BACKGROUND/INTRO (important concepts you should know):
Mendel’s 5 rules (two ofwhich are referred to as “laws”). Which “law” is routinely
broken and why?
1 law: law of segregation: during sex repro in hybrids with opposing traits,
half of the gametes receive one factor (A) and half receive the other (a)
2nd law: law of independent assortment: factors associated with different
traits segregate independently
During asexual reproduction, factors do not segregate
Inheritance of factors is bi-parental
Fertilization is random with respect to which factor is carried by a gamete
The most frequently encountered exception, violating the second law of
independent assortment is linkage because linkage causes some factors to segregate
more with each other than others. Trisomy and things violate the first law of
Meiosis – stages of first meiotic prophase / timing and primary function of meiotic
Stages of First Meiotic Prophase:
1. Leptotene – thin threads
2. Zygotene – homologue pairing first evident
3. Pachytene – meiotic recombination occurs & synaptonemal complex may be
4. Diplotene – double thread – chiasmata visible
5. Diakinesis – continued chromatin condensation
Bridges’ proof that genes are on chromosomes (chromosomal theoryof
There was already an indication that genes paralleled chromosomes in some
To prove this, had to show that if there was a change in how chromosomes
behaved, there is a corresponding change in inheritance patterns
Bridges found that when chromosome behavior was changed through XX + Y
or X + XY pairing, inheritance patterns paralleled in X-lined mutants. The three point test cross – linked genes (a b c/ +++ x a b c/a b c) . Know how
to correctly calculate map distances and quickly establish gene order.
(Single recombinants + 2x double recombinants)/ total
The Chi square ( test and the limitations of the Chi square, ie. when/how can we
Must be used on raw numerical data, not percentages
Cannot be used for experiments where the expected frequency of any
phenotypic class is < 5
X^2 assumes that all progeny are equally viable
Deviance is due to chance (n2ll hypothesis)
SUM((observed – expected) /expected)
Relationship between physical genetic maps and recombination genetic maps –
what does this tell us about recombination rates along the length of a chromosome?
Physical maps come from polytene chromosomes (arrive through a modified
cell cycle) that form light and dark bands. These bands may reflect genes
very low recombination at the centromere and telomere locations
(suppressed), highly repetitive heterochromatin
What are the general characteristics of heterochromatin compared to euchromatin?
Constitutive Heterochromatin: highly repetitive, inactive, recombination
reduced, gene density is low
Facultative Heterochromatin: only in certain situations and perhaps
places in the body does it become inactive
Euchromatin: active DNA
BACKGROUND FOR GENETICS IN MODEL GENETIC ORGANISMS:
What general attributes (attribute = quality, property, or characteristic) make an
organism highly suitable for genetic analysis in the lab?
Easy & inexpensive to keep in the lab (small)
Short generation time
Large number of progeny
Easy to control mating
Diploid – rather than haploid
Small number of chromosomes (linkage groups) Balancer chromosomes – for what are these used and what characteristics make a
chromosome a “balancer chromosome”?
Balanced stock: genotype of the progeny must be the same as that of the
If you have a lethal mutation that survives in hetero but lethal in homo, to
create a balanced stock, you need to get rid of the homo wild-type condition
to leave all hetero mutations.
Balancer Chromosomes: useful for maintaining any mutation which is
lethal or sterile when homozygous in the heterozygous condition
o Carry multiple inversions to prevent crossovers
o Carry a visible dominant marker
Mutation / classes of mutation and genetic tests that help determine class (null,
hypomorph, hypermorph, antimorph, neomorph)
Mutation: a mutation is a stable and heritable change in DNA sequence
Recessive mutation: m/+ appears wildtype and m/m displays phenotype
Dominant mutation: M/+ shows phenotype, mutant is dominant
Nullomorphic: a mutant with no remaining gene function
o Phenotype: null = deletion
o Test: protein nulls fail to produce protein product from antibody
o Classify: Df/m nul= m nu/m null
Hypomorphic: lower or residual activity compared to wildtype
o Test: Df/m hypoor m /ml hypo> m hyp/m hypo
Hypermorphic: hyperactive protein or too much protein produced
o Test: m hyp/m hype> m/+ > m hyp/Df
Antimorphic: produces protein that acts as a poison to the wildtype protein
anti anti anti
o Test: M / M >= M /Df
o Should be able to reduce the effect of an antimorph by increasing
Neomorphic: causes a gene to inappropriatelyactivate either in the
incorrect place or at the incorrect time
o Test: M ne/+ = M neo/+/+ etc…
o You cannot reduce the effect of a neomorph by increasing the
In what different ways can mutations be associated with dominant phenotypes?
