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23 Cards in this Set

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RNA Alternative Splicing

- way to generate different proteins in cells through single gene


- splicing done in 3' UTR and 5' UTR allows for different regulations of the protein


- skipped/cassette exon, alternative 5' splice site selection, alternative 3' splice site selection, mutually exclusive exons


- need regulatory proteins and general and regulatory splicing factors



Sex determination in flies

Sex determination: x: a ratio, 1= female, .5 =male


- sex lethal is sensitive to ratio and initiates cascade of alternative splicing resulting in diff proteins in males and females


- in females, early activation of sex lethal gene due to the ratio allows for the protein made to splice the late activation of sex lethal mRNA







Sex determination in flies: Females

- Females:


1. x: a ratio = 1, sex lethal does early activation and regulates sex lethal mRNA, exon 3 skipped (outcompetes U2AF for b.s. before exon 3) = more sex lethal protein


2. Sex lethal binds to a.s. in front of exon 2 in transformer and it's skipped (stop) so transformer protein made


3. Transformer protein includes exon 4 (AUUAAA) in doublesex. Hexanucleotide causes cleavage/polyA tail via CPF


4. Transformer helps bind u2FA to exon 2 (stop) in fruitless so it's included. No functional fruitless protein


5. Only shorter doublesex



Sex determination in flies: Males

- Males:


1. x: a ratio = .5, sex lethal can't regulate mRNA, exon 3 included (stop) = nonfunctioning sex lethal protein2.


2. No sex lethal so exon 2 included in transformer, stop codon so nonfunctional transformer protein


3. No transformer, no exon 4 in doublesex, so no cleavage/polyA tail at that site. Occurs at exon 6 where it's cleaved with polyA tail


4. No transformer, no exon 2 in fruitless (weak b.s.) which is stop so functional fruitless protein


5. Longer doublesex and fruitless



Regulation of translation

- Once mRNA is made, will the protein be made


- eggs storing mRNA for development + beyond


- sucrose gradient: polysomes + mRNPs (polyA)


- mRNPs: proteins that mask mRNA + preserve


- proteins assoc with mRNAs in the 3' UTR: need w.t. 3' UTR for mRNPs to work

Translational control: storage and activation

Broad based mechanism:


- protein synthesis at egg activation during fertilization due to pH change leads to altered interactions btw RNA and masking proteins




RNA specific mechanisms: temporal tsl


- cytoplasmic polyadenylation


- PTGS

Cytoplasmic polyadenylation

short polyA tail: no tsl, long polyA tail: tsl


mRNPs: prevent tsl and keep mRNA in storage


- oocyte maturation from primary to secondary: TPA only tsl in secondary oocyte- short to long A


- two mutants: d10 and d24: both w/ AAUAAA


- CPE sequence required for polyadenylation


- hexanucleotide allows for cleavage and polyadenylation in the nucleus. if just hexanucleotide, transported out of nucleus, tsl, dedenylated and degrated.


- with CPE, gets transported, dedenylated, no tsl, readenylated in cytoplasm and then tsl. proteins bind to 3' UTR


- CPEB interacts with maskin which binds to 4E initiation factor and so 5' end is bound up causing preventing of initiation of tsl. brings in dedenylase and thus short polyA tail + no tsl


- phosphorylation of CPEB, brings in CPSF, binds to unbound hexanucleotide, polya polymerase, long polyA tail. 4g also comes in and outcompetes maskin for 4E. tsl occurs.


