The DNA StoryThe DNA Story

Discovering the Discovering the material for heredity material for heredity

Accel Bio 2014

““Ancient” HistoryAncient” History By the 1920's By the 1920's chromosomeschromosomes were suspected to be were suspected to be

the carriers of genetic information based on the carriers of genetic information based on observations of mitosis through a light observations of mitosis through a light microscope.microscope.

Biochemical studies of chromosome composition Biochemical studies of chromosome composition demonstrated that they were composed of demonstrated that they were composed of 30-50% 30-50% nucleic acid nucleic acid andand 50-70% protein 50-70% protein..

It was generally believed that PROTEINS would It was generally believed that PROTEINS would prove to be the carriers of genetic information. prove to be the carriers of genetic information. WHY? WHY? (multiple reasons really…think about info on the next slide (multiple reasons really…think about info on the next slide and about protein structure)and about protein structure)

The Genetic Material had to:The Genetic Material had to: Carry information from one generation to the nextCarry information from one generation to the next Somehow “code” for the heritable traits of an Somehow “code” for the heritable traits of an

organismorganism Be easily and accurately copied Be easily and accurately copied

(but (but ableable to change as well, to allow for mutations) to change as well, to allow for mutations)

How are proteins appropriate for these functions? Why How are proteins appropriate for these functions? Why did so many scientists who were knowledgeable about did so many scientists who were knowledgeable about protein structure suspect they were the genetic material?protein structure suspect they were the genetic material?

Time Line of DiscoveryTime Line of Discovery Frederick Griffith (1928)Frederick Griffith (1928) discovers that bacteria can change discovers that bacteria can change

from one form to another (a process called from one form to another (a process called transformationtransformation). ). Oswald Avery & colleagues (1944)Oswald Avery & colleagues (1944) follow up Griffith’s follow up Griffith’s

earlier discovery and conclude that the transforming factor is earlier discovery and conclude that the transforming factor is DNA.DNA.

Rosalind Franklin & Maurice Wilkins (1950) Rosalind Franklin & Maurice Wilkins (1950) provide provide evidence that DNA is in the form of a double helix.evidence that DNA is in the form of a double helix.

Erwin Chargaff (1951)Erwin Chargaff (1951) publishes that the nitrogenous bases of publishes that the nitrogenous bases of DNA occur in a ratio, with equal amounts of adenine and DNA occur in a ratio, with equal amounts of adenine and thymine, and equal amounts of cytosine and guanine.thymine, and equal amounts of cytosine and guanine.

Alfred Hershey & Martha Chase (1952)Alfred Hershey & Martha Chase (1952) conduct experiments conduct experiments which further prove that DNA was the hereditary material, which further prove that DNA was the hereditary material, sufficient to code for the growth of a new organism.sufficient to code for the growth of a new organism.

James Watson & Francis Crick (1953)James Watson & Francis Crick (1953) publish the three publish the three dimensional structure and composition of DNA.dimensional structure and composition of DNA.

Genetic Transformation DiscoveredGenetic Transformation Discovered

Fred GriffithFred Griffith unwittingly discovered unwittingly discovered transformationtransformation. He showed that some . He showed that some “active genetic substance” could be “active genetic substance” could be transferred from dead bacteria capable of transferred from dead bacteria capable of causing disease to live harmless bacteria – causing disease to live harmless bacteria – making these live bacteria dangerous.making these live bacteria dangerous.

How did he accomplish this?How did he accomplish this?

Griffith’s ExperimentGriffith’s Experiment

Griffith was attempting to develop a vaccine for Griffith was attempting to develop a vaccine for StreptococcusStreptococcus pneumoniae pneumoniae (a type of bacteria).(a type of bacteria).

There were two strains of There were two strains of StreptococcusStreptococcus, one of , one of which was harmless to people. The other strain which was harmless to people. The other strain caused pneumonia. caused pneumonia.

The term for the ability of an organism to cause The term for the ability of an organism to cause disease is disease is virulencevirulence. Such a disease-causing . Such a disease-causing organism could also be called organism could also be called pathogenicpathogenic..