Haplo-insufficiency: single copy of wild-type gene is insufficient
Product of mutant gene is poisonous
Mutation causes gene to be misexpressed (at times or places that are weird)
Overexpression or over-activity In what different ways can mutations be induced or recovered in model genetic
organisms in the laboratory setting?
Irradiation: x-rays or gamma rays
o Free radicals, DNA strand breaks
o Best for producing gross chromosomal rearrangements
Chemical: alkylating agents and crosslinking agents cause transitions and
Transposable genetic elements: preferred method. Molecular tag, known
o can disrupt coding sequence, regulatory sequence,
Targeted gene disruption (homologous recombination)
How has the Drosophila P element been engineered so as to be a useful method for
1) Control transposition (jumping) via genetic crosses:
o P element mediated germline transformation: Using P elements
that have inverted terminal repeats and gene of interest and marker
(transformation plasmid) alongside P elements that have transposase
functionality (delta-2-3 helper plasmid)
Defective elements can only be mobilized when crosses to a strain that
produces transposase (delta-2-3)
What is “plasmid rescue”? What is imprecise excision?
Plasmid rescue: method of inserting a transposon
o 1) P element engineered to carry ORI and selectable marker
o 2) engineer so the transposon is cut once within and one out
o 3) re-ligate into plasmid and then replicate in E.coli
o The P element may transpose imprecisely and remove some flanking
DNA in the process creating a small Df Details of Drosophila as a model genetic organism
(genome size, chromosome number, number ofgenes, generation time, care, setting
up crosses, keeping balanced stocks, sex determination, polytene chromosomes…)
Crosses: requires collection of virgin females
Generation time: 9 days (@25 degrees)
Lifespan: 45 – 60 days
No. progeny: over 100 per single female
Genome Size: 120 Mb euchromatic, 60 Mb heterochromatic
Genes: 13,601 genes
Chromosome number: 2n = 8 (X;2;3;4 + Y)
Sex Determination: X chromosome to autosome ratio ( 1 = female, 0.5 =
Polytene Chromosomes: easy mapping of rearrangements, transposon
*There is no recombination during meiosis in male drosophila
P element based techniques and methods
(enhancer traps, GAL4/UAS, mosaics using FLP/FRT, PTT exon traps, GAL4 +
GAL80 for marking mosaic clonal patches)
Enhancer Trapping: p element transposon can be used with delta-2-3 to
insert in between an enhancer and coding region so that the enhancer
expresses your gene of interest. You can look for tissue specificity
GAL4/UAS: by crossing GAL4 with UAS-goi, theGAL4 protein produced
induces UAS to express your goi. EP element is a variation of this
Somatic mosaics: useful in bypassing lethality in early development and
examine patches of mutant tissue later. Can also give you progenitor counts
(can tell you how many cells were destined to form the tissue of interest at
the time of somatic recombination)
FLP/FRT: FLP recombinase mediates site specific recombination between
FRT sequences. You can incorporate these FRT sequences using P elements.
