©2000 Timothy G. Standish John 5:37 37And the Father himself, which hath sent me, hath borne...

©2000 Timothy G. Standish

John 5:37

37 And the Father himself, which hath sent me, hath borne witness of me. Ye have neither heard his voice at any time, nor seen his shape.


Structure and AnalysisStructure and Analysisofof

DNA and RNADNA and RNATimothy G. Standish, Ph. D.

DNA

mRNA

Transcription

IntroductionIntroduction

The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology

Cell

Polypeptide(protein)

TranslationRibosome



OutlineOutline1 How we know DNA is the genetic

material2 Basic structure of DNA and RNA3 Ways in which DNA can be

studied and what they tell us about genomes


Transformation Of BacteriaTransformation Of BacteriaTwo Strains Of Two Strains Of StreptococcusStreptococcus

Capsules

Smooth Strain(Virulent)

Rough Strain(Harmless)


Experimental

Transformation Of BacteriaTransformation Of BacteriaThe Griffith’s 1928 ExperimentThe Griffith’s 1928 Experiment

- Control

+ Control

- Control

OUCH!


Avery, MacLeod and McCartyAvery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty repeated

Griffith’s 1928 experiment with modifications designed to discover the “transforming factor”

After extraction with organic solvents to eliminate lipids, remaining extract from heat killed cells was digested with hydrolytic enzymes specific for different classes of macro molecules:

NoNuclease

YesProtease

Transformation?Enzyme

YesSaccharase


The Hershey-Chase The Hershey-Chase ExperiementExperiement

The Hershey-Chase experiment showed definitively that DNA is the genetic material

Hershey and Chase took advantage of the fact that T2 phage is made of only two classes of macromolecules: Protein and DNA

HOH

P

O

OH

HO ONH2

Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur.

H

OH

OH2N CC

CH2

SH

H

OH

OH2N C

CH3

C

CH2

CH2

S Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous.

CysteineMethionine

Using SUsing S3535Bacteria grown in normal non-radioactive media

T2 grown in S35 containing media incorporate S35 into their proteins

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

Is protein the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe supernatant is radioactive, but the pellet is not.

Did protein enter the bacteria?

Using PUsing P3232Bacteria grown in normal non-radioactive media

T2 grown in P32 containing media incorporate P32 into their DNA

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

Is DNA the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe pellet is radioactive, but the supernatant is not.

Did DNA enter the bacteria?

OH

OCH2

Sugar

H

HH

A NucleotideA NucleotideAdenosine Mono Phosphate (AMP)Adenosine Mono Phosphate (AMP)

OH

NH2

N

N N

N

BaseP

O

OH

HO O

Phosphate

2’3’

4’

5’

1’Nucleotide

Nucleoside

H+

-

Pyrimidines

NH2

O

N

N NH

N

Guanine

N

N

Adenine

N

N

NH2

N O

NH2

N O

NH2

NCytosine

Purines

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

NO

H

NO

N

NH C

ytosine

H

O

NN

N

N

N

H

H

Guanine -+

+

+

-

-

Base PairingBase PairingGuanine And CytosineGuanine And Cytosine

CH 3

N

O

N

ON

H+

- ThymineN

NN

N

HN H

-

+Adenine

Base PairingBase PairingAdenine And ThymineAdenine And Thymine

Base PairingBase PairingAdenine And CytosineAdenine And Cytosine

NO

H

NO

N

NH C

ytosine-

+

-

N

NN

N

HN

H

-

+

Adenine

Base PairingBase PairingGuanine And ThymineGuanine And Thymine

CH

3

NO

N

O

NH+

- Thymine

H

O

NN

N

N

N

H

H

Guanine

+

+

-

SU

GA

R-P

HO

SP

HA

TE

BA

CK

BO

NE

H

P

O

HO

O

O

CH2

HOH

P

O

O

HO

O

O

CH2

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NH2N

NH

N

N

N O

NH2

N

B A

S E

S

DDNNAA

OH

P

O

HO

O

O

CH2

HO

O

H 2N

NHN N

N H

H

P HO

O

O

CH2

OO

N

O

H 2N

NH

H2O

H OH

P

O

HO

O

O

CH2

CH 3

O

O

HNN

H2O

5’Phosphate group

3’Hydroxyl group

5’Phosphategroup

3’Hydroxyl group


The Watson - Crick The Watson - Crick Model Of DNAModel Of DNA

3.4 nm1 nm

0.34 nm

Majorgroove

Minorgroove

A T

T AG C

C G

C GG C

T A

A T

G CT A

A TC G

--

-

-

---

--

--

--

-

--

--

-

---

--

--

--

-

-


Forms of the Double HelixForms of the Double Helix

0.26 nm

2.8 nmMinorgroove

Majorgroove

C GA T

T AG C

C G

G CT A

A T

G CT A

A TC G

A T

G C

1.2 nm

A DNA

1 nm

Majorgroove

Minorgroove

A T

T AG C

C G

C G

G CT A

A T

G CT A

A TC G

0.34 nm

3.9 nm

B DNA

+34.7o Rotation/Bp11 Bp/turn

-30.0o Rotation/Bp12 Bp/turn

+34.6o Rotation/Bp10.4 Bp/turn

C GG C

G CC G

C G

G CG C

G CC G

G CC G

0.57 nm

6.8 nm

0.9 nm

Z DNA


C-DNA:– Exists only under high dehydration conditions– 9.3 bp/turn, 0.19 nm diameter and tilted bases

D-DNA:– Occurs in helices lacking guanine– 8 bp/turn

E-DNA:– Like D-DNA lack guanine– 7.5 bp/turn

P-DNA:– Artificially stretched DNA with phosphate groups found inside

the long thin molecule and bases closer to the outside surface of the helix

– 2.62 bp/turn

Even More Forms Of DNAEven More Forms Of DNA

B-DNA appears to be the B-DNA appears to be the most common form most common form in in vivovivo. However, under . However, under some circumstances, some circumstances, alternative forms of DNA alternative forms of DNA may play a biologically may play a biologically significant role.significant role.


