Discoveries on the nature of gene

1860 --- discovery of discrete unites of inheritance (Mandel)

1880 --- discovery of chromosomes
1903 --- discovery of homologous chromosomes
1909-1911 --- discovery of crossing over
1911 --- discovery gene could be mapped in order along length of chromosomes
1944-1952 --- discovery of DNA as genetic materials
1953 --- discovery of DNA structure

Gene as a unit of inheritance

Mandel experiments – cross breeding pea plants over generations and observed
different plant characteristics

Characteristic governed by distinct units of inheritance called gene

Each individual has 2 copies of a gene, one from each parents(inheritance) -> allele
Can be dominant(phenotype)or recessive
Homozygous: same copy AA
Heterozygous: different copy Aa

Reproductive cell(gamete) contained one copy of the gene for a particular trait

Mandel’s law of inheritance

Pair of allele can be separated during reproduction(formation of gamete/meiosis)

Law of segregation: an organism’s two alleles segregate from one another during gamete
formation. One gamete carries one allele for each trait

Law of independent assortment: Segregation of a pair of alleles for one particular trait has
no affects in the segregation of other alleles. (increase genetic diversity)
 Lead to number possible genetic arrangement
Chromosomes are carries of genetic information
1880s: ‘material’ governing the inheritance would have to be passed from cell to cell and
from generation to generation

Walther Flemming: observed chromosomes

Walter Sutton: establish concept of homologous chromosomes

Each diploid cell has pair of chromosomes(homologs) which associated with each other
- Called tetrads or bivalents
- First meiotic division separates the pair into different cell(reduction division)
- Mitosis separates tow chromosomes into two cells

The correlated with Mandel’s law of inheritable pair hypothesis -> chromosomes are
physical carriers of genetic factors

Gene on chromosome may be linked (e.g. always inherited together-linkage group) but
not always the case

Eukaryotic nuclear chromosomes are linear

Prokaryotic chromosomes are circular

Crossing over/recombination – allow reshuffling of gene

Homologous chromosomes can break and exchange with each other
The point at which crossed called chiasma(plural chiasmata)

Frederick Griffith in 1923 study streptococcus pneumonia

R = non-virulent
S = virulent
Heat killed S bacteria, but injected mixture of R with heat-killed S bacteria, it killed the
mice
‘Transformed’

Oswald Avery, Colin MacLeod and Maclyn McCarthy in 1944 isolating substance to
determine which(RNA, protein, DNA, lipid, carbohydrate[cellular fractions]) causing
transformation in R-strain bacteria.
Conclusion: DNA carries heritable information
Alfred Hershey & Martha Chase in 1952 experimented bacteriophages by injected their
genetic material into bacterial cells by making virus with radioactively labelled
protein(S35) and labelled DNA(P32)

DNA
Double strand, double helix
ATCG

Nucleoside = sugar + nitrogenous base

Nucleotide = nucleoside + phosphate groups
- Nitrogenous base is bonded to 1’ carbon of sugar
- Phosphate group is bonded to 5’ carbon of sugar
- Ribose in RNA, deoxyribose in DNA

DNA is a polymer of deoxy-nucleotides

- Nucleotides joined together by 3’ – 5’ phosphodiester linkages
- 3’ hydroxyl to 5’ phosphate of adjoining nucleotide
- This give directionality to each DNA strand

Chargaff’s rule for DNA base composition:

# of A = # of T
# of C = # of G
Number of A+T ≠ number of C+G

DNA structure: function

1. Storage of genetic information
2. Replication and inheritance
3. Expression of genetic message

Watson-Crick Model of DNA (James Watson & Francis Crick )

- Right handed helices
- Two strand comprise one double helix
- Two strand run opposite direction(antiparallel)
- The major/minor grooves of the helix are transcription factor binding site
DNA come apart – denature
come together – renature
Important for DNA replication and RNA synthesis(transcription)

DNA supercoiling
- The double stranded DNA molecules can also twist on itself, called supercoiling
when DNA unwound with no supercoiling, is not stable
- Important to make DNA molecules compact
- Important for unwinding sections of DNA (gene expression)
Positive supercoiling – helix is overwound
Negative supercoiling – helix is underwound
Topoisomerases- enzymes regulate supercoiling of DNA in cell

Complementary base pair in the DNA double helix: A-T & C-G, the bases can pair in
this way only if two polynucleotide chains that contain them are antiparallel to each
other.

DNA as a template for its own duplication: the ability of each strand of a DNA
molecules to act as a template for producing a complementary strand enables a cell to
copy, or replicate, its genome before passing it on to its descendants.

