©2000 Timothy G. Standish Isaiah 33:22 22For the Lord is our judge, the Lord is our lawgiver, the...

©2000 Timothy G. Standish

Isaiah 33:22

22 For the Lord is our judge, the Lord is our lawgiver, the Lord is our king; he will save us.


Molecular Basis Molecular Basis Of InheritanceOf Inheritance

Timothy G. Standish, Ph. D.


OutlineOutline

1How we know DNA is the genetic material

2When DNA is replicated

3How DNA is replicated

4How DNA is maintained


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 ExperimentThe Griffith 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”

Extracts from heat-killed cells were digested with hydrolytic enzymes specific for different classes of macromolecules:

NoNuclease

YesProtease

YesLipase

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?


When DNA Replication When DNA Replication OccursOccurs

Typically DNA replication only occurs when cells are preparing to divide (there are some exceptions)

The cell lifecycle is well defined and can be divided into four stages:– Gap 1 (G1) - The growth phase in which most cells are found

most of the time– Synthesis (S) - During which new DNA is synthesized– Gap 2 (G2) - The period during which no transcription or

translation occurs and final preparations for division are made– Mitosis - Cell division


G1G1

MM

G2G2

SS

The Cell LifecycleThe Cell LifecycleGap 1 - Doubling of cell size. Regular cellular activities. Transcription and translation etc.

Synthesis of DNA - Regular cell activities cease and a copy of all nuclear DNA is made

Gap 2 - Final preparation for division

Mitosis - Cell division

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

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

Purines

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


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

--

-

-

---

--

--

--

-

--

--

-

---

--

--

--

-

-


DNA Replication:DNA Replication:How We KnowHow We Know

There are three ways in which DNA could be replicated:

+

NewOld

+

Old

N

ewOld

N

ew

+

Old +

N

ewOld +

N

ew

OldConservative - Old double-stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together

OldSemiconservative - Old strands serve as templates for new strands resulting in double-stranded DNA made of both old and new strands

Old

Dispersive - In which sections of the old strands are dispersed in the new strands


The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

The Meselson-Stahl experiment demonstrated that replication is semiconservative

This experiment took advantage of the fact that nucleotide bases contain nitrogen

Thus DNA contains nitrogen

OH

HOH

P

O

HO ONH2

N N

N N

The most common form of Nitrogen is N14 with 7 protons and 7 neutrons

N15 is called “heavy nitrogen” as it has 8 neutrons thus increasing its mass by 1 atomic mass unit


After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge

Disper

sive m

odel

predict

ion

Conservativ

e

model pre

diction

Semico

nservativ

e

model pre

diction

The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

Prediction after 2 or more replications

Bacteria grown in N15 media for several replications

Transfer to normal N14 media

X

X

XThe conservative and dispersive models make predictions that do not come true thus, by deduction, the semi-conservative model must be true.


Stages of ReplicationStages of Replication Replication can be divided into three stages:

1 Initiation - When DNA is initially split into two strands and polymerization of new DNA is started

2 Elongation - When DNA is polymerized

3 Termination - When the new strands of DNA are completed and some finishing touches may be put on the DNA

Both elongation and termination may involve proofreading of the DNA ensuring that mutations are not incorporated into newly formed DNA strands


Tools of ReplicationTools of ReplicationEnzymes are the tools of replication:DNA Polymerase - Matches the correct

nucleotides then joins adjacent nucleotides to each other

Primase - Provides an RNA primer to start polymerization

Ligase - Joins adjacent DNA strands together (fixes “nicks”)


More Tools of ReplicationMore Tools of Replication

Helicase - Unwinds the DNA and melts itSingle-Strand Binding Proteins - Keep the DNA

single stranded after it has been melted by helicaseTopisomerase - Relieves torsional strain in the

DNA moleculeTelomerase - Finishes off the ends of DNA

strands


InitiationInitiation Initiation starts at specific DNA sequences called origins

(Ori C = origin in E. coli chromosomes) Long linear chromosomes have many origins First the origin melts (splits into two single strands of

DNA) Next primers are added Finally DNA polymerase recognizes the primers and

starts to polymerize DNA 5’ to 3’ away from the primers


Initiation - Forming the Initiation - Forming the Replication EyeReplication Eye

3’ 5’

3’5’5’

5’

3’

3’

Origin of Replication

5’

3’

3’

5’

5’3’

5’

5’

5’

3’

3’3’


Large Linear Chromosomes Have Large Linear Chromosomes Have Many Origins Of ReplicationMany Origins Of Replication

5’3’

3’5’

5’3’

3’5’

3’5’

5’3’

3’5’

5’3’

3’5’

5’3’

Origins of Replication


Leading StrandLeading Strand

Lagging StrandLagging Strand

3’

5’3’

5’

Extension - The Replication ForkExtension - The Replication Fork5’

5’5’3’

3’

5’3’3’

5’

Single-strand binding proteins - Prevent DNA from re-annealing

DNA Polymerase

Okazaki fragment

RNA Primers

Primase - Makes RNA primers

5’3’

5’

Helicase - Melts DNA


Extension - Okazaki FragmentsExtension - Okazaki Fragments

The nick is removed when DNA ligase joins (ligates) the DNA fragments.

3’ 5’5’ 3’

RNA PrimerOkazaki Fragment

3’ 5’5’ 3’

RNA PrimerRNA and DNA Fragments

Nick

3’ 5’5’ 3’

RNA Primer

DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction.

DNA Polymerase falls off leaving a nick.

DNAPol.

DNAPol.

Ligase


MutationMutationWhen Mistakes Are MadeWhen Mistakes Are Made

5’ 3’

5’

DNAPol.

5’

5’ 3’

5’ 3’

5’

DNAPol.

DNAPol.

Mism

atch

3’ to 5’ Exonuclease activity


Thim

ine

Dimer

MutationMutationExcision RepairExcision Repair

3’

5’ 3’

5’

5’ 3’

3’ 5’

Endo-Nuclease


5’ 3’

3’ 5’

5’ 3’

3’ 5’


3’

5’ 3’

5’

Endo-Nuclease

NicksDNAPol.


5’ 3’

3’ 5’


3’

5’ 3’

5’

5’ 3’

3’ 5’

DNAPol.

Endo-Nuclease


5’ 3’

3’ 5’

5’ 3’

3’ 5’


3’

5’ 3’

5’

DNAPol.

Ligase

Endo-Nuclease

Nicks

Nick

Ligase



Problem 1Problem 1 Question:

– If an organism’s DNA is 32 % adenine, what percent guanine, thymine, and cytosine are found in the DNA?

Answer:– As adenine always pairs with thymine, there must be 32 %

thymine– % GC = 100 % - (T% + A%) = 100 % - (32 % + 32 %) = 36 % – The proportion of guanine to cytosine has to be equal as they pair

with one another thus G and C % = 36 % / 2 = 18 %– G = 18 %, T = 32 % and C = 18 %


Problem 2Problem 2 Question:

– Given the following sequence of one strand of DNA, write out the complementary strand.

–5’AATACGCGATGCTGGTATC3’ Answer:

–5’AATACGCGATGCTGGTATC3’–3’TTATGCGCTACGACCATAG5’

Date post:	02-Jan-2016
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©2000 Timothy G. Standish Isaiah 33:22 22For the Lord is our judge, the Lord is our lawgiver, the...

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