evidence that dna can transform bacteria

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Evidence that DNA can transform bacteria

• Frederick Griffith (1928) – Streptococcus pneumoniae bacteria – transformation

• Mouse Experiment

• Experiment proved that transformation can happen

• Avery and colleagues (1944) – announced transformation agent was DNA


Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria?

Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:

Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by anunknown, heritable substance from the dead S cells.

EXPERIMENT

RESULTS

CONCLUSION

Living S(control) cells

Living R(control) cells

Heat-killed(control) S cells

Mixture of heat-killed S cellsand living R cells

Mouse dies Mouse healthy Mouse healthy Mouse dies

Living S cellsare found inblood sample.


Evidence that viral DNA can program cells

• Alfred Hershey and Martha Chase (1952) – bacteriophages or phages (viruses that infect bacteria) – discovered DNA is the genetic material NOT protein

• Blender experiment


Figure 16.3 Viruses infecting a bacterial cell

Phagehead

Tail

Tail fiber

DNA

Bacterialcell

100

nm


Additional evidence that DNA is the genetic material

• Erwin Chargaff (1947) – Chargaff’s rules – The equivalences for any given species between the number of A and T and G and C bases are equal.

• Analyzed DNA from different organisms

– Humans 30.3% of bases were A’s

– E. Coli 26% of bases were A’s


• Rosalind Franklin – (1950’s) – X-ray diffraction photo of DNA – helped Watson and Crick develop their model of DNA structure


Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA

(a) Rosalind Franklin Franklin’s X-ray diffractionPhotograph of DNA

(b)


Structure of DNA

• Watson & Crick – (1953) – 1 page paper in the British journal Nature “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic acids”


Figure 16.1 Watson and Crick with their DNA model


Figure 16.5 The structure of a DNA strandSugar-phosphate

backboneNitrogenous

bases

5 endO–

O P O CH2

5

4O–

HH

OH

H

H3

1H O

CH3

N

O

NH

Thymine (T)

O

O P O

O–

CH2

HH

OH

HH

HN

N

N

H

NH

H

Adenine (A)

O

O P O

O–

CH2

HH

OH

HH

H

H H

HN

NN

OCytosine (C)

O

O P O CH2

5

4O–

H

O

H

H3

1

OH2

H

N

NN H

ON

N HH

H H

Sugar (deoxyribose)3 end

Phosphate

Guanine (G)

DNA nucleotide

2

N


Figure 16.7 The double helix

O

–O O

OHP

H2C

O

–O O

OP

H2C

O

–O O

OP

H2C

O

–OO

OP

O

OH

O

O

OT A

C

GC

A T

O

O

O

O

OH

CH2

O O–

OOP

CH2

OO–

OOP

CH2

OO–

OO

P

CH2

OO–

OOP

5 end

Hydrogen bond3 end

5 end

3 end

C

T

A

A

T

CG

GC

A T

C G

AT

AT

A T

TA

C

TA0.34 nm

3.4 nm

(a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model

G

1 nm

G

H2C

G


Figure 16.8 Base pairing in DNAH

N H O CH3

N

N

O

N

N

N

N H

Sugar

Sugar

Adenine (A) Thymine (T)

N

N

N

N

Sugar

O H N

H

NH

N OH

H

N

Sugar

Guanine (G) Cytosine (C)


Unnumbered Figure p. 298

Purine + Purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: widthConsistent with X-ray data


DNA Replication Section 16.2

• Semi-conservative model – each of the two daughter molecules will have one old strand, derived from the parent molecules, and one newly made strand


Figure 16.9 A model for DNA replication: the basic concept (layer 1)

(a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

A

C

T

A

G

T

G

A

T

C




(b) The first step in replication is separation of the two DNA strands.

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C





(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C





(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C


Figure 16.10 Three alternative models of DNA replication

Conservativemodel. The twoparental strandsreassociate after acting astemplates fornew strands,thus restoringthe parentaldouble helix.

(a)

Semiconserva-tive model.The two strandsof the parentalmolecule separate, and each functionsas a templatefor synthesis ofa new, comple-mentary strand.

(b)

Dispersivemodel. Eachstrand of bothdaughter mol-ecules containsa mixture ofold and newlysynthesizedDNA.

