Lecture #2: Mendelian inheritance II, extension to mendelian inheritance I

The punnett square is a simple way to visualize the segregation and random union of alleles
- Each F1 hybrid produces 2 kinds of gametes in a 1:1 ratio
- F2 progeny
- 3:1 ratio of phenotypes
- 1:2:1 genotypic ratio
- ¼ will breed true for the dominant trait
- ½ will be hybrids
- ¼ will breed true for the recessive trait
Mendel's law
- Law of segregation
- Law of independent assortment
**looked at traits which will give you more than one phenotype**
Mendel's dihybrid crosses revealed the law of independent assortment
**two genes which will control two different traits**
- Mendel tested whether 2 genes in dihybrids would segregate independently
- First, he crossed true breeding yellow round pears with true breeding green wrinkled peas
to obtain dihybrid F1 plants. Then the dihybrid F1 plants were selfed to obtain F2 plants
- YY RR x yy rr → F1 Yy Rr (followed the rules of the monohybrid crosses)
- F1 Yy Rr x F1 Yy Rr → F2
- Mendel asked whether all of the F2 progeny would be parental types (yellow round and
green wrinkled) or would some be recombinant types (yellow wrinkled and green round)
A dihybrid cross produces parental types and recombinant types
- Each F1 dihybrid produces 4 possible gametes in a 1:1:1:1 ratio
- Yy Rr → ¼ YR, ¼ Yr, ¼ yR, ¼ yr
- 4 phenotypic classes occurred in the F2 progeny
- 2 are like parents
- 2 pare recombinant
Independent assortment in crosses of F1 dihybrids produces a 9:3:3:1 phenotypic ratio
- Note that in these F2 progeny, there is a 3:1 phenotypic ratio of dominant to recessive
forms
- 9/16 YR, 3/16 yyR, 3/16 Yrr, 1/16 yyrr
Mendel's law of independent assortment
- During gamete formation, different pairs of alleles segregate independently of each other
- Y is just as likely to assort with R as it is with r
- y is just as likely to assort with R as it is with r
**during formation of gametes, certain pairs of alleles will segregate independently of one
another**
Following crosses with branched line diagrams
- Progeny phenotypes for each gene are shown in different columns
Testcrosses on dihybrids
- Testcross dihybrids to individuals that are homozygous for both recessive traits; we can
use these to identify if the individual that we have is homozygous or heterozygous
Mendel's laws can be used to predict offspring from complicated crosses
- To calculate the possible number of gamete genotypes from a hybrid, raise 2 to the power
of the number of different traits (this works when you have a hybrid)
- Ie. Aa Bb Cc Dd → 2^4 = 16 types of gametes
- To do a punnett square with this cross involving 4 genes, you would need 16
columns and 16 rows
- An easier way is to break down a multi hybrid cross into independently assorting
monohybrid crosses
In humans, pedigrees can be used to study inheritance
Pedigrees: orderly diagrams of a family's relevant genetic features
**used to look at relevant genetic features within a family; looking at the specific trait you can
see if it is a dominant or recessive trait**
- Includes as many generations as possible (ideally, at least both sets of grandparents of an
affected person)
- Pedigrees can be analyzed using Mendel's laws
- Is a trait determined by alternate alleles of a single gene?
- Is a trait dominant or recessive?
Symbols used in pedigree analysis

**consanguineous = blood relative marriage**

A vertical pattern of inheritance indicates a rate dominant trait (Huntington disease)
- Every affected person has at least one affected parent
- Mating between affected person and unaffected person is effectively a testcross
**what you will see; one of the parents of the affected person is affected as well**
A horizontal pattern of inheritance indicates a rare recessive trait (Cystic fibrosis)
- Parents of affected individuals are unaffected but ae heterozygous (carriers) for the
recessive allele
How to recognize dominant traits in pedigrees
 - 3 key aspects of pedigrees with dominant traits
1) Affected children always have at least one affected parent
2) As a result, dominant stairs show a vertical pattern of inheritance
3) Two affected parents can produce unaffected children, if both parents are heterozygotes
- 4 key aspects of pedigrees with recessive traits
1) Affected individuals can be the children of two unaffected carriers, particularly as a result
of consanguineous matings
2) All the children of two affected parents should be affected
3) Rare recessive traits show a horizontal pattern of inheritance
4) Recessive traits may show a vertical pattern of inheritance if the trait is extremely
common in the population
Extension to mendelian inheritance I
Some phenotypic variation poses a challenge to mendelian analysis
- Ie. lentils come in an array of colours and patterns
- Crosses of pure breeding lines can result in progeny phenotypes that don't appear to
follow Mendel's rules
- Explanations for some traits
- No definitively dominant or recessive allele
- More than two alleles exist
- Multiple genes involved
- Gene-environment interactions
Extensions to Mendel for single gene inheritance
- Dominant is not always complete, there are different variations
- Incomplete dominance
- Codominance
- A gene may have >2 alleles
Pleiotropy: one gene may contribute to several characteristics
- Recessive lethal alleles
- Delay lethality
Summary of different dominance relationships
- The phenotype of the heterozygote defines the dominance relationship of 2 alleles
Complete dominance: hybrid resembles one of the two parents (will be one colour or the other)
Incomplete dominance: hybrid resembles neither parent (will have a blend of the two traits, will
not look exactly like either parents)
Codominance: hybrid shows traits from both parents (will show traits from both parents, but in a
pattern)
Flower colour in snapdragons is an example of incomplete dominance
- Crosses of pure breeding red with pure breeding white results in an all pink F1 progeny
- Phenotype ratios reflect the genotype ratios
In codominance, the F1 hybrids display traits of both parents
 - Phenotype ratios reflect the genotype ratios
- The hybrid display of poth parental traits is shown
Dominance relationships between alleles do not affect transmission of alleles
- Type of dominance (complete, incomplete dominance, codominance) depends on the type
of proteins encoded and by the biochemical functions of the proteins
- Variation in dominance relations do not negate Mendel laws of segregation
- Allele still segregate randomly
- Interpretation of phenotype/genotype relations is more complex
A gene can have more than two alleles
- Multiple alleles of a gene can segregate in populations
- Each individual can carry only to alleles
- Dominance relations are always relative to a second allele and are unique to a pair of
alleles
**relation between the two should be kept as pairs of alleles**
ABO blood types in humans are determined by three alleles of one gene
- I^A = A type sugar, I^B =B type sugar, i = no sugar
- 6 genotypes produce 4 blood types
- Dominance relations are relative to a second allele
- I^A and I^B are codominant
- I^A and I^B are dominant to i
Medical and legal implications of ABO blood group genetics
- Antibodies are made against type A and B sugars
- Successful blood transfusions only occur with matching blood types
- Type AB are universal recipients, type O are universal donors
Seed coat patterns in lentils are determined by a gene with 5 alleles
- 5 alleles for C gene; spotted, dotted, clear, marbled-1, marbled-2
- Reciprocal crosses between pairs of pure breeding lines is used to determine dominance
relations
- marbles-1>marbles-2>spotted = dotted>clear
Human histo-compatibility antigens are an extreme example of multiple alleles
- 3 major genes (HLA-A, HLA-B, HLA-C) encode histocompatibility antigens
- Cell surface molecules present on all cells except RBCs and sperm
- Facilitates proper immune response to foreign antigens
- Each gene has 20-100 alleles each
- Each allele is codominant to every other allele
- Every genotype produces a distinct phenotype
- Enormous phenotypic variation
Mutations are the source of new alleles
- If mutations occur in gamete-producing cells, they can be transmitted to offspring
- Frequency of gametes with mutations is 10^-4-10^-6
 - Mutations that result in phenotypic variants can be used by geneticists to follow gene
transmission
Nomenclature for alleles in populations
Allele frequency: the percentage of the total number of gene copies for one allele in a population
- Most common allele is usually the wild type (+) allele
- Rare allele is considered a mutant allele
- Gene with only one common type type allele is monomorphic
- Agouti gene in mice - only one allele in while populations, many alleles in lab
mice
- Gene with more than one common allele is a polymorphic
- High frequency alleles of polymorphic genes are common variants
The mouse agouti gene controls hair colour; one wild type allele, many mutant alleles
- Wild type agouti allele produces yellow and black pigments in hair
- 14 different agouti alleles in lab mice, but only A allele in wild mice
One gene may contribute to several characteristics
Pleiotropy: single gene determining several distinct and seemingly unrelated characteristics
- Ie. many aboriginal Maori men have respiratory problems and are sterile
- Defects due to mutations in a gene required for functions of cilia (failure to clear
lungs) and flagella (immotile sperm)
- With some pleiotropic genes
- Heterozygotes can have a visible phenotype
- Homozygotes can be invisible
The A^Y allele produces a dominant coat colour phenotype in mice
- A^Y allele of agouti gene causes yellow hairs with no black
The A^Y allele is a recessive lethal allele
- A^Y is dominant to A for hair colour, but is recessive to A for lethality
- 2:1 ratio is indicative of recessive lethal allele (pure breeding yellow A^YA^Y
mice cannot be obtained because they are not viable)
Pleiotropy of sickle cell anemia; dominance relations vary with the phenotype under
consideration
 Lecture #5: linkage and recombination
Gene linkage and recombination
- Genes linked together on the same chromosome usually assort together
- Linked genes may become separated by recombination (can affect phenotypic and
genotypic ratios)
- Two themes in this chapter
1) The further apart two genes are, the greater the probability of recombination
2) Recombination data can be used to generate maps of the relative locations of genes on
chromosomes
Detecting linkage by analyzing the progeny of dihybrid crosses: X linked genes
Syntenic genes: genes located on the same chromosome
- X linked genes in drosophila
- w (+) - red eyes, w - white eyes
- y (+) - brown body, y - yellow body
- Y represents Y-chromosome
- F1 males; X chromosome from their mothers
- F1 females are dihybrids
- Compare allele configurations in F2 to P generation
- Deviation from 1:1:1:1 segregation in F2 indicates the genes are linked
- There is a large number of F2 males resembling the parental generation (99% look
like the male, 1% has the recombinant type; the segregation of gametes does not
follow independent assortment)
**note that i this cross involving X linked genes, only the F2 male progeny were counted**
Designation of parental and recombinant relate to past history
- Note that the parental configurations in these two crosses are opposite of eachother
Autosomal traits can also exhibit linkage
- Bateson and punnett observed ratios that were significant departure from the expected;
- Crosses; true breeding purple flowered, long pollen plants (PPLL) and true
breeding red flowered, round pollen plants (ppll)
- There is a complete dominant relationship
- F1 progeny were all purple flowered with long polen (PpLl)
- Dihybrid F1 individuals yielded F2 progeny that are not in the expected 9:3:3:1
ratio, but this is not what is observed during this experiment.
**linkages between genes can happen at any chromosome for any traits; as long as they are close
when they are assorting, meaning they are not assorting independent. We see this when the
parental pheno/genotypes are overrepresented in the F2 progeny**
The 9:3:3:1 ratio is altered when genes A and B are linked
- For linked genes, the F2 genotypic classes produced most often by parental gametes
increase in frequency at the expense of the other classes
- In the AB/ab dihybrid cross shown here, the A-B- and aa bb classes in the F2 generation
will occur at higher frequencies, and the other two classes (A- bb and aa B-) at lower
frequencies than predicted by the 9:3:3:1 ratios
Autosomal genes can also exhibit linkage
- A test cross shows that the recombination frequency for the eye colour (pr) and wing size
(vg) pair of drosophila genes is 11%. Because parentals outnumber recombinants, the pr
and vg genes are genetically linked and must be on the same autosome
**not an X linked gene, they are autosomal**
Evidence that recombination results from reciprocal exchanges between homologous
chromosomes
**if there were no crossovers, the parental genes would be passed to the progenies and would
represent exactly the parental chromosomes; if there was recombination, we see different
phenotypes and changes on the chromosomes. On one of the X there is a discontinuity added,
and the other has a region of Y chromosome. If there is no recombination, the discontinuity and
Y chromosome will be on different X chromosomes, but if there is recombination during
meiosis, then both discontinuity and Y chromosome will be found on one X chromosome, nad
the other will have neither. This experimented showed that recombination does take place**
Recombination during meiosis I visualized by light microscopy