More on More on StreptococcusStreptococcus……

Strain #1:Strain #1: S strainS strain– Was called S strain because Was called S strain because

it formed smooth colonies it formed smooth colonies on a petri dish culture.on a petri dish culture.

– Had a polysaccharide coat Had a polysaccharide coat that protected it from attack that protected it from attack by the immune system.by the immune system.

– Was Was virulentvirulent..

Still More on Streptococcus…Still More on Streptococcus…

Strain #2:Strain #2: R strainR strain– Was called R strain because it formed rough Was called R strain because it formed rough

colonies on a petri dish culture.colonies on a petri dish culture.– Did Did notnot have the polysaccharide coat that have the polysaccharide coat that

protected it from attack by the immune protected it from attack by the immune system.system.

– WasWas aavirulentvirulent (harmless).(harmless).

Griffith (and later Avery & his Griffith (and later Avery & his colleagues) performed the following colleagues) performed the following

experiment…experiment…

Live S strain injected into a mouse yields Live S strain injected into a mouse yields a a deaddead mouse. mouse.

Live R strain injected into a mouse yields Live R strain injected into a mouse yields a a livelive mouse. mouse.

Heat-killed S strain injected into a mouse Heat-killed S strain injected into a mouse also yields a also yields a livelive mouse. mouse.

BUT… what if you heat-kill the S strain BUT… what if you heat-kill the S strain and add it to the live R strain?and add it to the live R strain?

Griffith’s ResultsGriffith’s Results

Heat-killed S strain

Live R strain mixed w/ Heat-killed S

strainLiving R strain

Living S strain

““Something” (we now know this to be DNA) from Something” (we now know this to be DNA) from the virulent S strain had been able to the virulent S strain had been able to transformtransform the the harmless R strain into a mouse killer!harmless R strain into a mouse killer!

Avery, McLeod, & McCarthy’s Avery, McLeod, & McCarthy’s Experiments Build on Griffith’sExperiments Build on Griffith’s Avery and his colleagues repeated Griffith’s experiment Avery and his colleagues repeated Griffith’s experiment

but added an additional step.but added an additional step. First they added a First they added a protein-destroying enzymeprotein-destroying enzyme to the to the

heat-killed S strain (then mixed with live R-strain). heat-killed S strain (then mixed with live R-strain). Mice still died.Mice still died.

They repeated the experiment but the second time added They repeated the experiment but the second time added a a DNA-destroying enzymeDNA-destroying enzyme to the heat-killed S strain to the heat-killed S strain (then mixed with live R-strain). (then mixed with live R-strain). The mice didn’t die!The mice didn’t die!

The “transforming factor” had to be The “transforming factor” had to be DNADNA!!!!

Alfred Hershey & Martha ChaseAlfred Hershey & Martha Chase

Even after Avery’s experiments, Even after Avery’s experiments, scientists were still skeptical about scientists were still skeptical about the possibility that DNA was the the possibility that DNA was the “stuff of heredity” in more complex “stuff of heredity” in more complex organisms (other than bacteria)organisms (other than bacteria)

In 1952, Alfred Hershey & Martha In 1952, Alfred Hershey & Martha Chase performed an elegant series Chase performed an elegant series of experiments which proved that of experiments which proved that DNA was the genetic material – DNA was the genetic material – using a household blender!using a household blender!

Hershey and Chase used ???Hershey and Chase used ??? Hershey and Chase used the T2_____________, a Hershey and Chase used the T2_____________, a

virus which infects and kills bacterial cells.virus which infects and kills bacterial cells.bacteriophagebacteriophage

Virus StructureVirus Structure Protein “coat” or Protein “coat” or

____________ Nucleic acid Nucleic acid

(____________)(____________)

H & C knew T2 H & C knew T2 phages were made of phages were made of DNA and protein, DNA and protein, but they had no but they had no proof as to whether proof as to whether proteinprotein or or DNADNA was was the genetic material the genetic material of the viruses.of the viruses.

