The document describes experiments to identify genes that regulate miRNA activity in Arabidopsis thaliana plants. The experiments involved mutagenizing plants with T-DNA insertions, screening for mutants with altered responses to ABA and BASTA, identifying locations of T-DNA insertions, and analyzing effects on miRNA target gene expression. Several mutant lines (HOAT2-2, HOAT3-2, HOAT5-2) were identified that showed atypical responses to ABA and altered expression of the miRNA target gene GRF3, suggesting they may have mutations affecting miRNA activity.
Sequencing cannabis sativa and cannabis indica, Courtagen Life Sciences, Inc,...Copenhagenomics
1. Medicinal Genomics Corporation sequenced the genomes of several cannabis cultivars including Chemdawg and LA Confidential.
2. Analysis of the genome sequences revealed high levels of polymorphisms and identified several copies of THCA synthase genes with single nucleotide variants.
3. RNA sequencing of different tissues showed tissue-specific expression of potential cannabinoid synthase genes, including a novel root-expressed synthase with a different N-terminal domain.
4. A family tree analysis grouped the potential synthase genes into families across the different cultivars.
The document summarizes research into how mutations affecting the nucleotide state of the E. coli replication initiator protein DnaA alter its function as a transcription factor. Experiments showed that expressing a DnaA mutant (DnaAR334A) that preferentially binds ATP leads to overinitiation of replication, DNA damage, and differential expression of genes involved in DNA replication and the SOS response. In contrast, a DnaA mutant (DnaAT174P) that preferentially binds ADP showed underinitiation and different gene expression patterns. ChIP-seq and RNA-seq were used to identify direct transcriptional targets of DnaA and new genes regulated by its nucleotide state.
RT2 Profiler PCR Arrays allow researchers to analyze gene expression in biological pathways or disease states using real-time PCR. The document discusses:
1. PCR Arrays focus on profiling genes relevant to specific pathways or disease states. They provide a simple, reproducible, and sensitive way to simultaneously profile expression of many genes related to a pathway.
2. Examples are provided demonstrating how PCR Arrays have been used in cancer research to discover breast cancer biomarkers, in immunology research to monitor cytokine induction, and in toxicity research to determine drug toxicity profiles.
3. Key advantages of PCR Arrays are highlighted, including their simplicity, performance, relevance to specific pathways, and ability to analyze gene expression from small amounts
This document summarizes research into the germline-specific function of DAF-18/PTEN in the nematode C. elegans. It describes how DAF-18 is required for larval arrest in C. elegans under unfavorable conditions. The researchers found that germline-specific knockdown of DAF-18 results in defective larval arrest, and that rescuing DAF-18 function only in the germline can correct this. They aim to identify upstream regulators of DAF-18 by observing changes in DAF-18 reporter fluorescence under RNAi knockdown of candidate genes. Integrating DAF-18 specifically in the germline under a germline promoter may
This document discusses potential applications of nanotechnology in biomedicine, specifically targeted drug delivery, regenerative medicine, and medical imaging. It then summarizes research using in vivo and in vitro models to study inflammatory responses and the effects of environmental toxicants and bacterial infections on brain inflammation. This includes analyzing changes in pro-inflammatory cytokines and other markers. The document also presents a novel parallel-plate flow chamber design to model ischemia-reperfusion and analyze effects of flow on endothelial cell gene expression of inflammation markers.
1) The document describes a cell-based multi-pathway profiling array that was developed to rapidly and sensitively interrogate the functional and biological effects of siRNAs on key cell signaling pathways in vivo.
2) The array uses luciferase reporter technology to monitor the activity of 13 cell signaling pathways, including NFkB, PKC/Ca2+, Notch, Wnt/β-Catenin, cAMP/PKA, p53, E2F, TGFβ, MAPK, and JAK/STAT pathways.
3) Testing of 5 siRNAs on the array found it could provide robust data on both the specific on-pathway and off-pathway effects of siRNAs, as well as highlight potential
This document summarizes research on rust effectors and rust resistance genes in flax. It discusses how 20 flax rust resistance genes have been cloned and encode receptor components of the plant immune system. Four flax rust avirulence genes have been identified that encode small secreted proteins expressed in haustoria. Recognition of these avirulence proteins occurs through direct interaction with the corresponding resistance proteins. The document also discusses how stem rust effectors are delivered into plant cells and the potential application of knowledge from flax to cereal rust diseases.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
1) The document examines the effect of silencing the long non-coding RNA lincRNA-RoR using siRNAs delivered by polyamidoamine dendrimers on the expression of the neighboring WDR7 gene in breast cancer cells.