a) early prophase, b) leptotene and zygotene, c) diplotene

**chiasmata are the sites of crossover; and they are found in diplotene. The crossover between
the nonhomologous chromosomes takes place**
Recombination; a result of crossing over during meiosis
- Frans Janssens - 1909 observed chiasmata chromosomes during prophase of meiosis I
- T.H Morgan - suggested chiasmata were sites of chromosome breakage and exchange
- H. Creighton and B. McClintock (corn) and C Stern (drosophila) - 1931, direct evidence
that genetic recombination depends on reciprocal exchange of chromosomes
- Physical markers were used to identify specific chromosomes
- Genetic markers were used as points of reference for recombination
Recombination frequencies are the basis of genetic maps
- A.H Sturtevant - proposed that recombination frequencies (RF) could be used as a
measure of physical distance between two linked genes
- 1% recombination = 1 RF = 1 map unit (m.u) - 1 centimorgan (cM)
**lower recombination = closer together they are, the higher the number = further apart**the %
of recombination frequency = the length
Properties of linked VS unlinked genes
Linked genes: parentals > recombinants (RF<50%), linked genes must be syntenic (on the same
chromosome) and sufficiently close together or the same chromosome so that they do not assort
independently
Unlinked genes: parentals = recombinants (RF = 50%), occurs either when genes are on different
chromosomes or when they are sufficiently far apart on the same chromosome (in either case,
they will assort independently of one another)
Comparison of two point crosses establish relative gene positions
- Left right orientation of map is arbitrary
- Most accurate maps obtains by summing many small
intervening distances
**two point cross = comparing gene pairs**
Limitations of two point crosses
1) Difficult to determine gene order if two genes are close
together
2) Actual distances between genes do not always add up (sometimes does not take into
consideration the double crosses)
3) Pairwise crosses are time and labour consuming
Three point crosses provide faster and more accurate mapping

Analyzing the results of a three point cross

- Testcross progeny had 4 sets of
reciprocal pairs of genotypes;
- Most frequent pair has parental
configuration of alleles
- Least frequent pair results from
double crossovers
- Examination of double crossover
class reveals which gene is in the middle

**when comparing the parental and the

recombinant, you will know which one is in the middle based off of which one is different; there
will be 2 that are the same within the parental and the recombinant, and the third will be different
(they will be the same gene but one will be + )**the gene that is different from the parental is the
one which is found in the middle**
**you don’t include ones that resemble the parental strands in the equation; only the ones which
are different than the parental that you are using**you are finding the distance between the two
genes, therefore you only use 2 of the given genes**
Correction for double crossovers
- This calculation is not accurate because it fails to account for double crossovers;

- Correct calculation that accounts for double crossovers

Inferring the location of crossover event

- Examine numbers of progeny
- Compare configuration of allele at two genes at a time to parental configuration
**before data analysis, you do not know the gene order or allele combination on each
chromosome**
Chi square test pinpoints the probability that ratios are evidence of linkage
- Deviations from 1:1:1:1 ratios can represent chance events or linkage
- The chi square test measures ‘goodness of fit’ between observed and expected values
(tells us if the observed values are deviated significantly from the expected values, to use
Chi square we need a null hypothesis)
Null hypothesis: observed values are no different from expected values
- In linkage studies, the null hypothesis is no linkage
- If genes are not linked, expected 1:1:1:1 ratio in F2
- Chi-square test can reject the null hypothesis, but it cannot prove a hypothesis
**there is no ratio for linked genes; they are all different depending on the scenario - this is why
we cannot prove a hypothesis**the sample size plays an important role - the higher the sample
size, the more accurate the results are**
Information needed for the chi square tes
- Use data from breeding experiment
- Total number of progeny
 - How many classes of progeny (important because we need to know the degree of
freedom based on classes**
- Number of offspring observed in each class
- Calculate number of offspring expected in each class if there is no linkage (1:1:1:1
segregation)
Applying the chi square test

- Calculate the chi square;

- Consider degrees of freedom (df) in the experiment
- df = N - 1 (where N is the number of classes)
- Determine a p value using chi square value and df
- Probability that the deviation from expected numbers had occurred by chance
Critical chi square values
- Use p value of 0.05 as cutoff
- Chi-square values that lie in the beige region of this table allow rejection of the null
hypothesis with >95% confidence
- If null hypothesis is rejected, then linkage can be postulated

**larger sample size = more significant outcome**Applying the chi square test to see if genes
A and B are linked
DNA structure, replication and recombination
Two general themes to genes at the molecular levels
- The genetic functions of DNa flow directly from its molecular structure
- Knowledge of molecular structure of DNA makes it possible to understand
biochemical processes of genetics
- All of the genetics functions of DNA depend on specialized proteins that red the
information in DNA sequence
- DNA itself is chemically inert (does not perform a specific function, but has all
the guidelines for the functions that need to occur in the cell - unlike proteins
which can function on their own. DNA is considered to be the instructions for the
rest of the parts of the cell)
Chemical studies located DNA in the chromosomes
- F. Meischre (1869) extracted nuclein (nuclear material) from nuclei of human white
blood cells
 - Weakly acidic, phosphorus rich material
- Chemical analysis of nuclein revealed that is major component was deoxyribonucleic
acid (DNA)
- Contains deoxyribose, found in nucleus and is acidic
- Staining of cells revealed that DNA localized almost exclusively within chromosomes
(which are in the nucleus, but there is also found not in the nucleus - in the mitochondria)
- Schiff reagent - stands DNA red
The chemical composition of DNA
- DNA contains 4 kinds of nucleotides linked in a long chain
Phosphodiester bonds: covalent bonds joining adjacent nucleotides
Polymer: linked chain of subunits
Are genes composed of DNA or protein?
- DNA is made of only 4 different subunits
- Too simple to specify genetic complexity?
- Protein is made of 20 different subunits
- More potential for creating different combinations
- Chromosomes contain more protein than DNA
Bacterial transformation implicated DNA as the substance of genes
- F. Griffith (1928) did experiments with 2 strains of Streptococcus pneumoniae
- Differ in colony morphology and biological activity
- Smooth (S) strain - virulent
- Rough (R) strain - nonvirulent
- R cells could be transformed by genetic material transferred from dead S cells
**transformation = transferring genetic material from one strain into the cells**
- Avery, MacLeod, and McCarty (1944) provided evidence that DNA is the transforming
principle of S cells
**DNA is the transforming material; not protein - which means that DNA is where the genes are
found because you are able to change genes, however when you try to change proteins they just
go back to what they were before - because they are not transforming and they do not contain
genes**
A simple system to test whether protein or DNA is the genetic material
- Bacteriophages (phages) are viruses that infect bacteria
- Phage particles contain roughly equal amounts of protein and DNA
- Contain very few genes but able to replicate themselves inside bacterial host
- After infection, ghost of phage particle remains attached to outer surface of cell
(they do not have genetic material because the genetic material is injected into the
bacterial cell)
- Phage genetic material injected into bacterial cell (contain few genes, but when
they are in the host they can replicate which can end up killing the host; they use
the host as a self replicating machinery)
The basis of Watson-Crick double helix model of DNA
- R. Franklin and M. Wilkins (1952) solved X ray diffraction pattern of DNA (this was the
basis for the double helix model of Watson and Crick)
- DNA is helical structure with 20 A diameter
- Spacing between repeating units is 3.4 A
- Helix undergoes a complete turn every 34 A
- Detailed knowledge of chemical constituents of DNA
- 4 nitrogenous bases (G,A,C,T), deoxyribose, phosphate
- E. Chargaff - base composition of DNA from many organisms
- Ratios of bases - A;T is 1:1, G:C is 1:1
A detailed look at DNAs chemical constituents
- Nucleotides linked in a directional chain
- Phosphodiester bonds always form covalent link between 3’ carbon of on nucleoside and
5’ carbon of the next nucleoside (phosphate groups form the backbone of the DNA)
**note the 5’-3’ polarity**
- Attachment of base to sugar = nucleoside , when you add a phosphate you get a
nucleotide (A,C,G,T)
Chargaff’s data on nucleotide base composition in the DNA of various organisms
- In all organisms, ratios of A to T and G to C are roughly 1:1 (gives an idea of base
pairing between A-T and G-C)
Complementary base pairing
- Base pairs consist of hydrogen bonds (weak electrostatic bonds) between a purine and a
pyrimidine (G with C, A with T)
**G-C has 3 hydrogen bonds, A-T have 2 hydrogen bonds**G and A are purines, C and T are
pyrimidine**
- Consistent with Chargaff’s rule
- Each base pair has ~ the same shape
The double helix structure of DNA
- Strands are antiparallel
- Sugar-phosphate backbone on the outside
- Base pairs in the middle
- Two chains held together by H bonds between A-T and G-C base pairs
Z DNA is one variant of the double helix
- B-form DNA forms right handed helix and has a smooth backbone
- Z form DNA forms left stranded helix and has an irregular backbone
**most common form is the right handed helix (B form)**
4 questions about how DNA structure relates to genetic functions
- How does the molecule carry information?; base sequence
- How is that information copied for transmission to future generations? ; DNA replication
- What mechanisms allow the genetic information to change? ; recombination, mutations
 - How does DNA encode information govern the expression of phenotype? ; gene
functions and expression (transcription and translation)
DNA stores information in the sequence of its bases
- Most genetic information is read from unwound DNA (ie. synthesis of DNA or RNA)
- Some genetic information is accessible within double stranded DNA (ie. DNA binding
proteins that regulate gene expression), we need proteins to read the DNA; they bind to
the DNA and read it, which then allows us to have regulation of gene expression =
formation of RNA molecules which can then be translated into functional proteins. In
other cases, RNA molecules can also perform specific functions without being translated
into proteins.
Chemical constituents of RNA
- 3 major chemical differences between RNA and DNA
1) Ribose sugar instead of deoxyribose
2) Uracil (U) instead of thymine (T)
3) Most RNA are single stranded
Complex folding pattern of RNA
- most RNAs are single stranded but can form base pairs within other parts of the same
molecule; which allows them to fold into 3D structures (which means that some RNAs
can perform different functions)
**some bacteria have double stranded RNA**
The model of DNA replication postulated by Watson and Crick
- Unwinding of double helix exposes bases on each strand
- Each strand can act as a template for synthesis of new strands
- New strands form by insertion of complementary base pair
- Single double helix becomes two identical daughter double helices
**new dna is made from the template and the new strand**
3 possible models of DNA replication
Semiconservative: the Watson-Crick model
Conservative: parental double helix remains intact, both strands of daughter helices are newly
synthesized
Dispersive: both strands of both daughter helices contain original and newly synthesized DNA
The experimental approach to test the models of replication
- M. Meselson and F. Stah (1958) separated preexisting parental DNA from newly
synthesized daughter DNA
- Gew E coli in media containing N^15 (heavy isotope) then switched to media
containing N^14 (normal isotope)
**they used their isotopes to grow bacteria; which allowed nitrogen to go into the DNA**
- Purified DNA from cells and subjected it to equilibrium density gradient
ultracentrifugation
 - Cesium chloride (CsCl) forms stable gradient with highest density at bottom of
tube
- DNA forms a tight band at position where its density equal the CsCl density
The mechanism of DNA replication
- A. Kornberg and others worked out the biochemical aspects of replication in E coli
- Energy for DNA synthesis comes from high energy phosphate bonds associated with
dNTPs
DNA polymerase: catalyzed new phosphodiester bonds
- Highly coordinated process that has 2 stages;
1) Initiation: proteins open up the double helix and prepare it for complementary base
pairing
2) Elongation: proteins connects the correct sequence of nucleotides on newly formed DNA
strands
DNA synthesis proceeds in a 5’ to 3’ direction
- Template and newly synthesized strands are antiparallel
- DNA polymerase adds nucleotides to 3’ OH of the new strand
**nucleotides are added to the 3’ end**formation of phosphodiester bond is formed by the 5’
end with the 3’ end that has already been incorporated**you add new nucleotides to the 3’ end
which is available**
The mechanism of DNA replication: initiation
- Initiation begins at the origin of replication
- Initiator proteins binds to the origin
- Helicase unwinds the helix
- Two replication forks are formed (are present on both sides of the replication bubble, they
will move as the DNA is being synthesized)
- Preparation of double helix for complementary base pairing
- Single strand binding proteins keep the DNA helix open (single strand binding proteins
binds to the single strand of DNA)
- Primase synthesizes RNA primer
- Primers are complementary and antiparallel to each template strand
The mechanism of DNA replication; elongation
- The correct nucleotide sequence is copied from template strand to newly synthesized
strand of DNA
DNA polymerase III catalyzes phosphodiester bond formation between adjacent
nucleotides (polymerization)
- Leading strand has continuous synthesis
- Lagging strand has discontinuous synthesis
- Okazaki fragment is a short DNA fragment on the lagging strand
- DNA polymerase I replaced RNA primer with DNA sequence
- DNA ligase covalently joins successive Okazaki fragments together
**direction of synthesis is in opposite direction of replication fork**
The bidirectional replication of a circular bacterial chromosome; an overview
- Replication proceeds in 2 directions from a single origin
- Unwinding of DNA creates supercoiled DNA ahead of replication fork
- DNA topoisomerases relax supercoiled by cutting the sugar phosphate backbone bonds
strands of DNA
- Unwound broken strands thens sealed by ligase
- Synthesis continues bidirectionally until replication forks meet
**bacterial chromosomes only have one origin of replication, unlike humans which can have
multiple origins of replication**
Lecture #6: mutations
Mutations; primary tools of genetic analysis
- Mutations are heritable changes in DNA base sequences
Forward mutation: changes wild-type allele to a different alleles (going from wild type to
mutant)
Reverse mutation (reversion): changes a mutant allele back to wild type
- Forward mutation rate is usually greater than reversion rate
Classification of mutations by effect on DNA molecules
Substitution: replacement of abase by another base
**changes of one or more bases; the number of bases remains the same**
Transition: purine replaced by another purine, or pyrimidine replaced by another
pyrimidine
Transversion: purine replaced by a pyrimidine, or pyrimidine replaced by a purine
Deletion: block of 1 or more base pair lost from DNA
Insertion: block of 1 or more base pair added to DNA
Inversion: 180 degree rotation of a segment of DNA
**part of the DNA will invert and then be added to the sequence. The site of inversion will have
the same nucleotides but the order will be flipped. To have the inversion take place and have the
correct sequence at the end, take the top strand and invert it; then place it on the bottom - the
bottom strand will be inverted and placed on the top strand**you want 5’ and 3’ to remain the
same as the initial sequence**
Reciprocal translocation: parts of two nonhomologous chromosomes change places (happens
due to chromosomal breaks, which causes translocation on the chromosomes)
**result when there are chromosomal breaks due to damage done to the DNA; causes
translocation of the chromosome**part of chromosome 1 will go to chromosome 2, and part of
chromosome 2 goes to chromosome 1**can have severe effects on the cell**
Rates of spontaneous mutation
- Rates of recessive forward mutations at 5 coat colour genes in mic
- 11 mutations per gene every 106 (1 million) gametes
- Mutation rates in other organisms
 - 2-12 mutations per gene every 106 gametes
**spontaneous rate of mutation is very low**
Different genes, different mutation rates
- Mutation rates are <10-9 to >10-3 per gene per gamete
- Differences in gene size
- Susceptibility of particular genes to various mutants genetic mechanisms
- Average mutation rate in gamete producing eukaryotes is higher than that of prokaryotes
- Many cell divisions take place between zygote formation and meiosis in germ
cells
- More chance to accumulate mutations
- Can diploid organisms tolerate more mutations than haploid organisms?
- They can; because there can be recessive mutations, whereas in haploid
organisms, there are no recessive mutations (you only need one mutation
to change the phenotype)
**eukaryotic mutations are more common than prokaryotic mutations; due to multiple divisions
and more DNA**
Experimental evidence that mutations in bacteria occur spontaneously
- S. Luria and M. Delbruck (1943) - fluctuation test
- Examined origin of bacterial resistance to phage infection
- Infected wild type bacteria with phage
- Majority of cells die, but a few cells can grow and divide
- Had the cells altered biochemically?
- Did the cells carry heritable mutations for resistance?
- Did the mutations arise by change or did they arise in response to the
phage
Replica plating verifies that bacterial resistance is the result of pre existing mutations

