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LSM1102 Notes

Thanks to Prof Wu Jinlu, Prof Cynthia He Yingxin and Prof Chew Fook Tim for their
lectures and slides, of which these notes are based on.

Definition list
1. Centromere
a. It is the DNA part of the chromatids that is below the kinetochore
proteins, linking sister chromatids
2. Kinetochore
a. It is the protein found at the centromere, where spindle fibres attach
during cell division
b. It has an inner plate that interacts with the centromere, and an
outer plate which the positive end of the spindle fibre attaches
3. Centrosome
a. It is an organelle in animal cells that act as the microtubuleorganising center
b. Made from 2 centrioles
4. Centriole
a. A structure made from 9 sets of triplet microtubules, which is made
from tubulin
5. Chromosome
a. It is a structure, made from DNA and proteins, containing genetic
material of an organism
b. A pair of non-sister chromosomes are homologous, being nearly
identical in shape and size
c. Can be either chromatid or chromatin
i. Usually a pair of sister chromatids is called a chromosome
during division.
6. Chromatin
a. It is a chromosome during interphase, where it is loosely packed to
allow for transcription.
7. Chromatids
a. It is a chromatin that has replicated. It has 2 forms: thread-like
chromatids after S phase, and condensed chromatids after prophase
b. It is used in the context of sister or non-sister chromatids of a
chromosome during division
8. Tetrad/ bivalent
a. Both refer to the pair of homologous chromosomes associating with
each other.
b. However, tetrad can also mean when the 4 chromatids are clearly
visible at diplotene and diakinesis
9. Horizontal gene transfer
a. When genetic material is transferred from one organism to another
that is not its offspring
a. It is the transfer of genetic material between bacterial through
direct cell to cell contact
a. Cells that are undergoing conjugation, the DNA donor and recipient

a. Any two cells that have previously mated
a. The recombinant descendants of recipient exconjugants
a. The building block of DNA, consisting of a pentose sugar, a
nitrogenous base and a phosphate group
a. A subunit of chromatin, consisting of a histone octamer with DNA
wrapped around it.
a. Controls the phenotype of an organism
a. Alternate forms of a gene and is inherited from parents
a. The site of a specific gene or DNA sequence on a chromosome
a. An organisms collection of genes and their combination of alleles
a. Physical expression of a genotype that may or may not be visible
i. Outward appearance
ii. Proteins
iii. Behaviours
a. A diploid organism having two identical alleles at the target locus on
homologous chromosomes
a. A diploid organism having different alleles at the target locus on
homologous chromosomes
a. A diploid organism that has only one allele of a gene
i. Such as genes on the X-chromosome for males
24.Dominant allele
a. An allele that is expressed when at least one copy is present
25.Recessive allele
a. An alleles whose expression is masked by a dominant allele
a. A non-sex determining chromosome
27.Sex chromosome
a. A chromosome containing genes that specify sex
a. It is the proportion of a genotype that expresses a trait, even mildly
a. It is the intensity of the phenotype of individuals of a particular
genotype, out of those already affected by that genotype
a. A gene that causes multiple effects
b. Not all effects need be expressed in an individual
31.Multiple alleles
a. When a given gene has 2 or more alleles that exist in the gene pool

Chromosomes and their transmission

Cell division

1. Cell cycle consists of interphase, mitosis/meiosis and cytokinesis

a. Interphase has G1, S and G2 phase
i. Gap 1 phase is where the cell grows and functions normally.
Nearing the S phase, the cell size doubles and more
organelles are produced
Such as mitochondria and centrosomes
ii. Synthesis phase is where DNA duplicates, forming sister
chromatids that are still uncondensed.
iii. Gap 2 phase is where the cells continues to grow and prepare
for division.
2. Mitosis has 5 phases. Prophase, prometaphase, metaphase, anaphase,
a. It produces 2 genetically identical, diploid daughter cells
b. During prophase
i. Chromatin condenses into chromatids (or chromosome)
ii. Nuclear envelope dissociates into smaller vesicles
iii. Mitotic spindle apparatus starts forming
Consists of centrosome, spindle microtubules, and
associated proteins
c. During prometaphase
i. Centrosomes move to opposite ends of the cells, demarcating
the two spindle poles
ii. Spindle fibres grow from the two poles, by rapid
polymerisation of tubulin proteins
Astral microtubules grows in all direction
Positions and orientates the spindle fibre
Polar microtubules grows towards the opposite pole
microtubules. Contact forces pushes the poles
Kinetochore microtubules grows and attaches to
kinetochore at the centromere of sister chromatids
iii. The kinetochore on a pair of sister chromatids are attached to
kinetochore microtubules from both poles
They attach to the outer plate of the kinetochore
depolymerisation and retracts to the centrosome.
d. During metaphase
i. Pairs of sister chromatids align themselves independently
along the metaphase plate, moving by pushing and pulling
The kinetochore microtubules pulls the kinetochore
towards the centrosome
The astral microtubules that contacts with the arm of
the chromatid pushes it away from the centrosome
The balance of these two forces from both poles directs
the chromosome towards the metaphase plate, where
it oscillates awaiting anaphase.
ii. The attachment of kinetochore microtubules to the
kinetochore is complete

e. During Anaphase
i. The kinetochore no longer joins the sister chromatids
together at the centromere
Each chromatid is now only linked to one pole
ii. Anaphase A and B occurs concurrently, the centromeres
move apart while the microtubules shorten by degradation
Anaphase A is the shortening of microtubules at the +
end (the end furthest from the centrosome)
Either by ATP-driven motor protein causing both
depolymerisation and chromosome movement
Or the microtubule disassembly drives chromosome
iii. Anaphase B is the growth of polar microtubules towards each
other, pushing each other when the polar microtubules meet
in the middle
f. During Telophase
i. Chromatids reach respective poles and decondense into
ii. Nuclear membrane reforms around the chromatin, forming
two separate nuclei
iii. Cytokinesis occurs concurrently
Cleavage furrow forms and splits the parent cell into
3. Prophase I of meiosis has several sub-stages
a. Leptotene is the condensation of chromatin threads to form
b. Zygotene is when homologous chromosomes pair up to form
bivalents, with a synaptonemal complex forming
i. Homolog recognition occurs with a pairing centre, located at
sister chromatids at either the telomere or centromere.
Proteins in the nuclear membrane recognise and bind
to the highly repeated sequence.
The protein then moves around the membrane until it
contacts the other homologous pair
A synaptonemal complex then forms between the
c. Pachytene is the crossing over between non-sister chromatids,
forming a chiasma, with the synaptonemal complex fully formed
i. Synaptonemal complex causes the crossing over
d. Diplotene is the start of dissociation of the synaptonemal complex,
but chromosomes are still linked by the chiasma
e. Diaknesis is when the dissociation of the synaptonemal complex is
finished, and the dissolution of the nuclear membrane
i. After Diaknesis, the bivalent is still linked by the chiasma, and
move together to the metaphase plate, arranging themselves
with independent assortment.
4. Prometaphase I occurs similarly to mitosis. Centrosomes move, 3 types of
spindle fibre forms, attachment to kinetochore occurs
5. The rest of meiosis differs from mitosis
a. During metaphase I
i. Pairs of sister chromatids are aligned along the metaphase
plate in a double row instead of a single row

There is independent assortment of the two pairs in

the double row
ii. A pair of sister chromatids is linked to one of the poles, while
the other homologous pair is linked to the opposite pole.
b. During anaphase I
i. The pair of homologous chromosome separates from each
other, to opposite poles, while the sister chromatids of each
chromosome are still connected
c. During Telophase I
i. The nuclear envelope forms around the sister chromatids at
the poles
d. Meiosis II occurs similarly to mitosis, producing 4 genetically
different haploid daughter cells
6. Hybrids can be infertile when they have an odd number of chromosomes,
as during the meiosis checkpoint, the failure to find a homologous pair
arrests the process of meiosis.