Summary
- Genetic information is carried in the linear sequence of nucleotides in DNA.
- Each molecule of DNA is a double helix formed from two complementary antiparallel
strands of nucleotides held together by hydrogen bonds between G-C and A-T base
pairs.
- Duplication of the genetic information occurs by the use of one DNA strand as a
template for the formation of a complementary strand.
- The genetic information stored in an organism’s DNA contains the instructions for all
the RNA molecules and proteins that the organism will ever synthesize and is said to
comprise its genome.
- In eukaryotes, DNA is contained in the cell nucleus, a large membrane-bound
compartment.
Lecture 2

Genome: the entire genetic makeup of an organism -> all the genetic information that is
present in an organism
Eukaryotic cells: DNA present in the nucleus, mitochondria, chloroplast, plastids and
maybe some extra-chromosomal DNA(outside of nucleus)
Prokaryotic cells: DNA present in the chromosome(don’t have membrane-bound
organelles. DNA inside the cell in cytosol)
Viruses: double or single stranded DNA or RNA

Structure of the Genome

Humans: all the genetic information is in a single haploid set of chromosomes – 22
autosomes and 1 sex chromosomes.
Human are diploid organism
Human genome is more complex than viruses and bacteria

DNA denature and renature

- The ability of two DNA strand to unwind and separate into two individuals strands is
called denature
- Double strand DNA molecules is held together by hydrogen bonds between the bases.
- Complementary single stranded DNA molecules can re-associate - Renaturation
/Reannealing

Why is important?
- Key to transcription and DNA replication
- Key to nucleic acid base pairing
- Used for investigation into determining the complexity of the genome
- One of the most important observations made in molecular biology
(how gene expression, how DNA replicated, focus on how to use these properties and
figure out the complexity of the genome)

DNA denaturation
- When DNA dissolved in weak saline solution and slowly warmed to certain
temperature, DNA strand separation begins. Within a few degrees of initiation of
strand separation, two chains are completely separated from each other (driven apart
by electrostatic repulsion of the negative charges on the sugar-phosphate backbone).
- Thermal denaturation of DNA = DNA melting
- Different DNA sequences denature at different temperature
- The progress of thermal DNA denaturation(melting) is monitored by following the
increase in absorbance of the dissolved DNA (as DNA become single strand, the
absorbance increase)
- A-T come apart at low temp.
- G-C take longer time to come apart because the number of H-bonds.

Thermal denaturation of DNA

- The nitrogenous bases of a nucleic acid absorb UV radiation(max at 260 nm)
- 260 nm wavelength can be used to determine DNA concentration
- Single stranded DNA absorbs almost twice as much UV light as compared to double
stranded DNA
- Melting curve: UV absorbance on Y-axis, temperature on X-axis
- Tm: the temperature at which the shift in absorbance is half completed (50%)

- Originally thought to be solely 仅仅 due to the number of hydrogen bonds that hold
G/C base pairs together vs. A/T pair
- It also has to do with the strength of base-stacking interactions
- C/G have stronger stacking interactions than A/T
- The higher the CG content of DNA, the higher the Tm due to the extra hydrogen
bond between the C-G pairs
DNA renaturation
- Complementary single strands of DNA can reassociate (reanneal) into a stable double
stranded DNA molecules
- Occurs when denature DNA is allowed to cool slowly – complementary strands ‘find’
each other and base pairing begins
- After renaturation, DNA become a double stranded helical molecules
- Ex: comparing the rate of renaturation of different sized genomes(C0t plots)

C0t value
Combination of two values:
 DNA concentration
 Incubation time
X-axis:
Initial DNA conc.(C0) * times of reaction (t)
Y-axis:
fraction(%) of original DNA conc. that is renatured (double strand DNA)
Determining of genome complexity using C0t plots
Comparing the rate of renaturation of 3 different genomes:
1. Small virus MS-2 (4 x 10^3 bp)
2. Larger virus T4 (1.8 x 10^5 bp)
3. Bacteria E.coli (4.5 x 10^6 bp)
- Fragment (cut) DNA into smaller pieces(equal length and at equal concentration) ->
raise the temperature to denature the DNA -> slowly cool to see how long it takes for
the complementary strands to find each other and renature.
- Large genome = longer time to renaturation because of lower probability of
complementary pieces to find each other
- Smaller genome = shorter time to renaturation because of higher probability of
complementary pieces to find each other.
 The larger the genome, the lower the concentration of complementary fragments in solution and
the longer the time it would take for renaturation.
 Single symmetrical curves because all sequences are present at the same concentration
 Shape of the curve (S) indicates that the chromosomes contain genes in a linear array

1. Pink
2. Red
Blue
3. Blue

- C0t plots for eukaryotic genomes shows that the fragment

Red
reanneal at very different rates.
- 3 distinct steps that represent 3 broad classes of DNA sequence
Pink
which reanneal at different rates due to number of times the
nucleotide sequences are repeated within the population of
fragments.
- Eukaryotic DNA is not a simple progression of one gene after
another.
Complexity of eukaryotic genomes

Packaging the genome

Genome must be packaged in nucleus in order to fit:
- In humans, diploid cell has ~6.4 billion bp of DNA in 46 chromosomes
- Length of 1 bp = 0.34 nm -> 6.4 billion bp = approx. 2m
- 1 bp binds 6H2O -> expands volume
- All this has to fit in a nucleus that is 10 mm in diameter. (nucleus = 10mm)
Packaging must be compact 紧凑的 but DNA still needs to be accessible to enzymes and
regulatory factors.

