From Last Lecture

 Both genotype and environment determine phenotype.

 The methylation state of DNA affects gene expression. Gene expression (transcription-RNA
polymerase) is also affected by modifications to histones.
 Lifestyle (smoking, exercise, sleep habits) and diet have significant effects on the
epigenetic status of an individual.
 Epi-alleles are particularly sensitive to environmental input.
 In fact, the Avy phenotype is often used as a methylation reporter - Bio-sensor.
 Depending on the methylation state of the mice they could have different colors of coats and it
also affects obesity
 Yellow mice: hypomethylated
 High levels of methylation shut off the normal sequence
 These mice are used as biosensors, look at the effect of drugs on phenotype
 Whether on not a particular food suppresses methylation or supports normal levels of
methylation
 Epigenetic changes can affect both the penetrance and expressivity of phenotypic
traits.
 penetrance is expressed as the percentage of individuals of a specified genotype that show the
trait.
 Anything less than 100% is incomplete penetrance
 Forward genetics can be used to better understand gene function and
developmental/metabolic pathways.
 Forward genetics is system centric – what genes are involved in my process of
interest?
 Trait specific screen… mutagenizing population and screening for individuals
that have defects or modifications in that pathway
 reverse genetics is gene centric – I have a gene; what does it do?
 You have DNA sequence and you want to know what that DNA sequence does
 Following a forward genetic screen complementation testing can be used to
identify allelic (same gene) and non-allelic (different genes) mutations.
 Individuals with similar phenotypes are crossed & the F1 individuals
examined
 Mutations should be recessive to wild-type and ideally null alleles
 Double mutants can be made to assess interactions between genes that
participate in similar processes.
 looking for epistatic (more on this today) additive, or synthetic phenotypes – this is
assessed in the F2 population where 1/16 of individuals (from dihybrid crosses) will
be double mutants.
 What is the phenotype of that 1/16 individual?
 Epistasis has implications for evolution- selection of can't act upon hidden traits. And is
crucial for understanding complex diseases (diabetes, autism, schizophrenia, Alzheimer's)
 Many diseases have incomplete penetrance bc biological systems are inherently noisy and
there are other genes that affect the likelihood that affect whether that disease will
manifest (epistatic)

Deviations from a 9:3:3:1 ratio in the F2- an example of a synthetic phenotype

 Two different genes, two different loci
 F1 individuals: wt (complementation, eyes gone and eyeless are two different genes)
  Would expect 9:3:3:1 ratio of different genotypes
 9:6:1 ratio of different phenotypes (9 wt, 6 with no eyes, and 1-dead)
 Synthetic lethality: Synthetic interaction where the phenotype of the double mutant
doesn't look like the phenotype of the single mutants
 Suggests that both of these genes play a role in supporting an essential pathway. On
their own, neither is essential but when you lose both you lose this essential
pathway needed for the life of the fly

What is the phenotype of the double mutant

 Is it a new phenotype- dead?
 Identifies a new function
 Is it an additive phenotype? (branching)
 Genes aren't in a linear dependent pathway
 They're likely converging on a particular phenotype
 Is it like on of the single mutants?
 Epistasis, suggests that genes are functioning together in the same pathway
 Epistasis testing: homozygosity for null alleles, all of the assumptions relies on the fact
that you have null alleles

9:7 Ratio: Complementary Gene Action

 Bateson & Punnett epistasis: genotype at one locus masks the phenotype at another locus
 Bateson & Punnett were looking at epistasis and defining it as condition as double
mutants looking like one of the single mutants
 Don't rely on the ratios because they can mislead you… Understand the biology and see
what the different phenotypes tell you about the biology
 Fisher: anything from the deviation from the additive phenotypes
 Would also consider synthetic lethality epistasis (non classical definition)

Coat color in Labrador Retrievers

 Recessive epistasis
 3 color morphs: black, yellow, chocolate
 Black: at least one dominant gene (B gene)
 Chocolate: loss of function alleles of the B gene- bb
 Yellow: nose is chocolate color, homozygous for the loss of function alleles of the e
locus and the b locus (eebb)
 Yellow: nose is black,( eeB_)
 Coat color and the skin color are determined by the B locus, E locus only reflects the
coat color
 Coat color is achieved by the action of at least 2 genes
 Receptor sits at the plasma membrane, melanocortin receptor coded by the E gene and
binds different ligands such as agouti ligand.
 When agouti ligand binds, it shuts down that receptor and you will get yellow
 When melanocortin stimulating hormone binds, it activates the pathway and you
have activation of transcription that leads to the production of tyrosinase related
peptide 1 gene which is encoded by the B locus
 B locus is responsible for converting the chocolate colored compound into the black
colored compound
  Mutations in the B gene knock out the tyrosinase function so you only get brown
eumelanin form
 Black labs have a mutant form of the melanocortin 1 receptor, a neomorphic mutation so
the receptor is always on. It doesn't respond to agouti or the melanocortin 1 stimulating
hormone.
 If at least one wt copy on B locus, will make black eumelanin
 Yellow labs: have a lof at the MCR1 so the receptor will always be in off position, can
never activate TYRP1 expression, can never get the black eumelanin
 Golden phenotype is epistatic to the black or chocolate phenotype. It masks the
phenotype at the second locus and acts upstream of the two genes in the pathway. E
locus is upstream of the pathway.
 There are dogs that have wt alleles of the melanocortin 1 receptor. This receptor can be
turned on and off by the MSH or by agouti.
 Ex: dopamine fincher, black eumelanin & yellow/red regions where agouti is active
or the MSH is off.
 Has wt alleles of the e clous and B locus and can be turned on and off during the
development of the dog.
 e is epistatic to b, e is upstream and masks the b phenotype
 Indicative of epistasis when one crosses a BbEe dog to a BbEe dog you get 9:4:3 ratio of
black: yellow: brown coat color (yellow is epistatic to brown)
 Suggests recessive epistasis