GAL80 blocks GAL4 protein production (creates mosaics since some are
You can insert GFP by using Transposon-PTT splicing sequences-GFP and
inserting into genome in between introns randomly. The PTT and transposon
and normal introns are spliced out leaving a chimeric protein
see also the tutorial as it represents an application of much of the above MGO: C. ELEGANS
Details of C. elegans as a model genetic organism
(genome size, chromosome number, number ofgenes, generation time, food source,
,types of markers, setting up crosses, keeping balanced stocks, sex determination…)
Crosses: population is 99% “selfing” hermaphrodites
Generation time: 3 days
Lifespan: 2-3 weeks
No. progeny: 250 – 1000 per hermaphrodite
Balanced Stocks: balancer translocations
Genome Size: 97Mb
Genes: 19,099 genes
**Genome is smaller than drosophila but moregenes
Chromosome number: 2n =12 (1,2,3,4,5 + X)
Sex Determination: X chromosome to autosome ratio ( XX = hermaphrodite,
XO = males)
**A third of everything drosophila but genes 20,000 and chrome num =
C elegans has holocentric chromosomes - what are holocentric chromosomes? What
problem do holocentric chromosomes present during meiosis? How is this solved in
C. elegans and what consequence does this have on the C. elegans recombination
Holocentric chromosomes: differ in that their shape when being pulled to
the centrioles. Microtubules can attach to anywhere on holocentric
chromosomes. Appear straight and parallel to metaphase plate. No defined
During meiosis, holocentric chromosomes are disastrous because the
centrioles can tear the chromosomes. In C. elegans, they create localized
microtubule attachment sites during meiosis through ONE crossover. This
crossover allows the chromosomes to twist and align in a certain way to
allow pulling at the tips.
Part of the solution is that there is only one crossover per chromosome
towards one end! This explains the clustering of genes in the central
region of C. elegans recomb maps
In general, what strategy is used to clone a gene starting from a mutant and mutant
phenotype in C. elegans?
Cosmid injection into synsitial area is the common method of gene cloning
How is mosaic analysis achieved in C. elegans?
Using extrachromosomal arrays (cloned DNA with wild-type and marker) in
the worm injected in synsitial area of worm Homologous chromosomes have mutated gene
Extrachromosomal array anneals itself to the mutated gene to some of them
and not others? creates a mosaic
If you know the lineage, and see where GFP is functioning from the marker,
you can further refine the pattern of expression
You know that wherever green shows, the gene is required for whatever
In C. elegans screening for recessive mutations can be done in the F2 generation.
Why is this not possible in Drosophila?
Works in worms because of hermaphroditism.
Herm can be exposed to mutagen, progeny can herm themselves
Don’t need to worry about original males contribution
In drosophila, you need a third generation to mate brothers and sisters from
What strategy was developed to screen for maternal effect lethal mutations in C.
You can screen for maternal effect lethal mutations using the bagofworms
The rare plates will have 1/4 dead progeny that will not break out of mother.
The others will break out
MGO3 : YEAST
Details of yeast as a model genetic organism (chromosome number, size of genome,
number of genes, types of markers, mating system, recombination frequency…)
Generation Time: 90 min
Lifespan: mother produces 20 – 30 buds
No. progeny: unlimited
Crosses: haploid/diploid life cycle. Haploids can be crossed
o Diploid yeast will sporulate only when deprived of nitrogen
Chromosome Number: 16 linear chromosomes
Number of Genes: 6607 (only3.8% have introns)
Size of Genome: 12 Mb
Laboratory strains of yeast must be heterothallic – what does this mean?
Diploid yeast are always heterozygous at the mating-type locus (MATalpha
and MATa) – like male and female (MAT a produces a pheromone and
receptors for alpha pheromone and vice versa) Heterothallic strains of yeast are stable and do not switch their MAT (do not
randomly mate) Heterothallic strains do not have the HO gene.
Homothallic strains can switch mating types (activate their silent mating
cassettes initiated by the HO (homothallism gene)) Homothallic strains carry
at least one copy of this HO gene and grow as diploids instead of haploids and
What are the different type of shuttle vectors that are commonlyused in yeast
Ylp (integrative plasmids):
o cannot replicate autonomously
o One copy per cell
o *needs bacterial origin of rep
YEp (episomal plasmids):
o Replicate autonomously
o Many copies per cell
o *has own origin of rep
YCp (centromeric plasmids)
o Replicate autonomously
o One copy per cell
o segregates in meiosis like a chromosome
o *has yeast origin of rep
o *has yeast chromosomal centromere!
All derived from pBR322 and can grow in E. coli.
What is “cloning by complementation” in yeast?
Using some selectable marker on a plate (like the requirement of uracil)
Plate random YCp vectors with yfg and ura
Select colonies that can grow under restrictive conditions
The plasmid selected will have yfg because the only way the selectable
marker is maintained is if the plasmid is repaired with yfg mutant that was
Outline the strategy used to replace a yeast wild-type gene with a mutant gene (the
two step gene replacement).