-


-



PTGS: silencing

via microRNAs: RNA interference done by introducing double stranded RNA. RISK complex


- double stranded RNA recognized by dicer, leading to microRNAs which are complementary and anneal to mRNA of interest inhibiting tsl


- lin4 gets folded, diced, and made to microRNAs and anneals to lin14 gene


- not 100% complementary, bind to 3' UTR


- can lead to differential gene expression

PTGS: degradation

microRNAs don't generally anneal 100% 2 mRNA


- when viral RNA is present, and it's double stranded, dicer can make microRNAs out of it and these regions anneal 100% to other viral RNA allowing for complete degradation of mRNA


- homologous to intact regions of the RNA, RISK complex attaches again and degrades


- hybrids are 100% matches anywhere along the mRNA not just 3' UTR


- provides nucleotides to cells



Ed Lewis and Drosophila

- Bithorax and antennapedia: single gene mutations, affecting embryogenesis decisions


- homeotic transformations: bithorax, T3 turns to T2. takes identity of neighboring segment. genes operating to form basic body plan and morphogenesis


- model: cascade of gene regulation to progressively pattern the embryo


- based on phenotype observations and downstream/upstream target observations


- first form bilateral symmetry: a/p, d/v, l/r, then divide to segments, and then segment identity


- make pattern of larva early


- cytoplasmic polarity genes, segmentation genes (gap, pair-rule, segment polarity), homeotic transformation genes

Maternal effect/Cytoplasmic polarity genes

- set up polarity of the egg via localizing components


- before cell differentiation, after nuclei move to periphery


- egg stores proteins and mRNA for later


- four sets: anterior, posterior, D/V, terminal


- anterior/posterior involves quantitative opposing morphogen gradients







Maternal effect: anterior group genes

- anterior morphogen: bicoid


- nurse cells create cytoplasmic bridges and transcribes bcd and nos and pumps into oocyte


- microtubules oriented from + to - orientation


- bcd mRNA binds to dynein motor and tethered near entryway and nurse cells


- tsl: before fertilization, short polyA tail and after, long polyA tail so c. polyadenylation


- at fertilization, localized source of mRNA needed so once translated, proteins can diffuse


- nos and bcd gradients create opposing gradients of hunchback at anterior end and coddle at posterior end


- - mRNA tight patch of bcd at anterior end


- bcd is translational repressor (1) of cd and transcriptional activator (2) of zygotic hb


- bcd must bind to DNA and RNA. has homeodomain

Maternal effect: posterior genes

- posterior morphogen: nanos


- nos mRNA tethered to oskar scaffold which interacts with kinesin motor proteins


- oskar mutant: no nanos localization


- tsl: any nos mRNA not localized is repressed


- creates opposing gradients of hb at anterior end and cd at posterior end


- mRNA tight patch at posterior end of nos

Maternal effect: hb and cd

- hunchback: anterior, coddle: posterior


- made by nurse cells and transported into oocyte but not localized, mRNA everywhere


- negative regulation by bcd and nos: bcd represses tsl of coddle at anterior end and nos represses tsl of hunchback at posterior end


- high conc of hb at anterior end, high conc of cd at posterior end


- quantitative mechanisms of control bc gradients leading to differential gene exp.


- hb and cd: tsc factors


- hunchback: hb has hunchback bc hb mRNA is tsl at anterior end bc bcd represses cd but also bcd goes into nucleus and acts as a tsc factor to activate hb zygote gene tsc



maternal effect: dorsal/ventral genes

- both types of genes effect dorsal/ventral patterning


- central players coded for receptors/secreted stuff


- need interaction between egg and follicle cells in order to move Dorsal protein into nuclei


- ventral morphogen: Dorsal (activate gene expression + tsc in ventral nuclei)

Maternal effect: dorsal side

Gurken mRNA is localized in dorsal anterior side and after tsl, fuses with membrane and binds to torpedo receptors on follicle cells. binding causes follicle cells to take on a dorsal fate and second prevents synthesis of pipe


- no pipe, dorsal. pipe, ventral


- dorsal side is allowed to be dorsal bc no pipe is expressed



Maternal effect: ventral side

- In absence of grk on ventral side, pipe is expressed in ventral follicle cells


- Pipe initiates cascade of proteolytic cleavage in extracellular space on ventral side


