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10-26-11 Part A:
DNA, Chromosomes and Genomes
Artificial selection for certain physical traits in
domesticated animals and plants has been
used for thousands of years
The field of genetics began in the 19th century
Early in the 20th century, scientists recognized that
physical traits are inherited as discrete units
(genes)
Chromosomes were also identified as the carriers of
genetic information
Deoxyribonucleic acid (DNA) was eventually
recognized as the genetic information carrier
DNA structure was elucidated in 1953 by James
Watson and Francis Crick
Molecular biology emerged as a new science
DNA Structure: From Mendel’s Garden to Watson
and Crick
The scientific revolution leading to the DNA model
began with an Austrian monk, Gregor Mendel
Discovered basic rules of inheritance by cultivating
pea plants and published his results in the
Journal of the Brunn Natural History Society in
1865
His findings were ignored until the early 1900s because
few biologists could understand his
mathematics and had no frame of reference
Nuclein (Nucleic acid) was nearly simultaneously
discovered by Friedrich Miescher (1869)
Albrecht Kossel and P.A. Levene figured out the
chemical composition of DNA (1882–1897)
Scientists at the time had decided to focus on protein as
the genetic material, although they did know that
the genetic material resided in the nucleus
In 1928, Fred Griffith performed
a remarkable set of
experiments involving
pneumococcal strains:
smooth (S) and
rough (R)
Identified the
concept of
transformation,
though few
accepted his
discovery
In 1944, Oswald Avery and colleagues reported the
identification of Griffith’s transforming principle as
DNA
Some people of influence were still not convinced
It was not until 1952, when Hershey and Chase
demonstrated the different functions of protein
and DNA with their T2 bacteriophage experiment,
that DNA was accepted as the genetic material
Determining the structure of DNA became an obvious
priority
Investigators including Linus Pauling, Maurice Wilkins,
Rosalind Franklin, James Watson and Francis Crick all
worked toward this goal
Watson and Crick won the race for the double helix and
published their findings in the journal Nature in
1953; awarded the Nobel Prize in chemistry in 1962
Information used to construct the model of DNA:
1. Chemical and physical dimensions of deoxyribose,
nitrogenous bases, and phosphate
2. 1:1 Ratios of adenine to thymine and cytosine to
guanine (Chargaff’s rules)
3. X-ray diffraction studies of Rosalind Franklin
4. Wilkins and Stokes diameter and pitch estimates from
X-ray diffraction
5. Linus Pauling’s recent demonstration that proteins
could exist in a helical conformation
3.4 A between
bases
Helix
34 A
spacing
17-11
Scientists have studied how organisms organize
and process genetic information, revealing the
following principles:
1. DNA directs the function of living cells and is
transmitted to offspring
DNA is composed of two polynucleotide strands
forming a double helix
A gene is a DNA sequence that contains the base
sequence information to code for a gene
product, protein or RNA
The complete DNA base sequence of an organism is
its genome
DNA synthesis, referred to as replication, involves
complementary base pairing in two strands that
comprise the DNA helix
2. The synthesis of RNA begins the process of
decoding genetic information
RNA synthesis is called transcription and involves
complementary base pairing of ribonucleotides to DNA
bases
Each new RNA is a transcript; the total RNA transcripts
for an organism is its transcriptome
3. Several RNA molecules participate directly in the
synthesis of protein, or translation
Messenger RNAs (mRNA) specifies primary sequence
Transfer RNAs (tRNA) delivers the specific amino acid
Ribosomal RNAs (rRNA) are components of ribosomes
The proteome is the entire set of proteins synthesized
4. Gene expression is the process by which cells
control the timing of gene product synthesis in
response to environmental or developmental cues
Metabolome refers to the sum total of low molecular
weight metabolites produced by the cell
The Central dogma schematically summarizes the
previous information
Includes replication, transcription and translation
The central dogma is generally how the flow of
information works in all organisms, except some
viruses have RNA genomes and use reverse
transcriptase to make DNA (e.g., HIV)
DNA
RNA
Protein
DNA consists of two polynucleotide strands that
wind around each other to form a right-handed
double helix
Each DNA nucleotide monomer is composed of a
