Genetics Chapter 9: DNA and the Molecular Structure of Chromosomes
Functions of the Genetic Material:
- In 1865, Mendel showed that “Merkmalen” (now genes) transmitted genetic information.
Many studies were one that demonstrated that the genetic material must perform three
(1) The genotypic function, replication. The genetic material must store genetic
information and accurately transmit that information from parents to offspring,
generation after generation.
(2) The phenotypic function, gene expression. The genetic material must control the
development of the phenotype of the organism. That is, the genetic material must
dictate the growth of the organism from the single-celled zygote to mature adult.
(3) The evolutionary function, mutation. The genetic material must undergo changes to
produce variations that allow organisms to adapt to modifications in the environment
so evolution can occur.
- Other early genetic studies established correlation between patterns of transmission of
genes and the behaviour of chromosomes during sexual production, providing strong
evidence that genes are usually located on chromosomes.
- Chromosomes are composed of 2 types of large organic molecules called proteins and
- Nucleic acids are of 2 types: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA).
- During 1950’s results of elegant experiments established that genetic information is
stored in nucleic acids, not in proteins.
- In most organisms genetic information s encoded in structure of DNA, however in small
viruses’ genetic information is encoded in RNA.
Proof that Genetic Information is Stored in DNA:
- Most of DNA cells is located in chromosomes, while RNA and proteins are also
abundant in cytoplasm suggested that DNA harbors the genetic information of living
organisms. In addition to the fact that a correlation exists between amount of DNA per
cell and number of sets of chromosomes per cell.
Most somatic cells of diploid organisms contain twice the amount of DNA as haploid
germ cells of same species.
Molecular composition of DNA is same in all cells of organism, whereas composition
of both RNA and proteins is variable from one cell type to another
DNA is more stable than RNA or proteins. Since genetic material must store and
transmit info from parents to offspring, expect it to be stable, like DNA. However,
none of the above PROVES it! Proof that DNA Mediates Transformation:
- Frederick Griffith’s discovery of transformation in Streptococcus Pneumoniae
When Griffith injected both heat-killed Type IIIS bacteria (virulent when alive) and
live Type IIR bacteria (avirulent) into mice, many of the mice developed pneumonia
and died, and live Type IIIS cells were recovered from their carcasses. Something
from the heat killed cells- the “transforming principle”- had converted the live Type
IIR cells to Type IIIS.
- Sia and Dawson performed same experiment in vitro, showing that mice played no role in
transformation process. Sia and Dawson set stage for Avery, MacLeod and McCartys
demonstration that the “transforming principle” in S. Pneumoniae is DNA. Avery and
colleagues showed that DNA is the only component of the Type IIIS cells required to
transform Type IIR cells to Type IIIS.
- The most definitive experiments in Macleod, Avery and McCarty’s proof involved us of
enzymes that degrade DNA, RNA, or protein.
- Purified DNA from Type IIIS cells were treated with enzymes (1) DNase, which
degrades DNA, (2) RNase, which degrades RNA, or (3) Protease, which degrades
proteins. DNA was then tested for its ability to transform Type IIR cells to Type IIIS.
Only DNase treatment had an effect- it eliminated all transforming activity.
- Work of Avery and coworkers established that genetic information in Streptococcus is
present in DNA.
Proof that DNA Carries Genetic Information in Bacteriophage
- Alfred Hershey and Martha Chase demonstrated that DNA is genetic material in 1952.
Results showed that the genetic info of a bacterial virus (bacteriophage T2) was present
in its DNA.
- Viruses are smallest living organisms and their reproduction if controlled by genetic info
stored in nucleic acids, same process as cellular organisms. They can only produce in
appropriate host cells.
- Bacteriophage T2 is composed of about 50% DNA and 50% protein. Hershey and Chase
showed that the DNA of the virus particle entered the cell, whereas most of the protein of
the virus remained absorbed to outside of cell. Implication was that the genetic info
necessary for viral reproduction was present in DNA.
- DNA contains phosphorus but not sulfur, whereas protein contains sulfur but no
phosphorus. Thus, they were able to label either (1) the phage DNA by growth in a
medium containing radioactive isotopes of Phosphorus or (2) the phage protein coats by
growth in a medium containing radioactive sulfur in place of normal isotope.