Bacterial diversity
1. Bacterial usually reproduce asexually by binary fission, thus horizontal
gene transfer is needed to encourage genetic diversity
a. Conjugation involves direct cell to cell contact
b. Transduction involves viruses
c. Transformation involves uptake from the environment
2. During conjugation between a F + cell and a F- cell
a. Cells that have conjugative plasmids (that contain the fertility
factor) contain the genes required for conjugation
i. Such as for pilin protein needed for sex pilus formation
b. F cells form sex pili that initiates genetic transfer on contact with a
recipient cell
i. They are hollow structures on the cell surface
c. On contact, relaxosome, a protein complex, separates DNA strands
in the conjugative plasmid
i. Contains relaxase that cuts one strand of the plasmid DNA at
the origin of transfer
ii. This produces a strand called T-DNA which gets transferred
d. T-DNA-relaxase complex is recognised by the coupling factor which
moves it into the exporter
i. Both the coupling factor and exporter are part of the sex
pilus, and are encoded by the conjugation plasmid
e. T-DNA-relaxase complex then moves through the mating channel
into the recipient cell
i. The other conjugation plasmid DNA strand remains in the
donor cells
f. Relaxase then circularised the T-DNA in the recipient cell
g. DNA replication processes in each cell then replicates the single
stranded DNA into a F plasmid
i. Both cells are now F+
h. This process is similar even if the cell is F
i. F cells have chromosomal genes in their F plasmid
3. During conjugation between a Hfr cell and a F - cell
a. When a F+ cell has its F plasmid integrate into the bacterial
chromosome, it becomes a Hfr strain

i. High frequency of recombination

ii. This is because the F plasmid is an episome
It can replicate autonomously as a plasmid or be
integrated into the chromosome.
b. The original of transfer determines the starting point and direction
of the transfer
i. Chromosomal genes nearest to the origin are transmitted
most efficiently
ii. The likelihood of transfer of genes decreases with distance
from origin
The transfer can break at any time, thus the probability
of faraway genes getting transferred is small
c. Transferred gene may not recombine with the recipients bacterial
d. Unless the entire donor chromosome is transferred, the recipient
cell does not become Hfr too.
i. Recombination of the F plasmid genes are also needed
4. Interrupted mating technique is a method to map the order of bacterial
genes, based on their order of transfer into the recipient cell
a. A large number of Hfr and F- cells are mixed, allowing them to mate
b. Conjugation can be interrupted at specific times using a blender
c. Cells are then transferred to various dishes with different media to
test if certain genes are transferred successfully
i. All the dishes must have a media that would exclude the
growth of the Hfr strain. There must be a selective marker for
the two strains (usually resistance)
d. The time taken for the recipient cell to be able to grow in the
specific medium will determine the order of the cells and their
relative distances
i. To check for antibody resistant colonies, you need to replica
plate an antibody-free medium plate to a plate with antibody
Done with sterile velvet
ii. If the timing is the same, the colony count will determine
iii. To get distance, a graph can be plotted and the extrapolated
e. Minutes can be used as a unit of distance between genes, which the
time it takes for the gene to enter the recipient cell
i. The starting 0 minute point is assigned arbitrarily
f. As different Hfr strains have the fertility factor inserted into different
point and directions, the whole chromosome can be mapped
g. The whole chromosome is usually not transferred due to several
i. The mating channel is fragile and easily broken by
environmental changes
ii. The time needed may be more than the average lifespan for
the bacteria
iii. The recipient bacteria may lack the space for additional DNA
5. Transduction requires a bacteriophage to transfer DNA from one bacterium
to another
a. Generalized transduction can occur with both temperate and
virulent bacteriophages




i. During the lytic cycle, the bacterial chromosome is

fragmented and the virus uses the cells machinery to
manufacture more viruses
ii. A bacterial DNA fragment may be packaged into the viral
capsid instead of the viral DNA
Any piece of chromosomal DNA may be incorporated.
b. Specialized transduction requires temperate bacteriophages that
can undergo both lytic and lysogenic cycles
i. Specialized transduction only occurs if the lysogenic cycle
happens, as a prophage must form.
A prophage forms when the viral DNA integrates into
the bacterial chromosome
ii. When the lytic cycle occurs, the excised viral DNA may also
contain a piece of bacterial DNA due to error in excision.
c. Lentiviruses, a form of retrovirus, is used as a vector
i. Has a higher transduction rate as targets are specific due to
surface molecules on the virus recognising specific cells
Genes that are fairly close together can be mapped by cotransduction, as
more than 1 gene may be packed into the phage
a. The maximum distance depends on the maximum size of DNA that
can fit into the virus
i. P1 can pack 2% to 2.5% of E.coli chromosome
ii. P22 can pack up to 1% of the Salmonella typhimurium
b. Plating twice is needed as once is to check that the reference gene
is transduced, and the second to check that the target gene is
transduced as well.
c. Ratio of the cell colonies on both plates will give the distance
between the two genes
i. Colony count of cotransduced gene / Colony count of
reference gene
ii. If grown on more than one plate, can do ratio of plates
1 plate testing for reference gene, 50 plates testing for
target gene
See how many out of the 50 plates have colony growth
Conjugation determines relative order and distance of genes, while
cotransduction gives more accurate distances between the two genes
Transformation occurs when a bacterium uptakes extracellular DNA
a. Natural transformation occurs without outside help
i. Only competent cells can undergo transformation, as they
have genes coding for competence factors
The factors facilitate DNA binding to cell surface,
uptake and integration
Expression of genes depend on environmental
b. Artificial transformation occurs with use of special techniques
All the above techniques involve replacing transfer where the DNA taken
up undergoes homologous recombination with the homologous region of
the DNA
a. Additive transfer can also occur where the incoming DNA is
incorporated into the chromosome.

Chromosomes and their structure

1. Genome is all the chromosomes and DNA sequences an organism/ species
a. It technically includes mitochondria DNA, but by convention it is not
included unless specified
2. One nucleotide is 0.34nm long, and one turn of the DNA double helix is
3.4nm, containing 10 nucleotides
a. Diameter is 2nm
b. DNA usually coils anticlockwise, in the right handed direction
i. It is called B-DNA
3. For viruses
a. Their nucleic acid is surrounded by a capsid of proteins
i. RNA or DNA genome
b. They require hosts cells to replicate, and have a limited host range
i. Obligate parasites
c. Some viruses have a lipid bilayer taken from the host cell,
containing proteins that aid in infecting another host cell
4. For bacteria
a. Bacteria have circular chromosomal DNA
i. A cell can have a single type of chromosome but with
multiple copies
ii. Only one origin of replication on each chromosome
iii. Repetitive sequences have a role in DNA folding, replication,
gene regulation and genetic recombination
b. The bacterial chromosome is first packed with the formation of
looped domains, then then supercoiling within the loops further
compacts the DNA
i. The loop domains formation compacts about 10 times
ii. The supercoiling further compacts it to 100 times
Overwinding makes the supercoil positive, as it twists
the DNA in the direction of the double helix, which is
anticlockwise like a right hand turning inwards.
This is for B-DNA (most common)
Z-DNA is DNA twisted in the left-handed
Twisting a closed loop anticlockwise results in one
region overlapping another heading in a direction to
the top left
c. Supercoiling is regulated by two enzymes: DNA topoisomerase I and
i. DNA topoisomerase I has a counter interaction with DNA
topoisomerase II
DNA topoisomerase I increases the linking number by 1
making the supercoil more positive, without ATP
DNA topoisomerase II uses ATP and reduced the linking
number of the supercoil by 2, such as from linking
number of +1 to -1
DNA Gyrase is an example
It breaks the double stranded DNA using ATP,
then rejoins it in a different orientation, reducing
the linking number