DNA + Protein -> Chromatin -> forms chromosomes in dividing cells

2 types of proteins:
1. Histones: most abundant, small basic protein(high Lys/Arg, strong + charge)
2. Nonhistone proteins: diverse structural, enzymatic and regulatory proteins

Histone
- Rich in basic amino acids Lys, Arg -> gives protein high ‘+’ charge -> easily interact with ‘–‘
charge of DNA
- Five major types/classes depending on Arg/Lys ratio [H1, H2A, H2B, H3, H4]
- Highly conserved 保存 since they either interact with DNA or other histone -> most AA can’t be
replaced[important for evolution]
- Can be modified by phosphorylation and acetylation(post-translational modifications)

Chromatin organization with histone:

- When chromatin is treated with nonspecific nucleases (DNase I, enzyme), DNA is converted to
fragment of ~200bp
- When treated naked DNA(no protein) produced random sized fragment
 DNA in chromatin is protected (by the protein) from enzymatic attack except at periodic
sites
- Kornberg(1974) propose that DNA and histones are organized into repeating subunits called
nucleosomes.
Chromatin organization - Nucleosomes
Nucleosome -> DNA + histones in repeating subunits -> lowest level of chromosomal
organization
 8 histones form a central core(octamer)
 H2A, H2B, H3, H4 -> two each
 146 bp of negatively supercoiled DNA wound around this
core(1.8 times)
 Histone H1 (linker histone) binds linker DNA that connected
one nucleosomes to another.
 Histone H1 helps pack the nucleosomes very tightly but without
H1, nucleosome look like ‘beads on a string’.
 Overall, packaging ratio of DNA in nucleosome is 7:1
Experiment:
- Remove H1 with low salt solution (low ionic strength)
- Digest DNA with non-specific nuclease
- Remove core histones with high salt solution
- ~200 bp fragment of DNA after non-specific nuclease digestion

- Dimer formation is mediated by C-terminal domain(alpha-helices)

- N-terminal forms long, flexible tail that extend outward.

Higher levels of chromatin structure – 30nm fiber

- Histone H1 + core nucleosome -> highly compact 30 nm fibers that increase the DNA-packaging
ratio 6-fold further
- Nucleosomes line up end-to-end into two stacks of nucleosomes that form a zig-zag structure
- The fiber is packaged ~6 nucleosomes per turn with H1 facing the inside of this structure
- 30 nm fiber can be seen under EM at physiological [salt] condition
- Maintenance of the 30 nm fiber depends on interactions between histone molecules if neighboring
nucleosomes
- Histone tails are required for this condensation into 30 nm fiber
- Chromatin regions that are not being actively transcribed are present mostly in either 30 nm fiber
or even higher ordered structure(looped domain)

Looped domain
- 30 nm fiber may further be gathered into a series of large, supercoiled loops/domains to form even
thicker fibers -> 80-100 nm
- These loops are attached to nuclear proteins that are part of the nuclear matrix(scaffold) e.g.
topoisomerases(control supercoiling of DNA, reduce tangling)
- Normally not visible because they’re spread out
- Can be visualized by removing histones from mitotic chromosomes
· Cells prepare for mitosis -> looped domains get further compacted into chromosomes
· DNA- packaging ratio in a chromosome is 10,000 fold
· Overall, 10 mm diameter nucleus can pack 200,000 times this length of DNA

- Nucleosomes Are a basic Unit of Eukaryotic Chromosome Structure

- Chromatin: As illustrated, chromatin consists of DNA bound to both histone and non-histone
proteins. The mass of histone protein present is about equal to the total mass of non-histone
protein, but—as schematically indicated here—the latter class is composed of an enormous
number of different species. In total, a chromosome is about one-third DNA and two-thirds
protein by mass.
- Structural organization of the nucleosome: A nucleosome contains a protein core made of eight
histone molecules. In biochemical experiments, the nucleosome core particle can be released from
isolated chromatin by digestion of the linker DNA with a nuclease, an enzyme that breaks down
DNA. (The nuclease can degrade the exposed linker DNA but cannot attack the DNA wound
tightly around the nucleosome core.) After dissociation of the isolated nucleosome into its protein
core and DNA, the length of the DNA that was wound around the core can be determined. This
length of 147 nucleotide pairs is sufficient to wrap 1.7 times around the histone core.