Life Science 3 Lecture 2

Front 1

Hershey-Chase Experiment

Back 1

-Uses radioisotopes and bacteriophages to show that DNA is the genetic material
-He labeled bacteriophage T2 with two different radioisotopes: distinguish between phage and host macromolecules as well as DNA from protein [DNA-labeled with 32P; protein-labeled with 35S]
-T2 much smaller than bacteria: 40%DNA, 60% protein
-Experiments designed to determine what happens to DNA and protein upon infection of bacteria
-In the experiment he would grow bacteria in 32PO4, infect with T2, centrifuge to separate phage from lysed cells, and use these 32P labeled phage to infect unlabeled bacteria
-He then determined what was in the supernatant [liquid portion] and what was in the pellet. In experiments where they used 35S there was little found within bacterial cells. however, in the 32P experiments there were more material recovered in the pellet.

Conclusion: DNA must contain genetic information.
Only genetic material can carry information to direct the synthesis of progeny phage; progeny phage are made in host cells; DNA injected in host cell; DNA must carry genetic information

Front 2

Radioisotopes

Back 2

-Elements that are defined by the number of protons they posses. [electrons=protons in non-ionized forms; neutrons can vary leading to different isotopes]
-When you add too many neutrons the molecule is too unstable and will spontaneously decay

Front 3

32P/ 35S

Back 3

32P: Important for labeling nucleic acids and phosphorylated proteins
-35S: important for labeling proteins

Front 4

Radioactive decay

Back 4

-Various particles are emitted
-Use detection methods to detect the decay: this includes geiger counters [emissions in air], scintillation counters [radioactive emissions are converted to light], autoradiography [X-ray film that helps determine where the radioactivity is]

Front 5

beta emitters

Back 5

-most biological radioisotopes are beta emitters:
--equivalent to an electron
--vary in energy: 3H low energy and 32P high energy
--half life (t1/2): time at which 1/2 of substance has decayed: 3H-12.3 years and 32P-14.3 days

Front 6

Initial understanding of DNA structure

Back 6

-Known to be a long polymer
-Made up of phosphate, deoxyribose and four bases [adenine, guanine, cytosine, and thymine]
-%adenine=%thymine; %guanine=%cytosine
-DIDN'T KNOW STRUCTURE

Front 7

Double Helix Model

Back 7

-Proposed in 1953 by James Watson and Francis Crick
-Based on data from Chargaff [who claimed equal A-T and C-G ratio]
-Also used preliminary X-rya diffraction data [discovered by Rosalind Franklin and Maurice Wilkins]
-Crick: worked on crystallography and was a chemist
-Watson: geneticist who emphasized double strand structure of DNA
-earlier models were a alpha helix [bases sticking out]; Watson said bases must face inward to correlate with replication of DNA

Front 8

Base vs. Nucleoside vs. Nucleotide

Back 8

Base: adenine, thymine, guanine, cytosine
Nucleoside: base+ sugar
Nucleotide: base+ sugar+ phosphate group

Front 9

Purine

Back 9

Two ringed structures: guanine and adenine

Front 10

Pyrimidine

Back 10

One ringed structures: cytosine and thymine

Front 11

Predominant forms of bases

Back 11

The predominant forms are most commonly seen forms. There are other forms [change the hydrogen bond characteristics]; when this happens and there is replication often times this can cause problems with base pairing and errors in replication

Front 12

Ribose vs. deoxyribose

Back 12

The existence of a hydroxyl or H group on carbon 2

Front 13

Importance of C in deoxyribose

Back 13

1': position is where base is connected
2': is missing the OH in DNA (deoxy)
3': is involved in phosphodiester bonds
4': where the ring O originates
5': used to form diester bonds

Front 14

C'2 vs. C'3 endo

Back 14

C'2 endo=predominates in DNA
C'3 endo=predominates in RNA

Front 15

dCMP [explain what it stands for]

Back 15

D: deoxy-DNA
C: cytosine-name of base
M: mono-number of phosphates attached

overall: deoxycytidine-5'-phosphate

[5' is location of phosphate attachment to carbon]

also for dAMP, dCMP, dGMP, dTMP

Front 16

AMP, ADP, ATP

Back 16

changes in number of phosphates:
nucleoside mono/di/tri phosphate

Front 17

DNA basic structure

Back 17

DNA is a polymer made of monomers: it is connected by a phosphodiester bond [at 3' and 5']. The structure goes from 5' [phosphate at top] to 3' [sugar on bottom]
There is a sugar phosphate backbone: alternate between the two
The bases are perpendicular to the helix
Two strands wrap together to form a double helix

Front 18

Right vs. left handed double helix

Back 18

Right handed double helix tends to be more common:
-Can determine whether right or left handed by wrapping hand underneath where they cross. Strands are antiparallel

Front 19

DNA Double Helix strands

Back 19

-anti parallel strands [5'--3' and other is 3'-->5'
-Strands are held together by hydrogen bonding between base pairs and opposite strands.
-Also by hydrophobic stacking interactions between base pairs on the same strand.
-Also large number of weak bonds contribute to stability.
-This is important because strength of hydrogen bonds is equivalent to strength of water hydrogen bonds; DNA in aqueous environment; this allows water to be excluded from the double helix

Front 20

Base flipping

Back 20

Base flipping occurs when bases change from facing the double helix to facing the aqueous environment; VERY unfavorable; this happens when different proteins or enzymes interact with DNA

Front 21

Purines/Pyridines flipped

Back 21

Purines and pyridines can be flipped: overall net size of each base pair is identical and fit perfectly in between two strands

Front 22

Hydrogen bond acceptors/donors

Back 22

-Hydrogen bond donors donate H to bond
-Hydrogen bond acceptors donate a pair of e- to bond
-G pairs with C: three H bonds
-A pairs with T: two H bonds
Explains why A/T and C/G are paired together
-C/G> A/T hydrogen bond

Front 23

Double helix in depth structure

Back 23

-10.5 bp per turn (34 A)
-sugar phosphates on outside
-bases inside
-major groove [perfectly fits an alpha helix from a protein' allows protein to interact with DNA]
and minor grooves.
-Phosphate makes DNA an acid
-ribbon model of DNA highlights the backbone of DNA; bases are perpendicular to axis of helix
-Major groove: 22a, Minor groove: 12A
-3.4 A per base pair; 34 A per turn

Front 24

Major groove and minor groove

Back 24

-Space filling model of DNA highlights the stacking of base pairs
-In every base pair part is in major groove and part in minor groove

Front 25

B-DNA
A-DNA
Z-DNA

Back 25

B-DNA: one we tend to see
A DNA: different grooves; bases stacked differently [RNA double helixes]
Z-DNA: squiggly

Front 26

Linear representation of DNA

Back 26

Just write the bases [phosphate/sugar assumed]
-One strand is complementary to other and its sequence may be deduced from base pairing rule
-A pairs with T and G pairs with C
-usually can write oen strand

Front 27

Replication [early explanation

Back 27

Double helix could separate and each helix serves as template to make new double helix. End with two identical sequences. Mechanism is much more complicated!