Module 1 BIO112, Lectures 2-5

Oswald Avery, Colin Macleod, Maclyn McCarty

Hypothesized that griffeth’s transformation principle is not protein based but DNA based.

Oswald Avery, Colin Macleod, Maclyn McCarty EXPERIMENT

Isolated the macromolecules of Dead S-cells

Separated them into 3 tubes containing (DNA-RNA, DNA-PROTEIN, RNA-DNA)

Found that only the living R-Cells exposed to DNA-containing test tubes transform

Hershy and Chase Experiment

Radio labeled the protein and DNA sections of T2 bacteriophage.

Mixed both the only DNA-labeled and only protein-labeled phages with bacteria. Only labeled DNA was found in the insides of bacteria.

Shows that DNA is the transmissible genetic information

Fredreich Meischer

Isolated Nuclein from nuclei of whiteblood cells

Phoebus Levene

Discovered the nucleotide bases ACTG and proposed the tetranucleotide hypothesis.

Chargaff’s Principle

ACTG abudance varies between species and that A=T and C=G

Rosiland Franklin adn Maurice Wilkins

Used Xray crystallography to find the Doble helical strucuture

Linus Pauling

Discovered a-helix and b-sheets

Triple Helix strucuture

Formation of the peptide bond

DNA Size and Shape

distance between nucleotides 3.34 A

diameter of double helix 20 A

10.5 bp per complete turn

Watson and Crick

Discovered double helical structure of DNA

Things needed to start DNA replication

-Primer

-Template strand

-dNTP

Reiji Okazaki’s experiment process

Believed that DNA could be semi-discontinuous or discontinuous

Grew bacteria with radio-labeled T

-isolated DNA and measured the length of DNA at different time points

Reiji Okazaki’s experiment results

With Ligase, had longer strands after 2 minutes

without ligase found more short strands or okazaki fragments

conclusion→semi-discontinuous

Arthur Kornberg 

DIscovered DNA pol 1 - Urochester

Eukaryote replication 

Multiple replication origins (can form replication bubbles)

bidrectional replication from each orgin

Linear DNA

Prokaryotes replication

Circular chromosome

Designated Ori

forms theta shape intermediate

Sliding Clamp and Clamp loader

Keeps DNA Polymerase in place

loaded using clamp loader 

1000x processivity of DNA pol

Fidelity of DNA Replication

DNA recognizes incorrect base pairs

DNA polymerase has 3’-5’ exonucleases - removes wrong nucelotides

mismatch repair

DNA pol recognizes wrong base pair and moves the strands to E site

MutL + MutS

binds to nicks in the DNA caused by mismatched base pairs and triggers DNA degradation

Why are telomeres only found in Eukaryotes?

End Replication Problem

After every replication in Eukaryotic DNA small gaps are left due to RNA Primers being removed at the end and DNA pol not being able to finish the strand completely.

Muller and Micklintok

Muller with Drosophila - After fracturing chromosomes, found that the internal parts stick together, but the natural ends were not sticky. (no head tot ail or head to head fusion)

McClinktok with Maize -  Similar conclusions, no head to tail or head to tail refusion. 

Telomere “Seal the ends” and prevents

Degrediation

End to end refusion and rearrangements

false alarm apoptosis

First disovered in tetrahymena - telomere sequence

Rich in GC’s many copies

Jack Szostak and Elizaberth Blackburn

Tried to introduce human DNA in yeast, but was unsuccessful until adding “Tetrahymena telomeres”

rDNA termini (essential telomeres) function as telomeres in yeast

Chromsomes overhang

Overhang rich in G 50-400 bp long

t-loop

3’overhang loops around to the ds region binding and forming a loop with telomeric repeats TTAGGG

D-loop vs R loop

both the tiny loop formed between the overhang and the opened ds dna. But D-loop is for ssDNA and R loop is for RNA

Issue with Circular chromsomes 

During Meiotic Recombination circular chromesomes dicentric formation may cause uneven when pulling apart

Carol Greider

Discovered telomerase (RIbonuceloprotein Enzyme) 

Telomerase

RIbonucleoprotein

is Reverse transcirptase

Adds telomeric repeats to 3’ G overhang

Leonard Hayflick and the Hayflick limit

40-60 regular cell divison for somatic cells,

apoptosis afterwards

TERT (Human Telomerase)

not active in somatic cells.

Inhibited by P53 tumor

Immortal cells are dangerous I.e. cancer

Central Dogma

DNA-mRNA-protein

James Watson’s RNA from DNA hypothesis

believed in potential total chemcial covnersion

unlikeluy because its very costly to convert every Thymine to uracil and every deoxyribose to ribose.

Instead→DNA as a template for RNA synthesis

Francais crick and Microsomal particles

Found that amino acids past rapidly through this Microsomal particle (ribosome) 

The Coding Problem

How 4 bases code for 20 amino acid

How many bases code for one amino acid?

overlapping or non overlapping code?

Trinucleotide hypothesis 

Accounts for the 20 nucleotides b/c 64 possible combinations

Evidence for non-overlapping genetic code

If overlapping 1 base ppair change would result in multiple codon codon and amino acid changes

However, experiment showed that only 1 codon and amino acid changed. 

Deletion experiments(Francis Crick)

In an amino acid sequence deleted 1, 2, and 3 nucleotides

Deletion of 1 and 2 resulted in frameshift and lost of protein function

Triple deletion - some amino acids changed but general function of portein persists.

Nature of genetic code

Tripe codex

non-overlapping code

degenerate code - one amino acid can be made by more than on codon

Base read from a fixed starting point

Adaptor Hypothesis

-Amino acids carried to the templete by an adaptor molecule

at least 20 adaptors exist

Evidence for tRNA

Rat experiment - radio labled PH5

this labled RNA was fgound to be able to transfer amino acids

Robert Holley

-Sequencing of yeast alanine tRNA and liked DNA to protein synthesis like

-Secondary structure of RNA looks like a clover-leaf 

Marshall nirenburg and mathaii (cracking the genetic code)

Organized 20 test tubes with a radio labled amino acid in each of them and tRNA in all of them.

Inserted poly-u (UUUUUU) chain into each test tube. 

Only polypetitde that synthesized was PHEPHEPHEPHEPHE so therefore 

UUU was found to the sequence for PHE