Transcript Tetrad Genetics

Tetrad Genetics
Drosophila melanogaster
Todd Nystul, Ph.D.
UCSF Depts. of Anatomy & OBGYN-RS
Center for Reproductive Sciences
Eli and Edythe Broad Center of
Regeneration Medicine and Stem Cell Research
Gene function is conserved from Drosophila to mammals
Expression of the master regulator,
eyeless, causes ectopic eyes to form
Expression of mouse eyeless (called
Pax6) produces a Drosophila eye!
Halder, Callaerts and Gehring, Science 1995
The life cycle
The Life Cycle
Embryogenesis
Larval development
Model for studying:
Model for studying:
•
The cell cycle
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Growth control
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Morphogen signaling
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Signal transduction
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Embryogenesis
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Developmental neurobiology
•
Epithelial morphogenesis
•
Hematopoiesis
•
Epithelial polarity
The Life Cycle
Model for studying:
Model for studying:
•Organogenesis
•
Stem cell biology
•Hormonal cues
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Homeostasis and aging
•Circadian rhythms
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Nutrition and fat storage
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Behavior and neurobiology
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Cancer biology
The Drosophila karyotype
4 pairs of chromosomes
X Chromosome is telocentric; 2 and 3 are metacentric; 4 is small and mostly
heterochromatic
The equal left and right arms are called 2L and 2R, and 3L and 3R
Each arm carries ~20% of the gene of the fly
Y is heterochromatic – few genes, fertility factors XO is a viable sterile male
Sex is determined by the X:autosome ratio (not the presence of a Y, as
in humans)
There is recombination in females, but NOT in males
Means that genes on the same chromosome behave as if they are 100%
linked in males.
The illustrious history of Drosophila genetics
1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is
sex-linked.
Why was the first mutation he isolated sex-linked?
In class Q1: Red-eyed male: w+/Y x White-eyed female: w-/w-
☿
w-
w-
w+
w+/wRed
w+/wRed
Y
w-/Y
White
w-/Y
White
♂
The illustrious history of Drosophila genetics
1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is
sex-linked.
Why was the first mutation he isolated sex-linked?
In class Q2: wts1; ry506/+ male x wildtype female:
wts1/+; ry506/+ or
wts1/+; ry+/+
The illustrious history of Drosophila genetics
1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is
sex-linked.
Why was the first mutation he isolated sex-linked?
1913: Sturtevant constructed the first genetic map.
1914-1916: Bridges discovers non-disjunction in XXY females, providing
first proof that chromosomes must contain genes. Chromosome theory of
heredity (1933 Nobel Prize)
w+/Y x w-/wX X non-disjunction
All females have red (w+) eyes
X(w-) X(w-) Y are
white-eyed females
The illustrious history of Drosophila genetics
1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is
sex-linked.
Why was the first mutation he isolated sex-linked?
1913: Sturtevant constructed the first genetic map.
1914-1916: Bridges discovers non-disjunction in XXY females, providing
first proof that chromosomes must contain genes. Chromosome theory of
heredity (1933 Nobel Prize)
The illustrious history of Drosophila genetics
1927: Muller showed that x-ray irradiation causes gene mutation, including
chromosomal rearrangements (1946 Nobel Prize).
1935-38: Bridges published polytene physical maps of such accuracy that
they are still used today.
1978: E. B. Lewis’s characterization of the bithorax complex (a Hox gene
cluster) provides foundation for understanding genetic regulatory elements.
(1995 Nobel Prize)
1980: Nusslein-Volhard and Wieschaus complete a systematic genomewide mutational screen to attempt to identify all genes involved in embryonic
axial patterning (1995 Nobel Prize).
1981: Rubin and Spradling make transgenic flies with the use of
transposable element vectors.
1993: Brand and Perrimon create a two-component transgenic system for
controlling ectopic gene expression.
2000: Drosophila genome sequenced (www.flybase.org).
Nomenclature
Genes are traditionally named for phenotype and given 1-4 letter abbreviations.
Gene name is italic and protein name is roman and capitalized (e.g. hedgehog
encodes for Hedgehog
First letter is lowercase for recessive alleles, uppercase for dominant alleles.
