Meeting 17

• 1. Mutagenize lots of individuals
• 2. Screen for interesting phenotypes
• 3. Clone the gene that carries themutation
• 4. Deduce protein structure, function
• (can be done in Yeast, worms, flies, zebrafish, human genetics)

• 1. Start with a protein
• 2. Deduce gene sequence
• 3. Make a mutant in the gene
• (can do in Mice, but now sequenced genomes and RNAi make this available to other species)

• 1. Establishment of polarity
• -Anterior-posterior
• - Dorsal-ventral
• 2. Specification of body plan
• 3. Organization of particular structures in specific
• positions (aka a head being anterior)

model systems: C. elegans, D. melanogaster, D. rerio (zebrafish), A. thaliana

• -embryos are easily accessible/visible
• -they have short generation times: worms: 2-3 days, flies: 10 days
• -small genome: (ex. flies have 4 chromosomes) don’t want too many genes; and you also want an organism where the genes are conserved in the organism and in humans

1. chemical agents (EMS, ENU): EMS adds an alkyl group to G, which itself pairs with T during replication; in subsequent round of DNA replication, this changes GC's to AT's: results in (silent, missense, nonsense) point mutations in coding regions; this is done predominantly in MALES so that they can easily spread the mutation

2. Radiation (X/gamma-rays): more serious defect because it causes breaks in the chromosome that can result in deletions/inversions/translocations

3. inserting a transposon: useful when it disrupts a coding sequence; this is because you know the sequence of the inserted transposon and so therefore can find it and as a result use it to find the surrounding sequence it interupted

-CyO is a dominant marker (therefore it takes the place of the general 'WT' when crossing heterozygous recessive mutants)

-when homozygous, CyO is lethal (aka CyO/CyO)

-but when heterozygous, CyO/+ flies have curly wings (this is how you know they're carrying the CyO gene and ONLY have one copy)

1st: unbalanced; because it is */+, so for every crossing with either a +/+ or */+ organism, the * mutant gene will still eventually be canceled out by the + WT gene

• 2nd: BALANCED; CyO/CyO flies (which is the analog to +/+ flies in an unbalanced cross) will DIE; therefore the * mutation will die out just as many times as the CyO mutation will
• 

ask if these two boxes are ACTUALLY showing the same thing

-during meiosis, homologous chromosomes pair up and crossing over can occur

-if this happens, then one chromosome will have both mutations and the other will be WT for both (this is BAD)

-if the double mutant is mated with the double WT, both mutations will be lost in subsequent generations

·need to prevent recombination between two chromosomes carrying different mutations

• ·during meiosis, chromosomes PAIR (aka synapse) on the basis of sequence similarity
• ·if you invert one chromosome, then its homolog fails to form a synapse its nucleotide order does not match the order of its corresponding homolog (almost as if they aren't homologs at all)

-chromosomes can’t recombine

·therefore CyO and * mutations remain present in future crosses

• in inverted homolog used to prevent crossing over (recombination) between homologous chromosomes during meiosis; the inversions are made by x-irradiation; allow populations of flies with heterozygous mutations to be maintained; have three important properties:
• 1) they suppress recombination with their homologs
• 2) carry dominant markers (ex. CyO)
• 3) negatively affect reproductive fitness when carried homozygously......?

the make-up of balancer chromosomes is multiple chromosomal inversions so that synapsis between homologous chromosomes is disrupted; IF crossing over occurs (during meiosis) between balancer chromosome & balancer's homolog, each chromatid ends up lacking some genes and carrying two copies of others: progeny carrying these types of chromosomes die

*367/CyO male crossed with CyO/Dominant female

• ·you known that if the gene has the dominant marker, you don’t want it...
• ·so CyO/NOT dominant is BY DEFAULT the progeny you want to keep; others have markers that rule out the presence of *367 or the CyO (curly wings) [both traits you want]

-the next step from here is to cross these flies together (*367/CyO)

• *367 = some recessive mutation
• CyO = lethal dominant marker
• Dominant = some other dominant marker (but not lethal) that's easily identifiable, like eye color

• there are two possible explanations for why two mutations lead to the same phenotype:
• 1) both mutations occur in the SAME gene
• 2) the mutations occur in different genes but affect the same PATHWAY

• 1) cross two strains each containing a 'different' mutation
• -1/4 of the progeny will DEFINATELY die because they'll be CyO/CyO
• -1/2 of the progeny will have curly wings and either the *61 or *524 recessive mutation
• -what happens to the REMAINING 1/4 of progeny determines whether the mutations are on the same gene:
• -because you know the mutations are lethal (you just do), if the 1/4 remaining is DED, then *61 and *524 FAIL to complement each other meaning they are on the same gene (silly that you gave the same mutation two different names)
• -BUT! if the remaining 1/4 of progeny (*61/*524) is alive, then you know the two mutations are on DIFFERENT genes and they complement each other

Easy.

• k = # of clones/mutations expected to be represented
• λ = average # of ______ per each position

• -so for the first question, λ was average clones per 1 genome, aka 1 clone/1genome
• -for the second, λ = mutations per each gene, 3000mutations for every 1000 genes = 3