Transcript two ald “mutations”

Gene Interaction
“Standard” interpretation
of complementation test
Hawley & Gilliland (2006) Fig. 1
“Mutation” of a gene might be due to changes elsewhere!
•ald is Drosophila mps1 homolog; isolated four mutations (all rescued by ald+ transgene)
•two ald alleles cause meiotic and mitotic defects (ald sequence changes)
•two ald “mutations” cause only meiotic defects (normal ald sequence)
•both contain Doc element insertion into neighboring gene
(silences transcription of neighboring genes in germline cells)
Hawley & Gilliland (2006) Fig. 2
“False positive” of transgenic rescue
•Ku and Dmblm genes both involved in DNA repair and closely linked on the chromosome
•Old mutations of mus309 map to the region genetically
•DNA lesions of mus309 lie in Dmblm, but can be rescued with extra copies of Ku (provided on a
transgene)
Shared regions between genes
updYM55
upd3d232a
Df(1)os1a
updYM55
os1
upd3d232a
Df(1)os1a
Lethal
OS
WT
Lethal
OS
OS
Lethal
Exceptions to “Non-Complementation = Allelism”
Intragenic complementation (usually allele-specific)
•Multi-domain proteins (e.g., rudimentary)
•Transvection – pairing-dependent allelic complementation (stay tuned!)
Second-Site Non-Complementation (“SSNC”)
•“Poisonous interactions” – products interact to form a toxic product
(usually allele-specific)
•“Sequestration interactions” – product of one mutation sequesters the
other to a suboptimal concentration in the cell (usually one allelespecific)
•Combined haplo-insufficiency (allele non-specific)
Intragenic complementation in multi-domain proteins
Transvection:
synapsis-dependent allele complementation
E. Lewis (1954) among BX-C mutations in Drosophila
Numerous other genes in Drosophila and similar phenomena
observed in Neurospora, higher plants, mammals
Most due to enhancer elements functioning in trans (allele-specific)
Exceptions to “Non-Complementation = Allelism”
Intragenic complementation (usually allele-specific)
•Multi-domain proteins (e.g., rudimentary)
•Transvection – pairing-dependent allelic complementation
Second-Site Non-Complementation (“SSNC”)
•“Poisonous interactions” – products interact to form a toxic product
(usually allele-specific)
•“Sequestration interactions” – product of one mutation sequesters the
other to a suboptimal concentration in the cell (usually one allelespecific)
•Combined haplo-insufficiency (allele non-specific)
Example of a “Poisonous interaction” SSNC
Hawley & Gilliland (2006) Fig. 4
(after Stearns & Botstein (1988) Genetics 119: 249–260)
Non-complementation of
non-allelic mutations
A model for synthetic lethality
Figure 6-23
A model for recessive epistasis
Figure 6-19
Dominant epistasis due to a white mutation
Figure 6-21
Recessive epistasis due to the yellow coat mutation
Suppression: a form of epistasis whereby the
expression of one mutation (the “suppressor” mutation)
normalizes the phenotype of another mutation (the
“suppressed” mutation). The suppressor mutation may
display no other phenotype.
Intragenic suppression: “pseudo-reversion”; can be same
codon or different/interacting region of gene.
Intragenic suppression by compensatory missense
mutation:
Example:
•p53 tumor suppressor* gene; DNA-binding protein; numerous mutations catalogued
•Yeast reporter system (p53 binding site-UAS-URA3) requires p53 binding
•Expressed mutant human p53 (does not drive URA3 expression)
•Created variety of second-site mutations within p53, using gap-repair-mediated
replacement of mutagenic PCR fragments into p53-containing plasmid
(site-directed/not random mutagenesis)
•Screening for URA3 expression identified array of second-site mutations
• identified suppressors for each of three original p53 mutations
(Ura+ and FoaR phenotypes)
• most suppressing mutations were obtained multiple times
• luciferase-reporter transfection assays in mammalian cell lines gave similar results;
p53-induced apoptosis assays in mammalian cells also gave similar results
• created structural models for basis of suppression
Val contributes to hydrophobic core of β sandwich
Asn>Asp may shift H-bonds to maintain β sandwich
Gly “fits” within DNA contact loop
Asn>Tyr may provide new DNA contact (Y with PO4)
Ser>Asn may provide new stabilizing H-bond to loop
Arg is within H-bond network stabilizing DNA contact loop, eliminates quanidinium
His>Arg may create new quanidinium interaction
Thr>A/P is known to enhance DNA-binding affinity of wild-type p53
Extragenic suppression: suppressor mutation is in
different gene than suppressed mutation
Classic example: tRNA anticodon mutations that suppress nonsense/frameshift
mutations in other genes
Classic example: eye color suppression in Drosophila
A molecular mechanism for suppression
Figure 6-22
The Wnt Signaling Pathway
Ommatidium:
8 photoreceptors (R1-8)
12 accessory cells
sev mutations remove R7 photoreceptor
(cell develops as non-neuronal cone cell)
*non-lethal: functions only in precursor
of R7 photoreceptor!
sev encodes transmembrane protein tyrosine kinase
closely related to mammalian c-ros
Created sev ts mutation based on known v-src ts
mutants, using in vitro mutagenesis