History of DNA Flashcards
Mendel
1865- Plant Genetics paper
Friedrich Miescher (Swiss)
1869- purified nuclei of pus cells (later used salmon sperm)
gelantinous material - organic PO4 –> nuclein
70% protein (histones)
Discovery of nucleic acid
Albrecht Kossel
nuclein had organic bases, sugar
Richard Altman (student of Miescher)
1889 deproteinized yeast DNA –> started name nucleic acid
A. Ascoli
1901 uracil in yeast RNA
P.A. Levene
1908-1929 P.A. Levene - 2’-deoxy-D-ribose as DNA sugar
tetranucleotide theory
(very influential biochemist)
1910
DNA, RNA separate molecules
Levene & Bass
1931 - tetranucleotide theory
- 4 bases in a plane
- incapable of carrying genetic info
nucleic acids attracted very little attention
Fred Griffith 1928
Used Streptococcus pneumonia
transforming principle, rough and smooth (capsules) cultures
rough cells plus heat-killed smooth cells –> dead mouse
Initial reasoning: R cells restored viability to the S cells - not true
since he used cell-free extract of S cells and still changed R cells to S cells
Avery, MacLeod, McCarty (15 yrs later)
Partially purified the transforming principle
Demonstrated it was DNA, used modified known techniques
1. purified S culture DNA plus R cells –> plated it out
Results: culture with both S and R colonies isolated colonies,
replated - pure colonies
2. Attempted to use polysaccharide material as transforming material - no results
DNA extraction method
Avery, MacLeod, McCarty
- very impure (other evidence required)
1. chemical analysis, COP, nucleic acid
2. physical measurements very viscous - nucleic acid
3. Enzyme treatment
not lost with proteolytic enzymes - trypsin chymotrypsin
not lost with ribonuclease
lost when treated with DNase
no pure DNase - therefore, elaborate tests to confirm
Difficult time convincing the scientific community (published in 1944)
Avery, MacLeod, McCarty
Reasons
- tetranucleotide
- not enough variation with only 4 bases
- genes- chromosomal protein
a. TP (transforming principle) was actually a contaminating protein
b. DNA had a regulatory effect on capsule manufacture
Erwin Chargaff - 1952
Disproved tetranucleotide theory
Reason for tetranucleoside eukaroyotic cells - ATGC equimolar
used paper chromatography and UV absorbance
isolated DNA from wide variety prokaryotes
showed that molar concentrations of bases varied widely between different organisms
showed enough variation for DNA to be genetic material
Hershey and Chase
Blender experiment
Blender experiment
E. coli phage T2 (DNA in a protein shell)
DNA - phosphorus
Proteins - (methionine, cysteine) sulfur
grow with 32PO43- phosphate, radioactive “hot” DNA
35SO43- sulfate, radioactive “hot” proteins
hot or labeled phage 32P not 35S injected
blendor - tear off phage from bacteria see what goes inside
Blender experiment Proceedure
- label phage
- attach phage and centrifuge
- resuspend
- blendor then centrifuge
- 80% 35S in supernatant
20% 35S in pellet (tail fragments attached)
or
70% 32P in pellet
30% 32P in supernatant (breakage of bacteria during blending)
or defective phage
Transfer Experiment (confirmation experiment)
one step further - isolate phage progeny
half 32P was transferred to progeny, no 35S
Structure of the DNA molecule
Early experiments established:
- large MW
- extended chain
- highly ordered structure
1945 Astbury
- proposed a single chain molecule
1950 Chargaff
disproved tetranucleotide [A]=[T]=[C]=[G],
actually [A+G]=[T+C] or purines = pyrimidines
1952 Pauling
- helix with bases on the outside
Cavendish laboratory at Cambridge Univ.
Astbury -
primative x-ray diffraction 1. bases stacked 2. sugars in same plane as bases 3. single chain 4. internucleotide spacing 3.4 Å, 20 layers complete turn 20 Å diameter
Maurice Wilkins and Rosalind Franklin
high quality photos x-ray diffraction
Watson and Crick
own x-ray work - saw Franklin’s photos
model building
Watson and Crick Proposals
- double helix (density determinations)
- hydrogen bonds between AT GC
- antiparallel chain structure (sugars 3’ or 5’ on chain)
- worked out a method of replication
DNA STRUCTURE
a. Purines/pyrimidines
b. Sugars (differences)
c. Backbone
d. Complimentary base pairing
DNA STRUCTURE -bonds
a. Attachment N H (of base to sugar)
b. Ester bond or phosphdiester bond (phosphate in backbone 5’)
c. N-glycosylic bond, base to sugar
Nucleoside
base linked to sugar
Nucleotide
base, sugar + PO4
Polynucleotide
several nucleotides
3’ ______ terminusand 5’_______ terminus
OH terminus
-PO4 terminus
Two forces that hold DS-DNA together
- H bonds - base pairing
2. Hydrophobic interactions
Antiparallel 3’OH - 5’PO4
- each type of terminus at each end allows enzyme activity at either end
- strand orientation - different replication implications
DNA ___ _____ structure
Double helix
space models __, ___, ___ forms
B, C, Z
Forms of the Double Helix
majority are right-handed helices (natural forms)
B formm
92% relative humidity, low ionic strength
major groove-protein contact
minor grove -
Diameter 20 A
Watson & Crick model, standard form in organisms
10.4 bp/turn, in vivo cell form
A form
75% R.H., counter ions (sodium, cesium)
11 bp/turn, wider diameter
major groove much smaller (proteins inaccessible)
very close to DS-RNA (2’ hydroxyl group causes physical constraints)
Z form
left handed helix (in vitro –> in vivo)
most bp/turn 12, least twisted
diameter - 18 Å
name from Z form of backbone
only single groove, greater density negative charges
alternating purines & pyrimidines (CGCG…) but not always
possible in vivo conversion from B –> Z, B