2) The study showed that lincRNA-RoR expression was significantly decreased after transfection with siRNA encapsulated in PAMAM dendrimers. WDR7 expression correspondingly increased significantly upon lincRNA-RoR silencing.
3) This study demonstrates that PAMAM dendrimers can effectively deliver siRNAs to silence lincRNA-RoR and upregulate the adjacent WDR7 gene, suggesting their potential as a non-viral gene therapy vector
Sequencing cannabis sativa and cannabis indica, Courtagen Life Sciences, Inc,...Copenhagenomics
1. Medicinal Genomics Corporation sequenced the genomes of several cannabis cultivars including Chemdawg and LA Confidential.
2. Analysis of the genome sequences revealed high levels of polymorphisms and identified several copies of THCA synthase genes with single nucleotide variants.
3. RNA sequencing of different tissues showed tissue-specific expression of potential cannabinoid synthase genes, including a novel root-expressed synthase with a different N-terminal domain.
4. A family tree analysis grouped the potential synthase genes into families across the different cultivars.
The document summarizes research into how mutations affecting the nucleotide state of the E. coli replication initiator protein DnaA alter its function as a transcription factor. Experiments showed that expressing a DnaA mutant (DnaAR334A) that preferentially binds ATP leads to overinitiation of replication, DNA damage, and differential expression of genes involved in DNA replication and the SOS response. In contrast, a DnaA mutant (DnaAT174P) that preferentially binds ADP showed underinitiation and different gene expression patterns. ChIP-seq and RNA-seq were used to identify direct transcriptional targets of DnaA and new genes regulated by its nucleotide state.
RT2 Profiler PCR Arrays allow researchers to analyze gene expression in biological pathways or disease states using real-time PCR. The document discusses:
1. PCR Arrays focus on profiling genes relevant to specific pathways or disease states. They provide a simple, reproducible, and sensitive way to simultaneously profile expression of many genes related to a pathway.
2. Examples are provided demonstrating how PCR Arrays have been used in cancer research to discover breast cancer biomarkers, in immunology research to monitor cytokine induction, and in toxicity research to determine drug toxicity profiles.
3. Key advantages of PCR Arrays are highlighted, including their simplicity, performance, relevance to specific pathways, and ability to analyze gene expression from small amounts
This document summarizes research into the germline-specific function of DAF-18/PTEN in the nematode C. elegans. It describes how DAF-18 is required for larval arrest in C. elegans under unfavorable conditions. The researchers found that germline-specific knockdown of DAF-18 results in defective larval arrest, and that rescuing DAF-18 function only in the germline can correct this. They aim to identify upstream regulators of DAF-18 by observing changes in DAF-18 reporter fluorescence under RNAi knockdown of candidate genes. Integrating DAF-18 specifically in the germline under a germline promoter may
This document discusses potential applications of nanotechnology in biomedicine, specifically targeted drug delivery, regenerative medicine, and medical imaging. It then summarizes research using in vivo and in vitro models to study inflammatory responses and the effects of environmental toxicants and bacterial infections on brain inflammation. This includes analyzing changes in pro-inflammatory cytokines and other markers. The document also presents a novel parallel-plate flow chamber design to model ischemia-reperfusion and analyze effects of flow on endothelial cell gene expression of inflammation markers.
1) The document describes a cell-based multi-pathway profiling array that was developed to rapidly and sensitively interrogate the functional and biological effects of siRNAs on key cell signaling pathways in vivo.
2) The array uses luciferase reporter technology to monitor the activity of 13 cell signaling pathways, including NFkB, PKC/Ca2+, Notch, Wnt/β-Catenin, cAMP/PKA, p53, E2F, TGFβ, MAPK, and JAK/STAT pathways.
