Transcript Document

1
2
3
4
5
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.
6
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
7
The researchers also recovered recombinants from a U-tube experiment
8
Background: viral infection
9
10
A generalized bacteriophage lytic cycle
11
12
Phage plaques
Micro -> Macro?
With plaques.
13
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.
14
I. Mapping phage chromosomes using phage crosses
h clearness (herecolor) of plaque
r size of plaque
h-r+ x h+rh-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
15
I. Mapping the genome of a virus
16
(h+ r+) + (h- r-)
RF =
total plaques
II. Transduction, and mapping of the Bacterial genome
The mechanism of generalized transduction
17
IIa. Generalized Transduction
Score co-transductants , derive order and relative distance
thr
azi
leu
P1 can only cotransduce genes less then
approximately 1.5 min apart on the
E.coli chromosome map
18
OR
thr
leu
azi
thr
leu
azi
Virulent phage
19
Temperate phage/prophage
Specialized Transduction: Via a phage that integrates at ONE site
site-specific recombination
NOT homologous recombination
20
IIa. Specialized Transduction: Via a phage that integrates at ONE site
21
IIa. Specialized Transduction: Via a phage that integrates in one site
22
23
F’ - duction
24
F’
F-
F’-duction
25
26
27
The 1963 genetic map of E.coli.
units are in minutes, based on interrupted-mating experiments
(position of approximately 100 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)
28
September 1997-The complete sequence of the E. coli genome
29
Correlation of the genetic and physical maps
Genetic map
Physical map (open reading frames – gene sequences)
30
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
31
James D. Watson
1928 (age 84)
Francis Crick
1916-2004
Maurice Wilkins
1916-2004
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".
32
“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”
33
Rosalind Franklin X-ray
Diffraction analysis
1920 –1958
Of DNA
34
35
36
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.”
37
‫רוזי‪ ,‬כמובן‪ ,‬לא העבירה לנו ישירות את‬
‫הנתונים שבידיה‪ .‬למעשה אף אחד במכללת‬
‫קינג לא ידע שהנתונים בידינו‪.‬‬
‫‪38‬‬
"[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."
39
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
40
DNA is the genetic material: Frederick Griffith –
Discovery of bacterial transformation
The bacterium
Streptococcus pneumonia
Rough
appearance
1879-1941
Smooth
appearance
Somehow, the
cells debris of the boiled S cells
had converted the live R cells into live S cells
(transformation)
41
DNA is the genetic material of which genes and chromosomes are made
“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]
Oswald Avery
1877-1955
42
The demonstration that DNA is the transforming principle
was the first demonstration that genes (the hereditary material)
are composed of DNA
43
DNA is the genetic material: Avery et al. - transformation
44
45
DNA is the genetic material: Hershey & Chase - transduction
Alfred Day Hershey
(1908-1997)
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.
46
Martha Cowles Chase
(1927-2003)
47
Genetic map
Physical map (open reading frames – gene sequences)
48
In vitro
49 above, where the DNA must recombine and replace the endogenous copy,
NOT like
BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.)
Lyse bacteria, collect the multiple
Copies of plasmid DNA
-Sequence the DNA
of the inserted gene B
50 above, where the DNA must recombine and replace the endogenous copy,
NOT like
BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.)
Genetic map
Physical map (open reading frames – gene sequences)
51