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Discovery of transduction
Joshua Lederberg 1925 – 2008 Norton Zinder 1928 – 2012 Discovered that bacteriophage can carry genes from on bacterium to another. Initial experiments were carried out in Salmonella. Lederberg and Zinder named this process transduction.
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Discovery of transduction
1. Started with phe-, trp-, tyr- and met-, his-, from the bacterium Salmonella typhimurium 2. After the two strains were mixed wild type prototrophs appeared at the frequency of about 1 to 105 (similar to E. coli) 3. In this case the researchers also recovered recombinants from a U-tube experiment 4. By varying the size of the pores in the filter, they found that the agent responsible for gene transfer was the same size as a known phage of salmonella called phage P22
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The researchers also recovered recombinants from a U-tube experiment
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Background: viral infection
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A generalized bacteriophage lytic cycle
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Phage plaques Micro -> Macro? With plaques.
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I. Mapping phage chromosomes using phage crosses
h-: can infect two different E.coli strains (strains 1 and 2) h+: can infect only strain 1 r-: rapidly lyses cells, thereby producing large plaques r+: slowly lyses cells, producing small plaques Mixed infection Strain 1 is infected with both parental T2 phage genotypes. After an appropriate incubation period, the phage lysate is then analysed by spreading it onto a bacterial lawn composed of a mixture of E. coli strains 1 and 2.
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h-r+ x h+r- h-r+ h+r- h-r- h+r+
I. Mapping phage chromosomes using phage crosses h clearness (here- color) of plaque r size of plaque h-r+ x h+r- h-r+ h+r- h-r- h+r+ 1. Large plaques indicate rapid lysis (r-) 2. Small plaques slow lysis (r+) 3. Phage plaques with allele (h-) will infect both hosts, forming a clear plaque 4. Phage plaques with the allele (h+) can infect only strain 1, forming a cloudy plaque
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(h+ r+) + (h- r-) total plaques
I. Mapping the genome of a virus (h+ r+) + (h- r-) total plaques RF =
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II. Transduction, and mapping of the Bacterial genome
The mechanism of generalized transduction
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thr azi leu leu thr azi IIa. Generalized Transduction
Score co-transductants , derive order and relative distance thr azi leu OR P1 can only cotransduce genes less then approximately 1.5 min apart on the E.coli chromosome map leu thr azi
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Virulent phage Temperate phage/prophage
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Specialized Transduction: Via a phage that integrates at ONE site
site-specific recombination NOT homologous recombination
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IIa. Specialized Transduction: Via a phage that integrates at ONE site
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IIa. Specialized Transduction: Via a phage that integrates in one site
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F’ - duction
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F’ F- F’-duction
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The 1963 genetic map of E.coli.
units are in minutes, based on interrupted-mating experiments (position of approximately 100 genes)
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(1990, total of more then 1400 genes)
Linear scale drawing of a 5-minutes section of the 100-minute 1990 E.coli linkage map (1990, total of more then 1400 genes)
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September 1997-The complete sequence of the E. coli genome
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Physical map (open reading frames – gene sequences)
Correlation of the genetic and physical maps Genetic map Physical map (open reading frames – gene sequences)
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Molecular Genetics First step: DNA is the genetic material
DNA, NOT protein of the chromosomes/chromatin PROVEN WITH TRANSFORMATION AND TRANSDUCTION, THAT WE JUST MASTERED
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James D. Watson 1928 (age 84) Francis Crick Maurice Wilkins The Nobel Prize in Physiology or Medicine 1962 was awarded jointly to Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material".
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X-ray Rosalind Franklin Diffraction analysis 1920 –1958 Of DNA
“We wish to suggest a structure for the the salt of deoxyribose nucleic acid (D.N.A) this structure has a novel features which are of considerable biological interest” Rosalind Franklin 1920 –1958 X-ray Diffraction analysis Of DNA
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Ten years after Rosalind Franklin's death, James Watson in his best-selling book
The Double Helix wrote that: "By choice she did not emphasize her feminine qualities There was never lipstick to contrast with her straight black hair, while at the age of thirty-one her dresses showed all the imagination of English blue-stocking adolescents. So it was quite easy to imagine her the product of an unsatisfied mother who unduly stressed the desirability of professional careers that could save bright girls from marriages to dull men Clearly Rosy had to go or be put in her place. The former was obviously preferable because, given her belligerent moods, it would be very difficult for Maurice [Wilkins] to maintain a dominant position that would allow him to think unhindered about DNA The thought could not be avoided that the best home for a feminist was in another person's lab.”
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רוזי, כמובן, לא העבירה לנו ישירות את הנתונים שבידיה
רוזי, כמובן, לא העבירה לנו ישירות את הנתונים שבידיה. למעשה אף אחד במכללת קינג לא ידע שהנתונים בידינו.
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"[I am] inherently gloomy about the prospect of Africa [because] all our social policies are based on the fact that their intelligence is the same as ours—whereas all the testing says not really.” (October 2007) "[the] historic curse of the Irish, which is not alcohol, it's not stupidity. But it's ignorance.” (2013) “Stupidity is a disease and the "really stupid" bottom 10% of people should be cured” “beauty could be genetically engineered, "People say it would be terrible if we made all girls pretty. I think it would be great.” (2003) extracts of melanin – which gives skin its color – had been found to boost subjects' sex drive. "That's why you have Latin lovers," he said, according to people who attended the lecture. "You've never heard of an English lover. Only an English Patient."
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What was known about genes and DNA before Watson and Crick solved the structure of DNA?
1. Genes-the hereditary “factors” described by Mendel-were known to be associated with specific character traits, but their physical nature was not understood. Similarly, mutations were known to alter gene function, but precisely what a mutation is also was not understood. 2. The one-gene-one protein theory postulated that genes control the structure of proteins 3. Genes were known to be carried on chromosomes. 4. The chromosomes were found to consist of DNA and protein
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Streptococcus pneumonia
DNA is the genetic material: Frederick Griffith – Discovery of bacterial transformation The bacterium Streptococcus pneumonia Rough appearance Smooth appearance Somehow, the cells debris of the boiled S cells had converted the live R cells into live S cells (transformation)
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DNA is the genetic material of which genes and chromosomes are made
Oswald Avery “The most deserving scientist to not receive the Nobel Prize for his work” (though he was nominated for the award throughout the 1930s, '40s and '50s).[)2]
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The demonstration that DNA is the transforming principle
was the first demonstration that genes (the hereditary material) are composed of DNA
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DNA is the genetic material: Avery et al. - transformation
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DNA is the genetic material: Hershey & Chase - transduction
Alfred Day Hershey ( ) Nobel Prize winning in Physiology or Medicine in 1969, shared with Salvador Luria and Max Delbrück for their discovery on the replication of viruses and their genetic structure. Martha Cowles Chase ( )
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Genetic map Physical map (open reading frames – gene sequences)
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In vitro NOT like above, where the DNA must recombine and replace the endogenous copy, BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.)
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Lyse bacteria, collect the multiple Copies of plasmid DNA
-Sequence the DNA of the inserted gene B NOT like above, where the DNA must recombine and replace the endogenous copy, BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.)
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Genetic map Physical map (open reading frames – gene sequences)
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