fig. 16-1. dna – lots of it in a small space dna – a historical perspective 1865 – gregor...

Fig. 16-1

DNA – lots of it in a small space

DNA – A Historical Perspective

• 1865 – Gregor Mendel – “Father of Heredity”

• 1869 – Johann Miescher (Swiss biochemist) – isolates DNA from WBC

• 1902 – Walter Sutton – American Geneticist – Columbia U. Theory of the Chromosome

• 1928 – Frederick Griffith – British Bacteriologist – discovers transformational factor

• 1944 – Oswald Avery et al. - Canadian-born American physician – shows that the transformational factor was not a protein but DNA

• 1952 – Alfred Hershey & Martha Chase – provide conclusive evidence that DNA is the transformational factor

• 1952 – Rosalind Franklin & Maurice Wilkins – use x-ray diffraction to analyze DNA

• 1953 – James Watson & Francis Crick construct double helix model of DNA

Johannes Friedrich Miescher 1844-1895

In 1869, first to isolate a substance he called nuclein from the nuclei of leucocytes or WBC

Collected these from pus he obtained from bandages at nearby hospitals.

He found that nuclein contained phosphorus and nitrogen, but not sulfur

http://en.wikipedia.org/wiki/File:Friedrich_Miescher.jpg

Walter Sutton (1877-1916)

American geneticist & physician – Columbia University

Boveri-Sutton Chromosome Theory

Made connection – Mendel’s Laws of Heredity could be applied to chromosomes at the cellular level of living organisms

http://search.live.com/images/results.aspx?q=picture+of+Walter+Sutton&FORM=ZZIR9

Thomas Hunt Morgan (1866-1945)

American geneticist and embryologist – Columbia U.

Studied the mutations in fruit flies, Drosophilia melanogaster

demonstrated that genes are carried on chromosomes and are the mechanical basis of heredity

Nobel Prize in Physiology or Medicine in 1933

http://en.wikipedia.org/wiki/Gene

Frederick Griffith 1871 - 1941

What is the transformational factor??? Is it DNA or Protein???

Griffith’s research, working with two strains of a bacterium, one pathogenic and one harmless, addresses this vital question

In 1941, Griffith was killed at work in his London laboratory as a result of an air raid in the London Blitz.

DNA – A Historical Perspective

Griffith and Transformation

1928 – British pathologist was researching

How certain types of bacteria produced pneumonia

He isolated 2 different strains: R which was harmless and S - virulent

Live S-strain kills mouse

Injection of Rough Colonies ( R)

Results in Live Mice

Heat-killed Smooth colonies (S)Result in Live Mice

Heat-Killed S + Live R =Dead Mice

Fig. 16-2

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells and living R cells

Mouse diesMouse dies Mouse healthy Mouse healthy

Living S cells

RESULTS

EXPERIMENT

Oswald Avery and DNA (1944)

Working along with Colin Macleod & Maclyn McCarty

Repeated Griffith’s work with modifications

Which molecule in the heat-killed was the transformational factor?

The components of the Ground up S were isolated, each mixed with R and injected into mice

In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2

To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection

They concluded that the injected DNA of the phage provides the genetic information

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Animation: Hershey-Chase ExperimentAnimation: Hershey-Chase Experiment

Alfred Hershey and Martha Chase. 1953

Fig. 16-3

Bacterial cell

Phage head

Tail sheath

Tail fiber

DNA

100

nm

Fig. 16-4-1

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Fig. 16-4-2

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA



Empty protein shell

Phage DNA

Fig. 16-4-3

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA



Empty protein shell

Phage DNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterial cells and contents)

Radioactivity (phage protein) in liquid

Radioactivity (phage DNA) in pellet

Fig. 16-6

(a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

Erwin Chargaff (1905-2002)and “Chargaff’s Rules”

The bases were not present in equal quantities

They varied from organism to organism.

No matter where DNA came from — yeast, people, or salmon — the number of adenine bases always equaled the number of thymine bases and the number of guanine always equaled the number of cytosine bases.

He published a review of his experiments in 1950, calling the ratios — which came to be known as Chargaff’s Rules

Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases


Chargaff’s Rule

• American biochemist discovers that % of G and C bases are almost equal in any sample of DNA.

• The same thing is true for A and T

• [A]=[T] and [G]=[C]

Fig. 16-8

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

Fig. 16-UN2

Sugar-phosphate backbone

Nitrogenous bases

Hydrogen bond

G

C

A T

G

G

G

A

A

A

T

T

T

C

C

C

Fig. 16-7a

Hydrogen bond 3 end

5 end

3.4 nm

0.34 nm

3 end

5 end

(b) Partial chemical structure(a) Key features of DNA structure

1 nm

Fig. 16-5Sugar–phosphate

backbone

5 end

Nitrogenous

bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

DNA nucleotide

Sugar (deoxyribose)

3 end

Phosphate

Purines•Adenine•Guanine

PurAsGold

Pyrimidines•Cytosine•Thymine•Uracil

PyCUT

Carbon 1 – bonds to nitrogen base

Carbon 3 – bonds to next nucleotide

Carbon 5 – bonds to phosphate group

Fig. 16-UN1

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

Additional Evidence That DNA Is the Genetic Material

• It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group

• In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next

• This evidence of diversity made DNA a more credible candidate for the genetic material


Animation: DNA and RNA StructureAnimation: DNA and RNA Structure

Building a Structural Model of DNA: Scientific Inquiry

• After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role

• Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure

• Franklin produced a picture of the DNA molecule using this technique


• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical

• The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases

• The width suggested that the DNA molecule was made up of two strands, forming a double helix


Animation: DNA Double HelixAnimation: DNA Double Helix

• Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA

• Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior


• At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width

• Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray


• Watson and Crick reasoned that the pairing was more specific, dictated by the base structures

• They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C)

• The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C


Concept 16.2: Many proteins work together in DNA replication and repair

• The relationship between structure and function is manifest in the double helix

• Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material


Fig. 16-9-1

A T

GC

T A

TA

G C

(a) Parent molecule

Fig. 16-9-2

A T

GC

T A

TA

G C

A T

GC

T A

TA

G C

(a) Parent molecule (b) Separation of strands

Fig. 16-9-3

A T

GC

T A

TA

G C

(a) Parent molecule

A T

GC

T A

TA

G C

(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand

(b) Separation of strands

A T

GC

T A

TA

G C

A T

GC

T A

TA

G C

The Basic Principle: Base Pairing to a Template Strand

• Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication

• In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules


Animation: DNA Replication OverviewAnimation: DNA Replication Overview

Fig. 17-5Second mRNA base

Fir

st m

RN

A b

ase

(5

en

d o

f co

don

)

Th

ird

mR

NA

bas

e (3

e

nd

of

cod

on)

Enzymes involved in DNA Replication & Transcription

Enzyme FunctionHelicase “molecular zipper” – unwinds double helix;

breaks hydrogen bonds that holds base pairs together

Topoisomerase (gyrase) “molecular swivel”- relieves overwinding stress on DNA strands by working ahead of helicase and breaking, swiveling and rejoining small sections of the DNA molecule

DNA polymerase Using a parent DNA strand, adds free-floating nucleotides (A, T, G, & C’s) covalently to the new strand being constructed.

ligase “molecular glue” – joins fragments of the New DNA strand together

RNA polymerase (used in transcription) Uses one strand of DNA as a template to construct mRNA – adds free-floating nucleotide

Editase Fixes mistakes on DNA molecule

fig. 16-1. dna – lots of it in a small space dna – a historical perspective 1865 – gregor...

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