Denaturation and RenaturationDenaturation and Renaturation Heating double stranded DNA can overcome the

hydrogen bonds holding it together and cause the strands to separate resulting in denaturation of the DNA

When cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures

TACTCGACATGCTAGCACATGAGCTGTACGATCGTG

Double stranded DNA

TACTCGACATGCTAGCACATGAGCTGTACGATCGTG

Double stranded DNA

Renaturation

TACTCGACATGCTAGCAC

ATGAGCTGTACGATCGTG

Denatured DNA

Denaturat

ion

Single stranded DNA


Denaturation and RenaturationDenaturation and Renaturation DNA with a high guanine and cytosine content has

relatively more hydrogen bonds between strands This is because for every GC base pair 3 hydrogen bonds

are made while for AT base pairs only 2 bonds are made Thus higher GC content is reflected in higher melting or

denaturation temperature

Intermediate melting temperature

Low melting temperature High melting temperature67 % GC content -

TGCTCGACGTGCTCGACGAGCTGCACGAGC

33 % GC content -

TACTAGACATTCTAGATGATCTGTAAGATC

TACTCGACAGGCTAGATGAGCTGTCCGATC

50 % GC content -


Determination of GC ContentDetermination of GC Content Comparison of melting temperatures can be used to

determine the GC content of an organisms genome To do this it is necessary to be able to detect

whether DNA is melted or not Absorbance at 260 nm of DNA in solution provides

a means of determining how much is single stranded Single stranded DNA absorbs 260 nm ultraviolet

light more strongly than double stranded DNA does although both absorb at this wavelength

Thus, increasing absorbance at 260 nm during heating indicates increasing concentration of single stranded DNA


Determination of GC ContentDetermination of GC Content

OD260

0

1.0

65 70 75 80 85 90 95

Temperature (oC)

Tm = 85 oCTm = 75 oC

Double stranded DNA

Single stranded DNA

Relatively low GC content

Relatively high GC content

Tm is the temperature at which half the DNA is melted


GC Content Of Some GenomesGC Content Of Some Genomes

Phage T7 48.0 %

Organism % GC

Homo sapiens 39.7 %

Sheep 42.4 %

Hen 42.0 %

Turtle 43.3 %

Salmon 41.2 %

Sea urchin 35.0 %

E. coli 51.7 %

Staphylococcus aureus 50.0 %

Phage 55.8 %


HybridizationHybridization The bases in DNA will only pair in very specific ways, G

with C and A with T In short DNA sequences, imprecise base pairing will not be

tolerated Long sequences can tolerate some mispairing only if -G of

the majority of bases in a sequence exceeds the energy required to keep mispaired bases together

Because the source of any single strand of DNA is irrelevant, merely the sequence is important, DNA from different sources can form double helix as long as their sequences are compatible

Thus, this phenomenon of base pairing of single stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands which came from different sources


HybridizationHybridization

DNA from source “Y”

TACTCGACAGGCTAG

CTGATGGTCATGAGCTGTCCGATCGATCAT

DNA from source “X”

TACTCGACAGGCTAG

HybridizationHybridization


HybridizationHybridization Because DNA sequences will seek out and hybridize with

other sequences with which they base pair in a specific way much information can be gained about unknown DNA using single stranded DNA of known sequence

Short sequences of single stranded DNA can be used as “probes” to detect the presence of their complimentary sequence in any number of applications including:– Southern blots– Northern blots (in which RNA is probed)– In situ hybridization– Dot blots . . .

In addition, the renaturation or hybridization of DNA in solution can tell much about the nature of organism’s genomes


Reassociation KineticsReassociation Kinetics An organism’s DNA can be heated in solution

until it melts, then cooled to allow DNA strands to reassociate forming double stranded DNA

This is typically done after shearing the DNA to form many fragments a few hundred bases in length

The larger and more complex an organisms genome is, the longer it will take for complimentary strands to bum into one another and hybridize

Reassociation follows second order kinetics


Reassociation KineticsReassociation Kinetics The following equation describes the second order

rate kinetics of DNA reassociation:

11 + kCot

=CCo

Concentration of single stranded DNA after time t

Initial concentration of single stranded DNA

Second order rate constant (the important thing is that it is a constant)

Co (measured in moles/liter) x t (seconds). Generally graphed on a log10 scale.

Cot1/2 is the point at which half the initial concentration of single stranded DNA has annealed to form double-stranded DNA


Reassociation KineticsReassociation Kinetics

Fraction remaining single-stranded (C/Co)

0

0.5

10-4 10-3 10-2 10-1 1 101 102 103 104

Cot (mole x sec./l)

1.0Higher Cot1/2 values indicate greater genome complexityCot1/2


Reassociation KineticsReassociation Kinetics

0.5

Fraction remaining single-stranded (C/Co)

010-4 10-3 10-2 10-1 1 101 102 103 104

Cot (mole x sec./l)

1.0

Eukaryotic DNA

Prokaryotic DNA

Repetitive DNA Unique

sequence complex DNA


Repetitive DNARepetitive DNAOrganism % Repetitive DNA

Homo sapiens 21 %

Mouse 35 %

Calf 42 %

Drosophila 70 %

Wheat 42 %

Pea 52 %

Maize 60 %

Saccharomycetes cerevisiae 5 %

E. coli 0.3 %


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©2000 Timothy G. Standish John 5:37 37And the Father himself, which hath sent me, hath borne...

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