3) Testing of 5 siRNAs on the array found it could provide robust data on both the specific on-pathway and off-pathway effects of siRNAs, as well as highlight potential
This document summarizes research on rust effectors and rust resistance genes in flax. It discusses how 20 flax rust resistance genes have been cloned and encode receptor components of the plant immune system. Four flax rust avirulence genes have been identified that encode small secreted proteins expressed in haustoria. Recognition of these avirulence proteins occurs through direct interaction with the corresponding resistance proteins. The document also discusses how stem rust effectors are delivered into plant cells and the potential application of knowledge from flax to cereal rust diseases.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
1) The document examines the effect of silencing the long non-coding RNA lincRNA-RoR using siRNAs delivered by polyamidoamine dendrimers on the expression of the neighboring WDR7 gene in breast cancer cells.
2) The study showed that lincRNA-RoR expression was significantly decreased after transfection with siRNA encapsulated in PAMAM dendrimers. WDR7 expression correspondingly increased significantly upon lincRNA-RoR silencing.
3) This study demonstrates that PAMAM dendrimers can effectively deliver siRNAs to silence lincRNA-RoR and upregulate the adjacent WDR7 gene, suggesting their potential as a non-viral gene therapy vector
1. The document describes a miRNA PCR array experiment to analyze miRNA expression during osteogenesis and neurogenesis differentiation. Samples were collected from human mesenchymal stem cells (hMSCs) at different time points during differentiation and analyzed in triplicate using PCR arrays.
2. The data analysis plan involves first calculating the ΔCt for each gene of interest by normalizing to housekeeping genes. The ΔCts are then averaged for each gene within each sample group. Fold changes between groups will then be determined using the ΔΔCt method to identify differentially expressed miRNAs during differentiation.
RNA is emerging as an important molecule for medical treatment. It has three main types - DNA, proteins, and RNA. Recent research has uncovered new functions of RNA, including small RNAs that can silence genes. This has led to over 4,300 publications and 200 clinical trials on microRNAs (miRNAs) and small interfering RNAs (siRNAs) in the past 8 years. Researchers are now working to develop RNA-based therapies and several startups have received large investments to pursue promising new treatments.
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
The document discusses the central dogma of molecular biology and genetic changes that cause normal cells to become cancerous. It focuses on colorectal cancer, explaining that genetic changes in oncogenes and tumor suppressor genes can lead to uncontrolled cell growth. MicroRNAs are also discussed as small, non-coding RNAs that regulate gene expression and are aberrantly expressed in colorectal cancer, influencing metastasis and invasion. Several studies investigating specific microRNAs and their roles in colorectal cancer are summarized. The document concludes by outlining future research directions, including improved detection methods and therapeutic applications of microRNAs.
MicroRNAs and Ageing
Dr Kyriacos Felekkis presented on microRNAs and ageing. The following key points were discussed:
1. MicroRNAs are small non-coding RNAs that regulate gene expression and cellular pathways involved in processes like ageing.
2. Studies in models like C. elegans and mice show certain microRNAs are up or downregulated with age and can promote or delay ageing by targeting genes in pathways like insulin/IGF signaling.
3. In humans, microRNAs like miR-34a and miR-146 are altered with age and involved in processes like stress response and oxidative damage.
4. Understanding microRNA regulation may help identify biomarkers of ageing and
Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Ser...QIAGEN
This slideshow highlights the use of miRNA mimics, inhibitors and target protectors to increase, decrease and adjust the cellular concentration of miRNA and disrupt specific miRNA–mRNA interactions. A ready-to-use screening tool for identifying miRNA targets and info on how to predict mRNA targets using miRNA expression data are also highlighted.
Gene silencing and editing techniques allow for precise regulation of gene expression. RNA interference uses small RNA molecules like siRNA and miRNA to degrade target mRNA, decreasing protein production. Genome editing tools like CRISPR/Cas9 create targeted DNA breaks that are repaired, resulting in mutations like insertions or deletions that can knock out gene function. These techniques have applications in research including studying gene function and biotechnology to modify traits in crops.
The document discusses several topics related to DNA and genetics including DNA replication, restriction enzymes, gel electrophoresis, PCR, DNA sequencing, cloning, and gene expression. It provides figures to illustrate key concepts and techniques such as how restriction enzymes cut DNA, how gel electrophoresis separates DNA fragments by size, how PCR amplifies a target DNA sequence, and the process of cloning a gene into a bacterial plasmid.
