NOTES FOR BIOLOGY 1201
Section 001
Spring 2005
DR. STEVEN POMARICO
Evidence that DNA is genetic material
Mendel => heritable factors
Morgan => genes on chromosomes
>>>>>>The search for the genetic material
Griffith and Transformation - 6.1.3
In 1928, Griffith working with Streptococcus pneumoniae demonstrated that genetic material could be transferred from one bacterium to another.
-Smooth cells (with capsule)
-Rough cells (no capsule) are not pathogenic
-Heat killed smooth cells are not pathogenic
-Heat killed smooth cells plus rough cells are pathogenic
AND PRODUCE LIVE SMOOTH CELLS!
The trait from the smooth cells was transferred to the rough cells, and the rough cells were transformed.
---Transformation
But what was transferred??
-Chromosomes are composed of DNA and protein.
-Protein is denatured by heat.
-Maybe it’s the DNA. Many doubters.
>>>Evidence that viral DNA can program cells.
Avery, MacLeod, and McCarty/Hershey and Chase; DNA Wins! - 6.1.4
More evidence that DNA is the genetic material came from the study of bacteriophages.
---bacteriophage (a.k.a. phage)
-Bacteriophages quickly reprogram the bacterial cells they infect.
In 1952, Hershey and Chase discovered that the genetic material from bacteriophage T2 that reprograms the bacteria was DNA.
-They radioactively labeled the components of the phage.
35S-phage protein or 32P-phage DNA
-Infected bacteria with the labeled phage.
-Separated the phage from the bacteria and looked for which label was transferred
to the bacteria.
Concluded that the DNA was the genetic material in a virus.
BUT WHAT ABOUT EUKARYOTES??
>>>Additional evidence that DNA is the genetic material
Chargaff and Franklin and Wilkins: The DNA Story Begins - 6.1.5
Circumstantial evidence
-In a eukaryotic cell, the DNA content doubles prior to mitosis
-During mitosis the double DNA is divided equally between the two daughter cells.
-Diploid cells have twice the DNA content as haploid cells
-DNA is found to be less uniform than original thought
In 1947, Chargaff used paper chromatography to separate the bases of the DNA from different species
-Found that the base composition varied between species
-Adenine (A) + Thymine (T) / Guanine (G) + Cytosine(C)
ratio varies more diversity
-There was a relationship between the bases
Chargaff’s rules
A = T
G = C
>>>>>>Watson and Crick (and others) discovered the double helix
Watson and Crick: The Clues - 6.2.1
Remember the structure of the monomer (nucleotide) and the polymer (single strand DNA).
Based of the experiments of Hershey and Chase, along with the circumstantial evidence and the work of Chargaff, DNA was accepted as the genetic material.
Understanding the structure of DNA
-Physical structure evidence
X-ray crystallography - Rosalind Franklin
-Chemical structure evidence
Chargaff’s rules
Size of the bases
Lead to the double helix model
Watson and Crick: The Double Helix - 6.2.2
-2 strands are antiparallel
-specific-base pairing key to solving puzzle
=> A-T and G-C pairs, explained Chargaff's rules
-10 bases pairs/turn
>>>>>>DNA replicates by using itself as a template.
Replication: Meselson and Stahl - 6.3.1
The double helix model with it’s base pairing provides mechanism for replication
=> each strand can serve as a template
This type of replication follows the semiconservative model
-In this model each new DNA double helix would be made of a strand of old DNA and a strand of new DNA.
2 copies old-new paired
2 alternatives were possible
-conservative model => old-old paired and new-new paired
-dispersed model => mixture of old and new in each strand
Meselson and Stahl provided experimental support for the semiconservative model
-Labeled DNA with 15N (makes DNA denser)
-DNA replication in regular 14N media
-Both DNA double helixes have the same density
-Can’t be Conservative Model
-Second round of replication in regular 14N media
-Two different densities of DNA
-Can’t be Dispersive Model
>>>>>>The replication of DNA
DNA: Polymerization with Triphosphate Nucleotides - 6.3.2
The process of DNA replication is conceptually easy, but reality of the process very complex.
-Requires 20+ enzymes and accessory proteins
-Extremely rapid - prokaryote 500 nucleotides added per second
- humans 50 nucleotides added per second
-Accurate - About 1 in a billion chance of a mistake
>>>Where it starts
The origin(s) of replication
-In prokaryotes there is only one, also only one circular chromosome.
-In eukaryotes there are many per chromosome
At each origin of replication the DNA separates to from a replication “bubble”.
At each end of that replication “bubble” is a replication fork where the new strands are being synthesized.
---Replication forks
>>>Elongation of the strand
Two processes:
Strand separation
Synthesis of the new strands
strand separation
-enzymes called helicases unwind the helix
-strands are kept apart by proteins
synthesis of the new strands
-DNA polymerase
-synthesis occurs in 5' => 3' direction
-therefore synthesis is bidirectional
-energy for the synthesis comes from the high-energy phosphate
bonds of the nucleoside triphosphate
Because of the bidirectional nature of the replication one strand is synthesized in a continuous manner (leading strand) the opposing strand (Lagging strand) is synthesized in pieces (Okazaki fragments)
---Leading strand
---Lagging strand
---Okazaki fragments
The Okazaki fragments of the lagging strand are joined together by another enzyme called DNA ligase
>>>Priming the reaction
Because DNA polymerase can only add nucleotides on to a polymer, there must be a primer to start the reaction.
---Primer
The leading strand only has to be primed once at each replication bubble, after that synthesis is continuous. The lagging strand requires many primers.
The synthesis of theses primers is accomplished by another enzyme called primase.