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Lecture 9
Gene structure and function: a historical overview
1. Proof that DNA is genetic material
2. Initial insight into the nature of
mutations
3. Bacteriophage crosses
4. Intragenic recombination
5. Cis-trans test for complementation
6. Double helix
Streptococcus pneumoniae
Experiments of Griffiths in 1928 showed that bacteria can be
genetically altered by some chemical substances
(a discovery of bacterial transformation).
R type (rough colonies) – nonvirulent bacteria; S type (smooth colonies) – virulent bacteria
In 1944, Avery,
MacLeod and
McCarty showed
that virulence is
associated with DNA
Electron micrograph and diagram of bacteriophage T2
Life cycle of virulent phages T2 or T4
T4 phage genome: ~300 genes, 1700 bp
E. coli genome: 4,254 genes, 4.6 x 106 bp
In 1952 by Hershey and Chase
proved that genetic material is DNA
Hershey would
subsequently share the
1969 Nobel Prize in
Physiology or Medicine
for his work in discovering
the properties of DNA. But
Marta Chase (1927-2003),
who served as Hershey's
lab assistant during his
experiments and whose
name appears on the
paper, was snubbed. She
earned her Ph.D. in 1964
from the USC. A series of
personal setbacks
through the 1960s ended
her career in science. She
spent decades suffering
from a form of dementia
that robbed her of shortterm memory.
Alfred D. Hershey
1908-1997
Phosphorus is found in DNA, while sulfur in proteins
The experiment that shows that DNA, not protein, is genetic material
The injected genetic material contained phosphorus, therefore,
it is DNA, not protein
the structures not for memorization
Selection of E. coli resistant to phage T1 (tonr mutants)
Spread bacteria (108) over the plate
and incubate overnight at 37 oC
Mix bacteria (108) with phages (109)
and incubate overnight at 37 oC
bacterial lawn
(uniform growth
over the entire
surface)
only a few
individual tonr
colonies will grow
What is mutation?
 a stable and inheritable
physiological adaptation?
a cell tons  a cell tonr
 a spontaneous change?
allele tons  allele tonr
Luria-Delbrück experiment (1943) (also called the Fluctuation Test) demonstrates
that new mutations causing resistance to phage T1 arise spontaneously (in the
absence of any selection) rather than being a physiological change in response to
selection.
adaptation
spontaneous
change
In 1952 Joshua and Ester
Lederberg demonstrated that
auxotrophic mutations arise
spontaneously and are then
selected
(replica plating)
Some new terms:
Mutation rate
e.g.: 3 x 10-8 mutations per cell division
Mutation frequency
e.g.: 5.7 x 10-7 mutations per cell
these are actual data for
tonS  tonR new mutations
Radiation (X-ray and other types) cause mutations
Hermann J. Muller
The Nobel Prize in
Physiology or
Medicine 1946
ionizing radiation
often cause chromosomal
breaks at high doses
1 rad = the dose causing 100 ergs
of energy to be absorbed by one
gram of matter; 1 krad = 1000 rads
(1 erg = 10−7 joule)
FYI
not for memorization
chemical mutagens predominantly cause point mutations
FYI
Phage colonies (plaques) grown on the lawn of E. coli cells
phage plaques
E. coli lawn
Phage phenotypes:
• appearance of plaques
• resistance to drugs
• host specificity
• various biochemical features
commercially available Petri dish full of E. coli cells
A cross of bacteriophages
Parental phages:
small turbid (h- r+)
large clear (h+ r-)
Recombinant plaques
RF m.u. =
%
Total plaques
Recombinant phages:
small clear (h+ r+)
large turbid (h- r-)
recombinant
phages
Seymour Benzer
in early 1950-ies
at Purdue Univ.
Bead theory of genes:
By the early 1950-ies that was the prevailing
view on the structure and arrangement of
genes on chromosomes.
Benzer proved it was wrong
some time later
Later in CalTech
Phage rII mutants could grow only with E. coli strain B
mutant phage:
no plaques
wild type phage:
E. coli strain K
(nonpermissive)
E. coli strain B
(permissive)
Phage T4
mutant rII
E. coli B
large
E.coli K
no plaques
w/t (r+)
small
small
Intragenic recombination in rII mutants
rII gene
rII gene
rII1
rII1,2
rII2
w/t
Can grow only
on E. coli B
Can grow on both
E. coli B and K
A rare inside-the-gene) crossover between two different rII mutants generates a double mutant and a w/t phage
Cross two
mutants by coinfection a
permissive
culture of E.
coli B cells,
obtain phage
progeny
Plate the progeny on a
lawn of non-permissive
K cells
Plate the progeny on a
lawn of permissive B
cells
Count the plaques.