Interpretation of Luria-Delbruck fluctuation test and replica plating

- Bacterial resistance arises from mutations that occurred before exposure to bactericide
(pre existing mutation which can be passed from generation to generation)
 - Bactericide becomes a selective agent
- Kills non resistant cells
- Allows survival of cells with pre existing resistance
- Mutations occur as the result of random processes
- Once such random changes occur, they usually remain stable
**different mutations can cause different DNA damage
How natural processes can change the information stored in DNA
Depurination: random base insertion (purine removed and replaced by another base)
Deamination of C: C changed to U, normal C-G → A-T after replication (during replication, the
error will be caught and the normal DNA will be restored; however if it is not caught, there will
be errors)
- X rays break the sugar-phosphate backbone of DNA (can cause deletions, translocations
on the chromosomes)
- Ultraviolet (UV) light causes adjacent thymines to form abnormal covalent bonds
(thymine dimers; 2 T beside each other, which creates bonds between the two base pairs
and creates a dimer - during replication, it can cause the replication proteins to stall)
- Irradiation causes formation of free radicals (ie. reactive oxygen) that can alter individual
bases (free radicals can change the nucleotide conformation)
- 8-oxodG mispairs with A instead of C
- Normal G-C → mutant T-A after replication
Mistakes during DNA replication
- Incorporation of incorrect bases by DNA polymerase is exceedingly rare (<10-9 in
bacteria and humans)
- Two ways that replication machinery minimizes mistakes
1) Proofreading: function of DNA polymerase, 3’-5’ exonuclease recognizes and excises
mismatches
2) Methyl-directed mismatch repair: corrects errors in newly replicated DNA
**process of repair requires 3 basic steps; recognition of damage, removal of that specific
damaged DNA/sequence, replace with the correct sequence**
DNA polymerases proofreading function
- Mispaired base is recognized and excised by 3’-5’ exonuclease (removes the mismatched
nuclease) of DNA polymerase (will then replace it will the correct base)
- Improves fidelity of replication 100 fold
Unequal crossing over can occur between homologous chromosomes
- Pairing between homologs during meiosis can be out of register
- Unequal crossing over results in a deletion of one homolog and a duplication on the other
homolog
Transposable elements (TEs) move around the genome
- TEs can jump into a gene and disrupt its function
- Two mechanisms of THE movement (transposition)
**TEs; regions that are found on DNA that occupy a large part of the genome, and these regions
can jump from one part to another part, they can cause mutations in that certain part**they are
removed from one region to another region; they can either change to be the same as the new
region that they occupy, or they can stay different**
Experimental evidence that mutations induce mutation
- H. J. Muller
- X ray dose above the naturally occurring level causes increase mutation rate in
Drosophila
- Exposed male Drosophila to X rays
- Mating scheme used genetically marked ‘balancer’ X chromosome
- Able to detect X linked genes that are essential for viability
**increasing X ray increases mutations; these mutagens can cause alterations in the DNA
(mutations are agents or factors which can cause changes at the DNA level - can be
environmental, physical, chemical etc)**
How mutagens alter DNA: chemical action of mutagen
- Replace a base; base analogs (chemical structure almost identical to normal base)
- Alter base structure and properties; hydroxylating agents (add an -OH group)
- Alter base structure and properties; alkylating agents (add ethyl or methyl groups)
**adding ethyl group will cause pairing between G and T; no triple bond between G and C, there
will be a double bond between G and T**
- Deaminating agents (remove amine -NH2 groups)
- Insert between bases; intercalating agents (ie. profilin which inserts itself into the DNA
and disrupt DNA replication and repair → causes insertion or deletion in the sequence)
- This was later used to study codons in translation machinery
DNA repair mechanisms that are very accurate
- Reversal of DNA base alterations
- Alkyltransferase: removes alkyl groups
- Photolyase: splits covalent bond of thymine dimers (that are produced due to UV)
- Homology: dependent repair of damaged bases or nucleotides
- Base excision repair
- Nucleotide excision repair
- Correction of DNA replication errors
- Methyl-directed mismatch repair
Base excision repair removes damaged bases
- Different glycosylases cleave specific damaged bases
- Particularly important for removing ucacil (created by cytosine deamination) from DNA
- Creates an AP site (apurinic); there is no base attached to the backbone in this
case
Nucleotide excision repair corrects damaged nucleotides
- UvrA - UvrB complex scans for distortions to double helix (ie thymine dimers)
 - UvrB - UvrC complex nicks the damaged DNA
- 4 nt to one side of damage
- 7 nt to the other side of damage
In bacteria, methyl-directed mismatch repair corrects mistakes in replication
- Parental DNA strand marked by adenine methylase (a methyl group is added to A on the
parental strand, and that's how you can identify the difference between the new and old
strand)
- Methyl group added to A in GATC sequence
- Newly replicated DNA isn't yet methylated
- MutS and MutL bind to mismatched nucleotides (identify and will bind to it)
- MutH nicks the unmethylated strand opposite the methylated GATC
- Gap made in unmethylated (new) strand by DNA exonucleases
- Gap filled in by DNA polymerase using the methylated (old) strand as template
DNA repair mechanisms that are error prone
- SOS system - bacteria (results in adding more mutations)
- Used at replication forks that stalled because of unrepaired DNA damage
- ‘Sloppy’ DNA polymerase used instead of normal polymerase (the polymerase is
not very accurate)
- Adds random nucleotides opposite damaged bases (which causes the mutations)
- Nonhomologous end-joining
- Deals with double-strand DNA breaks caused by X rays or reactive oxygen
(involves repairs with double stranded breaks - it is an error prone repair
mechanism which can cause mutations and inaccurate repair)
Repair of double strand breaks by nonhomologous end joining
- Unrepaired double strand breaks can lead to lethal chromosome rearrangement (ie.
deletions, inversions, translocations)
- Resection step can lead to loss of DNA (when the enzymes bind, they start making new
nucleotides on the end of the broken strands, which can cause mutations or errors in the
repair)
**certain proteins (PKcs, KU80, KU70)bind to the broken ends of the DNA and then ligases and
polymerases add new nucleotides to both strands; because there is no homology, the new
nucleotides could have mismatches or changes from the original template strand**
Health consequences of mutations in genes encoding DNA repair enzymes
- Xeroderma pigmentosum; (associated with mutations in DNA repair enzymes)
- Mutations in one of seven genes encoding enzymes involved in nucleotide excision repair
- Hereditary forms of colorectal cancer; mutations in human homologs of bacterial genes
(MutS and MutL) involved in mismatch repair
**cancer is associated with mutations in repair enzymes**mutations in BRCA1 increases
susceptibility to breast cancer**
Impact of unrepaired mutations
Germ line mutations: occur in gametes or in gamete precursor cells, transmitted to next
generation, provide raw material for natural selection
Somatic mutations: occur in non-germ cells, not transmitted to next generation of individuals,
can affect survival of individual, can lead to cancer
The Ames test identifies potential carcinogens
- Assay uses his” mutants in S. typhimurium
- Detects mutations that cause his” to his+ reversion
Complementation and gene function; mutations
What mutations tell us about gene structure
Complementation testing: reveals whether two mutations are in a single gene or in different
genes
Complementation group: synonymous with gene, collection of mutations that do not complement
each other
Drosophila eye colour mutations produce a variety of phenotypes
- Do these phenotypes result from allelic mutations or from mutations in different genes?
A complementation table for X-linked eye colour mutations in drosophila
- These results reveal 5 complementation groups (genes);
- Mutations in white, cherry, coral, apricot and buff are allelic (all affect the white
(w) gene)
- Mutations in garnet, rub, vermillion and carnation are not allelic with each other
or with white mutations
**question on exam; which ones are allelic/which ones form complementation group; + is wt
phenotype, look at which ones give you wt compared to white for example, 4 genes are wt
phenotype meaning they are in different gene compared to white**
What mutations tell us about gene function
- Garrod (1902); some human diseases result from inborn errors of metabolism
- Beadle and Tatum (1940s); the one gene, the one enzyme hypothesis
- Neurospora crassa; mutants in arginine synthesis
- Genetic dissection of a biochemical pathway
- Ingram (mid 1950s); mutations in a gene can result in amino acid substitutions that
disrupt the function of the encoded protein
- Missense substitution in hemoglobin 𝛽 causes sickle cell anemia
Alkaptonuria; an inborn error of metabolism
Beadle and tatum; the one gene, one enzyme hypothesis
- Screened for x ray induced mutations in Neurospora that disrupted synthesis of arginine
Prototroph: wild type strain that grows in minimal media without nutritional
supplements
Auxotroph: mutant strain that cannot grow in minimal media
- Recombination analysis used to map mutations to 4 different regions of
genome
- Each region contained a different complementation group
- 4 genes for arguing biosynthesis; ARG-E, ARG-F, ARG-G,
ARG-H
Experimental support for the one gene, one enzyme hypothesis
- Scheme used by beadle and tatum for isolation of arg- auxotrophs in
neurospora
**haploid spores grown on minimal media with only one specific amino acid,
mutants growing on that media confirms the mutant cannot make the amino
acid and requires it from the environment**
- Growth response if nutrients is added to minimal medium
- Inferred biochemical pathway; each ARG gene encodes and enzyme needed to convert
one intermediate to the next in the pathway
**one gene one enzyme is an oversimplification of biochemical pathways**
Proteins are chains of amino acids linked by peptide bonds
- 20 different amino acids
- R group is the side chain that is unique to each amino acid
- 4 groups of amino acid based on R group properties; COOH group and NH2 group of
adjacent amino acids are joined in covalent peptide bonds
- Polypeptides have N and C terminus
Amino acids with nonpolar R groups
Amino acids with uncharged R groups

