Group 1-1Josh Farhi Tyler Madden Group 1-2Youssef Neema Christine Schmidt Group 2-1Caitlin Carlisle Kourtney Gordon Nina Nissan Group 2-2Nicole Stabler Rachel Edgar Group 2-3Kathleen Shah Camille Fong Chih Kai Groups
Where do we do the heat shocks? Room 361 of the Western Science Center. How do I get there on Sunday if the front doors of WSC are locked? Tunnels. A) Middlesex College front door turn left down to the staircase in the middle of the hallway. Go down to tunnel that leads to the WSC. B) Natural Science Center to Physics and find a tunnel leading to WSC. C) Natural Science Center to B&G third floor to WSC
Functional allele wild-type allele Allele classification Active gene product
Loss-of-function allele (lf) Generally recessive Two classes important for this course
Many developmentally important genes are expressed in a spatially restricted pattern. The pattern of the expression is important for the phenotype of the organism. Non- or mis-expression of the gene can result in a phenotype. Antennapedia is expressed in the second thoracic segment where the second leg will form. Leg to antenna transformation.
Antennapedia can be misexpressed either by spontaneous mutation or by genetic engineering. Antenna to second leg transformation hsp Antpry +
Summary of experiments on Antennapedia Antenna primordia Leg primordia wild type Antp lf Antp gf antenna leg
Dominant negative (dn) antimorph Criteria The gene product works in a complex, either with itself or another protein, and the activity of all the proteins in the complex is essential for the total activity of the complex.
homodimer heterodimer Complex inactive Mutation must not affect the ability of the complex to form. X X X
A dominant negative allele reduces activity to a greater extent than a null allele when heterozygous. 1/2 wild type activity1/4 wild type activity X wt dn x x xx 1 : 2 : 1
Multiple copies of the dn allele or overexpression will strongly inhibit wild type activity. Only rarely will a complex of two wild type proteins form. X wt dn X X X xx xx xx xx x
Saturation screens An attempt to identify as many genes whose products contribute to the process that you are studying as is statistically and technically possible.
Genetic screen for leucine auxotrophic yeast Yeast cells mutagenesis Random pool of DNA sequence changes Replica plate to minimal media +leu-leu
Complementation analysis leu 1 X leu 2 leu 1 leu 2 leu 1 leu 2 leu + leu - leu + Non-complementation in same complementation group =same gene. Complementation XX XX
#hits (numbers of independent allele/ complementation group (gene)) # Complementation groups (genes) Example of 100 mutants and 1,000 genes required for leucine biosynthesis. 901 98 1 Poisson distribution zero group=e -m 0123 100 900
Example of 100 mutants and 10 genes required for leucine biosynthesis. e -10 =4.5X10 -5 # Complementation groups (genes) #hits (numbers of independent allele/ complementation group (gene)) 0 1020 1 2 3
Epistasis Epistatic interactions are assayed by comparing the phenotype of a double mutant organism with that of the singly mutant organisms.
Epistasis: Criteria for the two mutations A. Have related phenotypes growth control sex determination dorsal ventral axis determination B. Work on a pathway that makes a distinct decision growth/nongrowth male/female expression/nonexpression C. The two mutations have distinct/opposite phenotypes all males versus all females expression always ON versus always OFF Ventralized versus Dorsalized
Control of sporulation by sporulation inducing factor (sif) in a hypothetical fungus No sporulationSporulation
The pathway that controls sporulation -sif +sif Receptor protein kinase Kinase Inhibitor of sporulation Inducer of sporulation Spo genes Inactive OFF Inactive OFF Inactive OFF Inactive OFF Active ON Active ON Active ON Active ON No expression OFF Expression ON
Epistasis example Drosophila embryos have a dorsal and ventral side. Mutations exist where the mother lays eggs where the embryo develops with only the dorsal side lacking The ventral side (dorsalized). Mutations exist also that ventralize the embryo.
Loss-of-function allelesphenotype spzdorsalized Tolldorsalized pelledorsalized tubedorsalized dldorsalized cactventralized Double mutants spz cactventralized Toll cactventralized pelle cactventralized tube cactventralized dl cactdorsalized What does the above data tell you about the order of function? Draw out the pathway indicating positive and negative interactions.
Toll gf ventralized Toll gf spzventralized pelle Toll gf dorsalized tube Toll gf dorsalized dl Toll gf dorsalized What does this additional information tell you about the order of function?