- Presence of pipe -> Easter is cleaved


- Easter cleaves protein spatzle creating two smaller peptides, one of which is activated spatzle which acts as a ligand to receptor in egg plasma membrane


- Activated spatzle binds to Toll receptor which is in plasma membrane


- As a result of Toll signalling, Dorsal gets into the nuclei of ventral side as cactus lets the protein go


- Dorsal allows for activation of ventral specific genes after fertilization

Maternal effect: terminal group

anterior side: acron, posterior side: telson


- Torso codes for a transmembrane receptor protein that acts from signals from follicle cells


- as result of follicle cell signalling on both ends, terminal structures are formed


- one gene: two outcomes bc two diff locations


- bicoid mutants, two telsons. bcd needed for acron formation. torso + bicoid= acron, torso=telson


- torso receptor everywhere, ligand is localized not downstream targets. constitutive receptors caused gain of function mutation


(mutant phenotype in w.t. embryo)


- ligand = torso-like, made by follicle cells and two ends


- different things found in diff parts of embryo so two different outcomes

Segmentation genes: Gap genes

- occurs after early fertilization, no cells yet


- zygotic genes


- lay out broad domains in embryos


- after hb/cd gradients bc they're tsc factors, so hb/cd gradients etsablish these domains


- quantitative mechanism (lil bit qualitative)


- hb gradient: lot of hb, giant. less, kruppel, etc


- gap genes: tsc factors, zn-finger proteins. regulate each other (mostly negative). start inhibiting each other to form domains


- only create irregular, non-repeating units

Point of segmentation

- needed physiologically to divide up the coelom so it can specialize into different parts


- physiologically beneficial


- segmentation of musculature allows for more complicated movement


- understanding segmentation in embryogenesis allows for understanding how we go from unsegmented species to segmented species


- segmentation selected for over evolutionary time

Segmentation genes: Pair-rule genes

- creates evenly spaced repeated elements; diff collections of gap genes can activate same final gene


- Fugi-tarazu (ftz): mutants miss even segments


- diff combinations of tsc factors regulate one gene depending on location in embryo







Pair-rule genes: mechanism of tsc control

- Even-skipped expressed in odd segments


- took DNA regions flanking the gene and hooked it up to B-gal, and saw expression. 8kB of gene sequence needed for the 7 stripes. need 5' flanking DNA


- can get w.t. expression, 0 expression, decreased expression, or altered expression


- individual subdomains required for expression in individual regions


- need to look for binding sites + proteins present to see what is regulating the gene: bcd, hb, giant, kruppel


- high conc: positive regulation, low conc= neg


- bcd, hb: high conc, +. giant, kruppel: low, -


- diff nuclei have diff collections of tsc factors, dna has many diff binding sites with diff subdomains so allows for

Segmentation genes: segment-polarity genes

- engrailed and wingless provide anterior and posterior half of each segment


- mutations in segment polarity genes led to deformations in the cuticle, which has denticles anterior and nothing posterior. engrailed mutant had denticles on anterior + posterior side (dup)


- 14 lines of expression, 1 cell wide. determine what anterior + posterior halves look like


- pair rule genes critical for seg-pol expression, only need two diff ways: even and odd


- gap genes turn on wingless initially and ftz and eve repress wingless. everywhere except in places in between ftz and eve


- wingless and engrailed do positive regulation on each other: wingless anterior, engrailed posterior. maintain expression of each other


- wingless = vertebrate wint protein


- wingless is secreted and binds to frizzled receptor on engrailed cell, to disheveled, inhibits ZW3 (GSK3), binds to Armadillo (B-catenin), transcribes engrailed and hedgehog. hedgehog is secreted and binds to patched protein on wingless cell, binds to patched receptor, lets go of smoothened protein, tsc of wingless continued.


- B-catenin: tsc factor and structural factor


- draw a line behind engrailed, segment made


- gradients of wingless and hedgehog make dentricle and naked cuticle pattern


- engrailed leads to structural adhesive proteins