nitrogenous base, a deoxyribose sugar and
phosphate
Nucleotides are linked by 3′,5′-phosphodiester
bonds. These join the 3′- OH of one nucleotide to
the 5′- phosphate of another
Deoxyribose in DNA is the reduced form of ribose in RNA
RNA
DNA
The base sequence of DNA defines genetic content
( the “genome”)
The antiparallel nature of the two strands allows
hydrogen bonds to form between the nitrogenous
bases
Two types of base pair (bp) in DNA: (1) adenine (purine)
pairs with thymine (pyrimidine) and (2) the purine
guanine pairs with the pyrimidine cytosine
Major groove
Minor groove
The dimensions of crystalline DNA have been
precisely measured:
1. One turn of the double helix spans 3.4 nm (34 A) and
consists of 10.3 base pairs
2. Diameter of the double helix is 2.4 nm, only suitable
for base pairing a purine with a pyrimidine
3. The distance between adjacent base pairs is 0.34 nm
DNA is a relatively stable molecule with several
noncovalent interactions adding to its stability
Features of DNA structure:
1. Hydrophobic interactions - internal base clustering
2. Hydrogen bonds - formation of preferred bonds:
three between CG base pairs and two between AT
base pairs
3. Base stacking - bases are nearly planar and
stacked, allowing for weak van der Waals forces
between the rings
4 Hydration - water interacts with the structure of
DNA to stabilize structure
5. Electrostatic interactions - destabilization by
negatively charged phosphates of sugarphosphate backbone are minimized by the
shielding effect of water on Mg2+
DNA replication is
semi-conservative
DNA Replication
DNA replication must occur before cell division; the
mechanism is similar in all living organisms:
After two strands have separated, each serves as a
template for synthesis of a complementary strand
This process is referred to as semiconservative
replication
This was first demonstrated in 1958 in an experiment by
Matthew Meselson and Franklin Stahl
The experiment involved generating DNA with a greater
density by incorporating the heavy nitrogen
isoptope 15N
After 14 generations in the presence of 15N, all of the
DNA was of higher density as demonstrated by
density - gradient centrifugation
After one generation in 14N, all the DNA was medium
density
After two generations in 14N, the DNA was light and
medium density
This data was consistent with the semiconservative
replication model
DNA Structure: Variations on a
Theme
Watson and Crick’s discovery is
referred to as B-DNA (sodium salt)
Another form is the A-DNA, which
forms when RNA/RNA and
RNA/DNA duplexes form
A-DNA, B-DNA, and ZDNA
Z-DNA (zigzag conformation) is
left-handed DNA that can form as a
result of torsion during
transcription, viral infections
Z-DNA is a left handed double helix in which
the backbone phophoryl groups zigzag
DNA can form other structures, including cruciforms,
which are cross-like structures probably a result
of palindromes (inverted repeats)
Packaging large DNA molecules to fit inside a cell or
nucleus requires a process termed supercoiling
DNA Supercoiling
Facilitates several
biological processes:
DNA packaging
replication
transcription
Linear and circular DNA
can be in a relaxed or
supercoiled shape
Solenoidal
Plectonemic
When DNA is underwound, it twists to the right to
relieve strain, causing negative supercoiling
Most naturally occurring DNA is negatively supercoiled
Winds around itself to form an interwound supercoil and
has stored energy in the form of torque
This stored energy facilitates strand separation in replication
and transcription
Supercoiling that forms during strand separation can be
relieved by a class of enzymes called
topoisomerases
Make reversible cuts that allow the supercoiled
segments to unwind
Chromosomes and Chromatin
DNA contain genes that are
packaged into chromosomes
Prokaryotic and eukaryotic
chromosomes differ
The E. coli
Chromosome
Prokaryotes - the E. coli
chromosome is a circular
DNA molecule that is
extensively looped and coiled
Supercoiled DNA complexed
with a protein core
In the nucleoid, the chromosome
is attached to the protein
core in at least 40 places
This structural feature limits the
unraveling of supercoiled
DNA
Eukaryotes have extraordinarily large genomes when
compared to prokaryotes
Chromosome number and length can vary by species
Each eukaryotic chromosome consists of a single, linear
DNA molecule complexed with histone proteins to
form nucleohistone
Chromatin is the term used to describe this complex
Nucleosomes are formed by the binding of DNA and histone
proteins, have a beaded appearance when viewed by