Allele name is superscript (eag1 or eagEY00714). Wild-type is a plus sign (+)
Homozygote: single allele written by itself implies homozygosity: eag101
Heterozygote: alleles are above and below a line or separated by a slash (/):
eag101/eagEY00714 or eag101/+
Hemizygote: For X-linked in males, single allele is written over Y: w/Y
Multiple alleles:
Alleles on the same chromosome are separated by a comma
Alleles on different chromosomes are separated by a semi-colon:
y, w, eag1; shgR21; ry506/TM6
Chromosomes listed in order (X, II, III) and anything unlisted is assumed to be
wildtype: f ; cn bw ; TM2, Ubx130 / tra
Balancers
A balancer is a chromosome that has been massively reorganized (by inducing many
translocations) to prevent recombination. It is one of the two homologs, not an extra
chromosome.
Balancer chromosomes provide two main advantages:
1.They preserve disadvantageous alleles in a stock automatically!
2.They make it easier to trace alleles of interest through multiple generations
Preserving disadvantageous alleles:
1.Autosomal balancers carry recessive lethal mutations
A stock in which the lethal allele, EGFRco is balanced by the balancer CyO:
♂
☿
EGFRco
CyO
EGFRco
EGFRco/EGFRco
(dead)
EGFRco/CyO
CyO
EGFRco/CyO
CyO/CyO
(dead)
parental genotype is maintained
Balancers
Balancer chromosomes provide two main advantages:
1.They preserve disadvantageous alleles in a stock automatically!
2.They make it easier to trace alleles of interest through multiple generations
Preserving disadvantageous alleles:
1.Autosomal balancers carry recessive lethal mutations.
2.X chromosome balancers must be viable as hemizygotes but usually carry a
recessive female-specific sterile mutation.
Why?
♂
☿
dlg1
FM7
Y
dlg1/Y
(dead)
FM7/Y
FM7
dlg1/FM7
FM7/FM7
(sterile)
parental genotype is maintained
Balancers
Balancer chromosomes provide two main advantages:
1.They preserve disadvantageous alleles in a stock
2.They make it easier to trace alleles of interest through multiple generations
1. Preserving disadvantageous alleles:
A.Autosomal balancers carry recessive lethal mutations
B.X chromosome balancers must be viable as hemizygotes but usually carry a
recessive female-specific sterile mutation.
A stock in which the lethal allele, EGFRco is balanced by the balancer CyO:
♂
☿
EGFRco
CyO
♂
EGFRco
EGFRco/EGFRco
(dead)
EGFRco/CyO
CyO
EGFRco/CyO
CyO/CyO
(dead)
parental genotype is maintained
☿
Lgl1
EGFRco
Lgl1
Lgl1/Lgl1
(dead)
Lgl1/EGFRco
EGFRco
Lgl1/EGFRco
EGFRco/EGFRco
(dead)
parental genotype is maintained?
Balancers
1. Preserving disadvantageous alleles:
A.Autosomal balancers carry recessive lethal mutations.
B.X chromosome balancers must be viable as hemizygotes but usually carry a
recessive female-specific sterile mutation.
1
co
Lgl
Meiosis
EGFR
Lgl1
X
EGFRco
A.Balancers have chromosomal inversions that suppress meiotic recombination
a
b
c
d
e
f
g
h
a
b
c
d
e
f
g
h
a
b
c
d
e
f
g
h
b
a
d
c
f
e
h
g
balancer chromosome
wild type
a
b
c
d
e
f
g
h
a
b
c
d
e
f
g
h
selection pressure for wild type
chromosomes cause loss of
lethal mutation
acentric and dicentric chromosomes
cause lethality of gametes
Balancers
2. Tracing alleles of interest through multiple generations
X: FM7 (dominant marker: Bar eyes)
II: CyO (dominant marker: Curly wings)
III: TM3: (dominant marker: Stubbly back hairs)
IV: virtually no meiotic recombination so balancer is unnecessary.
Sensitized backgrounds to identify genetic interactions
•
A sensitized background is a genotype that works with other genetically
related mutationis to produce a phenotype or enhance a phenotype.
•
The alleles that interact in a sensitized context may or may not produce a
phenotype on their own
Erika A. Bach et al. Genetics 2003;165:1149-1166
The P-element revolution
1977: P-M Hybrid Dysgenesis (Kidwell, Kidwell and Sven)
wild “P” ♂ x lab “M” ☿
sterile progeny
lab “M” ♂ x wild “P” ☿
fertile progeny
1982: “P-elements”: Rubin, Kidwell, and Bingham demonstrate that
the “P” cytotype is due to transposable elements.
