DNA: The Molecular Basis of Inheritance

Hbio

Ms. Pagodin

Do Now:

Happy Pi Day! Grab your clickers Talk to your classmates and find out who

read the same article as you! In your article group, discuss the experiment

and conclusion!

Nuclear Composition?

1868- Johann Miescher Collected pus & fish sperm

Isolated and identified acidic compound with nitrogen and phosphorus…. Today we know it as Deoxyribonucleic acid

Molecule of Heredity

Is it Proteins or Nucleic Acid??

What makes up proteins?A. Nucleotides

B. Amino acids

C. Monosaccharides

Nucleotides

Amino acids

Monosa

ccharid

es

33% 33%33%

What makes up nucleic acids?A. Nucleotides

B. Amino acids

C. Monosaccharides

Nucleotides

Amino acids

Monosa

ccharid

es

33% 33%33%

How many amino acids are there?A. 4

B. 16

C. 20

D. 60

4 16 20 60

25% 25%25%25%

How many different DNA nucleotides are there?A. 4

B. 16

C. 20

D. 60

4 16 20 60

25% 25%25%25%

Identifying the Genetic Material 1928 Fredrick Griffith (English Bacteriologist)

Trying to find a vaccine for pneumonia Vaccine: prepared from killed/weakened microorganisms

introduced into the body to produce immunity Griffith worked with 2 strains of Streptococcus pneumoniae

bacteria S strain

Polysaccharide Capsule “Smooth” edged colonies Virulent – able to cause disease

R strain No Capsule “Rough” edged colonies Nonvirulent - does not cause disease

Griffith’s Experiment

Griffith’s Conclusion: Something had passed from heat killed bacteria to the nonvirulent R strain making them virulent… he called this the “transforming principal”

Griffith did not know what it was, but many scientists thought it was proteins

Today we know…

Transformation – cells take up foreign genetic material, changing their own genes (used for genetic engineering)

Heat killed S bacteria – enzymes were denatured therefore the DNA could not be copied

Proteins are denatured at 600C and DNA is denatured at 900C

DNA of heat killed S bacteria survived and transformed DNA of R bacteria

Virulent strainsA. Have a capsuleB. Cause diseaseC. Do not have a

capsuleD. Do not cause

diseaseE. A&BF. C&D

Have a capsu

le

Cause dise

ase

Do not have a ca

psule

Do not cause

disease A&B

C&D

17% 17% 17%17%17%17%

Transformation is the addition of genes to another organisms genome

A. True

B. False

True

False

50%50%

The Search for what caused the Transformation…

1944 – Oswald Avery, MacLeod, & McCarty (American Bacteriologists)

Experiment:1. Added protease to “R and heat-killed S” mixture

Result Mice died

2. Added DNAase to “R and heat-killed S” mixture Result Mice Lived

Conclusion: DNA, not protein, is the transforming factor in

Griffith’s experiment

More Evidence that DNA is the Genetic Material…

1952 – Alfred Hershey & Martha Chase (NY) Used T2 bacteriophages (phage) – virus

that infects bacteria Composed of nucleic acid surrounded by a

protein coat Viruses infect specific host Viruses are not living

Not composed of cells Cannot reproduce on their own Do not grow and develop

Background Info on Viruses

Which type of virus is chicken pox?A. Lytic

B. lysogenic

Lytic

lysoge

nic

50%50%

Which type of virus is the flu?A. Lytic

B. lysogenic

Lytic

lysoge

nic

50%50%

Hershey & Chase Experiment Experiment:

1. Grew T2 w/radioactive Sulfur 35S (protein coat takes in 35S)2. Grew another group of T2 w/ radioactive Phosphorus 32P (DNA

takes in the 32P)3. 35S-labeled and 32P–labeled phages were used to infect E.Coli

bacteria4. Separated phages from bacteria using a blender and a

centrifuge… the bacterial cells at bottom and viral parts at the top

Results: 35S-labels still in viral parts 32P-labels mostly in the bacterial cells, and new phages also

contained 32P DNA Conclusion:

Viral DNA (not protein) enters bacteria and carries instructions on how to make more phages

Without a doubt, DNA is the hereditary material!

Hershey & Chase Experiment

A bacteriophageA. Is a virus that

infects bacteria

B. Is a virulent bacteria

C. Cannot be used for experiments

Is a vi

rus t

hat infects

bac...

Is a vi

rulent b

acteria

Cannot be use

d for e

xp...

33% 33%33%

Structure of DNA?

Linus Pauling Nobel prize for deducing structure of protein

Collagen

If protein structure could be determined and modeled, why not DNA?