Amino acids with charged R groups

- Basic R group;

- Acidic R group;
The molecular basis of sickle-cell anemia
 - Glu → Val substitution at 6th amino acid affects the 3D structure of the hemoglobin 𝛽
chain
- Abnormal protein aggregates causes sickle shape of red blood cell
Levels of polypeptide structure
- Interactions that determine the 3D conformation of a polypeptide
- 1 prime structure is the amino acid sequence
- 2 prime structure is the characteristic geometry of localized regions
- 3 prime structure is the complete 3D arrangement of a polypeptide
Quaternary structure; multimeric proteins are complexes of polypeptide subunits

- Identical subunits;

- Non-identical subunits;
One gene, one polypeptide
- One gene, one enzyme concept is not broad enough
- Not all proteins are enzymes
- Some proteins are multimeric and subunits are encoded by different genes
- Complex pathways can be dissected through genetic analysis
- Different mutations in a single gene can produce different phenotypes
- Different amino acid substitutions can have different effects on protein function
 - Mutations can affect protein function by altering the amount of normal protein
made

**to do these questions; look at the rows and columns (does not matter which one) and find
which combinations will result in a phenotype that is not wild type. Look at the rows and
whichever ones are negative will give you a complementation group, if you go down the rows
(ie. go to row C) and the C column has already been incorporated into a group, you can skip over
that row**
Ie. for this example; complementation groups are ACEH (from row A), BFD (from row B), G
(from row G)
Lecture #7: gene expression
4 general themes for gene expression
- Pairing of complementary bases is the key to the transfer of information from DNA to
RNA and from RNA to protein
**all molecules in this process have polarity, ie. 5’ and 3’, N and C terminus for proteins**
- Polarities of DNA, RNA and polypeptides help guide the mechanisms of gene expression
- Gene expression requires input of energy (energy from nucleotides themselves, and
proteins get energy from ribosomal enzymes) and participation of specific proteins and
macromolecular assemblies
- Mutations that change genetic information or obstruct the flow of its expression can have
dramatic effects on phenotype (change of structure, change of expression by increasing or
decreasing the expression rate within the cell)
**gene expression refers to transcription and in some cases translation of the gene**how
information goes from DNA to protein**
Gene expression; the flow of genetic information from DNA via RNA to protein
- RNA polymerase transcribes DNA to produce an RNA transcript
- Ribosomes translate the mRNA sequence to synthesize a polypeptide
- Translation follows the genetic code
**RNA transcript; serves directly as mRNA prokaryotes processed to become mRNA in
eukaryotes**DNA → transcription → mRNA → translation → polypeptide chain**
Triplet codons of nucleotides represent individual amino acids
 - 61 codons represent the 20 amino acids (there are more codons than there are amino
acids, there are multiple codons which encode for the same amino acid**
- 3 codons signify stop (nonsense or stop codon**
Studies of frameshift mutations showed codons consist of 3 nucleotides
- F. Crick and S. Brenner (1955)
- Proflavin-induced mutations in bacteriophage T4 rIIB gene
- Intercalates into DNA
- Causes insertions and deletions
- 2nd treatment with proflavin can create a 2nd mutation that restores wild-type
function (revertant)
**used intercalating agents (agents that insert themselves into the DNA sequence, resulting in an
insertion or deletion mutation)**they found that sometimes 2 treatments of the intercalated agent
will restore the wildtype function; if there are insertions it will change the coding frame. If the
first treatment is a deletion and the second mutation is an insertion, it can change the DNA back
to what it was before and restore the reading frame**keeps a functional mutation**their study
was to identify the number of nucleotides in a codon**
Different sets of T4 rIIB mutations generate either a mutant or a normal phenotype
- Codons must be read in order from a fixed starting point
- Starting point establishes a reading frame
- Intragenic suppression occurs only when wild type reading frame is restored
**they induced mutations and found that when they had insertion or deletion → mutant, 2
insertion or 2 deletion → mutant, insertion and deletion → wildtype, 2 insertion and 2 deletion
→ wild type. When you have the same amount of insertion and deletion it will create a
wildtype**
Codons consist of 3 nucleotides read in a defined reading frame
- Counterbalancing of mutations (ie. +1 and -1) can restore the reading frame
- Intragenic suppression occurs when mutations involve multiples of 3
**even if it changes the amino acid sequence, it may not change the function of the protein.
Sometimes it does not even change the amino acid sequence**
Cracking the code; discovery of mRNA
- 1950s, studies in eukaryotic cells
- Evidence that protein synthesis takes place in cytoplasm
**DNA is found in the nucleus but protein synthesis takes place in the cytoplasm; so there has to
be an intermediate between these 2. DNA does not move by itself through the nucleus; this is
how they found the mRNA**
- Deduced from radioactive tagging of amino acids
- Implies that there must be a molecular intermediate between genes in the nucleus and
protein synthesis in the cytoplasm
- Discovery of mRNA molecules for transporting genetic information
**study to find which nucleotides encode what amino acids**
Using synthetic mRNAs and in vitro translation to crack the genetic code
- 1961; Marshall Nirenberg and Heinrich Mathaei
- Added artificial mRNAs to cell free translation systems
**they made translation machinery outside of the cell so that they could look at it; they started
with the basic RNA (UUU) and found what amino acid is encoded from this sequence**after
finding the amino acid for one code, they change the sequence and analyze the new amino acids
which are in the codon table**
Cracking the genetic code with mini mRNAs
- Nirenberg and Leder (1965)
- Resolved ambiguities in genetic code
- In vitro translation with trinucleotide synthetic mRNAs and tRNAs charged with
a radioactive amino acid
**they used radioactive amino acids which they put through a filter and based on that it will find
out which amino acid will create that specific polypeptide chain and bind to the specific tRNA**
Correlation of polarities in DNA, mRNA and polypeptide
- Template strand of DNA is complementary to mRNA and to the RNA-like strand of
DNA
- 5’ to 3’ in the mRNA corresponds to N to C terminus in the polypeptide
**process requires a template strand, which is used in transcription and produces the
mRNA**polymerase binds to the template strand and copies it creating a complementary strand,
which has the same sequence as the mRNA except T is replaced with U**transcription is 5’ →
3’**5’ is the N and 3’ is the C**
Summary of the genetic code
- Genetic code has triplet codons
- Codons are nonoverlapping
- 3 nonsense codons do not encode an amino acid; UAA (ocher), UAG (amber), UGA
(opal)
- Genetic code is degenerate
- Reading frame is established from a fixed starting point - codon for translation initiation
is AUG
- mRNAs and polypeptides have corresponding polarities
- Mutations can be created in 3 ways; frameshift, missense and nonsense
Genetic code is almost, but not quite, universal
**what we have in the codon table remains the same for almost all the organisms**codon bias;
some organisms prefer to use specific codons do encode specific amino acids**the genetic code
evolved very early on in history, evolutionary this has been set at the very beginning of time**
- Virtually all cells alive now use the same basic genetic code
- In vitro translational systems from one organism can use mRNA from another
organism to generate protein
 - Comparisons of DNA and protein sequence reveal perfect correspondence
between codons and amino acids among all organisms
- Genetic code must have evolved early in history of life
- Exceptional genetic codes found in ciliates and mitochondria
Transcription; from DNA to RNA
- RNA polymerase catalyzes transcription (adds the nucleotides to the growing chain of
nucleic acids)
**polymerase binds to the 3’ end of the DNA**
Promoters: DNA sequences that provide the signal to RNA polymerase for starting transcription
(guide polymerase to start transcription)
- RNA polymerase adds nucleotides in 5’ to 3’ direction
- Formation of phosphodiester bonds using ribonucleotide triphosphates (ATP, CTP,
GPT, UTP)
- Hydrolysis of bonds and NTPs provides energy for transcription (same for DNA
but it comes from hydrolysis of DNTPs)
Terminators: RNA sequences that provide the signal to RNA polymerase for stropping
transcription
Transcription in bacterial cells, Initiation; the beginning of transcription
- RNA polymerase binds to promoter sequence located near beginning of gene
**promoter is close to the start of transcription but is not part of the final transcript**
- Sigma factor binds to RNA polymerase ( → holoenzyme)
**increases binding of polymerase to the promoter region, without the sigma factor the affinity
between these two is not very high. Sigma factor increases the affinity**
- Region of DNA is unwound to form open promoter complex
- Phosphodiester bonds formed between first two nucleotides
Transcription in bacterial cells, Elongation; an RNA copy of the gene
- Sigma factor separates from RNA polymerase ( → core enzyme)
- Core RNA polymerase loses affinity for promoter, moves in 3’ to 5’ direction on template
strand
- Within transcription bubble, NTPs added to 3’ end of nascent mRNA
Transcription in bacterial cells, Termination; the end of transcription
- Terminators are RNA sequences that signal the end of transcription
- Two kinds of terminators in bacteria
1) Extrinsic: require rho factor
2) Intrinsic: dont require additional factors
- Usually form hairpin loops (intramolecular H bonding)
**affinity between promoter and polymerase is much lower at termination which allows for a
release of polymerase from the template strand**terminator region is part of the final RNA
transcript**
The product of transcription is a single stranded primary transcript
**the primary transcript in eukaryotic cells undergoes modifications to become the final mature
mRNA molecule**the sequence of the transcript is similar to that of the coding strand, and is
complementary to the template strand**polymerase binds to the 3’ end**
The promoters of 10 different bacterial genes
- Most promoters ar upstream to the transcription start point
- RNA polymerase makes strong contacts at -10 and -35
**transcription begins after promoter region**
Structure of the methylated cap at the 5’ end of eukaryotic mRNAs
- Capping enzyme adds a backward G to the 1st nucleotide of a primary transcript
**in this case, a G that has a methyl group on it is added to the 5’ end of the mRNA molecule,
this is important in guiding the translation process of gene expression**
Processing adds a tail to the 3’ end of eukaryotic mRNAs

