Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant...
-
Upload
alice-lynch -
Category
Documents
-
view
218 -
download
2
Transcript of Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant...
![Page 1: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/1.jpg)
Changes to Syllabus:
Quizzes put back:Change Oct. 3 to Oct. 17Change Nov. 7 to Nov. 14
FastPlant lab delayed:Oct. 4 and 5.
![Page 2: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/2.jpg)
![Page 3: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/3.jpg)
Advantages of C. elegans:
1. rapid life cycle
2. hermaphrodite
3. prolific reproduction
4. transparent
5. only ~1000 cells
6. laser ablation
7. complete cell lineage map
8. genetics
![Page 4: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/4.jpg)
![Page 5: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/5.jpg)
![Page 6: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/6.jpg)
![Page 7: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/7.jpg)
![Page 8: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/8.jpg)
Advantages of C. elegans:
1. rapid life cycle
2. hermaphrodite
3. prolific reproduction
4. transparent
5. only ~1000 cells
6. laser ablation
7. complete cell lineage map
8. genetics
![Page 9: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/9.jpg)
![Page 10: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/10.jpg)
RNA interference:
A type of gene regulation Involving small RNA molecules and induced by double stranded RNA
![Page 11: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/11.jpg)
Adding a Chalcone synthase gene from Petunia fused to a strong viral promoterto transgenic Petunia interfered with expressionof the native homologous’gene.
Gene Silencing
![Page 12: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/12.jpg)
Gene Silencing
1.More common from strong promoters. 2. More common from inverted repeat inserts
(could make a double stranded RNA).
3. Induced by RNA viruses (have double stranded RNA replication intermediates).
![Page 13: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/13.jpg)
Tobacco plant expressing GFP proteinInfected with RNA virus with GFP geneVirus infection travels through veinsGFP expression inhibited starting at veins.
RNA viruses can block expression of a transgene if a copy of the transgene has been added
![Page 14: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/14.jpg)
Gene silencing and RNA virusesshare potential to produce dsRNA
Fire and Mello used C. elegans to prove that dsRNA inhibits expression of endogenous genes homologous to that dsRNA
![Page 15: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/15.jpg)
Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans
GFP expressed in nuclei After adding dsRNA for GFP
![Page 16: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/16.jpg)
Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans
GFP expressed in nuclei After adding dsRNA for GFP
How would you design a genetic screen to identify the genes involved in the RNAi mechanism?
![Page 17: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/17.jpg)
Features of RNAi
Induced by dsRNA with homology to exons
Catalytic: very small amounts of ds RNA are sufficient
Spreads: injection into gut silences genes in embryos
Small RNAs produced
![Page 18: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/18.jpg)
The real genetic screen for C. elegans genes essential for RNAi
![Page 19: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/19.jpg)
Mechanism of RNAi
Dicer binds dsRNAAnd cleaves makingsiRNA
siRNAs direct Risc to copy homologous mRNA
Dicer binds shortstretch of dsRNA and cleaves it.
![Page 20: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/20.jpg)
Catalysis: RdRP copies mRNA making more ds RNA. Dicer cuts that generating more siRNA
More RdRP is activated and more dsRNA is made.
Spread: dsRNA transported to other cells.Not in Drosophila or mammals
![Page 21: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/21.jpg)
What is the function of RNAi mechanism in non-transgenic organisms?
Protection against viruses
Keep Transposable elements inactive
Gene regulation
![Page 22: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/22.jpg)
Gene regulation by small RNAs
SiRNAs degrade mRNAto stop gene expression quickly
StRNAs prevent translation to stop gene expression quickly
![Page 23: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/23.jpg)
MIRNA gene
HASTY
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
AAA
Pol II
DCL1
HEN1, HYL1
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
AGO1
AGO1
CUAUAACCGCGCCGAGUUAGU
UAUUGGCCUGGUUCACUCCACU
AGO1
CUAUAACCGCGCCGAGUUAGU
AAA ..... .............
