Cycle 2 BMP Workshop Flashcards
Define:
Gene expression
Process when a gene is turned into a function
How does a gene become an RNA transcript?
Transcription
How does an RNA transcript become a protein?
Translation
Describe:
Northern Blot Technique
Tells us if a gene is transcribed (mRNA)
Explain:
The acronym SNOW DROP
Southern Blot - DNA
Northern Blot - RNA
OOOOOOOO - OOO
Western Blot - Protein
Describe:
Steps of Northern Blot
- Extract mRNA
- Gel Electrophoresis (separate mRNA strands by size)
- Transfer gel to a membrane
- Probe the membrane
- Visualize the probes
True or False:
In Gel Electrophoresis, the smaller mRNAs will move to negative end faster
False, the smaller mRnas will move to positive end faster/further than the larger mRNAs
Describe:
Step 3 in Northern Blot
Step 3: Transfer gel to a membrane (since it’s hard to see all mRNA on the gel as it is so thick)
* Place gel in salt solution
* Wick all the mRNAs from gel
* Get onto membrane
Describe:
Step 4 of Northern Blot
Step 4: Probe the membrane
* Use single-stranded DNA probes with fluorescent radioactive label (denatured to become single strand)
* Mix DNA probes with the membrane
* ssDNA probes will complementary base-pair with GAL mRNA
How is the fluorescent/radiactive label transferred in Northern Blot?
By using an x-ray film
True or False:
Darker lines in Northern Blot means lots of transcript abundance
True
Define:
Gene
A DNA sequence that codes for an RNA strand
True or False:
All genes code for mRNA
False, also codes for rRNA and tRNA
Which RNA codes for proteins?
Only mRNA
True or False:
mRNA is the largest portion of RNA in the cell
False, it is the smallest portion of RNA in the cell
How does information transfer occur?
Information in DNA is transribed into RNA and then mRNA is translated into proteins
What proteins are involved in each step of information transfer?
Enzymes
What shape is DNA? How is this achieved?
A double helix, formed by hydrogen bonding
What shape is RNA? How does it achieve this?
Can be various shapes, can bond with itself using hydrogen bonding to form these shapes
What shapes are proteins?
Varies; depends on function
What is generally thought to have evolved first?
RNA
Define:
Ribozyme
A class of catalytic RNA molecules
What are two examples of ribozymes?
Ribosome: Key organelle that plays a role in the synthesis of proteins
* 2/3 rRNA and 1/3 Protein
Spliceosomes: Involved in removing introns
Define:
Transcription
DNA to RNA
Define:
Translation
mRNA to Protein
What are the 2 ways to measure gene expression?
- Transcript Abundance: How much mRNA corresponding to a specific gene is in the cell
- Protein Abundance: How much of a protein is in the cell
How is transcript abundance measured?
Northern Blots (RNA blot hybridization)
Transcription rate and rate of mRNA breakdown
How is protein abundance measured?
Western Blots
Translation rate and rate of protein breakdown
State the similarities and differences between:
DNA and RNA
Similarities
* Polymers of nucleotides
* Carriers of information
Differences
* DNA contains a deoxyribose sugar; RNA contains a ribose sugar
* DNA does not have OH at carbon 2
* RNA is less stable and therefore more reactive
* RNA is also attacked by enzymes called ribonucleases
Describe:
A Nucleotide
5 Carbon sugar + Phosphate + Nitrogenous base
Why is it good that RNA degrades more quickly than DNA?
Good temporal regulation
We do not need mRNA present all the time if unnecessary as this is a waste of cellular resources
True or False:
RNA degrades quickly, so we need to ensure than RNA samples are intact prior to experimentation
True
Define:
3 types of expression
- Induced: Up-regulation in gene expression
- Constitutive: Expression of the protein or transcript will always be the same
- Repressed: Down-regulation in gene expression
State:
The groups of “omics”
Genomics
Transcriptomics
Proteonomics
Metabolomics
What is the purpose of “omics”?
Allows us to look at whole genome, transcriptome, proteome etc.
In gel images:
What is the RNA and how can we tell size?
The glowing rods are RNA, and the further down the strip the smaller it is
How is homology proven using molecular info?
Look at the genes that make up each homologous structure
State the types of:
Expression at level of transcription
- Basal expression: Normal level
- Increased expression: Transcription factors enhance
- Decreased expression: Transcription factors inhibit
State the types of:
Stability at level of mRNA
- Basal stability
- Increased stability, factors bind to end of mRNA (stabilizing it)
- Decreased stability, factors bind to end of mRNA (destabilizing it)
What is the difference between regulation at level of promoter and regulation by stability?
Regulation at the level of promoter controls transcription
Regulation by stability is at level of mRNA, independent of transcription
What are transcription factors?
Proteins, binds to DNA promoter to inhibit/promote expression
What binds to the promoter of DNA?
RNA polymerase
What is the half-life of mammalian mRNA? Protein?
mRNA: about 9 hrs
Protein: about 46 hrs
What is post-translational modification?
Modifications made after translation to make the protein functional
What is an example of post-translational modification?
- When opsin binds to retinal, it has been modified
- Functional rhodopsin protein has formed
- Remember, retinal is not a protein therefore it was not coded from a gene!
How do we link a genotype to a phenotype?
We mutate the gene and see what happens to the phenotype
Compare:
Forward genetics and Reverse genetics
Forward genetics: We start with a phenotype, and identify the underlying gene
Reverse genetics: We start with a specific gene which is altered, and identify the phenotype
How do we generate a population of mutants?
- Insert a gene randomly into the genomes of organisms
- Use methods to get the phenotype of interest
How does one prove a gene is responsible for a phenotype?
After conducting knocking out gene, we rescue the mutant by putting the wild-type copy of the gene back into the mutant
If that gene is responsible for the mutant phenotype, we should get a wild-type phenotype when we rescue it