**at the 3’ end, addition of poly a tail ; a long A sequence tail**

RNA splicing removes introns
Exons: sequences found in a genes DNA and mature mRNA (expressed regions)
Introns: sequences found in DNA but not in mRNA (intervening regions)
- Some eukaryotic genes have many introns
**RNA splicing removes introns and keeps exons which are then translated to produce the final
protein**after this occurs, it is considered a mature mRNA**
The human dystrophin gene; an extreme example of RNA splicing
- Splicing removes introns from a primary transcript
**a large portion of genes are introns which are all removed**
RNA processing splicing out introns and joins adjacent exons
- Short sequences in the primary transcript dictate where splicing occurs
- Two sequential cuts remove an intron
**the process of splicing is driven by specific sequences that are found in the intron and exon
region; those sequences are binding sites for spliceosome which are involved in the splicing of
mRNA**once the introns are spliced out, those regions are degraded and the nucleotides are
used in different processes**
Splicing is catalyzed by the spliceosome

Alternative splicing cna produce two different mRNAs from the same gene

**advantage of splicing creates alternative splicing; which can produce different proteins from
the same gene**
Translation; from nRNA to protein
**change from nucleotide to amino acid**
Transfer RNAs (tRNAs): mediate translation of mRNA (used as a template to produce the final
proteins) codons to amino acids
 - Translation takes place on ribosomes that coordinate movement of tRNAs carrying
specific amino acids
- tRNAs are short single stranded RNAs of 74-95 nucleotides
- Each tRNA has an anticodon (which recognizes the codon on the RNA molecule
and is complementary to the codon sequence) that is complementary to an mRNA
codon
- A specific tRNA is covalently coupled to a specific amino acid (charged tRNA)
- Base pairing between an mRNA codon and an anticodon of a charged tRNA
directs amino acid incorporation into a growing polypeptide
Some tRNAs contain modified bases

3 levels of tRNA structure

1) Nucleotide sequence is the primary structure (the sequence of the tRNA)
2) Secondary structure (cloverleaf shape) is formed because of short complementary
sequences within the tRNA (2D structure, you can see the amino acids, anticodon region
and the codon region on it)
3) Tertiary structure (L shape is formed by 3D folding)
**anticodon region is very important**
Aminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAs
- Each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural
features of its corresponding tRNA
**each aminoacyl-tRNA synthetase has one amino acid**the number of unique aminoacyl
synthetases is not equal to the number of tRNAs**
Base pairing between an mRNA codon and a tRNA anticodon determines which amino
acid is added to a growing polypeptide
**what decides the amino acid that is added depends on what is found on the anticodon region of
the tRNA**what is attached to the tRNA does not play a role in what will be added during the
process of translation**
Wobble; some tRNAs recognize more than one codon for the aminoacido they carry

**wobble effect; some tRNAs recognize more than one codon for amino acids, usually the last
nucleotide is different in those codons**because of this, mispairs at that position (wobble
position - last position of the anticodon) is common ; could be tolerated because in many cases,
changing the last nucleotide is not going to change the amino acid sequence of the
polypeptide**the 5’ end does not have to be specific to the tRNA**
A ribosome has two subunits composed of RNA and protein
**ribosomes have 2 different subunits; small and large. Both composed of rRNAs and
proteins**eukaryotic ribosome has more members compared to the prokaryotic but the structure
and function are very similar**translation is a highly conserved process so it is similar in
eukaryotes and prokaryotes**
Mechanism of translation
Initiation stage: start codon is AUG at 5’ end of mRNA. In bacteria, initiator tRNA has
formylated methionine (fMet)
Elongation stage: amino acids are added to growing polypeptides. Ribosome move in 5’ to 3’
direction along mRNA. 2-15 amino acids added to C terminus per second
Termination stage: polypeptide synthesis stops at the 3’ end of the reading frame. Recognition of
nonsense codons. Polypeptide synthesis halted by release factors. Release of ribosomes,
polypeptide and mRNA
Different parts of a ribosome have different functions
- Small subunit binds to mRNA
- Large subunit has peptidyl transferase activity
(where the peptide bonds will be formed), the small
subunit is where there is binding of the mRNA
molecule**
- 3 distinct tRNA binding areas; E, P and A sites

Translation of mRNAs on ribosome; initiation phase in prokaryotes

- Ribosome binding site consists of a Sine-Dalgarno sequence (binds to a region in the 5’
untranslated region) and an AUG
- 3 sequential steps
1) Small ribosomal subunit binds first
2) fMet-tRNA positioned in P site (attachment at the P site only for the initiator, everything
else goes to the A site)
3) Large subunit binds
 - Initiation factors play a transient role
**there are untranslated regions on the 5’ and 3’ regions**
Translation of mRNA on ribosomes; initiation phase in eukaryotes
- Small ribosomal subunit binds to 5’ cap (required for binding of ribosomal subunit to the
RNA) then scans the mRNA for the first AUG codon
- Initiator tRNA carries Met (not fMet)
Translation of mRNAs on ribosomes; elongation phase
- Addition of amino acids to C terminus of polypeptide
- Charged tRNAs guided into A site by elongation factors
**formation of polypeptide bond at A site**
Polyribosomes consist of several ribosomes translating the same mRNA
- Simultaneous synthesis of many copies of a polypeptide from a single mRNA
**multiple ribosomes will be on the RNA at the same time which allows them to produce
multiple proteins at once**
Translation of mRNAs on ribosomes; termination phase
- No normal tRNAs carry anticodons for the stop codons
- Release factors bind to the stop codons
- Release of ribosomal subunits, mRNA and polypeptide
**release factor hydrolyzes the growing polypeptide chain and the ribosomal subunit
disassembles which stops the process of translation**
Posttranslational processing can modify polypeptide structure

- Cleavage may remove an amino acid;

- Cleavage may split a polyprotein;

 - Chemical constituent addition may modify a protein;

Differences between prokaryotes and eukaryotes in gene expression

- Prokaryotes;
- No nucleus, transcription and translation take place in the same cellular
compartments, and translation is often coupled to transcription
- Genes are not divided into exons and introns
- Transcription;
- One RNA polymerase consisting of 5 subunits
- DNA sequences needed for transcription initiation are located close to the
promoter
- Promoters are not wound up in chromatin
- Primary transcripts are the actual mRNAs; they have a triphosphate stat at
the 5’ end and no tail at the 3’ end
- Translation;
- Unique initiator tRNA carries formyl methionine
- mRNAs have multiple ribosome binding sites and can thus direct the
synthesis of several different polypeptides
- Small ribosomal subunit immediately binds to the mRNAs ribosome
binding site
- Eukaryotes;
- Nucleus separated from the cytoplasm by a nuclear membrane. Transcription
takes place in the nucleus, while translation occurs in the cytoplasm. Direct
coupling of transcription and translation is not possible
 - The DNA of a gene consists of exons separated by introns; the exons are defined
by post transcriptional splicing, which deletes the introns
- Transcription;
- Several kinds of RNA polymerase, each containing 10 or more subunits;
different polymerases transcribe different genes
- Enhancer sequences far from the promoter are often needed for
transcription initiation
- Transcription initiation requires promoters to be cleared of chromatin to
allow access to RNA polymerase
- Primary transcripts undergo processing to produce mature mRNAs that
have a methylated cap at the 5’ end and a poly-A-tail at the 3’ end
- Translation;
- Initiator tRNA carries methionine
- mRNAs have only one start site and can thus direct the synthesis of only
one kind of polypeptide
- Small ribosomal subunit binds first to the methylated cap at the 5’ end of
the mature mRNA and then scans the mRNA to find ribosome binding site
Types of mutations in the coding sequence of genes

Mutations in the coding sequence of a gene can alter the gene product
Missense mutations: replace one amino with another, there are 2 types
1) Conservative: chemical properties of mutant amino acid are similar to the original amino
acid
2) Nonconservative: chemical properties of mutant amino acid are different from original
amino acid
Nonsense mutations: change codon that encodes an amino acid to stop codon (UGA, UAG,
UAA)
Frameshift mutations: result from insertion or deletion of nucleotides with the coding region. No
frameshift if multiples of 3 are inserted or deleted
Silent mutation: do not alter the amino acid sequence, degenerate genetic code (most amino acids
have >1 codon)
Mutations outside the coding sequence cna disrupt gene expression