MC1R mutations are pleiotropic

 A lot of different phenotypes in humans
 Pleiotropic mutation: affects a lot of different phenotypes, eg affects both hair color in
humans
 Red hair: Changes in sensitivity to anesthesia and pain killers and opioids
Additional examples of recessive epistasis
 A group: homozygous for codominant A allele or heterozygous, make A antigen and
antibodies against the B antigen
 B group: B antigen, anti A antibodies
 AB group: A and B antigens
 O group: loss of function alleles, no antigens, anti A and Anti B
 Bombay phenotype: ) blood type child born to parents who don't have the O allele if a
recessive form of the allele for the H antigen also is inherited from both parents
 AB phenotype is affected by two different loci, AB antigens that are produced by
modifying immunoglobulin that's on the surface and the immunoglobulin that is
modified is produced by a locus called H
 H antigen
 Homozygous for loss of function alleles for the H locus
 H locus --> immunoglobulins, modified by the A and B alleles at the I locus
 Need at least one functional copy of the allele at the H locus in order to make the A
and the B antigens
 H antigen is a precursor to the A and B antigens
 The A and B allele must be present to produce the A and B enzyme that modifies the
H antigen to become the A or B antigen
  If only recessive alleles for H antigen are inherited, the H antigen will not be
produced so A and B antigens will also not be produced and it will look like type O
blood.
 If homozygous for the loss of function mutations at h, it's masking the phenotypes
at the I locus (Epistasis)

Epistasis in adrenaline biosynthesis

 Double mutant for dopa decarboxylase and hydroxylase --> build up of dopa
 Will look identical to the single mutant for dopa ecarboxylase
 In a biosynthetic pathway, the double mutant will always reflect the upstream mutant
Epistasis in a phage morphogenesis pathway
 Mutation in gene 2 is epistatic to mutation in gene 3, tells us that gene 2 is upstream of
gene 3 assuming that it's a linear pathway

Using epistasis testing to determine order of genes in a pathway

 Mutaganized pea plants in an F1 screen and isolated several different phenotypic classes
 All these different true breeding lines represent mutations in different genes and all of
these are null alleles
 Make double mutant b/w all of the individuals and score their phenotype. What is the
phenotype of the double mutant in the F2 generation.
 Table shows phenotype of the epistatic class
 Pink is epistatic to blue, white is epistatic to pink
  Yellow is epistatic to pink
 Looking at the deviation from the ratio, get 9:4:3 and whatever that 4 class is, your
double mutant is there
 2 pink, 1 red, 0 blue, 4 white, 3 yellow
 White --> yellow --> pink --> red --> blue --> violet
 Function of pink gene is to produce red pigment, function of red is to produce blue
etc
Tangerines
 Double mutant should be yellow but they got tangerine bc they weren't working with
completely nulls
 There's also feedback, compounds upstream for the gene responsible for the tangerine
phenotype upregulate PSY1 allele
 Bc the PSY1 allele wasn't a true null, when they lose CRTISO the other compounds were
enough to upregulate this to get tangerine color

Additive effects of pym and rfi mutations on trichome nuclear DNA content
 Other deviations from Mendelian ratios give us information
 Increase in trichome branching and endoreduplication
 These two genes act in a switch pathway bc they have both positive and negative
indicators
 They act in a linear pathway where one is upstream of the other… if this was the case, the
double mutant would be like one of the single mutants
 If it's the other pathway, additive phenotype
 9:6:1, the genes aren't functioning in a linear pathway, instead acting to converge
together on an end phenotype so that the phenotype we're seeing is additive

Mutations in a gene called C.. When you have loss of function mutations in C you get white
phenotype
When you have loss of function in P you get a white phenotype
The phenotype of the double mutant is white

Duplicate gene action

 Ex for rfi and polychome is not the same as duplicate gene action
 Gene A and Gene B are functionally redundant so if you have A or B you're wt
 You have to lose both A and B to have a phenotype (15:1 ratio)
 Both genes individually are sufficient for gene activity
 Neither rfi or polychome alone is sufficient enough to block endoreduplication

Go over 42 & 43 slides

Review the rest of the slides on eye color pathways in drosophila