But, why are wild females protected? Hmmm...
(notice how the use of italics creates suspense)
1982: Spradling and Rubin clone the P-element and demonstrate that it
can be used to generate transgenics.
1988: Cooley and Spradling publish a method for efficient generation
and screening of insertional mutants.
1993- present: Starting with Brand and Perrimon’s two-component
expression system, P-elements become the basis for many genetic
tools.
Useful features of P-elements
Natural P-element
Transposition and copy number can be controlled:
P-elements used in stable lines lack transposase, so they cannot hop.
P{ry+ Δ2-3} has transposase that can only be translated in the germline
but lacks the 31 bp repeats at each end that are essential for
transposition.
Why do they lack the 31 bp repeats?
To mobilize, cross a P-element source to Δ2-3 and then cross Δ2-3
away again. Score for presence/absence of P-element.
Lab strains lack endogenous P-elements so copy number can be tightly
controlled.
Enhancer traps
P-elements have a strong bias for inserting near the 5’ end of genes, but
otherwise transposition is somewhat random.
Enhancer traps are generated by P-elements carrying a reporter gene with
a minimal promoter
must land within the regulatory region of a gene
usually an approximation of the cellular expression pattern of the gene,
but not the subcellular localization of the protein
Typical enhancer
trap construct
LacZ
LacZ
Protein traps
Protein traps are generated by P-elements carrying a reporter gene that is flanked by splice acceptors and
donors are hopped around the genome.
Must land within the transcriptional unit of a gene to be expressed
Must be in frame to form fusion protein (otherwise, it disrupts normal protein translation
Fusion proteins are accurate reports of both cellular transcription pattern and subcellular protein
localization patterns.
Typical protein trap construct
Gal4/uas transcription system
Gal4 is a transcription factor that activates the UAS promoter.
Gal4 P-element can use endogenous enhancers (enhancer trap) or can include a promoter.
Gal4 can be driven by
a ubiquitous promoter (e.g. Tub-Gal4 or Ubi-Gal4)
a tissue-specific promoter (e.g. elav-Gal4 is expressed in all neurons and MHC-Gal4 is expressed in all
muscle cells)
Variations to provide temporal control:
Gal80 inhibits Gal4; Gal80ts is only active at permissive temp. (18° - 22°C)
Gal4ER is only active in the presence of an estrogen analog
In class Q1
ϕC-31 integrase
attB insertion sites in
the original collection
In the TRiP RNAi collection, RNAi constructs are inserted using ϕC-31 technology
In class Q2
MiMIC lines
Inserted DNA can:
1. Manipulate gene expression
2. Tag the protein (protein trap)
3. Utilize the enhancers of the gene (enhancer trap)
4. Mutate (or reverse a mutation of) the gene
Flp/FRT recombination
Flipase (FLP) is an enzyme that catalyzes recombination between two FRT sites.
FRT sites can be on the same chromosome (e.g. tub-FRT-STOP-FRT-Gal4) or homologous
chromosomes.
Flp can be controlled by a cell-type specific promoter (e.g. eyeless), or it can be
inducible (e.g. heat-shock)
The result is a genetically heritable rearrangement that positively or negatively labels cells.
When labeled cells divide they form a “clone” that is distinguishable from
surrounding unlabeled cells.
Cells in clone can become homozygous mutant for, or specifically overexpress, a gene of
interest.
Flp/frt recombination
Positive marking, all cells are
wildtype
Negative marking, labeled cells are
mutant
Tub FRT
FRT
LacZ
hs-Flp
GFP
hs-Flp
FRT
Tub FRT
FRT
FRT
LacZ
GFP
*
GFP+ wildtype cell
lethal mutation
FRT
GFP+ heterzygous cell
FRT
*
GFP- homozygous mutant
Planar cell polarity
Cell autonomous: only cells within the clone are
affected.
Cell non-autonomous: cells within the clone affect
cells outside the clone (or visa-versa)
Tumor suppressor screens
1967: Gateff and Schneiderman identify lethal giant larvae, the first in
vivo example of a tumor suppressor.
1995: Xu et al., identify the first component of the hippo pathway by
generating clones in larval imaginal discs and screening adults for mutations.
Dual clone technologies
Dual-marked clones
Tracking multiple lineages
simultaneously
Differentially labeling single cells
FSCs
FSCs
DNA
GFP
GFP
B-gal
DAPI
B-gal
Stem cell niche cells