capsidcapsid

DNA or RNADNA or RNA

Bacteriophage MicrographBacteriophage Micrograph

Bacteriophages on surface of bacterial cellBacteriophages on surface of bacterial cell

Virus Life Cycle Virus Life Cycle (in brief)(in brief) Viruses infect living cells and then multiply inside these Viruses infect living cells and then multiply inside these

cells, producing millions of copies of the virus which then cells, producing millions of copies of the virus which then explode (____) the cell, releasing these copies to go out and explode (____) the cell, releasing these copies to go out and _______________._______________.infect infect moremore cells cells

lyselyse

Experimental PredictionsExperimental Predictions

IfIf the virus carried the instructions for making the virus carried the instructions for making copies of itself (its genetic material) in the form copies of itself (its genetic material) in the form of _______, of _______, thenthen the virus would have to inject the virus would have to inject its _______ into the bacteria.its _______ into the bacteria.

IfIf the virus carried the instructions for making the virus carried the instructions for making copies of itself (its genetic material) in the form copies of itself (its genetic material) in the form of _____, of _____, thenthen the virus would have to inject its the virus would have to inject its _____ into the bacteria._____ into the bacteria.

proteinproteinproteinprotein

DNADNADNADNA

The Hershey-Chase ExperimentThe Hershey-Chase Experiment Use a batch of virus with radioactive capsid Use a batch of virus with radioactive capsid proteinsproteins

(labeled with (labeled with 3535SS).). Use a second batch of virus with radioactive Use a second batch of virus with radioactive DNADNA

(labeled with (labeled with 3232PP).). Allow each batch to infect bacteria (Allow each batch to infect bacteria (E. coliE. coli), then ), then

remove the viruses on the outside of the bacterial cells remove the viruses on the outside of the bacterial cells by agitating the cells…in a blender.by agitating the cells…in a blender.

Spin the bacterial culture tubes in centrifuge so the Spin the bacterial culture tubes in centrifuge so the dense bacterial cells sink to the bottom (forming a dense bacterial cells sink to the bottom (forming a pelletpellet) & the lighter viruses stay in the upper liquid ) & the lighter viruses stay in the upper liquid portion (called the portion (called the supernatantsupernatant))

Collect the Collect the E. coliE. coli bacteria and see whether they contain bacteria and see whether they contain 3535SS (radioactive protein) or (radioactive protein) or 3232PP (radioactive DNA). (radioactive DNA).

Hershey-Chase ExperimentHershey-Chase Experiment

Hershey-Chase ResultsHershey-Chase Results

E. coliE. coli bacteria in the pellet contained bacteria in the pellet contained virtually virtually nono 3535S. S.

The offspring of the virus (its progeny) The offspring of the virus (its progeny) contained contained lotslots of of 3232P-labelled DNA.P-labelled DNA.

ConclusionConclusion: : DNA carried the genetic DNA carried the genetic information!!information!!

What next??What next?? TheThe Hershey-Chase experiment Hershey-Chase experiment was strong evidence was strong evidence

that DNA was genetic material in virusesthat DNA was genetic material in viruses Fact that cells double amt DNA in cell prior to mitosis Fact that cells double amt DNA in cell prior to mitosis

& then distribute equally to daughter cells was & then distribute equally to daughter cells was circumstantial evidence that DNA was genetic material circumstantial evidence that DNA was genetic material in Eukaryotes as wellin Eukaryotes as well

Given these suspicions, what do you think was the next Given these suspicions, what do you think was the next question that came to mind to scientists?question that came to mind to scientists?

What would be your first step to figuring this out?What would be your first step to figuring this out?

(remember proteins…what is the key to understanding their function?)(remember proteins…what is the key to understanding their function?)

HowHow does DNA work? does DNA work?

Figuring out the STRUCTURE…so you can learn Figuring out the STRUCTURE…so you can learn something about FUNCTION!something about FUNCTION!