Most DNA are packed with negative supercoiling

topoisomerase, causing the chromosome to lose
its structure, arresting processes like replication

Eukaryotic genome organisation

1. There are several difference between genomes in eukaryotic and
prokaryotic cells
a. Eukaryotes have telomeres to protect the chromosome from
degradation, and the 3 end replication problem
i. It acts as a biological ageing marker, and leads to senescence
b. Eukaryote genome is much larger with several origin of replication
2. Chromosome organisation has 4 levels
a. Primary structure is like beads on a string, with nucleosomes linked
by linker regions
i. Each nucleosome consists of a histone octamer
2 of each type of histone: H2A, H2B, H3, H4
Each histone has a N-terminal tail, and a C-terminal
histone fold
N-terminal tails will determine the interaction
between histones, and can be disrupted by
methylation or promoted by other processes
The interaction between histones is like a
histone code that controls gene transcription. It
is affected by cell type and environment. It is
hereditary giving rise to epigenetics
The histone contains many positively charged amino
acids that bind to the phosphates along the DNA
Lysine and arginine are the amino acids
ii. The length is DNA around a histone is fixed at around
146/147 base pairs, but the length of linker regions varies
The diameter of the nucleosome is 11nm
The DNA makes 1.65 negative superhelical turns
around the core
iii. This structure was proven with digesting by DNase I
Fragments produced where in multiples of 200bp
Fragment consists of nucleosome DNA and linker DNA
b. Secondary structure is the organisation of nucleosomes to form a
30nm fibre
i. This involves histone (H1) and non-histone proteins
H1 is less tightly bound to DNA, and helps compact
adjacent nucleosomes.
ii. Salt concentration will affect the organisation as amino acids
are charged and will interact with the ions present
At moderate salt concentration, H1 is removed, leading
to primary structure

At low salt concentration, H1 remains bound and the

nucleosome associate together into a more compact
iii. The nucleosomes zig-zag, and have little face to face contact
with each other
c. Tertiary structure is radial loop model/ domain.
i. Formed from the interaction between the 30nm fibre and the
nuclear matrix
The Matrix-Attachment Regions (MAR) on the fibre gets
anchored to the nuclear matrix, creating radial loops
The nuclear matrix consists of nuclear lamina and
internal matrix proteins
Nuclear lamina is dense fibrillar network, which
consists of intermediate filaments (cytoskeleton)
and membrane associated proteins
d. Quaternary structure occurs during metaphase, with the nuclear
matrix being reorganised into a scaffold that the highly compacted
radial loop can anchor to
i. Histone and non-histone proteins help to compact the DNA
The compacted heterochromatin undergoes minimal
gene transcription
ii. Both cohesin and condensin are multiprotein complexes that
work like tongs to push the DNA together compacting them.
They have SMC proteins that use ATP to catalyses
changes in chromatin structure
Structural Maintenance of Chromosomes
Condensin helps with chromosome condensation
Condensin is usually in the cytoplasm and enters
the nucleus only during cell division
It binds to chromosomes and compacts the
radial loops, changing the diameter but not loop
Cohesin helps with sister chromatid alignment
At the end of S phase, cohesin binds to
uncondensed sister chromatids to hold them
They remain bound until the middle of prophase,
during which all the cohesins are released
except those at the centromere
During anaphase, the cohesins at the
chromatids to separate
Both differs from the synaptonemal complex that joins
non-sister chromatids.
They differ in function and location
3. During interphase, chromosomes have their own territory in the nucleus, a
region of space where they are usually found
a. There are two levels of organisation of chromosomes during
i. Euchromatin are less condensed regions of chromosomes
This region is transcriptionally active

Consists of 30nm fibres forming radial loop domains

ii. Heterochromatin
Generally, transcription does not occur at the
heterochromatin region, but it can occur at places like
the telomeres and centromere
The radial loop domains present are highly compacted
iii. There are also two types of heterochromatin
Permanently transcriptionally inactive
Usually contains highly repetitive sequences
Facultative heterochromatin can covert between
euchromatin and heterochromatin
For example Barr bodies in females (the inactive
X chromosome)
iv. By prophase, sister chromatids are entirely heterochromatin
4. Chromosomes can be banded by different methods
a. Banded by supercoiling regions, positive and negative
b. Banded by packed-ness, heterochromatin and euchromatin
c. Banded by staining of A-T rich regions

Molecular Genetics
Structure of DNA and RNA
1. Genetic material has several parts that meets its role
a. It contains the information necessary to code for an entire organism
b. It is hereditable and is passed from parents to offspring
c. It is replicable, to pass from within, and between organisms
d. It is variable, to be able to react to changes
i. To account for the phenotypic variation seen between species
2. The genetic material was first identified to be in the chromosome, which
consists of both proteins and DNA
a. While DNA had only 4 nucleotides, ATCG, proteins have 20 different
amino acids
b. There was more protein by weight that DNA in chromosomes
c. Variations in proteins were identified, that they are responsible for
different functions
3. Several experiments showed that the genetic material was DNA instead of
a. Frederick Griffith experiments with Streptococcus pneumoniae
i. Showed that genetic material survived heat treatment
ii. The bacterial cell type could change through the process of
Recessive, non-lethal type with heat-killed dominant,
lethal type, killed the lab rat
b. Aver, MacLeod and McCarty experiments
i. Showed that the genetic material was DNA, and not RNA or
ii. Added DNA of dominant phenotype of recessive cell type and
grew with various enzymes
None = grew
a. To show the extract contained genetic material
DNase = no growth
a. To show DNA was responsible
RNase = grew
a. To show RNA was not responsible
Protease = grew
a. To show proteins was not responsible
c. Hershey and Chase experiment with Bacteriophage T2
i. Showed that genetic material was DNA not protein
Bacteriophage T2 only has DNA and protein
ii. Either viral proteins or DNA were radioactively labelled with P
or S
To identify if the bacterial cell take up DNA or proteins
iii. Radioactivity of solution not taken up by bacterial showed
more S than P present
Conclude that bacterial cells take up DNA rather than
Thus DNA is the genetic material
4. Other research shows that RNA can also function as a genetic material





