Flow of Genetic Information 1
Transcript of Flow of Genetic Information 1
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UNIT 1
THE FLOW OF GENETIC INFORMATION
LECTURES:
1. DNA Structure and Chemistry2. Genomic DNA, Genes, Chromatin
3. DNA Replication, Mutation, Repair
4. RNA Structure and Transcription
5. Eukaryotic Transcriptional Regulation6. RNA Processing
7. Protein Synthesis and the Genetic Code
8. Protein Synthesis and Protein Processing
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THE FLOW OF GENETIC INFORMATION
DNA RNA PROTEIN
DNA
1 2 3
1. REPLICATION (DNA SYNTHESIS)2. TRANSCRIPTION (RNA SYNTHESIS)
3. TRANSLATION (PROTEIN SYNTHESIS)
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1. DNA Structure and Chemistry
a). Evidence that DNA is the genetic information
i). DNA transformationii). Transgenic experiments
iii). Mutation alters phenotype
b). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
ii). Structure of the DNA double helix3,5-phosphodiester bond
polarity of the polynucleotide chains
hydrogen bonding of the bases
specificity of base pairing
complementarity of the DNA strandsc). Chemistry of DNA
i). Forces contributing to the stability of the double helix
ii). Denaturation of DNA
hyperchromicitymelting curves and Tm
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i). DNA transformation: in vivo experiment
Mice are injected either with Type R, non-virulent
Streptococcus or with heat-killed, virulent Type S cells.
The mice are healthy.
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X
Mice are injected with both Type R, non-virulent and
heat-killed, Type S Streptococcus
DNA carrying genes fromthe virulent, heat-killed cells
transforms the non-virulent
bacterial cells, making them
lethal to the mice
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DNA transformation: in vitro experiment
Type R cells Type R colonies
Type S cells Type S colonies
Mixture of
Type R and Type S
colonies
Type R cells
+ DNAfrom
Type S cells
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Genotype:An organisms genetic constitution.
Phenotype:The observed characteristics of an organism,
as determined by the genetic makeup
(and the environment).
DNA from Type S cells (thus conferring the Type S genotype)
t ransformedType R cells into cells having the Type S phenotype
Listen to audio descriptions of genotype* andphenotype* (requires RealPlayer).
*Thanks to the NHGRI glossary of genetic terms.
http://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LCG/Audio/definition_of_genotype_277.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_phenotype_373.ramhttp://www.genome.gov/glossary.cfmhttp://www.genome.gov/glossary.cfmhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_phenotype_373.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LCG/Audio/definition_of_genotype_277.ram -
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ii). Transgenic experiments
Plasmid DNA carrying the
growth hormone gene
Injected into nucleus
of a fertilized mouse egg
Egg implanted into uterus
of surrogate mother mouse
Mother mouse gives
birth to transgenic mouse
Listen to audio descriptions of:
transgenic*
knockout*
mouse model*
(requires RealPlayer).
http://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_transgenic_292.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_knockout_325.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_mouse_model_330.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_mouse_model_330.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_knockout_325.ramhttp://www.nhgri.nih.gov/RealMediaMetaFiles/DIR/LGDR/Audio/definition_of_transgenic_292.ram -
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Mouse with growth
hormone transgene
Normal mouse
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iii). Mutation alters phenotype
Phenotypic differences between individuals
are due to differences between their genes
These differences have arisen by mutation of DNA
over many thousands of years
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Some inherited differences are less severe
rates of agingdrug responses
Others are more severedebilitating inherited diseases
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Thymine (T)
Guanine (G) Cytosine (C)
Adenine (A)
b). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
Purines Pyrimidines
5-Methylcytosine (5mC)
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[structure of deoxyadenosine]
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Nomenclature
Purines
adenine adenosineguanine guanosine
hypoxanthine inosine
Pyrimidinesthymine thymidine
cytosine cytidine
+ribose
uracil uridine
Nucleoside NucleotideBase +deoxyribose +phosphate
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polynucleotide chain
3,5-phosphodiester bond
ii). Structure of the
DNA double helix
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hydrogen bonding of the bases
A-T base pair
G-C base pair
Chargaffs 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
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base pairing during DNA synthesis
Parental DNA strands
Daughter DNA strands
base pairing during RNA synthesis
DNA that is over- or underwound is supercoiled
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DNA that is over- or underwound is supercoiled
positive supercoiling results from overwinding DNA
and normally occurs during DNA replication
negative supercoiling results from underwinding DNA
and normally occurs in the nucleosome
negative supercoiling can give rise to Z-DNA
Z-DNA is a left handed helix
with zigzagged (hence Z) phosphates
Z-DNA occurs where there are
alternating pyrimidines and purines (on one strand)
the transition of B- to Z-DNA isfacilitated by 5-methylcytosine
negative supercoiling may affect RNA synthesis
by promoting Z-DNA formation
by making it easier to separate the DNA strands
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5
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35carbon
3carbon
antiparallel polarity of the
polynucleotide chains
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nucleases hydrolyze phosphodiester bonds
Endonucleases cleave internally
and can cut on either side of a
phosphate leaving 5 phosphate
or 3 phosphate ends depending
on the particular endonuclease.
Exonucleases cleave at
terminal nucleotides.5
53
3e.g., proofreading exonucleases
e.g., restriction endonucleases
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c). Chemistry of DNA
i). 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)
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5
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3
Hydrophobic
core region
Hydrophilicphosphates
Hydrophilicphosphates
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Stacking interactions
Charge repulsion
Charger
epulsion
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Model of double-stranded DNA showing three base pairs
ii) D t ti f DNA
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ii). Denaturation of DNA
Double-stranded DNA
A-T rich regions
denature first
Cooperative unwinding
of the DNA strands
Strand separation
and formation ofsingle-stranded
random coils
Extremes in pH or
high temperature
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Electron micrograph of partially melted DNA
A-T rich regions melt first, followed by G-C rich regions
Double-stranded, G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
h h i it
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hyperchromicity
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
260
Absorbance
Single-stranded
Double-stranded
220 300
DNA melting curve
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100
50
0
7050 90
Temperature oC
Percenth
yperchromicit
y
DNA melting curve
Tmis the temperature at the midpoint of the transition
T i d d t th G C t t f th DNA
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average base composition (G-C content) can be
determined from the melting temperature of DNA
50
7060 80
Temperatureo
C
Tmis dependent on the G-C content of the DNA
Percenthyperchromicity
E. coli DNA,
which is 50% G-C,
has a Tmof 69oC.