- When T2 phage particles with sulfur (35S) were mixed with E. coli cells, most of the
radioactivity (thus proteins) could be removed. When T2 particles labeled phosphorus
were used the DNA was not subject to removal by shearing in a blender. - These results indicated that DNA of the virus enters host cell, whereas protein coat
remains outside cell.
The Structures of DNA and RNA
Nature of the Chemical Subunits in DNA and RNA:
- Nucleic acids are macromolecules composed of repeating subunits called nucleotides.
Each nucleotide is composed of :
(1) Phosphate group
(2) A five-carbon sugar
(3) A cyclic nitrogenous base
- In DNA the sugar is 2-deoxyribose and RNA is ribose.
- There are 4 major bases in DNA: adenine (A), guanine (G), thymine (T), and cytosine
(C). RNA uses uracil (U) instead of thymine.
- Adenine and guanine are double ring bases called purines, while cytosine, thymine and
uracil are single-ring bases called pyrimidines.
- RNA usually exists as a single-stranded polymer composed of long sequence of
nucleotides. DNA is usually a double-stranded molecule.
DNA Structure: The Double Helix
- In 1952 James Watson and Francis Crick deduced the correct structure of DNA. Watson
and Crick’s double-helix structure was based on two kinds of evidence:
(1) When Erwin Chargaff and colleagues analyzed composition of DNA they found that
the concentration of thymine was always equal to concentration of adenine and
concentration of cytosine was always equal to guanine. Results suggested that
thymine and adenine and cytosine and guanine in DNA had some fixed
interrelationship. Data also showed that total concentration of pyrimidines was
always equal to purines.
(2) When x rays are focused though fibers the rays are deflected by atoms of molecules
in special patterns, called diffraction patterns, which provides info about organization
of components of molecules. Watson and Crick used X-ray diffraction data on DNA
structure provided by Wilkins and Franklin. These data indicated DNA was a highly
ordered, two stranded structure with repeating substructures every 0.34 nanometers.
- Watson and Crick proposed that DNA exists as a right handed double helix in which two
polynucleotide chains are coiled in a spiral.
- Nucleotides are linked together by phosphodiester bonds, joining adjacent deoxyribose
moieties. The two polynucleotide strands are held together by hydrogen bonding between
bases in opposing stands. Adenine always pairs with thymine and guanine is always
paired with cytosine, thus all base pairs consist of one purine and one pyrimidine.
- The two strands of a DNA double helix are said to be complementary. This makes DNA
uniquely suited to store and transmit genetic info from generation to generation. - The sugar-phosphate backbones of the two complementary strands are said to be
antiparallel. The stability of DNA is due to hydrogen bonds and hydrophobic bonding
between adjacent base pairs.
- The two grooves of DNA are not identical; one, the major grove is much wider than the
minor groove. Some proteins bind to major, while others bind to minor groove.
- Supercoils are when one or both strands are cleaved and when complementary strands at
one end are rotated or twisted around each other with other end in place not allowed to
- Supercoiling causes DNA to collapse into a tightly coiled structure similar to coiled
telephone cord. They are introduced/taken away from DNA by enzymes that play
essential role in DNA replication.
- Supercoiling occurs only in DNA molecules with fixed ends that are not able to rotate.
Chromosome Structure in Prokaryotes and Viruses:
- Prokaryotes are typically monoploid; only one set of genes. In most viruses and
prokaryotes, the single set of genes is stored in a single chromosome, which in turn
contains a single molecule of nucleic acid.
- The large DNA molecule present in E. coli cell must exist in highly condensed
configuration. This structure is called the folded genome and is the functional state of a
bacterial chromosome. Within folded genome the DNA molecule is organized into 50 to
100 domains or loops, each independently negatively supercoiled. RNA and protein are
components of folded genome, which can be partially relaxed by treatment with either
DNase or RNase. RNase treatment will not affect supercoiling
- Bacterial chromosomes contain circular molecules of DNA
Chromosome Structure in Eukaryotes:
- Most eukaryotes are diploid, having two sets of genes. They contain much more DNA
than prokaryotes and it’s packed into several chromosomes, which is present in two or
Chemical Composition of Eukaryotic Chromosomes:
- Chemical analysis, electron microscopy, and x-ray diffraction studies of isolated
chromatin have provided valuable info about structure of eukaryotic chromosomes.
