doublechain
helix
polynucleotide
5’
3’
• polynucleotide chain
• 3’,5’-phosphodiester bond
A-T base pair
Hydrogen bonding of the bases
G-C base pair
Chargaff’s rule: The content of A equals the content of T,
and the content of G equals the content of C
in double-stranded DNA from any species
Double-stranded DNA
5’
3’
Major groove
Minor groove
“B” DNA
3’
5’
3’
5’
Chemistry of DNA
Forces affecting the stability of the DNA double helix
• hydrophobic interactions - stabilize
- hydrophobic inside and hydrophilic outside
• stacking interactions - stabilize
- relatively weak but additive van der Waals forces
• hydrogen bonding - stabilize
- relatively weak but additive and facilitates stacking
• electrostatic interactions - destabilize
- contributed primarily by the (negative) phosphates
- affect intrastrand and interstrand interactions
- repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins)
Charge repulsion
Stacking interactions
Charge repulsion
Model of double-stranded DNA showing three base pairs
Denaturation of DNA
Double-stranded DNA
Strand separation
and formation of
single-stranded
random coils
Extremes in pH or A-T rich regions
high temperature denature first
Cooperative unwinding
of the DNA strands
Electron micrograph of partially melted DNA
Double-stranded, G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
• A-T rich regions melt first, followed by G-C rich regions
Hyperchromicity
Absorbance
Absorbance maximum
for single-stranded DNA
Absorbance
maximum for
double-stranded DNA
220
260
300
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
Percent hyperchromicity
DNA melting curve
100
50
0
50
70
90
Temperature oC
• Tm is the temperature at the midpoint of the transition
Percent hyperchromicity
Tm is dependent on the G-C content of the DNA
E. coli DNA is
50% G-C
50
60
70
80
Temperature oC
Average base composition (G-C content) can be
determined from the melting temperature of DNA
Genomic DNA, Genes, Chromatin
a). Complexity of chromosomal DNA
i). DNA reassociation
ii). Repetitive DNA and Alu sequences
iii). Genome size and complexity of genomic DNA
b). Gene structure
i). Introns and exons
ii). Properties of the human genome
iii). Mutations caused by Alu sequences
c). Chromosome structure - packaging of genomic DNA
i). Nucleosomes
ii). Histones
iii). Nucleofilament structure
iv). Telomeres, aging, and cancer
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
k2
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
Faster,
zippering
reaction to
form long
molecules
of doublestranded
DNA
DNA reassociation kinetics for human genomic DNA
% DNA reassociated
Cot1/2 = 1 / k2
k2 = second-order rate constant
Co = DNA concentration (initial)
t1/2 = time for half reaction of each
component or fraction
0
50
Kinetic fractions:
fast (repeated)
intermediate
(repeated)
Cot1/2
Cot1/2
slow (single-copy)
Cot1/2
100
I
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fast
intermediate
slow
I
I
I
log Cot
I
I
I
106 copies per genome of
a “low complexity” sequence
of e.g. 300 base pairs
high k2
1 copy per genome of
a “high complexity” sequence
of e.g. 300 x 106 base pairs
low k2
Type of DNA
% of Genome
Features
Single-copy (unique)
~75%
Includes most genes 1
Repetitive
Interspersed
~15%
Interspersed throughout genome between
and within genes; includes Alu sequences 2
and VNTRs or mini (micro) satellites
Highly repeated, low complexity sequences
usually located in centromeres
and telomeres
Satellite (tandem)
0
~10%
fast ~10%
2
intermediate
~15%
50
slow (single-copy)
~75%
100
1 Some
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Alu sequences are
about 300 bp in length
and are repeated about
300,000 times in the
genome. They can be
found adjacent to or
within genes in introns
or nontranslated regions.
I
genes are repeated a few times to thousands-fold and thus would be in
the repetitive DNA fraction
Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu sequences)
GCTGAGG
GCTGAGG
GCTGAGG
Tandem repeats (e.g., microsatellites)
TTAGGGTTAGGGTTAGGGTTAGGG
Genome sizes in nucleotide pairs (base-pairs)
plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human
genome is ~ 3 X 109 bp;
almost all of its complexity
is in single-copy DNA.
amphibians
reptiles
birds
The human genome is thought
to contain ~30,000 to 40,000 genes.
104
105
106
107
mammals
108
109
1010
1011
Gene structure
promoter
region
exons (filled and unfilled boxed regions)
+1
introns (between exons)
transcribed region
mRNA structure
5’
3’
translated region
The (exon-intron-exon)n structure of various genes
histone
total = 400 bp; exon = 400 bp
b-globin
total = 1,660 bp; exons = 990 bp
HGPRT
(HPRT)
total = 42,830 bp; exons = 1263 bp
factor VIII
total = ~186,000 bp; exons = ~9,000 bp
Properties of the human genome
Nuclear genome
• the haploid human genome has ~3 X 109 bp of DNA
• single-copy DNA comprises ~75% of the human genome
• the human genome contains ~30,000 to 40,000 genes
• most genes are single-copy in the haploid genome
• genes are composed of from 1 to >75 exons
• genes vary in length from <100 to >2,300,000 bp
• Alu sequences are present throughout the genome
Mitochondrial genome
• circular genome of ~17,000 bp
• contains <40 genes
Alu sequences can be “mutagenic”
Familial hypercholesterolemia
• autosomal dominant
• LDL receptor deficiency
From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.
LDL receptor gene
Alu repeats present within introns
4
5 6
Alu repeats in exons
unequal
crossing over
4
Alu
5
Alu
6
X
4
Alu
5
Alu
6
one product has a
deleted exon 5
4
(the other product is not shown)
Alu
6
Chromatin structure
EM of chromatin shows presence of
nucleosomes as “beads on a string”
Nucleosome structure
Nucleosome core (left)
• 146 bp DNA; 1 3/4 turns of DNA
• DNA is negatively supercoiled
• two each: H2A, H2B, H3, H4 (histone octomer)
Nucleosome (right)
• ~200 bp DNA; 2 turns of DNA plus spacer
• also includes H1 histone
Histones (H1, H2A, H2B, H3, H4)
• small proteins
• arginine or lysine rich: positively charged
• interact with negatively charged DNA
• can be extensively modified - modifications in
general make them less positively charged
Phosphorylation
Poly(ADP) ribosylation
Methylation
Acetylation
Hypoacetylation
by histone deacetylase (facilitated by Rb)
“tight” nucleosomes
assoc with transcriptional repression
Hyperacetylation
by histone acetylase (facilitated by TFs)
“loose” nucleosomes
assoc with transcriptional activation
Nucleofilament structure
Condensation and decondensation
of a chromosome in the cell cycle
Telomeres are protective
“caps” on chromosome
ends consisting of short
5-8 bp tandemly repeated
GC-rich DNA sequences,
that prevent chromosomes
from fusing and causing
karyotypic rearrangements.
Telomeres and aging
Metaphase chromosome
telomere
centromere
telomere structure
telomere
<1 to >12 kb
(TTAGGG)many
(TTAGGG)few
young
senescent
• telomerase (an enzyme) is required to maintain telomere length in
germline cells
• most differentiated somatic cells have decreased levels of telomerase
and therefore their chromosomes shorten with each cell division
Class Assignment (for discussion on Sept 9th)
Botchkina GI, et al.
“Noninvasive detection of prostate cancer by
quantitative analysis of telomerase activity.”
Clin Cancer Res. May 1;11(9):3243-3249, 2005
PDF of article is accessible on the website