Integrate a Ylp plasmid with URA3 with mutant with the wildtype by one
The plasmid will be lost because no ORI and undergo recombination where
some will lose the URA3.
Single copy will be remaining, either wild type or mutant
FOA medium negative select for cells expressing URA3
The yeast two hybrid assay: what is this used for and how does it work?
If your gene of interest is working together in some complex and you want to
know what theother players are in that complex Based on the functional reconstitution of an intact transcription factor that
activates reporter gene expression
The “prey” bait protein has an activation domain and when fused with the
DNA binding protein, will cause expression of reporter gene.
The only way the prey can fused with the dna binding protein is if there is a
relationship and physical interaction between the gene of interest with its
other players (one player is attached to the activation domain, another is
attached to the dna binding domain)
This allows you to build protein-protein interaction maps
What is the purpose of the sectoring yeast lethality assay? How does this work?
What type of shuttle vector (YIp, YEp, YCp) does this assay require?
Requires a YCp shuttle vector to multiply onlyonce
YCp plasmid carries the gene mutant of interest and ADE2+
The areas that lose this plasmid turn red sectors, those that don’t turn white
Ade2- are red, ade3- are white, double mutant is white ADE3+ restores red
The plasmid carries ADE3+ and YFG2+ to rescue the mutant yfg2-
Another plasmid is transformed carring a second form of YFG2* without
If the new mutant can rescue the original function, there will be sectors
because the original YCp plasmid can be lost and still function. If it can’t it
will be all red because the new one cannot rescue
What is a YAC?
Yeast Artificial Chromosomes: allow the stable maintenance of extremely
large linear DNA fragments
They are circular in form and have restriction cut sites
Have many selectable markers
Needs homologous recombination of insert and YAC arms to maintain
ARS1, CEN4 and TEL allow long-term propogation
1) You should be able to apply the binomial distribution (I will give you the
equation) to predict the likelihood of the outcomes of simple genetics crosses.
Something like: given 8 progeny from the cross Aa x Aa, what is the liklehood that
exactly 2 progeny will be the recessive aa?
P(r successful outcomes in N trials)
P(r number of a given genotype among N progeny) =
N! . p . (1-p)-r
r!(N-r)! 2) From the metastasis paper you should understand the design of the genetic
screen and the main conclusion regarding how tumours associated with Ras
activation become metastatic.
Lack of cell polarity and Ras causes metastasis
Function of Ras has no link to cell proliferation,growth and survival
eyFLP/FRT: to drive recombinase in the eye imaginal disc for proliferation
GAL4/UAS and GAL80: to promote Ras and GFP
o Using UAS-Ras85D to cause hyper-proliferation
o UAS-GFP for viewing expression
What, in general, is tetrad analysis? What is the prerequisite for tetrad analysis?
In what organisms is it possible to perform tetrad analysis?
Tetrad analysis: the ability to isolate and test the four products of a single
meiosis for genetic mapping. In yeast for example, you can isolate the 4
haploid spores to determine their genetics. From this, you can analyze the
ratio between the different segregation types and determine linkage
A prerequisite for tetrad analysis is that the organism produces 4 distinct,
separable haploid components
It is possible to perform tetrad analysis in yeast, neurospora crassa and
Know how to identify the different tetrad types (parental ditype (PD), non-parental
ditype (NPD), tetratype (TT)).
From Dihybrid unlinked unordered:
PD = AB AB ab ab
NPD = Ab Ab aB aB
Tetratype = AB aB Ab ab (from crossover at 1 meiotic division)
In tetrad analysis how does one determine if two genes are linked/unlinked?
You will know if two genes are linked if the number of PD is approximately
equal to the number of NPD because 50-50 chance of arrangement in either
cis or trans. They are unlinked if there are many more NPD because these
only come from recombination
If two genes are not linked, but both are tightlylinked to their respective
centromeres, what is the overall expected ratio of PD:NPD:TT? In this case, crossovers need to happen between one of the genes and the
centromere which is unlikely since they are tightly linked to their
centromere. This is how you can determine separation between the genes
Expected ratio is 1:1 for PD:NPD
Consider tetrad analysis of diploids resulting from a yeast mutant strain “a” crossed