The document discusses how AP-2α induces apoptosis in cancer cells. It finds that AP-2α activates the intrinsic apoptosis pathway by binding to the Bcl-2 promoter and repressing its transcription. This allows Bax to translocate to mitochondria, disrupt membrane potential, and release cytochrome c, activating caspase-9 and caspase-3. Downregulation of anti-apoptotic Bcl-2 is important for AP-2α-induced apoptosis. Overexpressing AP-2α enhances cancer cell chemosensitivity to various drugs.
The document discusses DNA, genes, and the process of protein synthesis. It explains that DNA contains genes which code for proteins. There is a two-step process for protein synthesis: transcription copies DNA into mRNA in the nucleus, and translation uses mRNA and tRNA to assemble amino acids into proteins at the ribosome. The ribosome binds mRNA and tRNA to join amino acids together into polypeptide chains based on the genetic code, resulting in the production of proteins.
The document describes research into controlling the reproductive cycle of Azolla filiculoides, a small aquatic fern. Through next-generation sequencing of small RNA libraries from Azolla under different conditions, 15 conserved microRNAs were discovered, including miR156 and miR172. While miR172 was not found, miR156 and miR172 are known to regulate flowering time in plants and their presence in Azolla suggests they may play a role in controlling sporulation. Quantification of small RNA sequencing data found that miR156 and miR172 are present in Azolla.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
1. The document describes a miRNA PCR array experiment to analyze miRNA expression during osteogenesis and neurogenesis differentiation. Samples were collected from human mesenchymal stem cells (hMSCs) at different time points during differentiation and analyzed in triplicate using PCR arrays.
2. The data analysis plan involves first calculating the ΔCt for each gene of interest by normalizing to housekeeping genes. The ΔCts are then averaged for each gene within each sample group. Fold changes between groups will then be determined using the ΔΔCt method to identify differentially expressed miRNAs during differentiation.
RNA is emerging as an important molecule for medical treatment. It has three main types - DNA, proteins, and RNA. Recent research has uncovered new functions of RNA, including small RNAs that can silence genes. This has led to over 4,300 publications and 200 clinical trials on microRNAs (miRNAs) and small interfering RNAs (siRNAs) in the past 8 years. Researchers are now working to develop RNA-based therapies and several startups have received large investments to pursue promising new treatments.
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
The document discusses the central dogma of molecular biology and genetic changes that cause normal cells to become cancerous. It focuses on colorectal cancer, explaining that genetic changes in oncogenes and tumor suppressor genes can lead to uncontrolled cell growth. MicroRNAs are also discussed as small, non-coding RNAs that regulate gene expression and are aberrantly expressed in colorectal cancer, influencing metastasis and invasion. Several studies investigating specific microRNAs and their roles in colorectal cancer are summarized. The document concludes by outlining future research directions, including improved detection methods and therapeutic applications of microRNAs.
MicroRNAs and Ageing
Dr Kyriacos Felekkis presented on microRNAs and ageing. The following key points were discussed:
1. MicroRNAs are small non-coding RNAs that regulate gene expression and cellular pathways involved in processes like ageing.
2. Studies in models like C. elegans and mice show certain microRNAs are up or downregulated with age and can promote or delay ageing by targeting genes in pathways like insulin/IGF signaling.
3. In humans, microRNAs like miR-34a and miR-146 are altered with age and involved in processes like stress response and oxidative damage.
4. Understanding microRNA regulation may help identify biomarkers of ageing and
Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Ser...QIAGEN
This slideshow highlights the use of miRNA mimics, inhibitors and target protectors to increase, decrease and adjust the cellular concentration of miRNA and disrupt specific miRNA–mRNA interactions. A ready-to-use screening tool for identifying miRNA targets and info on how to predict mRNA targets using miRNA expression data are also highlighted.
Gene silencing and editing techniques allow for precise regulation of gene expression. RNA interference uses small RNA molecules like siRNA and miRNA to degrade target mRNA, decreasing protein production. Genome editing tools like CRISPR/Cas9 create targeted DNA breaks that are repaired, resulting in mutations like insertions or deletions that can knock out gene function. These techniques have applications in research including studying gene function and biotechnology to modify traits in crops.