(c)

Parent cellFirstreplication

Secondreplication


Figure 16.13 Incorporation of a nucleotide into a DNA strand

New strand Template strand

5’ end 3’ end

Sugar A TBase

C

G

G

C

A

C

T

PP

P

OH

P P

5’ end 3’ end

5’ end 5’ end

A T

C

G

G

C

A

C

T

3’ end

Nucleosidetriphosphate

Pyrophosphate

2 P

OH

Phosphate


Figure 16.12 Origins of replication in eukaryotes

Replication begins at specific siteswhere the two parental strandsseparate and form replicationbubbles.

The bubbles expand laterally, asDNA replication proceeds in bothdirections.

Eventually, the replicationbubbles fuse, and synthesis ofthe daughter strands iscomplete.

1

2

3

Origin of replication

Bubble

Parental (template) strand

Daughter (new) strand

Replication fork

Two daughter DNA molecules

In eukaryotes, DNA replication begins at many sites along the giantDNA molecule of each chromosome.

In this micrograph, three replicationbubbles are visible along the DNA ofa cultured Chinese hamster cell (TEM).

(b)(a)

0.25 µm


Parental DNA

DNA pol Ill elongatesDNA strands only in the5 3 direction. 3

5

5

3

35

21

Okazakifragments

DNA pol III

Templatestrand

Leading strandLagging strand

32 1

Templatestrand DNA ligase

Overall direction of replication

One new strand, the leading strand,can elongate continuously 5 3as the replication fork progresses.

The other new strand, thelagging strand must grow in an overall3 5 direction by addition of shortsegments, Okazaki fragments, that grow5 3 (numbered here in the orderthey were made).

DNA ligase joins Okazakifragments by forming a bond betweentheir free ends. This results in a continuous strand.

2

3

1

4

Figure 16.14 Synthesis of leading and lagging strands during DNA replication


Figure 16.15 Synthesis of the lagging strand


3

3

3

35

35

35

35

35

35

35

3 5

5

1

1

21

12

5

5

12

35

DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.

6 The lagging strand in this region is nowcomplete.

7

DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2.

5

After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off.

4

After reaching the next RNA primer (not shown), DNA pol III falls off.

3

DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

2

Primase joins RNA nucleotides into a primer.

1

Templatestrand

RNA primer

Okazakifragment


Table 16.1 Bacterial DNA replication proteins and their functions



Helicase unwinds theparental double helix.

Molecules of single-strand binding proteinstabilize the unwoundtemplate strands.

The leading strand issynthesized continuously in the5 3 direction by DNA pol III.

Leadingstrand Origin of replication

Laggingstrand

Laggingstrand

LeadingstrandOVERVIEW

Leadingstrand

Replication fork

DNA pol III

Primase

PrimerDNA pol III Lagging

strand

DNA pol I DNA ligase

1

2 3

Primase begins synthesisof RNA primer for fifthOkazaki fragment.

4

DNA pol III is completing synthesis ofthe fourth fragment, when it reaches theRNA primer on the third fragment, it willdissociate, move to the replication fork,and add DNA nucleotides to the 3 endof the fifth fragment primer.

5 DNA pol I removes the primer from the 5 endof the second fragment, replacing it with DNAnucleotides that it adds one by one to the 3’ endof the third fragment. The replacement of thelast RNA nucleotide with DNA leaves the sugar-phosphate backbone with a free 3 end.

6 DNA ligase bondsthe 3 end of thesecond fragment tothe 5 end of the firstfragment.

7

Parental DNA

53

43

21

5

3

Figure 16.16 A summary of bacterial DNA replication


Proofreading and repairing DNA

• Errors do occur

– 1 in 10 billion nucleotides on entire DNA

– 1 in 100,000 for incoming nucleotides

• Proofreading is done by DNA pol III as it attaches new nucleotides

• Mismatch repair cells use special enzymes to fix mismatched nucleotides

– A-C for example


Figure 16.17 Nucleotide excision repair of DNA damage

Nuclease

DNApolymerase

DNAligase

A thymine dimerdistorts the DNA molecule.1

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4

A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.

2


Telomeres

• Repeated units of bases

– TTAGGG in humans

• Do NOT contain genes

• They protect the genes from being eroded (getting shorter and shorter) through DNA replication rounds


Figure 16.18 Shortening of the ends of linear DNA molecules

End of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3


Figure 16.19 Telomeres

1 µm

evidence that dna can transform bacteria

Documents