Loss of function mutations result in reduced or abolished protein activity

- Loss of function mutations are usually recessive
Null (amorphic) mutations: completely block function of a gene product (ie. selection of an
entire gene)
Hypomorphic mutations: gene product has weak, but detectable activity
- Some loss of function mutations can be dominant
Incomplete dominance: phenotype varies with the amount of functional gene product
Haploinsufficiency: phenotype is sensitive to gene dosage
Gain of function mutations enhance a function or confer a new activity
- Gain of function mutations are usually dominant
- Enhance proteins function (more product, more efficiency), proteins gain novel
function, protein is expressed at the wrong time
Hypermorphic mutations: generate more gene product or the same amount of a more efficient
gene product
Neomorphic mutations: generate gene product with new function or that is expressed at
inappropriate time or place
Hypermorphic mutations
- Achondroplasia or dwarfism
- FGFR3 gene
- Encodes growth factor receptor, to get activated it binds to FGF
- Mutant FGFR3
- Contains Arg instead of Gly at amino acid 480, activated in the absence of FGF
Neomorphic mutation
- Antp promotes leg development
- Antm heterozygous mutants develop legs on the head
- Transcriptional mutations causes expression of Atp in antennae tissues
Mutations classified by their effects on protein function

The cellular components of gene expression

 - Mutations in genes encoding gene products for transcription, RNA processing, translation
and protein processing are often lethal
- Some mutation in tRNA genes can suppress mutations in protein coding genes
A nonsense mutation in a protein coding gene creates a truncated, nonfunctional protein

Nonsense suppression
- A second, nonsense suppressor mutation in the anticodon of tRNA gene allows
production of a mutant full length polypeptide

Lecture #8: gene regulations (prokaryotes)

RNA polymerases participates in all three phases of transcription
Initiation: core RNA polymerase plus sigma (σ) factor
- Core has four subunits make up RNA polymerase: two alpha (𝛼), one beta (𝛽), one beta
prime (𝛽’)
- DNA is unwound and polymerization begins
Elongation: core RNA polymerase without σ factor
- Continues until RNA polymerase recognizes termination signal (either rho dependent or
rho independent signals)
Termination: two kinds in bacteria
- Rho-dependent - Rho (p) protein binds to RNA polymerase and removes it from RNA
- Rho-independent - 20 nt sequence in RNA forms stem-loop
**rho is a helicase molecule**
Role of RNA polymerase in initiation and elongation phases of transcription
Two kinds of transcription termination in bacteria

Regulation of expression can occur at many steps

- Transcriptional control
- Binding of RNA polymerase to promoter (key step in regulation of prokaryotic
genes)
- Most critical step in regulation of most prokaryotic genes
- Shift from initiation to elongation
- Release of mRNA at termination
- Posttranscriptional control
- Stability of mRNA
- Efficiency of translation initiation
- Stability of polypeptide
Utilization of lactose by E. coli provides a model system of gene regulation
- Lactose utilization required 2 enzymes
- Permease: transports lactose into the cell
- Beta-galactosidase: splits lactose into glucose and galactose
- In the absence of lactose, both enzymes are present at very low levels
**lactose itself enhances the production of permease and beta-galactosidase; an inducer of these
two enzymes**
- Lactose is the inducer of the genes encoding permease and beta-galactosidase
- Induction: stimulation of synthesis of a specific protein
- Inducer: molecule responsible for induction
**e coli provides source of nutrients to be glucose, but in the absence of glucose it uses lactose**
Lactose utilization in an E. coli cell
Advantages of using lactose utilization by E. coli as a model for understanding gene
regulation
- Lac- mutants can be maintained on media with glucose and so lac genes are not essential
for survival
- If both glucose and lactose are present, E. coli cells will use glucose first (as long
as they have glucose, they will not use lactose as energy source)
- Simple assays for lac expression - use of ONPG or X-gal as substrates for
beta-galactosidase (colour change due to the production of beta-galactosidase)
- Lactose induces a 1000 fold increase in beta-galactosidase activity (due to the ONPG)
- Detection and characterization of hundreds of lac- mutants defective in lactose utilization
Studies of lac- mutants revealed the operon theory of gene regulation
- Jacques Monod and François Jacob - Pasteur institut
- Nobel prize in 1965 (with A. Lwoff) for their discoveries concerning genetic
control of enzyme and virus synthesis
- Compared the effects of many different types of lac mutants on induction and
repression of enzyme activity for lactose utilization
Operon theory: one signal can simultaneously regulate expression of several clustered genes
**in a bacterial cell; there are multiple genes involved in the same process which are all on one
mRNA, and use a single promoter. These genes will always be expressed together**
- Hypothesized that lac genes are transcribed together as a single mRNA
(polycistronic; have multiple genes on the mRNA) from a single promoter
The lactose operon in E. coli
- The players
- Three structural genes - lacZ, lacY, lacA (produce the proteins which allow us to
utilize lactose)
Promoter: site to which RNA polymerase binds
Cis-acting operator site: controls transcription initiation
Trans-acting repressor: binds to the operator (encoded by lacl gene)
Inducer: prevents repressor from binding to operator
**lacl produces the repressor**repressor protein binds to the operator which prevents synthesis
of the structural genes, resulting in genes not being produced**
Repression of lac gene expression
- In the absence of lactose, repressor binds to the operator and prevents transcription of
lacZ, lacY, lacZ
**if there is no lactose present in the system, there is no point in producing the genes that break
down lactose**when there is no lactose, the repressor protein is bound to the operator which
does not allow the genes to be produced**
- Lac repressor is a negative regulatory element
Jacob and Monod defined the roles of the lac genes by genetic analysis of many lac-
mutants
- Complementation analysis identified three genes in a tightly linked cluster
- lacZ encodes beta-galactosidase (breaks down lactose into glucose and beta
galactose)
- lacY encodes permease (protein that is found on the cell wall within the
membrane, and allows import of the lactose)
- lacA encodes transacetylase
- Most studies focus on lacZ and lacY
- Constitutive expression of beta-galactosidase and permease was caused by mutations in
the lacl gene
Constitutive mutants (lacl-): express the enzymes in the absence and presence of inducer
**mutant results in the production of the lacA, lacZ, lacY genes; does not allow the repressor to
bind to the operator creating a constant production of the genes**
Lacl- mutants have a mutant repressor that cannot bind to operator
- In lack- mutants, lac genes are expressed in the absence and the presence of inducer
(constitutive expression)
**genes will be expressed at all times; repressor is mutated and it is not able to bind to the
operator**

The PaJoMo experiment provided evidence that lacl encodes

a repressor
- Laco+ lacZ+ DNA transferred into laci- lacZ- cells
- Beta-gal levels increased initially
 - Beta-gal levels decreased as repressor accumulated
- Beta-gal accumulation resumed after addition of inducer
- Jacob and Monod proposed that lacl encodes a repressor that binds to an operator site
near the lac promoter
**inducer will inhibit the repressor and production will continue to increase**
Lacls mutants have a super repressor that binds to operator but cannot bind to the inducer
- In lacls mutants, lac genes are repressed in the absence and the presence of inducer

**always produces the repressor and will also have an active repressor even in the case and
presence of the inducer**the genes will always be repressed because the inducer is not able to
bind to the repressor; resulting in a hyperactive repressor in the cell**
LacOc mutants have a mutant operator that cannot bind the repressor
- In lacOc mutants, lac genes are expressed in the absence and the presence of inducer
(constitutive expression)

**repressor is not able to bind to the operator because the operator has a different shape than the
repressor needs; resulting in the constant production of the genes**mutant operator that the
repressor cannot bind to**
Proteins act in trans, DNA sites act in cis
- Jacob and Monod used partial diploids carrying different alleles of lac regulatory
elements and structural genes to identify trans-acting and cis-acting elements
- F’ lac plasmids were used to generate partial diploids
Trans-acting elements: can diffuse through the cytoplasm and act at target DNA sites on any
DNA molecule in the cell
**elements that are produced somewhere else in the cell and then bind to the DNA → regulate
that region**
Cis-acting elements: can only influence expression of adjacent genes on the same DNa molecule
**proteins act in trans because they are produced in the cytoplasm and then move to the DNA
molecule, DNA sites (ie. operator, promoter) are cis acting elements; they only influence
expression of genes that are in close proximity to them**
Lacl+ protein acts in trans
- Repressor expressed from the plasmid can diffuse through the cytoplasm and bind to the
operator on the chromosome
Lac repressor has two separate domains
- Mutated sequences in many different lacl- mutants clustered in the DNA-binding domain
**production of lacZ genes**
- Mutated sequences in many different lacls mutants clustered in the inducer-binding
domain
**hyperactive repressor that always inhibits production of lac genes*
- X-ray crystallography revealed the two separate domains
**there are 2 main domains; the DNA binding domain which binds to the operon and the other
domain is the inducer binding domain (inducer binds to and inhibits the enzyme from binding to
the operon - result in change of conformation of the enzyme)**enzymes that can change their
conformation are allosteric enzymes; repressor an example of this**
Further studies revealed more about regulatory proteins and sites
- Biochemical evidence for lac repressor binding to lacO
- X ray crystallography revealed the structure of repressor proteins
- Lac repressor has a helix-turn-helix (HTH) motif
**site that is found within the DNA binding site**
- Evidence that specific amino acids in the alpha-helices of lac repressor are required for
binding to lacO
- DNA sequences to which negative and positive regulators bind have a two-fold rotational
symmetry
- Ie. CRP-binding site of the lac operon
- Most DNA binding regulatory proteins are oligomeric, with two to 4 subunits
**have multiple subunits which could be dimer or tetramer; the different subunits bind to
different regions of the DNA**
DNA recognition sequences by helix-turn-helix (HTH) motif
- A protein with an HTH motif has two alpha-helical regions separated by a turn in the
protein
- The HTH motif fits into the major groove of DNA (as a result, incase of the repressor,
will inhibit the expression of genes downstream of the region)
- One of the alpha-helices recognizes a specific DNA sequence
The lac repressor binds to operator DNA
The lac operon of E. coli is regulated by both lactose and glucose
- When both glucose and lactose are present, only glucose is utilized (lac operator must be
shut down)
- Lactose induces lac mRNA expression, but only in the absence of glucose
- Lactose prevents repressor from binding to lacO
- Lac repressor is a negative regulator of lac transcription
- Lac mRNA expression cannot be induced if glucose is present (because glucose controls
levels of cAMP which is an effector that binds to the cAMP receptor protein; positive
regulator)
- Glucose controls the levels of cAMP
- cAMP binds to cAMP receptor protein (CRP)
- CRP-cAMP is a positive regulator of lac transcription
Positive regulation by CRP-cAMP
Catabolite repression: overall effect of glucose is to prevent lac gene expression
**with the presence of glucose; it decreases availability of cAMP, cAMP is a positive regulator
of the whole process**when there is no cAMP, the inducer will bind and it will result in the
repressor not being able to bind to the operator**positive regulator reduces the amount of
cAMP**under normal condition when the operon is active, cAMP will bind to CRP which binds
to promoter and is a positive regulator of production of the lac genes**in the presence of
glucose, the amount of cAMP is lower resulting in the positive regulator not being able to work
efficiently resulting in reduction of gene expression**there are 2 regulators; presence of lactose
(inducer) and positive regulation**positive regulation is inhibited when glucose is present**
CRP-cAMP binds as a dimer to a regulatory region
- CRP-binding sites have two-folding rotational symmetry
- CRP protein binds as a dimer
- CRP-binding site consists of two recognition sequences, one for each subunit of
the CRP dimer
LacZ fusion used to perform genetic studies of the regulatory region of gene X
- Conditions that regulate expression of the test regions from gene X will alter the
levels of beta-galactosidase
- Specific regulatory sites can be identified by construction and testing mutations in
the test regions of gene X