DNA Structure DNA Structure and and The Big RaceThe Big Race

What What diddid we know we knowabout DNA in the 1940’s?about DNA in the 1940’s?

We knew that it was composed of chains We knew that it was composed of chains of four __________ – containing four of four __________ – containing four different _______________.different _______________.

We knew Chargaff’s Rule.We knew Chargaff’s Rule.

nucleotidesnucleotidesnitrogenous basesnitrogenous bases

What is a Nucleotide?What is a Nucleotide? One nucleotide has three parts – a One nucleotide has three parts – a

sugar (deoxyribose), a phosphate sugar (deoxyribose), a phosphate (PO(PO44

--), and a nitrogenous base.), and a nitrogenous base. There are four different nitrogenous bases There are four different nitrogenous bases

that appear in DNA:that appear in DNA:– Adenine ( )Adenine ( )– Thymine ( )Thymine ( )– Cytosine ( )Cytosine ( )– Guanine ( )Guanine ( )

AA

TTCCGG

The Four Nitrogenous BasesThe Four Nitrogenous Bases

The bases with one ring The bases with one ring are are pyrimidinespyrimidines..– CytosineCytosine

– ThymineThymine

The bases with two rings The bases with two rings are are purinespurines..– AdenineAdenine

– GuanineGuanine

““AAnnggels are els are purepure in in heartheart.”.”((heartheart reminds you of two rings) reminds you of two rings)

http://www.sci.sdsu.edu/classes/bio100/Lectures/Lect08/Image196.gif

Chargaff’sChargaff’s RuleRule 1949 – Austrian Biochemist Erwin Chargaff noticed that 1949 – Austrian Biochemist Erwin Chargaff noticed that

in every analysis of DNA that he performed, in every analysis of DNA that he performed, the amount the amount of adenine always equaled the amount of thymine of adenine always equaled the amount of thymine andand the amount of cytosine equaled the amount of guanine.the amount of cytosine equaled the amount of guanine.

In other words:In other words: There could be different overall amount of C & G and A There could be different overall amount of C & G and A

& T – but these pairs of bases were always present in & T – but these pairs of bases were always present in equal ratios!equal ratios!

• • This is called This is called Chargaff’s Rule.Chargaff’s Rule.

A = TA = T andand G = C G = C

A + G = T + CA + G = T + C

http://img0.liveinternet.ru/images/attach/c/0//47/349/47349945_Erwin_Chargaff.jpg

http://www.nature.com/scitable/content/24234/sadava_11_7_FULL.gif

The Big Race The Big Race (early 1950’s)(early 1950’s)

Once DNA was proven to be the genetic Once DNA was proven to be the genetic material, an increasing number of material, an increasing number of scientists became passionate about trying scientists became passionate about trying to discover its structure…to discover its structure…

The most significant scientists who were The most significant scientists who were racingracing each other to be the first to discover each other to be the first to discover

the structure of DNA were:the structure of DNA were:– Linus PaulingLinus Pauling (CalTech) (CalTech)

– Rosalind FranklinRosalind Franklin & & Maurice Wilkins Maurice Wilkins

(Kings College, London)(Kings College, London)

– James D. Watson James D. Watson & & Francis CrickFrancis Crick

(Cambridge University)(Cambridge University)

James Watson

Francis Crick

Linus PaulinPaulin

gg

Maurice Maurice WilkinsWilkins

Rosalind Franklin & Rosalind Franklin & Maurice WilkinsMaurice Wilkins

Maurice Wilkins and Maurice Wilkins and Rosalind Franklin, a Rosalind Franklin, a talented X-ray talented X-ray crystallographer, crystallographer, developed X-ray developed X-ray diffraction images of diffraction images of DNA.DNA.

Franklin’s uniquely sharp Franklin’s uniquely sharp images indicated that images indicated that DNA existed in the form DNA existed in the form of of two twisted strandstwo twisted strands. .

James Watson & Francis CrickJames Watson & Francis Crick

Chargaff’s RuleChargaff’s Rule: In a : In a sample of DNA, adenine sample of DNA, adenine & thymine occur in equal & thymine occur in equal amounts, as do cytosine amounts, as do cytosine and guanine.and guanine.