a. A.Gierer and G.Schramm showed that some viral genome contains

only RNA, and extracted RNA can still infect cells
The basic unit of DNA is a nucleotide
a. It has a phosphate group
i. Bound to 5 carbon of one nucleotide, and 3 of another
b. It has a 5 member sugar ring
i. 4 carbon and one oxygen
ii. Another carbon attached to the 4 carbon, making this a
iii. Difference between Deoxyribose and Ribose is the presence
of a hydroxyl group on the ribose 2 carbon
c. It has a nitrogenous base
i. Purines are A,G, with 2 carbon rings joined together
ii. Pyrimidines are T,C and U with only one carbon ring
iii. It is always attached to the 1 carbon
d. A nucleoside is when the nitrogenous base binds with the pentose
sugar, without any phosphate group
DNA is built in the 5 to 3 direction, using an existing strand, and an
incoming nucleotide that is a triphosphate
a. The triphosphate group reacts with the 3 carbon at the 3 end,
forming a phosphodiester bond, and producing pyrophosphate
i. Pyrophosphate (PPi) is two phosphate groups joined together
b. The phosphate groups and pentose sugar forms the DNA backbone,
with nitrogenous bases projecting outwards in the same direction
Rosalind Franklins X-ray diffraction pattern of DNA fibres suggested that
DNA was helical and contains more than one strand, with 10 bases per
complete turn
Erwin Chargaff showed that all four bases in DNA are in roughly equal
a. Plus proportions of A=T, and as well as C=G
b. Became Chargaffs rule
In a DNA double helix
a. The two strands of DNA form a right-handed double helix
i. This B-DNA form is the predominant form in living cells
ii. Under in-vitro conditions, A-DNA and Z-DNA can form
While A-DNA is right handed, it is more compact as the
base pairs are not perpendicular to the helix-axis
Z-DNA has a left-handed double helical structure
b. The bases in opposite strands form hydrogen bonds according to
Chargaffs rule
i. A with T forms 2 hydrogen bonds
ii. C with G forms 3 hydrogen bonds
c. The two strands are anti-parallel as they have opposite 5 to 3
d. There are 10 nucleotides per complete turn of the helix of 3.4nm
e. Has major and minor groves
i. Major groves are where the backbones are far apart
ii. Minor groves are where the backbones are close together
iii. The distance is the vertical line distance along the backbone,
not the distance between base pairs, which is fixed
Due to the glycosidic bond between the nitrogenous
base and pentose sugar being 120 degrees
120 degrees portion forms the minor groves

The remaining 240 degrees forms the major groves

iv. The major and minor groves alternate, along the vertical line
of the backbone
10.Most proteins binding occurs at the major groove
a. Binding can be nucleotide sequence specific, or non-specific
b. The narrowness of the minor groves leads to the edges of the bases
major grove being more accessible
c. Proteins make contact with the sides of the bases at the major
groove and interact with them
d. A synthetic DNA strand can also bind to the major groove to form a
triple helix
i. This is a sequence specific binding
11.DNA expression can be regulated by the expression of DNA mimic proteins
a. AbrB (repressor) has the complementary shape to DNA, and will
bind to it to prevent transcription
b. AbbA (activator) has a shape similar to DNA, causing AbrB to bind to
it instead, allowing transcription to occur
c. Sort of like competitive inhibition due to the shape of DNA grooves
12.RNA strands have a similar structure like DNA, but with a ribose sugar
instead and U in place of T
a. Complementary base pairing can still occur to give secondary
i. Bulge loop
ii. Internal loop
iii. Hair-pin loop
These loops are formed from regions that are noncomplementary, and thus project out of the structure
iv. RNA junction, like a 4-way street with a hole in the middle
b. Base stacking, involving VDW forces between nucleotides, also help
to hold the secondary structure together

DNA replication and repair

1. A pulse-chase experiment has a labelled pulse, and the chase finds the
prevalence of the label in each fraction/ generation
a. Proves that DNA replication is semi-conservative, with the original
pulse as the parent strands being made from Nitrogen-15
i. The label is the heavier isotope of Nitrogen that is examined
through equilibrium density-gradient centrifugation
Separates DNA molecules containing denser strands
into its own band
2. DNA replication occurs with several proteins, in the 5 to 3 direction
a. Helicase first breaks the strands apart at the origin of replication
(oriC), creating a replication fork
i. Prokaryotes only have one oriC on their circular DNA
DNA synthesis occurs bidirectionally from the origin of
Chromosomal replication
Replication ends when the two replication forks meet,
at the ter sequence, separating the two chromosomes
ii. Eukaryotes have multiple oriC due to their length
DNA replication still processes bidirectionally from each
Eventually all the replication forks merge to give two
b. The single strands are kept apart by proteins
i. Prokaryotes have single-stranded binding proteins
c. RNA primase creates RNA primers, allowing replication in the 5 to
3 direction
i. Only one needs to be created at the origin of replication on
the leading strand
ii. Lagging strand requires continues synthesis of a RNAprimer
d. DNA polymerase synthesises a daughter strand, in the 5 to 3
direction, using the parent strand as a template, through
complementary base pairing
i. Prokaryotes have DNA polymerase I to V
I and III are involved in normal replication
I is a single polypeptide and removes RNA
primers and replaces them with DNA
5 to 3 exonuclease digest the DNA
Simultaneous 5 to 3 polymerase activity
to replace with DNA
III has 10 different subunits and is the main
replicating DNA polymerase
The alpha subunit in the DNA polymerase
III holoenzyme synthesises the DNA
The epsilon subunit does the proofreading
The theta subunits stimulates the
II, IV and V are used in DNA repair and replication of
damaged DNA
e. DNA ligase covalently links DNA okazaki fragments together,
removing the nicks

i. Uses energy from ATP or NAD

Topoisomerase (DNA gyrase) removes the supercoiling caused by
i. As helicase moves down the strand, there is positive
supercoiling as the double helix is broken into linear strands.
ii. DNA gyrase uses energy from ATP to alleviate the supercoil
g. Topoisomerase IV also helps separate the two chromosomes after
the termination of DNA replication
i. The two chromosomes are intertwined, forming a catenanes
3. Bacterial oriC has DNA sequences needed for its function
a. AT-rich regions
i. Strands are easier to separate as it has fewer hydrogen
bonds compared to CG
ii. Located next to the DnaA boxes
b. DnaA boxes
i. Several are found at the oriC
ii. DnaA proteins bind here to initiate replication
iii. They form a large complex that spans across all the boxes,
causing the DNA region to wrap around
Mechanical stress separates the nearby AT-rich region
iv. Allows helicase to bind to the oriC
c. GATC methylation sites
i. Regulates rates of replication and DNA replication mismatch
ii. Parent strands have the adenosine methylated, while the
daughter strand does not
Allows differentiation of strands to know the template
for mismatch repair during replication
iii. Another round of replication can only occur when both
strands have their GATC sequence methylated
Prevents concurrent replications
4. Opposite of the oriC is a pair of termination sequences (ter) on the DNA
a. The protein termination utilization substance (tus) binds to these
sequences to stop the movement of replication forks
b. They physically prevent further movement of helicase, which would
otherwise unwind the daughter from the parent strand
i. They can only block movement from one direction, thus you
need two in total
If the helicase comes from the other direction, it is
allows to pass to be stopped by the other tus protein
ii. As both tus protein blocks movement from coming from the
same side, the region between the tus proteins is only
separated once
5. As eukaryotes have linear DNA, they have telomeres at both ends
a. Telomere refers to the complex of telomeric DNA sequences, and
the associated bound proteins
i. Each telomeric DNA sequence contains multiple tandem
ii. They also have a 3 end overhang that is 12-16 nucleotides
Due to 3 end replication problem
b. Telomeres shorten with each round of replication

