Lecture 5
Article 1
- Briefly describe the experimental approach that is used to create a
gene expression map of the Arabidopsis root. With microarray you
can measure the expression of genes, they make protoplasts, cellwall is
removed. You get a mixture of cells and they sort the cells the cells are
labelled with GFP. You have to know the promoter and than GFP fusion.
Collect mRNA of cells and do a sequence. Using the root you can find
different developmental stages.
- Can you use this approach for an organism whose genome has not
been sequenced? Explain no, you can’t use this approach for an organism
whose genome has not been sequenced. You can’t make a microarray
without a sequence.
- And for an organism for which only a genome sequence is
available? Only a genome sequence available, that can be annotated but
you don’t have the tools difficult.
2 keysteps in the approach: 1. You have to know the genome/gene. 2. You need to
have plants that express GFP in a specific site.
- What other disadvantage does this method have? Sorting process is
disadvantage; you have to add enzymes that solve the tissue.
- Why is the output of this method called a digital in situ? It is called
digital in situ because it is expressed in a certain place at a certain time.
- How did they determine an error rate of 5.5%? Error rate is
calculated by false positives and by false negatives by comparing new data
with old date.
- In how many subzones was the root divided and how many genes
were differentially regulated across these sub zones? 15
subzones
- Why is LED 8 significantly overrepresented with genes involved in
nuclear organization, cell cycle, and mitosis (fig 3A/B)? With LED 8
all genes are in stage 1, these are overrepresented with genes in nuclear
organization and mitosis. This means that they are overexpressed in that
stage.
- Alternative for micro-array? Alternative for micro-array is RNA
sequencing. Here you have more reads that are higher expressed. Other cell
type profiling methods are for example isolating different types of tissue
with laser dissection;
1. Label the nuclei of the cell by making a fusion of GFP with protein that
goes to the nucleus. Isolate nuclei, you don’t have to sort them.
2. Label the ribosomes in that specific tissue, you can sequence the mRNA that is
made in the ribosome
, Article 2
- What is epigenome profiling? Epigenome; mutilation of DNA and
histones. Profiling; finding out what type of methylation occurs.
- What is chromatin immune-precipitation? You catch antibodies to
detect the mixture of protein and DNA
- What is a whole genome-tiling array? You make a micro-array that
covers the whole genome.
- How do the authors obtain the DNA that is used to hybridize to the
microarray? They take material from mixture of cells and isolate the
nucleus from the tissue. You have to shear the DNA (first crosslinking) ant
then add antibodies.
- Which two chromatin marks are mainly associated with
heterochromatin, and which 3 marks are found in both euchromatin
and heterochromatin? (Fig1A) chromatin marks associated with
heterochromatin are H4K20me1 and H3K9me2. Chromatin marks associated
both in euchromatin and heterochramatin are 5mC, H3K27me1 H3K27me2
and H3.
- Which of the chromatin states is mainly associated with TE’s, and
which two chromatin marks are mainly found associated with this
state? (Fig 2A/B)
- What is the relevance of epigenome profiling?
Article 3
- What is the function of AP1? AP1 is a transcription factor that regulates
flower development.
- What are the two major experimental approaches used in this
paper? 1. Micro-array to find genes. 2. Combine data by finding out
where it binds
- By which transcription factors is the AP1 gene regulated? Several
transcription factors have been identified that bind directly to the AP1
promoter and control the onset of its expression. These include the floral
meristem identity factor LEAFY (LFY), the basic leucine zipper (bZIP) protein
Article 1
- Briefly describe the experimental approach that is used to create a
gene expression map of the Arabidopsis root. With microarray you
can measure the expression of genes, they make protoplasts, cellwall is
removed. You get a mixture of cells and they sort the cells the cells are
labelled with GFP. You have to know the promoter and than GFP fusion.
Collect mRNA of cells and do a sequence. Using the root you can find
different developmental stages.
- Can you use this approach for an organism whose genome has not
been sequenced? Explain no, you can’t use this approach for an organism
whose genome has not been sequenced. You can’t make a microarray
without a sequence.
- And for an organism for which only a genome sequence is
available? Only a genome sequence available, that can be annotated but
you don’t have the tools difficult.
2 keysteps in the approach: 1. You have to know the genome/gene. 2. You need to
have plants that express GFP in a specific site.
- What other disadvantage does this method have? Sorting process is
disadvantage; you have to add enzymes that solve the tissue.
- Why is the output of this method called a digital in situ? It is called
digital in situ because it is expressed in a certain place at a certain time.
- How did they determine an error rate of 5.5%? Error rate is
calculated by false positives and by false negatives by comparing new data
with old date.
- In how many subzones was the root divided and how many genes
were differentially regulated across these sub zones? 15
subzones
- Why is LED 8 significantly overrepresented with genes involved in
nuclear organization, cell cycle, and mitosis (fig 3A/B)? With LED 8
all genes are in stage 1, these are overrepresented with genes in nuclear
organization and mitosis. This means that they are overexpressed in that
stage.
- Alternative for micro-array? Alternative for micro-array is RNA
sequencing. Here you have more reads that are higher expressed. Other cell
type profiling methods are for example isolating different types of tissue
with laser dissection;
1. Label the nuclei of the cell by making a fusion of GFP with protein that
goes to the nucleus. Isolate nuclei, you don’t have to sort them.
2. Label the ribosomes in that specific tissue, you can sequence the mRNA that is
made in the ribosome
, Article 2
- What is epigenome profiling? Epigenome; mutilation of DNA and
histones. Profiling; finding out what type of methylation occurs.
- What is chromatin immune-precipitation? You catch antibodies to
detect the mixture of protein and DNA
- What is a whole genome-tiling array? You make a micro-array that
covers the whole genome.
- How do the authors obtain the DNA that is used to hybridize to the
microarray? They take material from mixture of cells and isolate the
nucleus from the tissue. You have to shear the DNA (first crosslinking) ant
then add antibodies.
- Which two chromatin marks are mainly associated with
heterochromatin, and which 3 marks are found in both euchromatin
and heterochromatin? (Fig1A) chromatin marks associated with
heterochromatin are H4K20me1 and H3K9me2. Chromatin marks associated
both in euchromatin and heterochramatin are 5mC, H3K27me1 H3K27me2
and H3.
- Which of the chromatin states is mainly associated with TE’s, and
which two chromatin marks are mainly found associated with this
state? (Fig 2A/B)
- What is the relevance of epigenome profiling?
Article 3
- What is the function of AP1? AP1 is a transcription factor that regulates
flower development.
- What are the two major experimental approaches used in this
paper? 1. Micro-array to find genes. 2. Combine data by finding out
where it binds
- By which transcription factors is the AP1 gene regulated? Several
transcription factors have been identified that bind directly to the AP1
promoter and control the onset of its expression. These include the floral
meristem identity factor LEAFY (LFY), the basic leucine zipper (bZIP) protein