Rosalind Franklin’s data Rosalind Franklin’s data indicated that the DNA indicated that the DNA molecule was an anti-molecule was an anti-parallel parallel double-stranded double-stranded molecule.molecule.

What Watson & Crick What Watson & Crick knew…knew…

What they finally figured out…What they finally figured out…

DNA is a two-stranded (double) DNA is a two-stranded (double) helix -- like a twisted ladder.helix -- like a twisted ladder.

The The backbone backbone of the molecule is of the molecule is composed of the phosphates and composed of the phosphates and deoxyribose sugars of the deoxyribose sugars of the nucleotides (nucleotides (covalently bondedcovalently bonded).).

The The rungsrungs are composed of are composed of nitrogenous nitrogenous basebase pairspairs, which , which stick together by hydrogen stick together by hydrogen bonding. *These pairs consist of bonding. *These pairs consist of either A & T either A & T oror G & C. G & C.

DNA strands are Anti-parallel5’

3’ 5’

3’

Three Representations of DNAThree Representations of DNA

More Representations of DNAMore Representations of DNANote that the pictures are not oriented the same with respect to the 5’ & 3’ ends.

The reason for Chargaff’s Rule:The reason for Chargaff’s Rule: Base-Pair Hydrogen BondingBase-Pair Hydrogen Bonding

• • AdenineAdenine (a purine) (a purine) always pairs up with always pairs up with ThymineThymine (a pyrimadine). (a pyrimadine).

• • GuanineGuanine (a purine) (a purine) always pairs up with always pairs up with CytosineCytosine (a pyrimadine).(a pyrimadine).

• • Diameter of DNA helix is Diameter of DNA helix is constant because A-T & G-C constant because A-T & G-C base pairs have equal widthbase pairs have equal width

And the winner is…And the winner is…1953: Watson & Crick publish their findings in the journal Nature.

1962: The Nobel Prize for Medicine & Physiology was awarded to Crick, Watson, & Wilkins.

Franklin died in 1958 of ovarian cancer, at age 38. Because the Nobel Prize is not awarded posthumously, and because no more than three individuals can share the prize, we can only speculate about whether the committee would have recognized Franklin’s contribution to the discovery of the double helix.

Structure Shows ActionStructure Shows Action"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

-Watson and Crick in the scientific paper that was published in Nature, April 25, 1953.

Adding fuel to the fireAdding fuel to the fire19681968: Watson publishes : Watson publishes The Double HelixThe Double Helix, his , his (not-so-modest) account of his participation in the (not-so-modest) account of his participation in the race to find the structure of DNA and the role race to find the structure of DNA and the role Crick, Wilkins, & Franklin played as well.Crick, Wilkins, & Franklin played as well.

The book is widely read and popular, despite The book is widely read and popular, despite criticism from those involved of Watson’s criticism from those involved of Watson’s subjective depiction of the other scientists, subjective depiction of the other scientists, especially his unflattering portrayal of Franklin, especially his unflattering portrayal of Franklin, who was no longer alive to defend herself.who was no longer alive to defend herself.

That That OtherOther Nucleic Nucleic Acid: RNAAcid: RNA

What is RNA?What is RNA?

Is Is single-strandedsingle-stranded Uses Uses riboseribose sugar sugar

instead of instead of deoxyribose sugardeoxyribose sugar

Has four different Has four different nitrogenous basesnitrogenous bases::– AdenineAdenine– CytosineCytosine– GuanineGuanine– Uracil Uracil instead of instead of

ThymineThymine

Ribonucleic acidRibonucleic acid is a polynucleotide similar to DNA is a polynucleotide similar to DNA with a few key differences. RNA:with a few key differences. RNA:

An RNA nucleotideAn RNA nucleotide

DNA, with helix dimensionsDNA, with helix dimensions

More DNA, with 3’ & 5’ endsMore DNA, with 3’ & 5’ ends