i. Telomerase can lengthen the telomeres, preventing their loss

Contains proteins and RNA
The RNA is complementary to the repeating sequence
in the telomeric DNA sequence
Allows binding to the 3 end overhang
ii. Only the 3 end strand is lengthened by telomerase
Binding occurs through complementary base pairing
Extension occurs through reverse transcription of RNA
in telomerase
Translocation occurs to allow the telomeric DNA to be
further extended
iii. The other 5 end is lengthened by normal DNA replication
Primer, DNA polymerase, ligase
6. There is a low rate of mutation due to several factors
a. DNA polymerase has proofreading mechanisms
i. Configuration of DNA polymerase active site makes it unlikely
to catalyse bond formation between mismatched pairs
ii. Epsilon and Sigma subunits in prokaryotic DNA polymerase III
allow for proofreading, as mismatched pairs are unstable
3 to 5 exonuclease activity removes the incorrect
Unlike the 5 to 3 exonuclease in DNA poly I
The DNA polymerase II then moves back and resumes
DNA synthesis in the 5 to 3 direction
b. DNA repair mechanisms
i. There is instability of mismatched pairs, leading to
recognition by repair mechanisms
Mutation caused by change of nitrogenous base, or
deletion of base
Cytosine can be deaminated into uracil,
changing the complementary base pair from
guanine to adenine
Nitrogenous bases can also dimerise which leads to
deletion or change in base of the daughter strand
ii. Base excision repair removes a single mismatched base pair
DNA glycosylase breaks the glycosidic bond between
the pentose sugar and nitrogenous base, removing the
nitrogenous base
phosphodiesterase removes the associated sugar
The single nucleotide gap is then filled by DNA
polymerase and DNA ligase
iii. Nucleotide excision repair removes a section containing a
nitrogenous base dimer
Both eukaryotes and prokaryotes use DNA excision
nuclease and DNA helicase
In prokatyotes, the DNA excision nuclease first
cuts the sugar phosphate backbone around the

damage, then DNA helicase removes the

damaged portion
In eukaryotes, DNA helicase first unwinds the
DNA locally, followed by DNA excision nuclease
freeing the damaged portion
Prokaryotes remove around a 12 nucleotide portion,
eukaryotes remove around a 30 nucleotide portion
The gap is then filled by DNA polymerase and DNA
iv. Double-strand breaks repair joins blunt ends together
Nonhomologous end joining
The DNA portion at the site of the break is
damaged and is degraded
DNA ligase then seal up the gap, leading to a
deletion of bases
Homologous recombination
Unlike nonhomologous end joining, this restores
the original DNA sequence by using the sister
chromatid as a template
Only occurs when the damage happens after
DNA replication, but before cell division occurs
Is also used to join broken ends during
chromosome crossovers in meiosis
c. Coordination between DNA replication and cell division
i. Ensures DNA replication does not happen excessively, which
would increase the chances for mutation
ii. Controlled by availability of DnaA proteins manufactured by
the cell
As the number of DnaA boxes doubles after replication,
there is insufficient DnaA proteins needed to initiate
another round of replication
iii. Also controlled by GATC methylation sites
DNA adenine methyltransferase takes time to
methylate the adenine at the GATC methylation site on
the daughter strand
Having only one strand methylated does not efficiently
initiate replication

From DNA to protein

1. A gene is a transcriptional unit, and gets transcribed into RNA
a. Structural genes encodes for a polypeptide, and produces mRNA
when transcribed.
b. Nonstructural genes do not result in a protein product, as the RNA
transcript is the final product
i. Such as tRNA, rRNA, microRNA
ii. They have important cellular function
iii. They can either work along, or be part of a protein complex
with protein subunits
2. Transcription takes place along the template strand, producing a RNA
transcript that is similar to the coding/sense strand
a. RNA has Uracil instead of Thymine

3. During transcription in prokaryotes

a. Initiation
i. Transcription factors bind to the gene promoters
There is a consensus sequence gathered from all the
genes in the organism.
The closer the promoter sequence resembles this, the
greater the rate of transcription
ii. The transcription factors recruits RNA polymerase to bind to
the promoter, forming a closed promoter complex
RNA polymerase holoenzyme consists of 6 subunits

A core enzyme of

2 '

A sigma factor of

The sigma factor recognises and binds to the DNA

It recognises the -10 (Pribnow box)and -35
RNA synthesis occurs in the 5 to 3 direction
iii. The RNA polymerase then breaks the hydrogen bonds in DNA,
forming an open promoter complex
b. Elongation
i. RNA polymerase slides along the DNA in an open complex to
synthesise the RNA transcript
ii. The first amino acid (N-terminal) is always methionine
c. Termination
i. Eventually a termination signal is reached, causing RNA
polymerase to dissociate from the DNA
4. As prokaryotes do not have a nucleus, the RNA transcript is exposed to the
cytoplasm as it is transcribed, allowing translation to take place
a. Coupling of the two process occurs as the ribosome will attach to
the RNA 5 end once it is long enough
b. A polyribosome forms as the mRNA transcript has multiple
ribosomes bound to it, translating it
5. Translation occurs only with the mRNA produced from transcribed
structural genes
a. The genetic code is written in codons of 3 nucleotides
i. The codons are degenerate as amino acids are coded for by
more than one codon
ii. The first two nucleotides determine the amino acid in most
cases, thus the third nucleotide can vary
iii. Some codons are translated faster than others, between
similar and different amino acids
b. Requires tRNA, mRNA and rRNA with ribosomes
i. tRNA is able to recognise a triplet codon in mRNA, and carries
the corresponding amino acid
They recognise using an antisense RNA sequence to
the codon
Aminoacyl-tRNA synthetases catalyses the binding of a
specific amino acid, to their appropriate tRNA
The amino acid is attached to the 3 end of tRNA
via an ester bond

20 types of synthetases for the 20 types of

amino acids
ii. The ribosome complex consists of a large and small subunit
Svedberg units is a measure of their rate of
In prokaryotes, the 30S and 50S subunits forms a 70S
ribosome complex
The 30S subunit binds to the Shine-Dalgarno
sequence on the mRNA, through complementary
base pairing with the 16S rRNA in the subunit
In eukaryotes, the 40S and 60S subunits forms a 80s
ribosome complex
iii. The ribosome complex has three sites for tRNA
E site for the exit of tRNA
P site holding a tRNA with the polypeptide chain
Peptidyltransferase catalyses the formation of
peptide bond
A site where a aminoacyl-tRNA enters and binds to
the mRNA
iv. The first amino acid is always a modified form of methionine
c. Termination occurs when the ribosomes A site reaches a stop
codon, UAG UAA or UGA
i. There are no tRNA complementary to them
ii. Release factors will enter the A site to dissociate the
ribosome complex from mRNA
The structure of the release factor is similar to the
shape of tRNA
6. Transcription in eukaryotes
a. Unlike prokaryotes, eukaryotes have three RNA polymerase instead
of one
i. RNA polymerase I transcribes all rRNA genes
ii. RNA polymerase II transcribes all structural genes and some
snRNA genes
Thus it produces all the mRNA in the cell
iii. RNA polymerase III transcribes all tRNA genes and the 5S
iv. However, they share a structural similarity and consists of
b. The RNA polymerase binds to the promoter which has consensus
i. The promoter contains at least 2 out of 4 elements
BRE element is located at -35 and recruits TFIIB
TATA element is located at -30 and recruits TBP
INR element is located at +1 and recruits TFIID
DPE element is located at +30 and recruits TFIID
ii. There is a basal level of transcription when RNA polymerase
binds to the core promoter
Additional regulatory elements either simulate or
inhibit transcription

