Topic 10, DNA structure and analysis
The genetic material must possess 4 major characteristics
A. Storage of information (to be discussed in topic 10)
B. Replication (to be discussed in topic 11)
C. Expression of genetic information (to be discussed in topics 12-15)
D. Variation by mutation (to be discussed in topic 17)
What is the genetic material
A. Who were the candidates?
1. It was known in the early part of the century (~1902) that
a. Genes are a part of the chromosome
b. Chromosomes are made up of protein and nucleic acids
c. Thus, either proteins or nucleic acids should be the genetic material
2. Proteins are long chains of amino acids
a. There are 20 different types of amino acids in living things,
so enormous diversity is possible
b. Over half of the dry weight of cells is protein
3. There are two kinds of nucleic acids, DNA and RNA
a. DNA is found in the chromosomes while RNA is found in chromosomes and also
in the cytoplasm, so it was felt that the genetic material was probably not RNA
b. DNA is composed of only 4 types of building blocks (nucleotides), A, T, G, and C
c. Most scientists at that time felt that DNA was too simple of a molecule to be
the genetic material
d. Also, a widely-held view was the tetranucleotide hypothesis (DNA was made up
of tetranucleotides of 1A, 1T, 1G, and 1 C) that were repeated in the DNA
molecule
4. Before 1950, most scientists believed that the genetic material was protein because
they thought that DNA was too simple of a molecule to contain the genetic information
B. An indirect evidence that nucleic acid was the genetic material
1. It was known that UV light could cause mutations
2. The action spectrum for mutation induction was determined;
the wavelength found to be most effective in producing mutations was 260 nm
3. The absorption spectra for nucleic acids and proteins were determined.
a. The absorption spectra for nucleic acids had a peak at 260 nm
(DNA and RNA have the same absortpion spectra)
b. The absorption spectra for proteins had a peak at 280 nm.
4. Because the action spectrum for mutation induction paralleled the absorption
spectrum for nucleic acids, this implied that nucleic acid was the genetic material
and tended to exclude the possibility that proteins were the genetic material
C. Three direct evidences that DNA (or RNA in some viruses) was the genetic material
1. Transformation in pneumococcus (Diplococcus pneumoniae)
a. Griffith discovered transformation using pneumococcus
1) S (smooth colony) pneumococcus produces smooth colonies because the cells
have a polysaccharide capsule. They are virulent (can cause disease)
2) R (rough colony) pneumococcus produces rough colonies because the cells
do not have a polysaccharide capsule.
They are avirulent (cannot cause disease).
3) Living S cells cause pneumonia in mice (a control)
4) R cells do not cause pneumonia in mice (a control)
5) Heat-killed S cells do not cause pneumonia in mice (a control)
6) A mixture of R cells and heat-killed S cells causes pneumonia in mice
(experimental)
a) Living S cells are recovered from the mice that died
b) Something in the heat-killed S cells transformed the R cells into S cells.
The process is called transformation and the substance that caused
the transformation is the transforming substance.
b. In 1931, Dawson found that transformation could occur in vitro (in a test tube,
did not have to take place- inside a mouse)
c. In 1933, Alloway found that an extract of S cells could transform R cells into
living S cells
d. Avery, MacLeod, and McCarty (1944) carried out experiments to identify the
chemical that caused transformation
1) They purified DNA from heat-killed S cells and found that this DNA could cause
transformation. They concluded that the transforming substance was DNA
(DNA was the genetic material).
2) To be absolutely certain that contaminating protein or RNA in the DNA preparation
was not causing transformation,
a) They treated the DNA extract with protease (which destroys proteins).
They found tat the extract could still cause transformation, so contaminating proteins were
not responsible for transformation.
b) They treated the DNA extract with RNase (which destroys RNA).
They found that the extract could still cause transformation, so contaminating RNA was
not responsible for transformation.
c) They treated the DNA extract with DNase (which destroys DNA).
They found that the extract could not cause transformation, so they concluded that
DNA was responsible for transformation. (DNA was the transforming substance)
3) This work was published in a somewhat obscure journal and most scientists
were not ready to accept this finding at this time. The scientific community still generally
believed that protein was the genetic material
2. The Hershey and Chase Experiment (1952) with bacteriophage T2
a. Bacteriophages (phage) are viruses that infect and reproduce inside bacteria
b. T2 phage has E. coli as its host
c. T2 is approximately half protein and half DNA, so either protein or DNA had to be
the genetic material
d. Discussion of life cycle of phage T2
e. Allowed phage to reproduce in bacteria growing in medium containing 32P (which is in DNA
but not proteins), harvested progeny phage, and allowed them to infect unlabeled bacteria.
They knocked the heads off of the bacteria and found that label (32P) was in the bacterial cells.
f. Allowed phage to reproduce in bacteria growing in medium containing 35S
(which is in proteins but not DNA), harvested progeny phage, and allowed them to
infect unlabeled bacteria. They knocked the heads off of the bacteria and found
that label (35S) was in the supernatant (not associated with the bacteria).
g. Because the DNA (labeled with 32P) entered the bacterial cells, they concluded that
DNA was the genetic material
h. This work was widely recognized and convinced the scientific community that
DNA must be the genetic material.