Structure of DNA By 1950’s most scientists were convinced that

Chromosomes carry genetic material Genes are on chromosomes Genes are made of DNA

Basic Structure of DNA Composed of nucleotides Nucleotides made of 3 parts deoxyribose, phosphate, N base 2 types nitrogen bases:

Purines – double ring of C and N Adenine Guanine

Pyrimidines – single ring of C and N Cytosine Thymine

Discovering DNA’s Structure Erwin Chargaff (NYC)

1947 – DNA composition varies among different species

1949 -Chargaff’s Rules- Discovered regularity of ratios: # Adenines = # Thymines

(ie. Humans A =30%, T=30%) # Guanines = # Cytosines

(ie. Humans G = 20%, C = 20%)

1952 Rosalind Franklin & Maurice Wilkins (England) Developed X-ray crystallography photographs of

DNA Suggested “helix” shape of 2-3 chains of

nucleotides

April 25th, 1953 James Watson & Francis Crick

(England) Built the 1st accurate 3D (tin

and wire) model of DNA “Double Helix” – spiral

staircase Purine is always linked by h-

bond to a pyrimidine 2 strands of DNA are

complimentary to each other 2 strands are anti-parallel 5’(phosphate end)

3’(deoxyribose end) 1962 Awarded the Nobel Prize

More on DNA

Ex. If the sequence of bases on one strand is AATGCGCAT, than the complimentary strand will be: ________________

Human DNA has 3 billion base pairs.. Less than 1% of our DNA makes us different from one another!

Which seems most likely?

Models of DNA Synthesis

Semiconservative ea/ daughter molecule

will have 1 new strand and 1old strand

Conservative Parent molecule reforms

Dispersive All 4 strands have a

combination of old and new strands

Assignment:

Propose an experiment to determine how DNA replication occurs

1950’s Meselson & Stahl

Cultured Ecoli on medium labeled w/ 15N nt

Transferred EColi to medium labeled w/ 14N nt

Centrifuge after each replication and analyze

Origin of Replication Prokaryotic Cell – single origin of replication where proteins

separate the 2 strands and create a replication bubble, replication proceeds in both directions from the replication fork

Eukaryotic Cells – hundreds or thousands of replication bubbles form to speed up the copying process, replication proceeds in both directions from the replication fork

http://sites.fas.harvard.edu/~biotext/animations/replication1.swf

DNA Replication Watson and Crick proposed that the complimentary strand of DNA serves

as a template for which the other strand is built…experiments confirmed this 5 years later

DNA Replication: Process of Synthesizing new molecules of DNA1. Helicases catalyze the breaking of H-bonds (driven by ATP) and

opens up the double helix forming replication forks (point at which DNA separates into single strands)

2. Topoisomerase temporarily bind to relieve strain ahead of replication fork

3. Single-strand binding protein – binds to unpaired DNA strands until they serve as templates for new complimentary strand

Elongation DNA pol adds 50 nt/sec in Euk cells Each nt is a nucleoside triphosphate

1. At the replication fork, DNA Polymerase III continuously adds complimentary nucleotides to exposed bases on 3’ end of new strand, this is called the leading strand

2. DNA polymerase III must work away from the replication fork on the other strand, the lagging strand, to follow the 5’-3’ direction creating short segments of DNA called Okazaki fragments. DNA Ligase joins the Okazaki fragments together.

3. Process continues until all DNA has been copied, end result is 2 new molecules of DNA each identical to the original and composed of one new and one old strand

Priming DNA Synthesis

DNA pol can not initiate – only add nt to 3’ end of existing chain

Primer – short chain (5-10nt) of RNA Primase – enzyme starts RNA chain from scratch

Leading strand – 1 primer needed

Lagging strand – 1 primer needed for ea/Okazaki fragment

DNA pol I replaces RNA nt of primers w/DNA versions

DNA Synthesis

http://www.dnai.org/a/index.html

Proofreading

DNA polymerase only moves to the next nucleotide if the previous nucleotide was a correct match

If mismatched, DNA Polymerase backs up, removes the mismatched nucleotide(s) and replaces it with the correct one(s).

Repair enzymes can recognize and repair damaged sites too

Only 1 error per 1 billion nucleotides!

DNA Replication & Aging Every time DNA is copied,

DNA polymerase cannot complete replication on the ends

Eukaryotic DNA has a non-coding, repeating nucleotide sequence on the ends called telomeres that protects genes from being eroded over successive replications

It is believed that telomeres are directly related to the aging process