Enhancers recruits activators that hasten the

assembly of the complex
Silencers recruits repressors that inhibit the
assembly of the complex
iii. While prokaryotes only need the sigma factor in RNA
polymerase, eukaryotes form a complex with the RNA
General transcription factors give the basal level of
Histone-modifying enzyme loosens the DNA from the
histones to allow easier access to the template strand
supercoiling caused by the unwinding of DNA by RNA
c. As there is a nucleus to separate the nucleoplasm from the
cytoplasm, RNA processing can take place
i. 5 capping and 3 polyA tailing helps stabilize the mRNA and
its translation
Different enzymes complexes add the cap and tail
ii. RNA splicing to remove introns and connect exons
Alternative RNA splicing give rise to variation is
proteins coded for by the same gene
Spliceosomes are a complex of snRNA and proteins
They are flexible to allows alternative splicing
iii. Defective mRNAs will be degraded by exosomes within the
Only fully processed mRNA can be exported through
the nuclear pore into the cytoplasm
7. Translation in eukaryotes
a. Initiation
i. Starts from the first AUG codon after the 5 cap
Both 5 cap and 5 polyA tail recruits initiation factors
(eIFs) for assembly of tRNA-mRNA-ribosome complex
The AUG codon follows Kozaks rule
An A or G at the -3 position
One G at the +4 position, immediately after the
+1 to +3 AUG
ii. Smaller, 40S subunit binds first to the mRNA, using 18S rRNA
60S subunit associates with the 40S to form 80S
ribosome complex
The first aminoacyl-tRNA complex is recruited to the A
Carries methionine instead of f-methionine
b. Elongation
i. Occurs similarly to prokaryotes
Catalysis of peptide bond followed by translocation
c. Termination
i. Unlike prokaryotes that use several types of release factors,
eukaryotes use one release factor, eRF1, to recognise all
three stop codons
8. Gene expression is regulated by all the processes between DNA to protein

a. Transcription
i. Rate of assembly of RNA polymerase complex
b. RNA processing
i. Effectiveness of modification
ii. Alternative RNA splicing
c. Exporting
i. Efficiency of export proteins
d. Translation
i. Rate of ribosome complex assembly
ii. Structure of mRNA
iii. Rate of degradation of mRNA
e. Protein modification
i. Molecular chaperones to help protein folding
ii. Covalent modification needed for proper protein folding
iii. Cofactors and other protein subunits
iv. Degradation by proteasome, especially for incorrectly folded

Methods to study gene expression and function

1. Classic genetics studies how the phenotype of an organism arises from its
a. First the genome of the organism is exposed to mutagens
i. Chemicals
ii. Irradiation through UV or X-rays
iii. Transposons that insert randomly into the genome
b. Then the offsprings are screened for phenotypical changes
i. Limitation is that only specific and distinct abnormalities can
be detected
c. Mutated offsprings are identified and the general location of the
mutation is identified
i. Through finding the recombination frequency of the mutation,
using known gene loci as references
d. The mutated region is then cloned and sequenced
2. Reverse genetics studies how a gene can affect the phenotype
a. Using a gene of known sequence, all functions that requires it are
b. The gene of interest or its expression is manipulated
i. Deleting the gene
ii. Changing the expression pattern of the gene
Expression levels
Expression location
Expression time in stage of development
iii. Tagging with a reporter
Green Fluorescence Protein
iv. Genome editing of a specific portion of the gene
Uses the repair mechanism for double stranded breaks
to introduce new genes or delete targeted ones
Nonhomologous repair to delete nucleotides
around the break

Homology-directed repair to add genes by

introducing a template with the new gene
In lab we introduced with heat shock
Template can also be introduced with
Gene coupled with reporter to indicate
successful uptake into the genome
Breaks in the original genome can be directed
Zinc finger nucleases
Zinc finger binds to specific nucleotide
Positions DNA endonuclease at target site
One required for each strand of DNA
Transcription activator-like effector nucleases
A protein with each di-peptide being able
to bind to a specific nucleotide
Positions DNA endonuclease at target site
One required for each strand of DNA
Uses bacterial CAS9 to target specific
locations in the genome
Targeting directed by supplied guide RNA
Guide RNA forms a complex with CAS9,
which can cause a double stranded break
c. The phenotypical changes are then noted and compared with
different gene manipulations
3. Green Fluorescence Protein is a good reporter due to its properties
a. It requires no cofactor, so it always gives a signal when translated
b. GFP has been modified from its wild type sequence to make it more
efficient in mammals
i. A Valine codon has been added after the starting methionine
to fulfil Kozaks rule
ii. Changed the codons of existing amino acids to their more
expressed form

Population genetics
Inheritance types
1. Factors influencing phenotypic ratios
a. Complete or incomplete or co-dominance or sex-influenced or sexlimited
i. Partial dominance leads to intermediate phenotypes
ii. Sex-influenced dominance will cause intermediate to have
different phenotypes based on gender
iii. Sex-limited will only have the phenotype expressed in one
b. Autosomal or sex-linked
i. More males are affected when sex-linked
c. Monohybrid or dihybrid

d. Lethal or non-lethal
2. Expected phenotypic ratios in F2, with opposite homozygous parents
(heterozygous F1)
a. Autosomal complete dominance monohybrid cross
i. 3 dominant: 1 recessive
b. Autosomal incomplete dominance monohybrid cross
i. 1 dominant: 2 intermediate: 1 recessive
c. Autosomal complete dominance dihybrid cross
i. 9 dominant-dominant: 3 dominant-recessive: 3 recessivedominant : 1 recessive-recessive
d. X-linked dominant
i. Male:
ii. Female:
e. X-linked recessive
i. Male:
ii. Female
f. Y-linked
g. Recessive epistasis of autosomal complete dominance dihybrid
i. 9 dominant-dominant: 3 dominant-recessive : 4 recessive-_
Because recessive-dominant looks like recessiverecessive
h. Dominant epistasis of autosomal complete dominance dihybrid
i. 12 dominant-_: 3 recessive-dominant : 1 recessive-recessive
Because dominant-recessive looks like dominantdominant
i. Complementary loci of autosomal complete dominance dihybrid
i. 9 dominant-dominant : 7 recessive
Because any recessive will give the same phenotype
j. Duplicate dominant genes of complete dominance dihybrid cross
i. 15 dominant : 1 recessive-recessive
Because any dominant will give the same phenotype
k. Duplicate genes with cumulative effect
i. 9 dominant-dominant : 6 dominant + recessive: 1 recessiverecessive
l. Dominant and recessive interaction
i. 13 dominant-_ : 3 recessive-dominant
Because dominant-_ looks like recessive-recessive
3. Reading pedigree
a. Determining type of inheritance
i. Autosomal dominant diseases
Affects both males and females equally
Does not skip generations
ii. Autosomal recessive diseases
Affects both males and females equally
Does skip generations
iii. X-linked dominant
More affected females than males
May be lethal in hemizygous males, causing
decreased male count

Affected father always gives affected daughter

Does not skip generations
iv. X-linked recessive
More affected males than females
Affected mother always produces affected sons
May skip generation
v. Y-linked
Only males are affected
Affected father always produces affected sons
b. Tips
i. If a generation is skipped, the inheritance is definitely

Mendelian Genetics
1. There are several reasons why Mendel used the pea plant for this
a. It has well defined characteristics
b. Pure-bred varieties are available
c. The flowers have male and females parts, allowing for selffertilisation
d. Both self and cross fertilisation are possible
e. Relatively short life cycle
f. Large number of offspring produced from each mating
2. Mendels law of segregation states that the gamete only contains one
allele of a gene
a. Thus both alleles of a parents gene are not inherited together in
one gamete
3. Mendels law of independent assortment states that each allele in the
parental pair, will combine randomly with either allele in the other
parental pair, when forming the offspring
4. Partial dominance occurs when each genotype has a distinguishable
phenotype, with the heterozygote having an intermediate trait between
the two homozygotes extremes
5. When considering





numbers of loci

types of gametes will be produced

types of offspring genotypes will be produced
types of offspring phenotypes will be produced when there is

complete dominance
i. Else there will be

types of phenotypes if all are

incomplete dominance
6. Testcross is used to identify the genotype of an organism, whether it is
heterozygous or homozygous for the gene
a. The organism is mated with a homozygous recessive tester stock
b. If only one offspring phenotype is observed, the original organism is
7. Reciprocal cross is used to determine if the gene is sex-linked
a. A homozygous dominant male is mated with a homozygous
recessive female and vice-versa

b. Autosomal genes will have equal results, while sex-linked genes will
have different ones
8. Males are more affected by recessive X-chromosome diseases, as they are
9. There is a criss-crossing of recessive X-chromosome alleles
a. Affected mothers will definitely give affected sons
b. Affected fathers will definitely give carrier daughters
i. Daughters may be affected if mother is also carrier or
10.Barr bodies are found in female cells, as one X-chromosome is inactivated
a. One of the two X chromosomes in female cells is in the
heterochromatin state
b. Usually females cells will only have one Barr body, but the number
can differ due to aneuploidy
11.Autosomal genes can be sex-influenced, with the heterozygotes having
different phenotypes based on gender
12.Autosomal genes can also be sex-limited, where only one gender gets
13.Having the genotype does not necessarily mean the individual is affected
a. Penetrance and expressivity measures this
b. Pleiotropy also leads to not all effects being caused
14.Multiple Alleles will cause an increase in the number of genotypes
a. Number of genotypes =