electron micrograph
Histone proteins: Five major classes, H1, H2A, H2B, H3, H4
A nucleosome is positively coiled DNA wrapped around a
histone core (two copies each of H2A, H2B, H3 and H4)
Each of the highly conserved histone core proteins contain a
common structural feature called the histone fold
Three a-helices separated by two short unstructured
elements
The N-terminal tails of the histones protrude from the
nucleosomes and can be covalently modified
(phosphorylation, acetylation and methylation)
These epigenetic modifications can modify the accessibility
of the DNA
The histone core forms when two sets of H2A and H2B and
H3 and H4 each form two sets of head to tail
heterodimers
The H3•H4 heterodimers associate and bind DNA
The H3•H4 heterodimers associate and bind DNA
The H2A•H2B heterodimers associate with the H3•H4
tetramer, completing nucleosome assembly
Histone H1 binds to the nucleosome where
the DNA enters and exits and acts
as a clamp that prevents unraveling
Approximately 145 bp are in contact with
the histone octamer
Connection between nucleosomes requires
approximately 60 bp of linker DNA
In anticipation of cell division, chromatin must be
compacted into chromosomes
Nucleosomes are coiled into the 30 nm fiber, which is
further coiled to form 200 nm filaments
200 nm fibers have numerous supercoiled loops attached to a
central nuclear scaffold
During interphase, chromatin can be in one of two forms:
heterochromatin (highly condensed)
or euchromatin (less condensed)
Organelle DNA - Mitochondria and chloroplast are
semiautonomous organelles that possess DNA
and their own protein-synthesizing machinery
These organelles, both of which can reproduce via
binary fission, require proteins expressed by their
chromosomes as well as nuclear DNA
Because mitochondria and chloroplasts are believed to
be descendents of free-living organisms, it is not
surprising that they are susceptible to antibiotics
(e.g., chloramphenicol)
Genome Structure
Size varies over an enormous range from 106
(Mycoplasma) to 1010 (certain plants)
Most prokaryotic genomes are smaller than
eukaryotic genomes
Genomes of organisms can vary widely in
complexity and gene density
Eukaryotes can have introns, pseudogenes and
genome-wide repeats
Prokaryotic Genomes - Investigation of E. coli has
revealed the following prokaryotic features:
1. Genome size - usually considerably less DNA and fewer
genes (E. coli 4.6 megabases) than eukaryotic genomes
2. Coding capacity - compact and continuous genes
3. Gene expression - genes organized into operons
4. Prokaryotes often contain plasmids, which are usually
small and circular DNA with additional genes (e.g.,
antibiotic resistance)
Eukaryotic Genomes - Investigation has revealed the
organization to be very complex
The following are unique eukaryotic genome features:
1. Genome size - eukaryotic genome size does not
necessarily indicate complexity
2. Coding capacity - enormous coding capacity, but the
majority of DNA sequences do not have coding
functions
3. Coding continuity - genes are interrupted by
noncoding introns, which can be removed by splicing
from the primary RNA transcript
Existence of introns and exons allows eukaryotes to produce
more than one protein from each gene
Alternative splicing allows for various combinations of exons
to be joined to form different mRNAs
Intergenic sequences are those sequences that do not code
for polypeptide primary sequence or RNAs
Of the 3,200 Mb of the human
genome, only 38% comprise
genes and related sequence
Only 4% codes for gene
products
25,000-40,000 genes, of
which about 2,500 code for
functional RNAs
The Human Genome
25% of known protein-coding
genes are related to DNA
synthesis and repair
21% signal transduction
17% general biochemical functions
Human ProteinCoding Genes
38% other activities
Over 60% of the human genome is intergenic sequences
Two classes: 1) tandem repeats and 2) interspersed genomewide repeats
1) Tandem repeats (satellite DNA) are DNA sequences in
which multiple copies are arranged next to each other
Certain tandem repeats play structural roles like
centromeres and telomeres
Some are small, like microsatellites (1-4 bp) and
minisatellites (10-100 bp)
Used as markers in genetic disease, forensic
investigations and kinship
2) Interspersed genome-wide repeats are repetitive
sequences scattered around the genome
Often involve mobile genetic elements that can
duplicate and move around the genome
Transposons and retrotransposones
LINEs (long interspersed nuclear elements) and
SINEs (short interspersed nuclear elements) are
two types of transposons