Module 2-1 Flashcards
Give the central dogma.
DNA–>[Transcription]–>RNA–>[Translation]–>Protein
What were possible genetic materials pre 1940’s?
Proteins and nucleic acids
- Proteins were leading contender
- -believed 4 nucleotides were too simple to be genetic material
- -20 amino acids were more probable due to potential variation
Describe the work done by Frederick Griffith (1927)
He analyzed virulence of various strains of Diplococcus pneumoniae
-Some strains caused pneumonia in humans and mice (virulent) and some did not (avirulent)
What caused the virulence of Diplococcus pneumoniae?
a polysaccharide capsule of bacterium
What were the characteristics of a virulent colony?
Had a capsule and were smooth, and shiny (s)
What were the characteristics of an avirulent colony?
Did not have capsules and were rough (r)
What was Griffith’s experiment referred to?
Transforming principle
True or false? There were many serotypes of rough and smooth colonies?
True. There were type I, type II, and type II
Which serotypes did he use?
II and III of rough and smooth strains (IIR and IIIS)
What are cell capsules and walls built by in the different serotypes?
Proteins and carbohydrates
True or false? Each type can switch from smooth to rough but not to a different serotype?
True
What happened if a mouse was injected with living IIIS (virulent)?
it died
What happened if a mouse was injected with living IIR (avirulent)?
it lived
What happened if a mouse was injected with heat-killed IIIS?
it lived
What was the critical experiment?
He injected heat-killed virulent IIIS bacteria and living avirulent IIR bacteria into a mouse and it DIED and living IIIS were recovered
What was Griffith’s conclusion about the fact that the mice died?
The heat-killed bacteria were transforming the live avirulent IIR bacteria into virulent IIIS bacteria
*Suggested the transforming principle might be something in the capsule
What did further transformation studies find?
that transformation could happen in vitro and that soluble filtrate of heat-killed S cells was sufficient to cause transformation
*suggested a chemical substance was responsible for transformation phenomena
What did the experiments of Avery, McLeod, and McCarty conclude?
That DNA was the transforming molecule
Describe how the Avery, McLeod, and McCarty experiment was set up?
IIIS cells were centrifuged and IIIS cells were spun to the bottom of the tube and were heat killed. These were homogenized and the IIIS filtrate was recovered and the Carbohydrates, lipids, and proteins were extracted. These were put into IIR cells and different enzymes were added.
What happened when Protease was added?
the proteins were degraded and the transformation occurred
What happened when RNase was added?
the RNA was degraded and the transformation occurred
What happened when deoxyribonuclease was added?
DNA was degraded and the transformation DID NOT occur, meaning DNA must be the transforming material
What did the Hershey-Chase Experiments provide?
convincing evidence that DNA is the Genetic Material
What was the model used in the Hershey-Chase experiments?
E. coli and T2 bacteriophage, a virus of E. coli that replicates within it following infection.
Proteins were labeled with radioactive 35S and 32P (DIFFERENTIAL LABELING)
Describe the Hershey-Chase experiments.
Phage were added to E. coli 32P and also a 35S medium. The phage progeny were labeled and the 32P labeled phages and the 35S labeled phages were used to infect unlabeled bacteria
Which radioactive medium labeled DNA?
32P
Which radioactive medium labeled Proteins?
35S
What were the first key results?
The only radioactive phage ghosts were those labeled with 35S
What were the second key results?
Only the phage that were produced from the 32P labeling were radioactive, meaning that DNA was the genetic material!!!!!!!
What were two forms of indirect evidence that DNA was the genetic material in eukaryotes?
- DNA found only where primary genetic function occurs, correlation of ploidy and the quantity of genetic molecule
- UV light is most mutagenic at wavelength that RNA and DNA absorb UV light the most (~260)
What are the three components of DNA?
nitrogenous base, pentose (5-carbon) sugar, and a phosphate group
What are the two types of nitrogenous bases?
- Purines = nine member double ring
2. Pyrimidines = six member single ring
What nitrogenous bases are purines?
Adenine and Guanine
What nitrogenous bases are pyrimidines?
Cytosine, Thymine, and Uracil
What is the difference between DNA and RNA nitrogenous bases?
DNA has Thymine where RNA has Uracil
What is the difference between a ribonucleoside and deoxyribonucleoside?
ribonucleoside has 2 -OH groups where deoxyribonucleoside has 1
How do you make a ribonucleoside?
Add ribose to purine or pyrimidine
How do you make a deoxyribonucleoside?
Add 2-deoxyribose to purine or pyrimidine in specific manner
How do you make nucleotides from nucleosides?
Add a phosphate group
How are polynucleotides formed?
phosphodiester bond between phosphate group and C3’ and C5’
What were the features of the Watson and Crick DNA model?
- 2 polynucleotide chains coiled around a central axis, forming right handed helix
- 2 chains are anti-parallel: 5’-3’ runs in opposite directions
- the bases lay flat perpendicular to the axis and are stacked on one another 3.4 angstroms apart
What forms the backbone of the DNA double helix?
Sugar phosphate bonds
What is the diameter of DNA?
20 angstrom
How long is one complete turn of the double helix?
34 angstrom
What 2 forms of DNA were known at the time of Watson and Crick?
A and B form
What bonds form between nucleotides?
hydrogen bonds
True or False? Most RNA is single stranded.
True
How are RNA and DNA quantified?
using the idea that the amount of UV light absorbed is related to the concentration of RNA or DNA
*A260 = 50ug/ml
Concentration = 50ug/ml x absorption
Describe the process of Nucleic acid centrifugation.
RNA’s differentiated by sedimentation behavior (called Svedberg coefficient)
*Higher S = larger molecule
Nucleic acid loaded onto concentration gradient and gradients are eluted and measured
What is another mode of nucleic acid separation?
Gel electrophoresis
Describe the process of gel electrophoresis.
Nucleic acid added to a gel and move proportional to their length (smaller moves farther)
How is DNA denatured/hybridized?
By breaking hydrogen bonds by heating
*lower temperatures reanneal DNA
How are DNA association/CoT curves found?
DNA is sheared and sonicated, renatured, have a slow initial association, then goes faster, and the last takes the longest
How is the Cot1/2 point found?
It is the point where 50% of the DNA is associated
True or false? Smaller genomes re-associate faster than larger ones?
True
Is there a linear relationship between Cot1/2 and genome size?
Yes
What are the characteristics of Eukaryotic DNA association?
- initial rapid reassociation (repetitive DNA)
- unique sequences take longer to find (curve flattens)
- the remaining sequences come together (faster)
* *DNA reassociation curves reflect the complexity of the genome