, where

is the number of

15.Epistasis occurs when the genotype at one locus suppresses the
expression of the alleles of another locus
16.Recessive epistasis requires for a homozygous recessive genotype to mask
the other gene
a. Loci for gene A has recessive epistasis over loci for gene B
b. Caused by the loss of production of intermediate, masking the
effect of the other gene
i. Like ability for coat to have colour
c. Caused by leaky allele, where two recessive alleles gives rise to an
effect similar to a dominant allele
17.Dominant epistasis requires only one copy of the allele at the epistatic loci
a. The expression of the dominant allele masks the expression of the
other gene
i. The dominant allele causes the phenotype regardless of the
allelic conditions of the other locus
b. Cause by the dominant allele degrading the precursor or product of
the other gene
18.Complementary loci (Duplicate recessive gene) is when the recessive
alleles for both genes give the same phenotype
a. Like colourness and purple vs white
b. Caused by the pathway having a colourless precursor and colourless
c. Caused by needing both products to form a functional dimer
19.Duplicate dominant gene is when the dominant alleles for both genes give
the same phenotype
a. Both genes lead to the same final product, and phenotype is
independent on the relative quantity of final produce produced

20.Expression of the gene can be regulated by epigenetic factors

a. Positive or negative control of inducers and repressors for
b. DNA topoisomerase can affect packing of DNA, affecting access to
c. DNA and chromatin modification, like methylation
d. RNA processing can affect stability and export
i. Alternative gene splicing produces different proteins
e. microRNA can silence the mRNA
f. Protein modification
g. Prions
h. Factors affecting enzyme activity
21.Lethal genes cause the death of the organism
a. Dominant lethal genes only allow homozygous recessive organisms
to survive
i. The heterozygote might not die immediately at birth, but only
develop lethal symptoms later in life
b. Recessive X-chromosome lethal genes will only affect males
i. Because an affected daughter cannot be born
There is no affected father to pass on the disease allele
ii. Carrier daughters can be born from their carrier mothers,
thus making their sons vulnerable
22.Nondisjunction can occur during either Pachytene of prophase I or
anaphase I or II
a. Either the Holliday junction is not resolved leading to a persistent
chiasma, or the spindle fibres fail to achieve proper attachment.
b. Variation in chromosome number is called aneuploidy
i. Monosomy is one chromosome missing
ii. Trisomy is one chromosome extra
23.Uniparental disomy occurs when only one parent contributes both the
allele of the gene
a. Cause by disjunction occurring in both parents, where one gives
both, and the other gives none
b. Or caused by trisomic rescue, where the cell randomly chooses to
eliminate the extra chromosome, removing the non-nondisjuction
allele with it.

Non-Mendelian Genetics
1. Maternal inheritance is non-Mendelian because the mitochondrial DNA can
only be passed down from mother to offsprings
a. Mitochondrial DNA undergoes high rates of mutation
i. Due to exposure to free radicals from respiration
ii. Due to no DNA repair during replication
iii. Due to its numerous copies in each mitochondria
b. Thus can be used for comparison between species that diverged
only a short time ago
2. Linked genes are non-Mendelian because they do not obey Mendels Law
of Segregation
a. The genes tend to be inherited together, affecting the offspring
genotypic ratio
i. Parental genotype will be greater than recombinant genotype

ii. Genes can be on the same chromosome but still be

genetically unlinked, if they are very far apart
b. Each chromosome is thus a linkage group, where genes are
inherited together
i. Thus humans have 23 linkage groups
3. The distance between two genes loci on homologous chromosomes are
measured in centiMorgan
a. 1 map unit or cM is equal to a 1% chance that random chiasma
formation will occur between the two gene loci on non-sister
i. Genes with greater cM have a chance of multiple chiamata
forming between them
b. 1 cM is roughly equal to 1 million base pairs
c. Genes that are more strongly linked have a lower cM
d. Maximum value for cM is not 50
i. Although recombination frequency maximum is 50%, it tends
to underestimate the actual distance, thus 50cM would give
rise to less than 50% recombinant frequency
4. Genetic distance is calculated from the proportion of recombinant
a. Recombination fraction ( ) =

Number of recombinant progeny

Total number of progeny

0 0.5

ii. 1 cM

0.01 (


They are not equal when the real distance between loci
is great
There will be less recombinant frequency than
expected, as even numbers of chiasmata formation
between the loci produces a parental gamete
iii. Since the Mendelian ratio is 1:1:1:1, which means 50% parent
and 50% recombinant
b. A value of 0.1 would imply that crossing over to form recombinant
gametes occurs once every 10 times
c. A value of 0.5 would imply that recombinant gametes are form half
the time meaning that the gene are either physically or genetically
5. For linked genes, a heterozygote (AaBb) can have different forms of the
same genotype
a. Depends on the parents genotype
i. AABB x aabb
ii. AAbb x aaBB
b. Trans arrangement is when the mutant alleles are carried by both
i. Ab + aB
c. Cis arrangement is when one chromosome carries the wild-type
allele, and the other carries the mutant allele
i. AB + ab
6. Linkage analysis can be done to associate a certain form of a marker with
the disease allele of a gene
a. Closely linked markers with the disease allele will cosegregate

b. Markers can be repetitive DNA sequences with the flanking DNA

sequence being unique
i. Microsatellites are repeats of 2 to 5 nucleotides
ii. Minisatellites are tandem repeats of 15 to 35 nucleotide

Population Genetics
1. A population has a large group of sexually-reproducing interbreeding
individuals with a common gene pool
2. Hardy-Weinberg equilibrium is achieved when a population has no changes
in genotype frequency, at a specific loci, over generations.
a. Actual numbers of individuals with the genotype may change, but
the overall genotype frequency remains constant
b. When

p2+ 2 pq+ q2=1

i. Where p is the frequency of one allele, and q is the frequency
of the other allele


ii. The equation means, that at a specific allele frequency, there

must be a specific number of organisms that are homozygous
dominant ( p
recessive ( q

), heterozygous ( 2 pq , and homozygous


iii. The equation is a symptom of Hardy-Weinberg equilibrium,

not a test, as the population can coincidently be at the
equilibrium ratio, but is still in the process of changing.
iv. If the gene has more than 2 alleles, the equation changes

p+q+ r=1

p2+ q2 +r 2 +2 pq+2 pr +2 qr =1

c. The values of the genotype frequency, when there is HardyWeinberg equilibrium, is not fixed
i. When conditions change from ABC to ABD, and back to ABC,
the equilibrium genotype frequency at the first ABC is not
the same as during the second ABC
3. Hardy-Weinberg equilibrium is obtained when certain conditions are
a. There must be a large population size, to minimise the effects of
genetics drift
i. Since mating and survival is random, the genotype of a small
population will fluctuate greatly
Fixation occurs when one allele is lost from the gene
This is more likely to occur in small populations
b. There needs to be random mating
i. Non-random mating leads to increases in the frequency of
homozygotes in the sub-population