AAA
DCL1
miRNA*
RISCcomponents
RISCmiRNA
Target
Cleaved target
57 unique miRNAs from 26 families
~100 MIRNA genes**
**Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others
m7GpppN AAAAA
5’ Cap
Introns
Poly(A) tailmiRNA Precursor
Transcript
MIRNA genes are PolII genes that encode a hairpin shaped mRNA.Dicer cleaves the double stranded portion to makea short dsRNA.That combines with the RISC complex and directs cleavage of a specific target mRNA
HYL1
![Page 24: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/24.jpg)
miRNAs and Targets in Animals
C. elegans 114Drosophila 78Zebrafish 362Mouse 245Human 321
Registered miRNAs
Fig. 3 from Lewis, Burge and Bartel (2005) Cell 120, 15-20
May target 1/3 of all genes
Development - e.g. timing, stem cell function, differentiationCell and organ identityCancer - mis-regulation, deletion, duplication of MIRNA genes
HerpesvirusesSV40
![Page 25: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/25.jpg)
Transcriptional gene silencing is initiated by RNA directed methylation of promoter regions
dsRNA homologous to promoters leads to methylation and inactivation by recruitment of chromatin remodeling enzymes.
![Page 26: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/26.jpg)
Imprinting is another form of epigenetic gene regulation
ICR – imprintingControl region
Differential methylationleads to differential expression of Maternal and paternal alleles
![Page 27: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/27.jpg)
RNA is a gene regulator as well as a carrier of information
![Page 28: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/28.jpg)
Plant miRNA Targets
Animal miRNA Targets
Single sites
miRNA:target high complementary
Usually in coding region
Multiple sites, combinatorial
miRNA:target low complementary
3’ UTR region
microRNA Targets in Plants and Animals
-------lin-14 mRNA 3’UTR
AAA
AGGAAUACAGGGAGCCAGGCA || |||||||||||||||||3' ACCGUAUGUCCCUCGGUCCGU 5
-
ARF10 mRNA
miR160
UUGCAC-UCUCAGGGA |::||| ||||||||| 3’ AGUGUG AGAGUCCCU
A C U 5’ C C CU lin-4 miRNA
AAA
![Page 29: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/29.jpg)
RDR6
DCL4
AGO
AGO
CUAUAACCGCGCCGAGUUAGU
DCL2
v-siRNA*
RISCv-siRNA
|||||||||||||||||||||||||||||||||||
||||||||||| ||||||||||| |||||||||||
|||||||||||
viral RNA
RDR1
v-siRNA duplex
Antiviral RNAi - Redundancy of DCL, RDR Factors
![Page 30: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/30.jpg)
MIRNA gene
HASTY
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
AAA
Pol II
DCL1
HEN1, HYL1
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
AGO1
AGO1
CUAUAACCGCGCCGAGUUAGU
UAUUGGCCUGGUUCACUCCACU
AGO1
CUAUAACCGCGCCGAGUUAGU
AAA ..... .............
AAA
DCL1
miRNA*
RISCcomponents
RISCmiRNA
Target
Cleaved target
Pol II
RDR6, SGS3
HEN1
AGO
AGO
CUAUAACCGCGCCGAGUUAGU
AGO
CUAUAACCGCGCCGAGUUAGU
AAA ..... .............
AAA
DCL4
ta-siRNA*
RISCcomponents
RISCta-siRNA
Target
Cleaved target
|||||||||||||||||||||||||||||||||||
||||||||||| ||||||||||| |||||||||||
|||||||||||
Trans-acting siRNA gene
Vazquez et al. (2004) Mol. Cell 16, 69-79; Peragine et al. (2004) Genes Dev. 18, 2368-2379
HYL1Poethig, Vaucheret/ D. Bartel labs
![Page 31: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.](https://reader035.fdocuments.net/reader035/viewer/2022062719/56649ee75503460f94bf7f92/html5/thumbnails/31.jpg)
MIR gene
HASTY
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
5’
3’
CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U
AAA
Pol II
DCL1
HEN1, HYL1
C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U
AGO1
AGO1
CUAUAACCGCGCCGAGUUAGU
UAUUGGCCUGGUUCACUCCACU
AGO1
CUAUAACCGCGCCGAGUUAGU
AAA ..... .............
AAA
DCL1
miRNA*
RISCcomponents
RISCmiRNA
Target
Cleaved target
Pol II
RDR6, SGS3
HEN1
AGO
AGO
CUAUAACCGCGCCGAGUUAGU
AGO
CUAUAACCGCGCCGAGUUAGU
AAA ..... .............
AAA
DCL4
ta-siRNA*
RISCcomponents
RISCta-siRNA
Target
Cleaved target
|||||||||||||||||||||||||||||||||||
||||||||||| ||||||||||| |||||||||||
|||||||||||
Trans-acting siRNA gene
HYL1