Cycle 2: Central Dogma Flashcards
What makes the difference between different types of cells?
gene expression, not different genes, genes are identical
What is the use of RNA Blot Analysis?
measure mRNA (transcript) abundance in different media
How is RNA Blot analysis performed?
- cells are grown in different media
- purify mRNA from cells
- run them in a gel in an electrophoresis gel which seperates the mRNA types to find the desired gene
- the gel has a membrane/paper on top and transfer (wick) the mRNA onto the membrane
- put membrane in bag with DNA probe (segment extracted from gene)
- single strand DNA labeled with fluorescence put in bag so that it comp pairs with the gene transcript
- allow bag to set for several hours to allow DNA to complementarily pair with RNA
- place X-ray film on top (seen from fluorescence
- dark spots represent binding of DNA with the immobilized RNA
- dark spot/larger = greater is the level of transcription of gene
What is a gene?
DNA sequence that is copied into RNA
What are the RNA types? how much of each is present?
messenger RNA - 5%
transfer RNA - 10%
ribosomal RNA - 85%
What is the function of messenger RNA?
used to synthesize proteins - instructions
What is the ratio of rRNA to protein?
2/3 RNA, 1/3 protein
how much energy in the cell is used by protein synthesis from ribosomes?
60%
is transcription due to convergent evolution?
no! it is a highly conserved process across all forms of life
What is transcription? Where does it occur?
DNA information copied into RNA (tRNA, mRNA, or rRNA) in nucleus
what determines the transcript abundance?
rate of transcription compared to half life of RNA/rate of RNA breakdown
what determines the protein abundance?
rate of protein synthesis compared to protein degradation
What is translation?
ribosomes take mRNA outside the nucleus and turn them to polypeptides
what is the difference between polypeptides and proteins?
polypeptide is linear
when folded to 3D structure it becomes functional –> protein
Which is more easily degraded: RNA or DNA? Why?
RNA
–> RNA is more reactive
–> ribonucleases
Why is RNA more reactive?
ribose sugar has an OH group and DEOXYribose sugar does not have oxygen = more reactive with oxygen = O wants to bond with phosphate group = cuts in the backbone = RNA gets cut into smaller pieces = RNA degradation
What are ribonuclease?
enzyme that catalyzes break down of RNA
Why is it good that mRNA breaks down?
temporal regulation of gene expression (you don’t always need mRNA so timing is tight so energy isn’t wasted)
Which molecules are present in DNA and RNA and also in TAP?
nitrogen (bases) phosphorus (phosphate group)
Why is it important to make a gel of total RNA prior to experimentation?
check RNA degradation
How does an intact RNA gel compare with a degraded rNA gel?
intact = bright bands of rRNA (mRNA is only 5% so it is not shown)
degraded = all dark bands
What is heat shock?
cellular protective mechanism activated when an organism is exposed to high temperatures
–> highly conserved response
How is heat shock tested?
chlamy cells harvested at rm temp and isolated - some is kept separate and the temp of the rest is increased to 40ºC and cells are harvested at various time points and RNA is isolated
what is the heat shock protein?
molecular “chaperone” protein that helps other proteins stay functional at high temperatures and is not itself harmed by heat
What are the three types of gene expression?
constitutive - doesn’t change
induced - increased transcript/protein abundance
repressed = genes switched off
What is actin?
a constitutive protein that doesn’t fluctuate with stress, keeps the cell working, and is necessary for mitochondria, nucleus and gene expression
What did the results of the heat shock experiment show about the heat shock protein?
the number of heat shock mRNA floating outside the nucleus increased (transcript abundance) until 4 hours and then decreased
the number of heat shock PROTEIN increased just after the mRNA (protein abundance)
What is omics?
characterization of groups of molecules that provides a more global picture to better understand biological processes
What are genomics, transcriptomics, proteomics, metabolomics? Which are more useful?
G - studying genome
T - mRNA/transcript abundance
P - protein aduncance
M - molecules - ATP, hormones
P and M are more useful bc they relate to function
What is the function of a promoter?
controls transcription of RNA but isn’t part of RNA so its doesn’t get transcribed itself
what are transcription factors?
positively or negatively impact the rate of transcription by binding to promoter (from basal to increased expression)
what are translation factors?
binds to RNA and slows down the rate at which mRNA breaks down (regulates mRNA stability)
How does the half life of mRNA compare to protein?
protein is much more than mRNA
What are the possible genes defective in the TB12 mutation (that causes blindness in mice)?
DNA mutation to gene that codes opsin protein
What does the TB12 mutation cause?
much less rhodopsin molecule/rod cell = blindness
is there a gene for retinal that could cause blindness?
no, retinal is not a protein, it is a cofactor
How can retinal cause blindness since it is a cofactor?
enzymes (proteins) that breaks down beta carotene to retinal have a gene mutation which causes defect in retinal after translation
How do you identify the gene(s) responsible for a particular phenotype?
through a gene’s mutant phenotype
What is the issue with using mutant phenotypes to figure out normal phenotypes through genes?
naturally occurring mutants are rare
How is the fact that naturally occurring mutants are rare fixed?
implement mutations purposefully
What is forward genetics?
use phenotype to figure out the mutated gene (insertional mutagenesis)
What is reverse genetics?
intentionally make a mutation and put the gene back in the organism to see its effect through RNA interference/CRISPR
What is insertional mutagenesis?
produce a population of mutagenized organisms by randomly inserting a site of a gene that has its own promotor
Why is only one site of gene insertion allowed?
if there are two sites, it is difficult to tell which gene is causing the phenotypic expression
Where does the gene inserted go>
randomly destroys the section of the wild-type gene it takes the place of
Can the cell die from an insertion of a gene?
Yes; if the randomly inserted mutagen gene has disrupted a gene that is essential for life
What are the steps for forward genetics?
- generate a mutagenized population of cells
- screen the mutant population
- identify mutated gene
- rescue the mutant
How is a mutagenized population of cells generated?
- WT cells electroporated (high voltage electric charge) to insert DNA of antibiotic resistant gene (bleomycin-resistance)
- agar plate with bleomycin containing WT and mutagenized cells allowed to rest for 2 weeks
3. WT cells have died and mutant cells survived
how is the mutant population screened?
with a 96-well plate the mutagenized cell are plated with one row of WT cells and one row of mutant that was previously characterized for comparison
- take the cells that match the mutant and see where their site of infection is
How do you reuse that mutant?
need to prove that the gene you believed is mutated has caused the phenotype of flagella defect by taking the gene in the wild type of flagella development back into mutant and it should develop flagella properly and have antibiotic resistance