Like population stratification by religion or ethnicity

Like assortative mating where those with desired traits
are more popular
Positive assortment, when like only mates with
like, increases frequency of homozygous
Homozygous individuals, while more likely
to expresses desirable traits, are also
more likely to expresses undesirable traits
Negative assortment, when like strictly does not
mate with like, increases frequency of
heterozygous genotypes
Like inbreeding
c. There needs to be no net migration of genotypes
i. Movement of individuals can increase or decrease gene
ii. Or even introduce new genes through gene flow
d. There must also be no net mutations
i. The spontaneous mutations of an allele into the other form
for an offspring, needs to be counterbalanced by the death of
an organism carrying the alternative allele.
ii. No new allele should be formed also, else it will definitely
change genotype frequency when passed to an offspring.
e. Lastly, there needs to be no net selection pressure
i. Favouring the successful reproduction chances of a genotype
will lead to an increase in the frequency of that genotype
over time.
And vice-versa
ii. Selections against a genotype can be offset by new
4. Hardy-Weinberg equilibrium is obtained after 1 generation if all
assumptions hold true.
a. If all conditions are true for the current parent generation, their
offspring will have the exact same genotypic frequency as them,
showing that there is Hardy-Weinberg equilibrium
b. This equilibrium is maintained so long as the environment does not
change in a way to affect the targeted locus.
5. For sex-linked loci, the allele frequency in males will be equal to the allele
frequency in females of the previous generation
a. Males will chase after females, because they are hemizygous and
inherit the X-chromosome from the females
6. Mutations will change the alleles of genes, introducing genetic variation
a. Non-recurrent mutations are rare events, and not important for
population genetics
i. The probability of the rare allele successfully propagating
through the population over generations is unlikely
b. Recurrent mutations will introduce new alleles into the population,
and sustain them
i. New allele is sustained by a changeable mutation rate from
wild type to mutant

If the mutation is one way and recurrent, eventually all

the wild type allele will be converted to the mutated
The allele frequency of the wild type after n

pn= p0 ( 1 )

is the mutation rate

This leads to an eventual loss of genetic

If the mutation is two way and recurrent, a balance
between the two forms will be reached
At equilibrium, there will be no net change in
gene frequencies,

q e = p e
is the rate of mutation of

allele A1, whose frequency is

q , and

is the rate of mutation of allele A 2,

whose frequency is

If given the mutations rates, to find the allele

frequency at equilibrium:

q e=

, pe =

Note that the numerator is the mutation

rate of the other allele
This maintains the genetic variation in a
7. Natural selection will select for fitter organisms to survive to reproduce
a. The fitness of a genotype is
i. Where


is the coefficient of selection (selection intensity),

the proportionate reduction in gametic contribution of the


ii. The fittest genotype has a fitness value of 1 ( s=0 )

iii. A fitness of 0.5 means that for every one offspring produced
by the less fit genotype, 2 offspring are produced by the
fittest genotype
Thus the population has more of the fittest genotype
b. To find the allele frequency when it is selected against
i. Assume that the population is in Hardy-Weinberg equilibrium,
such that the observed number are equal to the expected
ii. Calculate the current allele frequency of all alleles
iii. Calculate the gametic contribution of each genotype
Genotype frequency times fitness
iv. Derive the allele frequency over one generation

q1 =

Gametic contribution by homozygous+ Gametic contribution of allele by hetero

Total gametic contributionby all genotypes

Note that heterozygous will only contribute the

allele half the time.
v. Derive a general formula for allele frequency
c. If there is overdominance, where the heterozygotes has the greatest
fitness, the population can obtain equilibrium

s 1 p e=s 2 q e


pe =

Because at this level of heterozygotes, the number of

homozygous organisms produced is equal to the
number that dies, thus maintain the frequency

, qe = 1
s 1+ s 2
s 1+ s 2

8. When analysing a population to check for Hardy-Weinberg equilibrium

a. Calculate the frequency of the first allele ( A 1 )

Number of organisms with A 1 A1 + ( Number of organisms with A1 A 2)
Totalnumber of organisms
b. Calculate the frequency of the second allele ( A 2 )

Number of organisms with A2 A 2 + ( Number of organisms with A 1 A 2)
Total number of organisms
c. Calculate the expected frequency of each
population is in Hardy-Weinberg equilibrium

A 1 A 1= p , A 1 A2=2 pq , A 2 A 2=q




d. See if the number of organisms corresponds to the expected

numbers should the population be in Hardy-Weinberg equilibrium
i. Expected




A1 A1


( p2 total number of organisms)

ii. Repeat for the other genotypes

iii. Perform


( observed numbersexpected numbers )2

Expected numbers

iv. Compare


value with table



number of genotypenumber of alleles


This is because the allele frequencies are not

independent of each other, and fixing the frequency of
one determines the other


9. When deriving an unknown genotype frequency in a population, from a

given genotype frequency
a. First assume that the population is in Hardy-Weinberg equilibrium,
and that the observed numbers equal the number expected of such
a population in Hardy-Weinberg equilibrium
b. Use the equation

p2+ 2 pq+ q2=1

to work out the frequencies of

the other two genotypes or other allele

c. If the gene is sex-linked
i. Assume that there are equal numbers of male and females in
the population
ii. Frequency of known allele in entire population =

( Allele frequency females )+ ( Allele frequency males)

A population in equilibrium should have allele

frequency in entire population = allele frequency in
females = allele frequency in males
10.Z-score is used to compare allele frequency between population to see if it
significantly differs

p1 p 2

a. Z-score =

p 1 ( 1p 1 ) p2 ( 1 p2 )

i. Z-score is significantly different if the value is greater than

11.Polymorphism occurs when a population has two or more alleles that are
maintained in high enough frequencies (greater than 1% of population)
that the rarest of them cannot be maintained by just constant mutation
and selection
a. Other factors play a role in keeping the rarer allele in the gene pool
i. Heterozygote advantage
There will be an equilibrium geneotype frequency
which is at intermediate levels (above 1%)
ii. Frequency dependent selection
Selection intensity for a genotype constantly changes,
thus maintaining a genotype frequency range over
time, keeping the rare alleles around
iii. Heterogeneous selection
The environment of the population changes, thus
changing selection and mutation rates
The environment change is either over time, or
by population movement
iv. Transition
Explains some currently observed polymorphisms

Due to recent environmental changes, the selection

and mutation values have just changed, and the
polymorphism will be lost over time.
v. Neutral mutations
The polymorphism confers no selection advantage
Thus polymorphism is maintained by random mutation
and random death
Polymorphic by chance alone
Greater chance when population size is larger
12.The founder effect is when genetic drift occurs through a new population
branching off the original population, bringing with them a subset of
alleles of the original population
a. The new population has different genotype frequencies depending
on who left the original population
13.A bottleneck event is when the variation in a populations gene pool is
greatly reduced due to environmental events like natural disasters or
a. Like the Black death
b. Alleles can be lost, and the genotype frequency changes
1. Can the prophase of mitosis be divided into Leptotene and Diaknesis as
2. As the microtubules radiate from the centrosome, are they all considered
astral until they contact the kinetochore (making them kinetochore MT) or
microtubules from the opposing pole (making them polar MT)
3. Will secondary nondisjunction always occur for those with trisomy?
1. Chromosome and Kinetochore