The document discusses several topics related to DNA and genetics including DNA replication, restriction enzymes, gel electrophoresis, PCR, DNA sequencing, cloning, and gene expression. It provides figures to illustrate key concepts and techniques such as how restriction enzymes cut DNA, how gel electrophoresis separates DNA fragments by size, how PCR amplifies a target DNA sequence, and the process of cloning a gene into a bacterial plasmid.
The document discusses how AP-2α induces apoptosis in cancer cells. It finds that AP-2α activates the intrinsic apoptosis pathway by binding to the Bcl-2 promoter and repressing its transcription. This allows Bax to translocate to mitochondria, disrupt membrane potential, and release cytochrome c, activating caspase-9 and caspase-3. Downregulation of anti-apoptotic Bcl-2 is important for AP-2α-induced apoptosis. Overexpressing AP-2α enhances cancer cell chemosensitivity to various drugs.
The document discusses DNA, genes, and the process of protein synthesis. It explains that DNA contains genes which code for proteins. There is a two-step process for protein synthesis: transcription copies DNA into mRNA in the nucleus, and translation uses mRNA and tRNA to assemble amino acids into proteins at the ribosome. The ribosome binds mRNA and tRNA to join amino acids together into polypeptide chains based on the genetic code, resulting in the production of proteins.
The document describes research into controlling the reproductive cycle of Azolla filiculoides, a small aquatic fern. Through next-generation sequencing of small RNA libraries from Azolla under different conditions, 15 conserved microRNAs were discovered, including miR156 and miR172. While miR172 was not found, miR156 and miR172 are known to regulate flowering time in plants and their presence in Azolla suggests they may play a role in controlling sporulation. Quantification of small RNA sequencing data found that miR156 and miR172 are present in Azolla.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
Similar to miRNA Activity in Arabidopsis thaliana (6)
2. Purpose
To identify potential genes that regulate miRNA
activity, especially in regards to growth and
development.
3. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
4. either too slow (ABA hypersensitive) or too fast (ABA hyposensitive), these collection of
Mutagenize with T-DNA
mutant plants will be highly enriched in mutations that affect miRNA activity. In sum, we
will direct our studies on screening for mutations that enhance or diminish the response
of Arabidopsis plants to the plant hormone abscisic acid (ABA). This approach has
insertion
already resulted in the identification of a number of interesting genes, and will hopefully
lead to identification of a number more within this semester's class.
5. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
6. Screen using BASTA
I1. Mutagenesis strategy using T-DNA containing three tandem strong
rview of procedure Brian Gregory used to prepare the mutagenized seed th
screen. B) Cartoon depicting thegerminated is inserted into the plant genom
Ten plants T-DNA that on BASTA.
ns. Shown is a random gene that is induced by the insertion (green arrow, r
borders of the T-DNA (RB, LB, orange), the herbicide resistance gene exp
r
DNA that we will use to select mutagenized seedlings (BASTA , purple), and
enhancers (35S enhancers, red).
7. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
8. Screen using ABA
Six plants displayed mutated responses to ABA ,
specifically they displayed hypersensitive root
growth.
9. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA
phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
13. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
14. Find location of T-DNA
insertion
TAIL PCR was
performed.
AD1/AD2
This gel shows
that AD primers
AD2
are about 100 bps.
15. Find location of T-DNA
insertion
TAIL PCR product for
HOAT2-2AD1
HOAT2-2AD2
HOAT2-2AD3
HOAT2-2AD2 and
HOAT3-2AD1
HOAT3-2AD2
HOAT3-2AD3
HOAT5-2AD1
HOAT3-2AD2 are
about 750 bps.
Lower bands are AD
primers.
16. Find location of T-DNA
insertion
Heavy upper bands could be
HOAT5-2AD2
HOAT5-2AD3
due to precipitated DNA.
Lower bands are AD primers.
17. Find location of T-DNA
insertion
DNA sequence analysis
performed.
HOAT3-2:
Had greater or equal to 200
as alignment score.
Expected value was 0.0.
18. Find location of T-DNA
insertion
AT3G09720 codes for a
upstream
protein with a p-loop.
Binds nucleotides and
upstream
ATP
Helicase functions
downstream
AT3G09730 has no
known function.
19. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes
are affected
Germination assay
BASTA assay
20. Determine whether miRNA
target genes are affected
GRF3 is a known target of miRNA.
Look at amount of mRNA transcribed from GRF3 to
determine if miRNA activity is potentially altered.
21. Determine whether miRNA
target genes are affected
HOAT2-2
HOAT5-2
HOAT3-2
Col-0
Non-degraded RNA has
the following bands: 28s
rRNA and 18s rRNA.
28s rRNA
Create cDNA for HOAT3-2 18s rRNA
and HOAT5-2.
28. Germination Assay
0.5 μM ABA • Mimics 0 ABA control
100
90 • T-value of 0.5275
80
70
Percent
60 • Don’t see too much
50
Germination
40
30 from this plate
20
10
0
wt 2-2 3-2 5-2
Experimental
30. Germination Assay
1.5 μM ABA • Similar to 1.0 μM ABA
100
90 • 3-2, 5-2
80
70
60
Percent
50
Germination
40
30
20
10
0
wt 2-2 3-2 5-2
Experimental
31. Germination Assay
Seeds from HOAT 2-2 may have a T-DNA
insertion but are not hyper/hyposensitive to ABA!
32. Overview
To identify those potential genes:
Mutagenize with T-DNA insertions
Screen using BASTA
Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay
BASTA assay
33. BASTA Assay - Theory
HYPOTHESIS: ABA hyper/hyposensitivity
caused by T-DNA insertion.
GIVEN:
T-DNA insertion confers BASTA resistance
ABA-hypersensitive plants on BASTA – should all
have T-DNA insertion
Plated surviving plants from ABA treatment onto
BASTA plates
34. BASTA Assay
Survival on BASTA • Data does not confirm
100
80 theory
60
Percent Col-0 • Most surviving plants
Survival 40 HOAT 2-2
HOAT 3-2
20 have BASTA resistance
HOAT 5-2
0
0.5 1 1.5
[ABA] in μM
35. BASTA Assay - Conclusions
Seeds from HOAT 2-2 HAVE a T-DNA insertion
but are not hyper/hyposensitive to ABA!
Contrary to original screen
2-2 originally identified as hypersensitive
36. Possible Explanations
Chance from first hyper/hyposensitivity assay
(n = 1)
NOT chance from this experiment
n = large (despite contamination)
Possibly growth conditions
BOTTOM LINE: We have a lot of factors at work
here that we don’t know about!
37. Possible Explanations
Possible interaction
Survival on BASTA
100
between ABA and
80
Percent
60
Col-0
BASTA
Survival 40 HOAT 2-2
HOAT 3-2
20
HOAT 5-2
0
0.5 1 1.5
[ABA] in μM
39. Conclusions
HOAT 3-2, HOAT 5-2
Increase in GRF3 expression – KNOWN miR396-
REGULATED GENE
Decrease in ATG09720 gene expression T-DNA
insertion inside gene.
Future work: How do changes in these gene
expressions explain original phenotype?
40. References
Earley, et al., An endogenous F-box protein regulates
ARGONAUTE1 in Arabidopsis thaliana Silence 2010, 1:15
Rodriguez RE, Mecchia MA, Debernardi JM, Schommer
C, Weigel D, Palatnik JF. 2010. Control of cell proliferation in
Arabidopsis thaliana by microRNA miR396. Development
137, 103–112.
Dr. John Wagner, personal communication.
Allison Lesher, personal communication.
NCBI BLAST database: gene identification
TAIR database: gene information
Editor's Notes
Should not be severalleaves near cauline leaf (should only be one)
Add circles for the HOAT2-2 and 3-2 for AD2
Fix 9720
SEEDS FROM
Conf. level -> same as control?Null hypothesis same as control. T-stat does not disprove null hypothesis. Also can examine by visual examination – same as 0 ABA.SEEDS FROM
SEEDS FROM
SEEDS FROM
We know it has T-DNA insertion from
If we were to plate…All plants having hypersensitive ABA phenotype are now dead. According to this theory their phenotypes were caused by T-DNA insertions.Remainder should not have T-DNA insertions and thus should not survive on BASTA.
2152643099
1st: process
Akin to giving a sick person and a healthy person the same vaccine
1a) original phenotype was not seen in the most recent germination/BASTA assay