Ethyl methanesulfonate - EMS- mutates stuff- nucleotide substitution assay: measure the amount or functional activity of a target entity

female IGF2- x WT male = normal WT female x IGF2- male = small 2 point mapping: If same chrom: 1/4 mm, 1/2 het, 1/4 wt/wt; if diff chrom: 9/16 wt;wt// 3/16 wt;rol// 3/16 m;wt// 1/16 m;rol (distinguishes)

SiRNA and miRNA

OHHH! we can transform the specific inverted repeat... of a human cell growing in a dish & shut off a particular gene miRNA- downregulates something downstream (IR is converted to single strand RNA to bind to mRNA). miRNA conserved between species extremely well. can have multiple targets. has nucleus stuff, no cleavage (perf match), and endogenous all the way. binds and blocks transcription & stability of mRNA IRON REG- IRP only binds in absence of iron. IRP binds to transferin mRNA=protects from degradation, makes transferrin to bring iron in; binds to ferritin mRNA=ribosome can't bind and no translation. can be removed by iron so that translation can proceed so that makes ferritin (iron storage protein) Low iron High iron Transferrin + - (high lvl inside, don't need more) Ferritin + (store more!) trans: IRP usually on, prevents deg ferr: IRP usually on, blocks transc protein usually interacts directly w/ mRNA -affects stability of mRNA -translatability of mRNA RdRP- RNA dependent RNA polymerase RdRP goes through two rounds to get RNA for packaging 16HA/ 9NA H1N1 and H3N2 circulate in humans coinfection= 256 possible combinations genetic shift= major change by genetic reassortment genetic drift= slow accumulation of point mutations in pop

females make Sxl late protein In the tra gene, Sxl binds to

male gets the stop codon in the exon again, lol -> Dsx (double sex) Tra gene binds to 4th exon. forces splicing of 3-4, default is 3-5 rather than cause skip DSX-F 1234AAA, DSX-M 123456AAA= transcription (not splicing) factors activate or repress sup35 locked up in prion aggregate Prion QQQQ (glu rich) region FLY DEVELOPMENT: class of mutants: maternal effect

siRNA- in drosophila, we can turn on the hairpin in any cell we want & look at the loss of function in spec gene in 1 cell

Trp: translation/ transcription are closely paired. after 1/2 are made by RNA pol, they pause so that a ribosome can attach on (and transl leader reg). if the ribosome stops viruses can be dna-dna, rna-dna, rna-rna anywhere but the stop codon (near reg 2) in SEX DETERMINATION the leader reg (starved/ stop codon)-> 2-3 in drosophila, 1:1 x:auto for female; 1/2:1 regulation at repressor level: trpR translates a x:auto for male Trp rep. binds ot O. high trp= binds. UNLIKE sisterless homodimer is active but lac which allolac removes) sisterless/deadpan heterodimer is inactive in 3-4= transcription termination symbol males homodimer turns on early Sxl promoter -> Sxl (attenuation) if less stable, does not terminate as effectively protein in fem only In late Sxl, Sxl protein binds to exon 3 (which ara is a repressor and an inducer. rep: no ara, contains a stop codon) so that exon is skipped no need to turn on gene. also CAP site can't bind. inducer: ara is inducer, but repressor during splicing (2-4 fuse) stays on DNA unlike lac. so, males contain exon 3, which has the stop lactose -> galactose/ glucose by b-gal, codon = no functional product permease brings in

