Genetics: Genetics Lecture 18, 10/10 - Bacteriophages, Genetic Experiments

How bacteriaphages accomplish genetic recombination

Lederberg-Zinder experiment showed bacteriaphage can accomplish recombination.
Two strains both of which are autotrophs for two genes.

between the two strains place a filter: media passes through but not cells
Plate cells from both sides of the tube on minimal media
Plating cells from the LA-2 side there is no growth.
From the LA-22 side you get prototrophs.
(4:35) How do you get prototrophs from the LA 22 side? ANS: Possible presence of a filterable agent which facilitated this result. Made 3 observation about this filterable agent:

1.) The agent was only produced when LA-2 cells were grown in concert with LA22 cells. If they grew LA2 and then placed it with LA-22 there was no cell recombination
2.) DNAse: enzyme which digests DNA did not make the filterable agent inoperative.
3.) The filterable agent was dependent on the size of the pores.
Concluded that the filterable agent was a phage (P22 phage). It was associated with the LA22 cells. Phage starts with LA22 and crosses the filter and picks up DNA from the LA 2 cells. (10:30) It then passes back across the filter and infects the LA22 cells. When this happens it donates the DNA from the LA2 cell to the LA 22 cell.
(11:45) Lysogenic cell - cell thats infected with phage but not a large enought level to kill the cell.
The prototrophs from the LA22 side where lysogenic cells with the DNA from the LA2 cell to make the cell prototrophic.

Chapter 10 - DNA structure and analysis.

This molecule must have the ability to be replicated
It needs to be able to serve as a repository of information
Needs to have a mechanism to allow it to be expressed. (it needs to be converted to protein to cause a phenotype)
It needs to be able to be changed. Mutation facilitates diversity.

DNA or protein (protein was originally the favored choice). It came about this way due to an incorrect hypothesis --> Tetranucleotide hypothesis: The largest DNA molecule you could make was 4 nucleotides long. This structure would contain each of the four nitrogenous bases.

(34:10) 1927 Fredrick Griffith worked with two strains of diplococcus pneumoniae. III S strain (virulent) and the smooth strain. When he injected the IIIS strain into a mouse it died.

IIR strain - rough strain - avirulent strain. When he injected into the mouse it lived
Smooth strain (IIIS) - Heat killed it. He found that when he injected it lived.
GRIFFITH'S CRITICAL EXP.: mixed heat killed IIS with living IIR cells and injected it into a mouse and got a dead mouse. (two avirulent strains became virulent). When analyzing the tissue of the dead mouse he recovered living IIIS cells.
proposed the presence of transforming principle: a molecule which survived heat killing and able to convert the IIR cells to IIIS cells.
Transforming principle is a logical candidate for the genetic material.

(42:10) Avery, Macleod, McCarty, 1944. Wanted to figure out what the transforming principle was. They heat killed with IIIS cells and extracted lipids, some proteins and carbohydrates (cell membrane). Took this filtrate and mixed with IIR cells. Transformation occured IIS cells

(44:50) Treat the filtrate with protease (degrades proteing). Mix with IIR cells and transformation occurs. Therefore protein is NOT the genetic material.
TReat the filtrate with ribonuclease (degrades RNA). Mix with IIR cells and transformation occurs. RNA is not the genetic material.
Treat the filtrate with DNAse (degrade DNA). Mix with IIR cells and transformation DOES NOT OCCUR. DNA is THE genetic material.

Virus is a protein coat surrounding DNA
took e. coli cells and grew them in two media: p32 label nucleic acid AND s35 label protein. Put phage into each culture. Expect the phage to pick up your label.
take each of the phages and place them in new cultures without label. The genetic material will be injected into the cell. Expect that whichever molecule was serving as the genetic material, the progeny phages will be labeled.
p32 progeny phages were labeled
s35 progeny phages were not labeled
this supports the idea that DNA is the genetic material for VIRUSES. Check out wikipedia for a good explanation

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