Lecture 10 Flashcards
Fredrick Griffith
- English medical officer and geneticist.
- Discovery of the “transforming principle” in bacteria.
Avery, MacLeod and McCarty
- Carried out experiments, reported in 1944 to try to discover what cellular factor of S cell accounts for transformation.
- Grew up culture of S cells, broke up the cells to make an “extract” and then treated the extract with different enzymes, one by one, to get rid of certain classes of compounds i.e. protein, RNA, DNA.
- They then mixed the extract with a culture of R cells and incubated this mixture for some hours to allow any newly formed S cells to reproduce.
- They added anitbodies against streptococcus pneumoniae. The antibodies bind to R cells, causing them to clump together; but antibodies cannot bind to S cells, because of the capsule.
- The mixture was then centrifuged at low speed to remove the clumped R cells. Because they are in clumps the R type cells pellet quickly, but the S type cells, if any have formed, remain in suspension.
- They then spread-plated the supernatant (broth left over after clumped R cells were removed) on agar plates, incubated the plates and then looked for colonies.
Hershey and Chase Experimental Approach
- Grow up phages in a way that radioactivity labels either the protein or the DNA.
1) In preliminary work, grow the bacterial host, E. Coli, in a medium containing 35S-Methionine (take up of this makes all the protein in the cell radioactive). The bacteria incorporate the 35S-methionine into their proteins then, infect those bacteria with T2 phage, allow them to grow lytically, and collect the released phage particles. The proteins of these phages, obtained from the breakdown of host cell proteins to AAs, are no 35S-labelled. Use these phages in an experiment to test if protein is the gentic material. DNA no sulfer → no label.
2) Repeat with another culture of the a medium containing 32P-phosphate, which will be incorporated into the bacterium’s DNA use these phage for the next experiment, to test if DNA is the genetic material. Protein→no phosphate→no label.
Avery, MacLeod, McCarty Experimental Results
- R cells + S cell extract (components of S type cells: proteins, RNAs, and DNA) but no intact S type cells. Positive Control. Result: S type colonies form on plate.
- R cells alone, negative control. Result: No colonies
- R cells + S cell extract treated with protease. Results: S type colonies.
- R cells + S cell extract treated with RNase. Result: S type colonies.
- R cells + S cell extract treated with DNase Result: No colonies.
- Conclusion: Treatment of the S cells extract with protease or RNase did not destroy the cellular factor responsible for transformation but treatment with DNase did destroy the transforming factor from S cells.
- Interpretation: DNA is the cellular factor responsible for transformation.
Which macromolecule in the cell is the genetic material?
What is the genetic material that carries the detailed instructions for complex process of Metabolism, survival and Reproduction?
- Proteins: 20 or so AA → large molecules with complex AA sequences that vary amoung different proteins.
- DNA: four nucleotides → not enough for complex sequences to encode genetic material?
Hershey and Chase Actual Experiment
1) Label the T2 phage proteins with 35S. Infect E. Coli with the 35S-labelled T2 phage, let it inject its genetic material, then kick the page particles off of the cells (in a blender), pellet the cells by centrifugation. - Fraction that has the radioactivity? The supernatant, protein not the genetic material. 2) Label the T2 phage with the 32P-phospate. Infect E. Coli with the 32P-labelled T2 phage, let it inject its genetic material, then knock the page particles off of the cells, pellet the cells by centrifugation. - Pellet with the cells has the radioactivity. Conclusion: DNA, not protein, is the genetic material of the bacteriophage T2. Interpretation: DNA contains the instructions for the formation of new phages.
S strain
- Smooth colony form of Streptococcus pneumoniae. The cells make polysaccharide capsules. As a result, their colonies on agar are big, smooth, and shiny.
- In 1928, the relationship between the capsule, phagocytic cells of the immune system, and the disease was not known.
- Griffith was interested in why capsulated cells of S pneumoniae were lethal to the mouse and why non-capsulated cells were not lethal.
Griffith’s Experiment
- Capsule formers: when injected into the blood stream of the mouse, the bacterium survives and reproduces in the blood stream, infects the lungs; kills mouth.
- Non-Capsule formers: when injected into the blood stream, the bacteria are engulfed and destroyed by phagocytic cells; mouse survives.
- Heat-killed S strain: mouse lives; bacteria have to be alive to infect the mouse.
- Heat-killed S + Live R: Live S and R strains isolated from dead mouse.
Griffith’s Hypothesis
- Heat killed S strain cells carry or release a transforming principle that transforms R strain cells to the S phenotype.
- The nature of this transforming principle remained obscure for some years until another scientist, Avery, and his collegues, MacLeod and McCarty, became interested in finding out what it was.
Three sets of experiments that helped explain DNA as the Genetic Material
1) The bacteriological discovery of a “transforming principle” by Fredrick Griffith in 1928.
2) The biochemical identification of the transforming principle as DNA by Avery, MacLeod, and McCarty in 1944.
3) The demonstration with a virus that the genetic material is DNA by Hershey and Chase in 1952.
Hershey and Chase Experiments, 1952
- Bacterial viruses-reproduce in a lyse bacterial cells.
- Also called bacteriophages or phages.
ex) Phage T2, infects E. Coli. Attaches to cell, injects its genetic material into cell. The phages genetic material forces the bacterial cell to make many new phage particales. The cell lyses and releases the new phage particles. - Background Info: Phage T2 was known to be made up of protein, DNA and the lytic cycle of phage was known. It attaches to a cell, injects its genetic material, and later the cell lyses releasing many new phage particles, but, what is the genetic material?
Chance in scientific discovery?
- Griffith was studying Streptococcus pneumoniae to try to develop a vaccine against it, using the mouse as a model for human pneunonia.
- Griffith was working with two colony forms of the bacterium on agar plates, smooth (produce a polysaccharide capsule), rough (no capsule).
- The smooth (S) strain was virulent, and the rough (R) strain was not virulent.
- Capsule produced by the S strain prevents phagocytosis by cells of the immune system, so the S strain can suvive in the human body, and cause disease. The R strain, because it lacks a capsule, is phagocytosed and destroyed by the immune system cells.
Experiment series II, with purified S cell components.
- R cells + purified S cell polysaccharide → no colonies
- R cells + purified S cell proteins → no colonies
- R cells + purified S cell RNA → no colonies
- R cells + purified S cell DNA → S type colonies
- Conclusion: Provision of S cell DNA mixed with R type cells gave rise to S type cells, but not S cell polysaccharide, protein and RNA had no effects.
- Interpretation: DNA is the factor from S cells responsible for transforming R type cells into S type cells.
Sterptococcus Pneumoniae
- Gram-Positive, aerotolerant anaerobe.
- Normal member of human upper respiratory tract.
- Can become pathogenic if immune system is suppressed.
- Causative agent of streptococcal pneumonia (lung infection).
- Bacterial pneumonia, strong inflammatory response, reduced lung function, 30% lethality if untreated.
- Multi-drug resistant strains, dangerous “emerging pathogen”.
- Also causes acute sinusitis.
- Produces a capsule, which is antiphagocytic.
- Causes pneumonia in mice that is similar to the disease in humans.
- Just 10 cells injected into the blood is enough to kill some mice, 100 cells kills all injected mice.