- Chemical analysis of chromatin shows that it consists primarily of DNA and proteins
with lesser amounts of RNA. Proteins are two major classes:
(1) Basic (positively charged) proteins called histones
(2) Heterogeneous, largely acidic (negatively charged) group of proteins called
nonhistone chromosomal proteins. - Histones are present in chromatin of all eukaryotes in amount equivalent to amounts of
DNA. The histones consist of five classes of proteins: H1, H2a. H2b, H3 and H4 and are
present in almost all cell types.
- 4 of the 5 types of histones are complexed with DNA to produce the basic structural
subunits of chromatin, nucleosomes. They are highly conserved during evolution
- Most of the 20 amino acids in proteins are neutral in charge , they have no charge at pH
7. However, a few are basic and a few are acidic. The histones are basic because they
contain 20-30 % arginine and lysine, two positively charged amino acids. The positively
charged side groups on histones are important in their interaction with DNA, which is
polyanionic because of the negatively charged phosphate groups.
- Histones are important in chromatin structure (DNA packaging) and are only
nonspecifically involved in regulation of gene expression. Chemical modifications of
histones can alter chromosome structure which can enhance or decrease level of
expression of genes located in the modified chromatin.
- The nonhistone portion of chromatin consists of large number of heterogeneous proteins.
The nonhistone proteins probably don’t play central roles in the packaging of DNA into
chromosomes. Instead they’re likely to regulate expression of specific genes or sets of
Three Levels of DNA Packaging in Eukaryotic Chromosomes:
- Chromatin is found to consist of a serious of ellipsoidal beads joined by thin threads. The
bead or chromatin subunit is called the nucleosome. The complete chromatin subunit
consists of the nucleosome core, the linker DNA and the associated nonhistone
chromosomal proteins, all stabilized by the binding of one molecule of histone H1 to
outside of the structure. Three levels of condensation are required to package the 10 to
10 um of DNA:
(1) First level involves packaging DNA as a negative supercoil into nucleosomes, to
produce 11nm diameter interphase chromatin fiber. This involves an octamer of
histone molecules, two each of histones H2a, H2b, H3 and H4.
(2) Second level involved an additional folding or supercoiliing of the 11 nm nucleosome
fiber to produce 30nm chromatin fiber. Histone H1 is involved in this supercoiling.
(3) Finally, nonhistone chromosomal proteins form a scaffold that is involved in
condensing the 30 nm chromatin into the tightly packed metaphase chromosomes. Thi
third level appears to involved the separation of segments of the giant DNA
molecules present in eukaryotic chromosomes into independently supercoiled
domains or loop, this mechanism which third level occurs is unknown.
Centromeres and Telomeres:
- The two homologous chromosomes of each chromosome pair separate to opposite poles
of meiotic spindle during anaphase 1 of meiosis. During anaphase II of meiosis the sister chromatids of each chromosome move to opposite spindle poles and become daughter
chromosomes. These movements depend on the attachment of spindle microtubules to
specific regions of the chromosomes, the centromeres.
- The centromere of a metaphase chromosome can be recognized as a constricted region.
The production of two functional centromeres I a key step in transition from metaphase to
anaphase and a functional centromere must be present on each daughter chromosome to
avoid the deleterious effects of nondisjunction.
- The structure of centromeres in multicellular plants and animals vary greatly from species
to species. One feature they have in common is the presence of specific DNA sequences
that are repeated many times.
- It’s been known that telomeres (meaning end and part), or ends of eukaryotic
chromosomes, have unique properties. Muller who introduced the term telomere in
1983demonstrated that Drosophila chromosomes without natural ends were not
transmitted to progeny.
- In study of maize chromosomes Barbara McClintock demonstrated that new ends of
broken chromosomes are sticky and tend to fuse with each other. In contrast, the natural
ends of normal (unbroken) chromosomes are stable and show no tendency to fuse with
other broken ends. McClintock’s results indicated that telomeres must have special
structures different from the ends produced by breakage of chromosomes.