Only w/t progeny can
grow = half of all the
recombinants (double
mutants cannot grow
on K cells)
Count the plaques. All
kinds of phages can
grow = total progeny
RF = Rec. / Total
Determine RF
= 2 x w/t %
total
RFmin = 0.01%
But how many genes are there in the rII gene region:
just one or more? A complementation test
rII gene region with many individual
mutations that were accurately mapped
1. If there is just one gene, then no two mutations will complement each other when two
mutant phages coinfect the same cell: no rII gene product (an enzyme or other protein)
is produced by either mutant
rII1
Enzyme
rII2
No phage growth (no cell lysis)
= no complementation
1. If there are two genes, then mutations in different genes will complement each other
when two mutant phages coinfect the same cell: each mutant is deficient with respect
to one product, but when combined they will provide all the products necessary
rII1
rIIA
Enzyme A
rIIB
Enzyme B
rII2
rIIA
rIIB
Phage growth (cell lysis)
= complementation
Note: no recombination is involved here!
The progeny phages are the original mutants!
A test for complementation in phages
- despite the host being non-permissive!
- the original mutants
Complementation tests for determining the units of function in the rII region of phage T4; the nonpermissive host E. coli K12 () is
infected with two different rII mutants. (a) Complementation occurs. (b) Complementation does not occur.
A test for complementation – aka „cis-trans test‟
Cistron = gene
cis-configuration: mutations on the same chromosome
trans-configuration: mutations on different chromosomes
How can DNA perform genetic functions?
It should satisfy the following criteria:
- be able to store genetic information;
- be able to change (mutate)
- be able to pass this information from generation
to generation.
The answer to these questions required knowledge
of the detailed chemical structure of DNA.
Erwin Chargaff analyzed the nucleotide composition of
DNA from many organisms and found the regularities in
molar concentrations of nucleotides:
A=T
G=C
A+G = C+T = 50%
but A+T ≠ G+C
Rosalind Franklin and Maurice Wilkins studied X-ray diffraction
patterns of DNA
The famous “photo 51”, shown to James
Watson by Maurice Wilkins without
Franklin's knowledge, was the critical
evidence that led to the confirmation of the
postulated double helical structure of DNA,
(see more on:
http://en.wikipedia.org/wiki/Maurice_Wilkins)
Their data suggested that DNA is a long molecule and
represents some sort of a spiral or helix with periodicity
of a 3.4 nm. Franklin even suggested it is a double helix.
In 1953 Watson and Crick put these two sets of data (Chargaff‟s and
Franklin-Wilkins‟) together and produced the model of DNA threedimensional structure: the famous double helix
How can DNA perform genetic functions?
It should satisfy the following criteria:
- be able to store genetic information:
The information is stored in the form of nucleotide sequence.
A genetic code is used to define amino acids by threenucleotide combinations (triplet codons) in DNA.
- be able to change (mutate):
The information may change (a mutation may occur) by a
change of nucleotide sequence.
- be able to pass this information from generation
to generation
The information is passed on to progeny by the mechanism
of semiconservative replication of DNA, wherein each strand
of DNA is used as a template for synthesis of another
strand, as was first realized by Watson and Crick, who
wrote...
Semiconservative replication of DNA
The most famous understatement in
the histrory of science:
“...It has not escaped our notice that
the specific pairing we have postulated
immediately suggests a possible
copying mechanism for the genetic
material.”
Watson and Crick (1953) Nature, 171:737-738
"for their discoveries concerning the molecular structure of
nucleic acids and its significance for information transfer in
living material"
Rosalind Elsie Franklin
1920-1958
A 1962 photo shows Nobel Prize winners (from
left): Maurice Wilkins (medicine), Max F.
Perutz (chemistry), Francis Crick (medicine),
John Steinbeck (literature), James Watson
(medicine) and John C. Kendrew (chemistry).
Not pictured are winners Linus Pauling (peace)
and Lev Landau (physics).