Cycle 2: The Central Dogma of Molecular Biology Flashcards
(40 cards)
Purpose of RNA blot analysis…what is it you’re trying to show
RNA blot analysis, or Northern blotting, is used to check whether a certain gene in DNA has been transcribed into a corresponding mRNA pool, by checking whether the mRNA pool exists - essentially to determine whether gene expression has occured for a particular gene.
What are the steps to RNA blot analysis?
1) Extraction and purification of mRNA using electrophoresis (to separate fragments based on size)
2) Transfer mRNA from gel to piece of membrane
3) Mix membrane with [specific gene] DNA probes in solution to check for [specific gene] mRNA
4) X-ray film laid down on membrane / membrane viewed under fluorescence microscope - [specific gene] mRNA shows up as a dark blob whose thickness and density indicates the aount of [specific gene] mRNA present
Importance of the probe being “labelled” and single stranded.
The probes are labelled with fluorescence/radioactivity so that they will be visible on xRay film or under a fluorescence microscope.
The probes are denatured into single-stranded form so that they are capable of complementary base pairing with mRNA (marked for later analysis).
Concept of “complementary base pairing” this occurs between the single-stranded DNA probe and the RNA molecules (the GAL transcript) stuck on the ‘membrane”.
The nucleotides on the single-stranded DNA probes pair with their complementary bases on the RNA molecules via hydrogen bonding.
Complementary base-pairing is due to ____________ …have a basic understanding of that type of chemical bonding (see page F-12 in the ebook).
Hydrogen bonding, the interaction involving a hydrogen atom located between a pair of other atoms having a high affinity for electrons (very electronegative, i.e. N, O, F)
Definition of the term: GENE
A DNA sequence that is copied/transcribed into RNA.
Different types of RNA: mRNA, tRNA and rRNA and relative abundances.
mRNA (messenger, series of instruction to make protein) - 5%
tRNA (transfer, key role in protein synthesis) - 10%
rRNA (ribosomal, protein synthesis in ribosomes) - 85%
tRNA and rRNA are functional as themselves.
Information flow from DNA to RNA to Protein
DNA (Carries information) TO RNA (infomation + structure/function) TO Protein (form structure and function of cells)
Understand the role of hydrogen bonding in DNA versus RNA.
DNA has no atoms available for H bonding with other DNA molecules (complementary base pairing within the molecule is achieved through H bonding though), and so it can’t fold into 3D shapes. RNA is single stranded so it can form H bonds with itself, folding upon itself and acquiring 3D shapes.
Why we think RNA was the first of these three molecules to evolve.
RNA is the compromise and jack of all trades between DNA and protein.
Because RNA is capable of conveying both information AND having structure/function (they can acquire shapes via folding) but MOSTLY due to the discovery of RIBOZYMES, which are a class of RNA molecules that are CATALYTIC like enzymes!
Ribozymes are thought to be the very first catalytic molecules.
Promoter vs transcriptional unit and the factors that act at the level of transcription that alter transcript abundance.
The promoter is the regulatory part of the gene attached to the transcriptional unit. Without transcription factors, you get basal expression of the gene. With transcription factors (they interact with RNA polymerase to influence rate of transcription), you can induce expression (increase) or inhibit expression (decrease).
Factors (proteins) that act at the level of mRNA stability that alter transcript abundance.
Factors can also influence the rate of mRNA decay. Factors that cause increased stability results in increased expression and vice versa: factors that cause decreased stability results in decreased expression.
Understanding of the link between genes and biochemical pathways (e.g. retinal, chlorophyll, testosterone biosynthesis. [TB12 Mice]
Defective/mutated genes not only influence the synthesization of biological proteins (e.g. opsin), but also that of the enzymes that catalyze biochemical/biosynthetic pathways (that produce pigments, such as retinal).
Concept of post-translational regulation.
Post-translational regulation applies to proteins that require a non-protein (COFACTOR) to achieve its function.
Remind yourself of what we mean by the words genotype and phenotype.
Genotype: Actual changes in the genetic code of the specimen
Phenotype: Clinical/research presentation of specimen
Forward vs Reverse genetics…how are these approaches different.
Forward genetics: figuring out genetics from PHENOTYPE to GENOTYPE (e.g. cystic fibrosis, where patients’ DNA were analyzed to find common gene causing disease)
Reverse genetics: figuring out genetics from GENOTYPE to PHENOTYPE (i.e. altering a given gene to see if/how it changes phenotype)
Basics of reverse genetics
Intentionally mutate specific gene + Add to genetic code of specimen
e.g. RNA interference, CRISPR
Idea behind Insertional Mutagenesis as a forward genetics approach
Forward genetics DEPENDS on having a large study population with a specific mutant phenotype to be able to analyze their genotypes.
Insertional mutagenesis SOLVES this by intentionally mutagenizing specimen (based on the underlying genotype of a mutant) - a WT genome is inserted with an insertional mutagen to produce mutated genome.
IMPORTANT: There is usually only 1 insertion site and its location is random!!
4 steps needed for an Insertional Mutagenesis project.
-
GENERATE A MUTAGENIZED POPULATION OF CELLS.
(e.g. Insertional mutagen encoded for antibiotic resistance added to WT cells through electroporation, Chlamy cell opens and mutagen is inserted - very low prob 1%. WT and mutated cells added to plate, WT cells die off, left with mutated cells.) - SCREEN MUTANT POPULATION
- IDENTIFY MUTATED GENE USING PCR
- PROVE GENE CAUSES MUTATION (rescue the mutant)
Two: Screening for mutants in the phenotype you’re interested in (e.g. lack of flagella)
- Add Chlamy to wells, such that each well has a separate colony with a unique Chlamy mutation
- Check each well for expected phenotype, compare to mutated cells in previously chracterized {flagella} gene (isolate mutants for specific quality)
One: Generating a mutagenized population….you need to understand this.
- Insertional mutagen encoded for antibiotic resistance added to WT cells through electroporation
- Chlamy cell opens and mutagen is inserted - very low prob 1%
- WT and mutated cells added to plate,
- WT cells die off, left with mutated cells.
You need a “good screen”. That is the mutant phenotype is easily determined…this enables the researcher to assay 1,000s of mutants for a particular phenotype quite rapidly.
Screen must be EASY and QUICK to check
- Flagella, cells with WT flagella clumb at the bottom of cells (small controlled blob of green) and mutated cells look cloudy
- Chlorophyll, WT green and mutated not
Three: Determining the site of insertion (pretty easy…although I don’t tell you how).
Gene isolated using polymerase chain reaction (PCR), since insertional mutagen genetic code is known and can be used to determine sequence of neighbouring DNA.
Four: Rescuing the mutant phenotype…(also called GENETIC COMPLEMENTATION)…why is this important.
This step unequivocally proves that the interrupted gene caused the mutation (and not another random gene!)
Insert interrupted gene identified through PCR and sourced from WT cell to the mutant cell. If the mutant + WT gene presents a WT phenotype and not mutant phenotype, then proved!