can be allolactose too mRNA for enzymes have very short half-lives Oc - cis dominant inducer can't bind to operator P mutations- cis dominant operons regulated by operator-repressor protein, operation region and promoter lac I- transcribed constitutively, but it has a weak promoter- forms tetramer "leaky expression" generates a few molecules of the three enzymes allow initial transport of lactose in & first lac -> allol allosteric shift: allol binds to repressor and changes shape, allol also binds to free rep so they don't bind to O IS trans dominant, unable to be induced I-D trans dominant, blocks ability to bind to DNA lacQ or lacSQ: decrease efficiency of induction of lac op, can be induced at high lac concentrations CAP stuff ensures that lac will function at high levels only if only lac, not glu too (catabolite repression/ glucose effect) RNA pol cannot bind efficiently to promoter without CAP-cAMP (cAMP very important!!!!) cAMP= indicator of glu lvl ATP -(AC)-> cAMP bicoid- inolved in formation of anterior structure, represses translation of mRNA caudal caudal/ nanos mRNA evenly distributed before fertilization, but there is a gradient of proteins (protein) caudal protein: posterior. translation after fertilization nanos/caudal/bicoid/hunchback= maternal effect gene nanos protein= translational repressor for mRNA hunchback hunchback= in the anterior mutations in gap: deletion in regions consisting of one or more adjacent sections mutations in pair rule: deletion of every other segment homeotic gene specifies identify of each segment many more levels for exp of protein-coding to be regulated in eukaryotic cis reg elements: enhancers, silencers: proteins bind here to inhibit transl, insulators: isolate gene from enhancer transacting factor(DNA binding dom) interacts with major groove also, chromatin remodeling complex, uses ATP to open DNA methylation represses transcription (activators can't bind, recruit HDAC, block transcription) histone methylation can recruit HAT/HDAC methyl added to lysine residue GAL1- is recessive, GAL 1/10 complementation, GAL4- is recessive. GAL1/GAL10- will grow in glucose, just not galac transcriptional fusion promoter/enhancer + gene

Regulation of transcription initiation by activators: general transcription factors, recruit RNA pol. activators: stimulate transcription initiation (DNA binding domain (cis on DNA, finger)and transcription activation domain(RNA pol/proteins)). coactivators: does not bind directly with DNA but interacts with GTF and activators. activator bind -> coactivator -> GTF

Lin4 causes a small, non coding RNA that blocks lin 14 Pasha and Drosha cleave extra RNA miRNA blocks translation and messes up stability of mRNA, mRNA degradation can use miRNA to k/o gene or k/o by downregulating mRNA virus synthesis: 3-5 in cell, synthesize 5-3 by RdRP, snatch cap, translation. RdRP binds to RNA pol. cleaves mRNA, binds CAP. causes RNA pol to release genetic shift/reassortment: major genetic changes genetic drift: slow accumulations of point mutations for flu vaccines, look for stuff that increases transmissability androgen insensitivity syndrome sup35 is rec, sup psi is dom protein aggregates into fibril structure called DNA acetylation by HAT: removes pos amyloid charge on lysine by acetylating lysines, so genetic, sporadic, infection deattaches from neg DNA. HDAC removed genetic: you just are more likely to get forms added acetyl groups. can remodel by that will misfold sliding/exposing, restructuring, transfering the syncitium nucleosome maternal factors (polarity), gap, pair rule, epigenetic: heritable change in gene segment polarity expression w/o change in DNA seq (ie gene kruppel controlled by hb. pair ruled controlled silencing) HpaII won't cleave if methylated. by gap.each stripe controlled by spec element CpG islands repress transcription in promoter. each enhancer regulates a stripe. combination of activate/ repress, can get a larger stripe in the absence of some repressors segment polarity + Hox genes= specify identity of segment mutate Hox genes= homeotic transformation d2= xchrom= endogenous location of sevenless gene. b4= 3rd chrom= added as a transposon in a different position. EMS makes IGF2 expressed only in dad, imprinted in dad rare mutations. protein sev is used in other pathways so lethal, can never see what a too. insulator blocks activation of promoter recessive gene is like (xray!). once a mat/pat x chrom is inactivated for sex make a patch of homo mutant to see the rec compen, all the descendants inherit that phenotype. patch=WT, outside is mutant to activation pattern. the noncoding/regulatory RNA coats the chrom in both directions form see if protein could function outside of R7. XIC, triggering chromatin remodeling that silences the gene at the transcriptional level. Sxl must be active in females, must be off in males (msl) knockout dsx mutants have a mixture of male and female characteristics (lack of repression of male/female spec genes) MSL downregulates (sxl turns it off in females)

RNA interference pathway can alter the inverted repeat to get what we want to turn off can use siRNA to selectively shut off stuff