Using lacZ to identify sets of genes regulated by the same stimulus

- Transposition of promoterless lacZ coding region → library of clones containing lacZ
insertions at random sites → screen library to identify all the genes that express lacZ in
response to a signal
Use of fusions to overproduce a gene product
- Expression of gene X under control of the lac regulatory system
- Expression of human growth hormone in E. coli controlled by lac
control region

Lecture #9: gene regulation (eukaryotes)

Overview of eukaryotic gene regulation
- Eukaryotes use complex sets of interactions
- Regulated interactions of large networks of genes
- Each gene has multiple points of regulation
- Themes of gene regulation in eukaryotes
 - Environmental adaptation in unicellular eukaryotes
- Maintenance of homeostasis in multicellular eukaryotes
- Genes are turned on or off in the right place and time
- Differentiation and precise positioning of tissues and organs during
embryonic development
Compared to prokaryotes, eukaryotes have additional levels of complexity for controlling
gene expression
- Eukaryotic genomes are larger than prokaryotic genomes
**larger set of games and chromosomes adds a level of complexity to gene expression**
- Chromatin structure eukaryotes makes DNA unavailable to transcription machinery
**not found in prokaryotes, this structure itself limits the availability of the DNA by
transcription machinery (ie. transcription factors or RNA polymerase)**
- Additional RNA processing events occur in eukaryotes (post transcriptional
modifications)
- In eukaryotes, transcription takes place in the nucleus and translation takes place in the
cytoplasm
Key regulatory differences between eukaryotes and prokaryotes

Multiple steps where production of the final gene product can be regulated in eukaryotes
Control of transcription initiation
- 3 types of RNA polymerases in eukaryotes
1) RNA polymerase I: transcribes rRNA genes (housekeeping genes - found in all of the
cells, essential to all types of cells regardless of their function)
2) RNA polymerase II: transcribe all protein coding genes (mRNAs) and micro-RNAs
3) RNA polymerase III: transcribes tRNA genes and some small regulatory RNAs
**based on which gene is being transcribed, different polymerases are needed**
RNA polymerase II transcription
- RNA polymerase II catalyzes synthesis of the primary transcript, which is
complementary to the template strand of the gene
- Most RNA polymerase II transcripts undergo further processing to generate mature
mRNA
- RNA splicing: removes introns
- Addition of 5’ GTP cap: protects RNA from degradation and also helps in the
process of translation
- Cleavage of 3’ end and addition of 3’ polyA tail
Cis-acting elements; promoters and enhancers
Promoters: usually directly adjacent to the gen, include transcription initiation site, often have
TATA box (found in promoter region, binding site for transcription factors that facilitate the
process of transcription), allow basal level of transcription (minimum transcription of a gene that
requires transcription factors and polymerase)
**site of binding of transcription factors and regulators**
Enhancers: can be far away from gene, augment or repress the basal level of transcription
**on the 3’ or 5’ end; can increase or decrease level of expression of the genes**enhancer
regions require other elements that facilitate regulation of gene expression**
Trans-acting factors interact with cis-acting elements to control trascription
initiation
- Direct effects of transcription factors; through binding to DNA
**directly binding to cis elements**
- Indirect effect of transcription factors; through protein-protein interactions
**interact with other elements that directly bind to the cis elements**
Basal transcription factors
**assist in binding rna poly to the promoter**
- Basal transcription factors assist in building of RNA polymerase II to promoters
- Key components of basal factor complex;
- TATA box binding protein (TBP)
- Bind to TATA box
- First of several proteins to assemble at promoter
- TBP associated factors (TAFs)
- Bind to TBP assembled at TATA box (recruits RNA polymerase to the
promoter region)
- RNA polymerase II associated with basal complex and initiates basal level of
transcription
Basal factors bind to promoters of all protein encoding genes
- Ordered pathway of assembly at promoter
1) TBP binds to TATA box
2) TAFS bind to TBP
3) RNA polymerase II binds to TAFs
**usually allows a low level of expression of the gene, needs
activators to enhance expression**
Activators are transcription factors that bind to enhancers
- Activators are responsible for much of the variation in levels of transcription of different
genes (proteins that increase levels of transcription; trans acting factors)
- Increase levels of transcription by interacting directly or indirectly with basal factors at
the promoter
- 3D complex of proteins and DNA
- Mechanisms of activator effect on transcription
- Stimulate recruitment of basal factors and RNA polymerase II to promoters
(enhance the recruitment and allow basal factors and RNA to be at the promoter
region)
- Stimulate activity of basal factors already assembled on promoters
 - Facilitate changes in chromatin structure (chromatin structure itself does not allow
accessibility to the promoter region, so the activator proteins change the
chromatin structure and allow access to the basal transcription factors to the
promoter regions within the chromatin structures)
Binding of activators to enhancers increases transcriptional levels
- Low level transcription occurs when only basal factors are bound to promoter
- When basal factors and activators are bound to DNA, rate of transcription increases
**no activators = low level of expression**basal factors/activators can interact which causes
activators proteins binding to enhance region**mediator connects activator proteins to basal
factors, allowing the enhance of transcription level**
Domains within activators
- Activator proteins have two functional domains
- Sequence specific DNA binding domain
- Bind to enhancer
- Transcription activator domain
- Interacts with other transcriptional regulatory proteins
- Some activators have a third domain (specific for environmental signals)
- Responds to environmental signals (ie. steroid hormone receptors)
**binding of signals changes structure of activators which allows them to bind to the
enhancers**
DNA binding domains of activator proteins
- Interact with major groove of DNA
- Specific amino acids have high affinity binding to specific nucleotide sequence
- The 3 best characterized motifs;
- Helix loop helix (HLH)
- Helix turn helix (HTH)
- Zinc finger
Steroid hormone receptors are activators only in the
presence of specific hormones
- Steroid hormones do not bind to DNA but are coactivators of steroid hormone receptors
- In the absence of hormone, these receptors cannot bind to DNA and so cannot
activate transcription (activator cannot bind to the enhancer - basal level of gene
expression)
- In the presence of hormone, these receptors bind to enhancers for specific genes
and activate their expression
**hormones cant bind to the DNA but they can change the structure of the receptors which
allows it to bind**
Repressor proteins suppress transcription initiation through different mechanisms
**decrease levels of transcription to lower than basal levels**
- Some repressors have no effect on basal transcription but suppress he action of activators
- Compete with activator for the same enhancer OR block access of activator to the
enhancer
- Some repressors eliminate virtually all basal transcription from a promoter
- Block RNA polymerase II access to promoter OR bind to sequences close to
promoter or distant from promoter
Repressor proteins that act through competition with an activator protein
- Repressor binds to the same enhancer sequence as the activator
- Has no effect on the basal transcription level
**repressor competes with activator; does not have effect on the basal transcription level because
basal proteins still have access to the promoter**repressor binds to enhancer and blocks binding
of the activator**

Repressor proteins that act through quenching an activator protein

Quenchers: bind to the activator but do not bind to DNA

- Type I: repressor blocks the DNA binding domain

- Type II: repressor blocks the activation domain;

Complex regulatory regions enable fine tuning of gene expression
- Each gene can have many regulatory proteins and regions that the proteins can bind to
- In humans, 2000 genes encode transcriptional regulatory proteins
 - Each regulatory protein can act on many genes
- Each regulatory region can have dozens of enhancers
Enhanceosome: multimeric complex of proteins and other small molecules that associated with
an enhancer
- Enhancers can be bound by activators and repressors with varying affinities
- Different sets of cofactors and corepressors compete for binding to activators and
repressors
**different activators have different affinity of binding to specific regions**
Chromatin structure and epigenetic effects
- Chromatin structure can affect transcription
- Nucleosomes can sequester promoters and make them inaccessible to RNA
polymerase and transcription factors (causes a change in nucleosome and
chromatin structure to allow access to those regions)
- Histone modification and DNA methylation
- Chromatin remodeling and hypercondensation (can allow more or less access to
certain regions)
Epigenetic changes: changes in chromatin structure that are inherited from one generation to the
next, DNA sequence is not altered (inheritance that is not related to changes of the DNA
sequence, just changes that affect the gene expression)
Chromatin reduces transcription

Effects of chromatin structure on transcription; histone modification and DNA methylation

- N terminal tails of histone H3 and H4 (tails extended outside the nucleosome structure)
can be modified
- Methylation (in C-G dinucleotides and is associated with gene silencing),
acetylation, phosphorylation and ubiquitination
- Can affect nucleosome interaction with other nucleosomes and with regulatory
proteins (can change the whole chromatin structure)
**acetylation = increased, methylation = decreased expression**
- Can affect higher order chromatin structure
- DNA methylation occurs at C5 of cytosine in a C-G dinucleotide
 - Associated with gene silencing
Chromatin remodeling (can change structure of nucleosome and chromatin) can expose the
promoter
- Nucleosomes can be repositioned or removed by chromatin remodeling complexes
- After remodeling, DNA at promoters and enhancers becomes more accessible to
transcription factors
- Can be assayed using DNase (cuts the DNA at different sites and by doing so, the
digested DNA can be analyzed) digestion (DNase hypersensitive sites)
**by doing this, they can allow more access to promoter region for transcription factors**

The SWI-SNF remodeling complex

- One of the best studied remodeling complexes
- Uses energy from ATP hydrolysis to alter nucleosomes positioning

**stabilizes the structure of the chromatin and allows TFs to get to the promoter**increased
levels of transcription**similar to activators**requires energy to carry out their function**
Regulation after transcription
- Posttranscriptional regulation can occur at any step
- At the level of RNA
- Splicing, stability and localization
- Ie. alternative splicing of mRNA; generates more diversity of proteins,
common feature in eukaryotes
- At the level of protein
- Synthesis, stability and localization
Some small RNAs are responsible for RNA interference (RNAi)
- Specialized RNAs that prevent expression of specific genes through complementary base
pairing
- Small (21-30 nt) RNAs
- micro-RNAs (miRNAs) and small interfering RNAs (siRNAs)
- First miRNAs (lin-4 and let-7) identified in C elegans
 - Nobel prize to A. Fire and C. Mello in 2006
- Posttranscriptional mechanisms for gene regulation
- mRNA stability and translation
- May also affect chromatin structure
Two ways that miRNAs can down regulate expression of target genes
- When complementarity is perfect; target mRNA is degraded
**miRNA binds to complementary region on the mRNA, recruiting a set of proteins that are
‘risc’ which then degrade the RNA molecules into smaller pieces getting rid of its expression**
- When complementarity is imperfect; translation of mRNA target is repressed
**’risc’ complex binds and inhibits RNA which represses RNA expression**

