Flow of Genetic Information 1

8/13/2019 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

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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

53

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.

Flow of Genetic Information 1

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