3. Fraenkel-Conrat and Singer's 1975 experiments with TMV and HR
a. TMV (tobacco mosaic virus) and HR (Holmes ribgrass virus) both infect tobacco
b. These viruses are made up of RNA and protein
c. They separated the protein coats from the RNAs of both viruses and reconstituted
them in new combinations
d. The reconstituted viruses were used to infect tobacco plants
e. The protein coats of the progeny viruses corresponded to the RNA of the
reconstituted viruses and not the protein.
f. They concluded that RNA is the genetic material in these viruses
- Nucleic acid chemistry
A. Components of DNA
1. DNA is made up of repeating units, the nucleotides
2. A nucleotide is composed of a phosphate group, a 5-carbon sugar, and
a nitrogenous base
a. The 5-carbon sugar (a pentose) is deoxyribose in DNA and ribose in RNA
b. The nitrogenous (nitrogen-containing) bases are purines and pyrimidines
1) Purines have a double-ring with 9 members (guanine and adenine)
2) Pyrimidines have a single ring with 6 members
(cytosine and thymine in DNA and cytosine and uracil in RNA)
c. A nucleoside is composed of a 5-carbon sugar and a nitrogenous base
d. Nucleoside and nucleotide terminology
B. Chargaff's data on base ratios in DNA from various species (1949-1953
1. Chargaff et al. examined the base ratios in different species. They found that:
a. The relative proportions of the 4 nucleotides were the same in all members of a
species and in all tissues of an organism, but were different in different organisms
b. The % of A = the % of T, and the % of C = the % of G
c. This indicated that the DNA was arranged in an orderly manner in the DNA
C. X-ray diffraction patterns of DNA by Wilkins et al. (1953)
1. They prepared crystals of DNA from several species and determined the
X-ray diffraction patterns for these DNAs
2. Their conclusions are given on the handout
D. Watson and Crick's model for the structure of DNA (1953) (now called the B form)
1. Watson and Crick primarily used the chemical data of Chargaff and the physical data
of Wilkins to construct their model for the structure of DNA
2. The main features of the model are listed on the handout
3. This work stood the test of time, and Watson and Crick were awarded the
Nobel prize in 1962 for this work
E. DNA can exist in additional forms (A, C, D, E, and Z)
F. RNA chemistry, and a comparison of RNA with DNA
1. There are 3 major differences between RNA and DNA
a. RNA has ribose in place of deoxyribose in DNA
b. RNA has uracil in place of thymine in DNA
c. RNA is typically single-stranded whereas DNA is typically double-stranded
d. In addition, DNA is located in the nucleus and RNA is primarily in the cytoplasm
2. The three major classes of RNA
a. Ribosomal RNA (rRNA)
b. Messenger RNA (mRNA)
c. Transfer RNA (tRNA
3. Sizes of RNA molecules is typically given S (Svedberg) units which is a
sedimentation value in a centrifuge
4. Table of different RNA types, S values, and number of nucleotides in prokaryotes,
given in class and in table 11.4
- Some analytical techniques to investigate DNA and RNA
A. Absorption of UV light
1. Nucleic acids (both DNA and RNA) absorb most strongly at wavelengths of
254-260 nm.
2. The amount of UV light absorbed at 260 nm is proportional to the amount of
DNA or RNA present
B. Sedimentation behavior
1. Involves centrifugation
2. Sedimentation equilibrium centrifugation (density gradient centrifugation)
a. A gradient (usually CsCl) is prepared and the DNA is placed in it.
b. The DNA comes to equilibrium while it is being centrifuged
c. This procedure can be used to estimate the base ratios of DNA
(DNA with a higher G-C content has a higher buoyant density)
3. Sedimentation velocity centrifugation
a. Follow the migration of molecules down a tube during centrifugation
b. Typically quantitated in S (Svedberg) units)
C. Denaturing (melting) and renaturing nucleic acids
1. Occurs by breaking hydrogen bonds between the two complementary strands
of a DNA double helix
2. Melting temperature = the temperature at which half of the strands are separated
a. Can be used to estimate base ratios,
the higher the G-C content the higher the melting temperature
3. Renaturing DNA (upon slow cooling, the complementary strands come back together)
D. Molecular hybridization (reannealing of complementary nucleotide sequences)
1. It is the basis of many powerful techniques in molecular biology
2. Often carried out with DNA immobilized on a membrane,
usually detected by radioactivity
3. In situ molecular hybridization, hybridization on a chromosome
a. FISH (fluorescent in situ hybridization)
E. Reassociation kinetics of repetitive DNA, an extension of molecular hybridizaiton
F. Electrophoresis of nucleic acids
1. Electrophoresis is the movement of molecules through a stationary phase (such as a gel)
in response to an electrical field
2. Widely used in the analysis of nucleic acids
3. DNA molecules are separated out according to size, smaller pieces migrate more
rapidly than larger pieces
Updated 11/06/00