Transcript Using Yeast to study Eukaryotic Gene Function From Recombinant

1.Which is the most unlikely involved in the intronsplicing of pre-mRNA ?
[a. U1 RNP; b. 5' cap structure; c. polyadenylation
signal; d. secondary structure of pre-mRNA], give a brief
explanation if you are not sure your answer (5%)
U1A protein inhibits polyadenylation of its pre-mRNA
U1A protein binds U1snRNA: 7-base sequence-U1A
binding site
U1A protein inhibits polyadenylation of its pre-mRNA
U1A binding site
2 copies in its own mRNA ,
at 3' end near the poly(A) signal
prevent polyadenylation, but not the cleavage of pre-mRNA
rapidly degraded
U1A protein inhibits polyadenylation of its pre-mRNA
-
prevent polyadenylation, but not the cleavage of pre-mRNA
truncated mRNA without poly(A) tail, rapidly degraded
RNA editing in mammalian
apo-B (apolipoprotein B), serum protein of lipid
transporter,
CAA UAA, glutamine stop codon, in intestine
both liver and intestine forms transport lipid
only liver apo-B delivers cholesterol containing LDL
RNA editing in glutamate receptor mRNA
Na+, Ca+2 ion channel, learning and memory
CAG  CIG, glutamate  arginine, Ca +2 cannot pass
Both editing of apo-B and glutamate receptor by RNA deaminases
Iron dependent regulation of TfR mRNA stability.
when iron ion high, IRE -BP inactive, cannot bind IRE,
TfR mRNA is protected from degradation;
when iron is low, IRE-BP is active and binds IRE.
then TfR mRNA stability , TfR mRNA degraded
Iron dependent regulation of translation of ferritin stability.
ferritin is an intracellular protein that binds iron ions
ferritin, 5' IRE free of BP, translation occurs, more ferritin.
The IRE in ferritin mRNA has no AU rich
ferritin
Using Yeast to study Eukaryotic Gene Function
from Recombinant DNA (J Waston et al.) Ch 13
Size of genome in the selected organisms
Yeast biosynthetic genes are cloned by complementation of
E.coli mutations
Cloning yeast biosynthetic genes by complementation in
E.coli
Shuttle vectors replicate
in both E.coli and yeast
Classes of yeast vectors
Yeast genes can be
cloned by simple
complementation
Cloning a yeast gene
by complementation
Generation of temperature sensitive mutants of yeast
Replica plating
Homolgous recombination is a relative frequent event in yeast
Gene targeting by homologous recombination
Replacing a gene by transplacement
Tetrad
analysis
Cloning genes required for mating
reveals a signaling pathway similar
to that seen in higher organisms
Pheromone signaling pathway
This fragment DNA only works in high copies,
suggesting it was acting as a suppressor
Cloning of the GPA1 gene as a high-copy suppressor of pheromone sensitivity
Genetic experiments in yeast can answer precise biochemical question
The receptor swap experiment
U2 RNA base-pairs with an intron sequence
Base pairing between U2 and branch point (in yeast)
A genetic assay for protein-protein interactions
Genetic analysis
in yeast can be
exploited to
identify and study
genes from higher
organisms
Plasmid shuffle