Lecture #10: the genetics of Cancer

The relative percentages of new cancers in Canada that occur at different sites

Two unifying themes about cancer genetics

1) Cancer is a disease of genes
a) Multiple cancer phenotypes arise from mutations in genes that regulate cell
growth and division
b) Environmental chemicals increase mutation rates and increase chances of cancer
**disease of cell proliferation; cells defy communications with other cells and grow
uncontrollably**
2) Cancer has a different inheritance pattern than other genetic disorders
a) Inherited mutations can predispose to cancer but the mutations causing cancer
occur in somatic cells
b) Mutations accumulate in clonal descendants of a single cell (one cell that acquires
one mutation, and that mutation continues from that single cell as the cell divides
= creating a cancerous environment)
**can be inherited from germ line cells, but the cells that are actually the cancer cells are somatic
cells**environment plays a higher role in cancer than genetics and inheritance**
Evading cell control regulations
- Signals play a key role in regulation of cell division
- Cell division required growth factors
Contact inhibition: cells stop dividing when get in contact with other cells (there is no room for
more division, cancer cells defy this inhibition; when they contact other cells, they continue to
divide and grow)
Programmed cell death (PCD): cells that are not required, starved of growth factors or damaged,
trigger signalling pathways that result in cell death (cancer cells evade programmed cell death,
allowing them to continue division and grow much more than other cells)
Phenotypic changes that produce uncontrolled cell growth
Autocrine stimulation; cancer cells can make their own stimulatory signals (once they produce
the signals, it binds to the receptors on the cell which can allow the cell to grow or divide)
Loss of contact inhibition: growth of cancer cells doesn't stop when the cells contact each other
(regular cells form a monolayer and then stop dividing, in cancer cells; they do not have contact
inhibition so they don’t grow in a monolayer, they can create a large stack or clump - tumours)
Loss of cell death: cancer cells are more resistant to programmed cell death (apoptosis)
Loss of gap junctions: cancer cells lost channels for communicating with adjacent cells (cancer
cells don’t have gap junctions, which allows them to evade signals from surrounding cells)
**cancer cells do not need signals in order to grow and divide**allows them to grow in
abnormal places, and have high tolerance for damaging things, allows them to defy cell growth
that is found in normal cells**
Immortal cancer cell
- Telomerase expression attributes to cancer immortality
- Telomerases maintains length of telomeres at the end of chromosomes after DNA
replication
- Normal cell will lose dna regions causing lethality
**cancer cells have high expression of telomeres which allows them to go through cell division
for longer**
Phenotypic changes that produce genomic and karyotypic instability
- Defects in DNA replication machinery;
- Cancer cells have lost the ability to replicate their DNA accurately
- Increased mutation rates can occur because of defects in DNA replication
machinery
**genomic instability; mutations that take place on the genome, one mutation causes more
mutations as replication continues**after replication there might be mismatches or mutations
which are not repaired**if there is one mismatch in the first replication, as replication continues
the mutation rate increases**higher mutation rate = more unstable genome**
- Increased rate of chromosomal aberrations
 - Cancer cells often have chromosome rearrangements (translocations, deletions,
aneuploidy etc.)
- Some rearrangements appear regularly in specific tumour types
**as a result; there is a very different chromosomal arrangement in the karyotype**there can be
too many chromosomes of the same number, or not enough, they can be the wrong length**
Phenotypic changes that enable a tumour to disrupt local tissue and invade distant tissues
Ability to metastasize: tumour cells can invade the surrounding tissue and travel through the
bloodstream (for malignant tumours)
Angiogenesis: tumour cells can secrete substances that promote growth of blood vessels (no
blood vessels in one area of the tumour, they can promote the formation of blood vessels which
allows them to have a source of nutrients - they create their own microenvironment within the
body)
Cancer is thought to arise by successive mutations in a clone of proliferating cells

**all cancer cells arise from a single cell**genomic instability is what makes the mutation rate
increase throughout cell division**
Overview of the initiation of cell division
- Two basic type of signals that tell cells whether to divide, metabolize or die
Extracellular signals: act over long or short distances, collectively known as hormones (steroids,
peptides or proteins)
**could be from the environment, from the specific cell that is dividing, or signals from cells
surrounding**mitogens are important in growth as well as cell division**
Cell bound signals: require direct contact between cells
Cancer phenotypes result from the accumulation of mutations
- Mutations are in genes controlling proliferation as well as other processes (ie. integrity of
the genome)
- Result in a clone of cells that overgrows normal cells
- Cancer phenotypes include; uncontrolled cell growth (disregard environmental factors),
genomic and karyotypic instability (increased mutation rates), potential for immortality
(increased telomeres), ability to invade and disrupt local and distant tissues
Mutations in genes
Oncogene: gain of function mutations; mutations which result in hyperactivity of a protein.
Mutant alleles that have dominant effects. WT gene is proto-oncogene
**gain of function mutations, dominant alleles to the wild type**one copy of the gene is enough
to develop tumour cells because it is dominant**
Tumour suppressor: loss of function mutation causes inactivity of genes. Mutant alleles that have
recessive effect
**mutant alleles that have recessive effects**we need both copies of the gene to be mutated
because of the recessive nature**

The RAS oncogene is the mutant form of the RAS proto-oncogene

- Normal RAS is inactive until it becomes activated by binding of growth factors their
receptors
- Oncogenic forms of RAS are constitutively activated
**mutant of the proto oncogene RAS**normally ras gets signals from other cells and once the
RAS gets the signal, the GDP turned into GTP and RAS is activated and signals cell
proliferation**in cancerous cells; when RAS oncogene, it is constantly attached to GTP meaning
it does not require any signals from the environment or other cells for RAS to get GTP and be
active. It is always found in the active state, meaning it always signals for cell proliferation. The
genes become hyperactive**
Cancer can be caused by mutations that improperly inactivate tumour suppressor genes
- Function of normal allele of tumour suppressor genes is to control cell proliferation
- Mutant tumour suppressor alleles act recessively and cause increased cell proliferation
(results in increased cell growth)
- Tumour suppressor genes identified through genetic analysis of families with inherited
predisposition to cancer
- Inheritance of a mutant tumour suppressor allele
- One normal allele sufficient for normal cell proliferation in heterozygotes
- Wild type allele in somatic cells of heterozygote can be lost or mutated →
abnormal cell proliferation
**if there is one normal allele of the suppressor genes, that is enough for the cell to behave
normally**if there is a wildtype, it can be mutated over time and the tumour suppressor can be
mutated and a tumour can be created**
Some families have a genetic predisposition to certain types of cancer
- Ie. retinoblastoma caused by mutations in RB gene
- Individuals who inherit one copy of the RB- allele are prone to cancer of the retina
- During proliferation of retinal cells, the RB+ allele is lost or mutated
- Tumour develop as a clone of RB-/RB- cells
**mutations in both copies result in tumour of the retina**allele that is wildtype has a high
chance of being mutated; which makes it develop cancer cells**
Events causing loss of heterozygosity in somatic cells or RB+/RB- individuals

**loss of function mutation on the wildtype gene is due to mutations which makes the cells
develop cancer cells**
Cancer treatment
 - Chemotherapy and radiation therapy (most commonly used cancer treatments, not
targeted therapy; it impacts all of the cells in the body but only kills the cancer cells and
the regular cells stay alive mostly)
- Targeted therapies
- Inhibitory drugs
- Antibodies (target specific oncogenes on the cells)
- Immunotherapy (boost the immune system to identify cancer cells)
- Bone marrow transplant
Inhibitory drugs
- Targets specific proteins or enzymes inhibiting their activities
- Gleevec inhibits activity of tyrosine kinase Bcr/c-Abl
**if we can identify a protein that is involved in the development of cancer, we can make drugs
that bind to the protein and inhibit its function**
Antibodies
- Monoclonal antibodies recognize targets on the cancer cell surface
- Herceptin binds to Her2 (protein that is highly expressed in 20% of breast cancer cells, it
is one of the markers for breast cancer)
**in breast cancer, there is overproduction of Her2 receptors; they bind to each other and form a
dimer which allows them to signal for proliferation**
- Inhibits Her2 signaling cascade
- Recruits immune cells to cancer cells
**antibody binds to region on Her2 which inhibits it from forming the dimer and shutting off the
pathway that leads to cell proliferation**antibodies recruit immune system to the cancer
microenvironment; cancer cells evade immune systems by telling immune cells that they are all
normal cells - they mask the disease in doing so**when antibodies bind to the markers, they
bring immune cells to the cancer microenvironment**
Emerging strategies
- Oncolytic viral therapies
Difference between a pathogenic virus and an oncolytic virus
- Interferon sensitivity
- Use vaccine strain or attenuated viral strains
**use viruses that only grow in cancer cells; takes advantage of cancer cells being
immunocompromised**they do not grow in regular cells because they have immune response**
Advantages
- Specific to cancer cells
- Boost the immune response
- Combination therapy
Oncolytic viruses
- Approved oncolytic virus
- T-vec for melanoma (the only FDA approved)
 - Other viruses
- Vaccinia used to eradicate smallpox (clinical trial)
- Measles
- Maraba (clinical trial)
- Vesicular stomatitis virus (VSV)
Utilization of lactose by E coli provides a model system of gene regulation
- Lactose utilization requires two enzymes
- Permease: transports glucose into cell
- Beta-Galactosidase: splits lactose into glucose and galactose
- In the absence of lactose, both enzymes are present at very low levels
- Lactose is the inducer of the genes encoding permease and beta-gal
- Induction: stimulation of synthesis of a specific protein
- Inducer: molecule responsible for induction
The lactose operon in E coli
- 3 structural genes; lacZ, lacY, lacA
Promoter: site to which RNA polymerase binds
Cis-acting operator site: controls transcription initiation
Trans-acting repressor: binds to the operator (encoded by lacl gene)
Inducer: prevents repressor from binding to operator

Lac operon
- Encodes for proteins that digest disaccharide lactose
- In the absence of glucose, cAMP is produced and CAp is activated
- In the absence of lactose, lac repressor switches the operon off
Repression of lac gene expression
- In the absence of lactose, repressor binds to the operator and prevents transcription
- Lac repressor is a negative regulatory element
- lacZ encodes beta-galactosidase
- lacY encodes permease
- lacA encodes transacetylase
Lacl- mutants have a mutant repressor that cannot bind to operator
 - In lack- mutants, lac genes are expressed in the absence and the presence of inducer
(constitutive expression)
Lacls mutants have a super repressor that binds to
operator but cannot bind to the inducer
- In lacls mutant, lac genes are repressed in the
absence and the presence of inducer
Lacl+ protein acts in trans
- Lacl- → repressor cannot bind, enzyme produced
- Lacls → inducer cannot bind, enzymes not
produced
- Oc → repressor cannot bind, enzymes produced
- Repressor expressed from the plasmid can diffuse through the cytoplasm and bind to the
operator on the chromosome
Lacls; dominant in lacl+ in trans
Oc and O+; action in cis
Lac repressor has 2 separate domains
- Mutated sequences in many different lacl- mutants clustered in the DNA binding domain
- Mutated sequences in many different lacls mutants clustered in the inducer binding
domain
- X-ray crystallography revealed the two separate domains
Positive regulation by CRP-cAMP
Catabolite repression: overall effect of glucose is to prevent lac gene expression

CRP-cAMP binds as a dimer to a regulatory region

- CRP-binding sites have a 2 fold rotational symmetry;

- CRP protein binds as a dimer
- CRP-binding site consists of two recognition sequences, one for each subunit of
the CRP dimer