- Telomere also have unique structures because replication of DNA does not permit
duplication of both strands of DNA at the ends of the molecules. Telomeres must provide
(1) Prevent deoxyribonucleases from degrading the ends of linear DNA molecules
(2) Prevent fusion of the ends with other DNA molecules
(3) Facilitate replication of the ends of the linear DNA molecules without loss of material
- Telomeres have basic pattern “T-A-G”. Most terminate with a G-rich single-stranded
region. Telomeres in humans have been shown to form structures called t-loops, in which
the single strand at the 3’ terminus invades an upstream telomeric repeat (TTAGGG) and
pairs with the complementary strand, displacing the equivalent strand. DNA in these t-
loops is protected from degradation by DNA repair processes by a protein complex called
shelterin. Shelterin is composed of six different proteins. TRF1 and TRF2 bind to double
stranded repeat sequences, and POT1 binds to single stranded repeat sequences. Chapter 10: Replication of DNA and Chromosomes
- Synthesis of DNA involves three steps: (1) chain initiation, (2) chain extension or
elongation and (3) chain termination.
- Watson and Crick proposed that the 2 complementary strands of double helix unwind and
separate, and that each strand guides the synthesis of a new complementary strand. The
sequence of bases in each parental stand is used as a template and the base-pairing
restrictions within the double helix dictate the sequence of bases in the newly synthesized
strand. This mechanism is called semiconservative replication.
- In conservative replication, the parental double helix would be conserved, and a new
progeny double helix would be synthesized.
- In dispersive replication, segments of both strands of the parental DNA molecule would
be conserved and used as templates for the synthesis of complementary segments that
would subsequently be joined to produce progeny DNA strands.
- Results presented by Cairns, Meselson and Stahl showed that DNA replicates
semiconservatively in E. coli.
- Meselson and Stahl grew E. coli cells for many generations in a medium in which the
heavy isotope of nitrogen, N , had been substituted for normal, light isotope, N . Thus,
the DNA of cells grown on medium containing N will have a greater density than N . 14
Molecules with different densities can be separated by equilibrium density-gradient
centrifugation. They were able to distinguish between the three models by following
changes in density of DNA grown on 15 medium then transferred to 14.
All DNA isolated from cells after one generation had a density halfway between the
densities of “heavy” and “light” DNA, this intermediate is referred to as a hybrid.
After two generations of growth half of the DNA was hybrid and half was light.
Conservative replication would not produce and DNA molecules with hybrid density;
after one generation of conservative replication of heavy DNA in light medium, half
of the DNA still would be heavy and other half would be light.
If replication were dispersive they would have observed a shift of DNA from heavy
toward light in each generation (that is half heavy or hybrid after one generation,
quarter heavy after two generations and so forth.
Unique Origins of Replication:
- In prokaryotes and bacteria there is usually only one unique origin per chromosome. In
large chromosomes of eukaryotes, multiple origins control replication. The single origin
of replication, called oriC, in the E. coli chromosome has been characterized in detail.
These three repeats are rich in A:T base pairs, facilitating the formation of a localized
region referred to as the replication bubble. The two strands of AT-rich regions of DNA
come apart more easily, input of less energy. Visualization of Replication Forks by Autoradiography:
- Gross structure of replicating bacterial chromosomes was first determined by John Cairns
in 1963 by autoradiography.
- The autoradiography indicated that that the unwinding of the two complementary
parental strands and their semiconservative replication occur simultaneously or are
closely coupled. Some kind of “swivel” must exist since they must rotate 360 to unwind
each gyre of the helix. IT’s now known that this swivel is a transient single strand break
produced by action of enzymes called topoisomerases.
- Replication of E. coli chromosomes occur bidirectionally from unique origin of
replication. Each Y-shaped structure is a replication fork and the 2 replication forks move
in opposite directions.
- The phage (ʎ) chromosome has a single stranded region, 12 nucleotides long, at the 5’
end of each complementary strand. These single stranded ends, called “cohesive” or
“sticky ends” are complementary to each other. The cohesive ends of a lambda
chromosome can thus base-pair to form a hydrogen-bonded circular structure.
- One of the first events to occur after a lambda chromosome is injected into a host cell is
its conversion to a covalently closed circular molecule.
This conversion is catalyzed by DNA ligase, an enzyme that seals single strand
breaks in DNA double helices.
- Like the E. coli chromosome, the lambda chromosome replicates in its circular form
- When DNA molecules are exposed to high temps or pH, the hydrogen and hydrophobic
bonds that hold the complementary strands together in double-helix form are b