Cycle 2: The Central Dogma of Molecular Biology

Question 1

Purpose of RNA blot analysis…what is it you’re trying to show

Answer 1

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.

Question 2

What are the steps to RNA blot analysis?

Answer 2

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

Question 3

Importance of the probe being “labelled” and single stranded.

Answer 3

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).

Question 4

Concept of “complementary base pairing” this occurs between the single-stranded DNA probe and the RNA molecules (the GAL transcript) stuck on the ‘membrane”.

Answer 4

The nucleotides on the single-stranded DNA probes pair with their complementary bases on the RNA molecules via hydrogen bonding.

Question 5

Complementary base-pairing is due to ____________ …have a basic understanding of that type of chemical bonding (see page F-12 in the ebook).

Answer 5

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)

Question 6

Definition of the term: GENE

Answer 6

A DNA sequence that is copied/transcribed into RNA.

Question 7

Different types of RNA: mRNA, tRNA and rRNA and relative abundances.

Answer 7

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.

Question 8

Information flow from DNA to RNA to Protein

Answer 8

DNA (Carries information) TO RNA (infomation + structure/function) TO Protein (form structure and function of cells)

Question 9

Understand the role of hydrogen bonding in DNA versus RNA.

Answer 9

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.

Question 10

Why we think RNA was the first of these three molecules to evolve.

Answer 10

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.

Question 11

Promoter vs transcriptional unit and the factors that act at the level of transcription that alter transcript abundance.

Answer 11

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).

Question 12

Factors (proteins) that act at the level of mRNA stability that alter transcript abundance.

Answer 12

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.

Question 13

Understanding of the link between genes and biochemical pathways (e.g. retinal, chlorophyll, testosterone biosynthesis. [TB12 Mice]

Answer 13

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).

Question 14

Concept of post-translational regulation.

Answer 14

Post-translational regulation applies to proteins that require a non-protein (COFACTOR) to achieve its function.

Question 15

Remind yourself of what we mean by the words genotype and phenotype.

Answer 15

Genotype: Actual changes in the genetic code of the specimen

Phenotype: Clinical/research presentation of specimen

Question 16

Forward vs Reverse genetics…how are these approaches different.

Answer 16

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)

Question 17

Basics of reverse genetics

Answer 17

Intentionally mutate specific gene + Add to genetic code of specimen
e.g. RNA interference, CRISPR

Question 18

Idea behind Insertional Mutagenesis as a forward genetics approach

Answer 18

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!!

Question 19

4 steps needed for an Insertional Mutagenesis project.

Answer 19

1. 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.)
2. SCREEN MUTANT POPULATION
3. IDENTIFY MUTATED GENE USING PCR
4. PROVE GENE CAUSES MUTATION (rescue the mutant)

Question 19

Two: Screening for mutants in the phenotype you’re interested in (e.g. lack of flagella)

Answer 19

• 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)

Question 19

One: Generating a mutagenized population….you need to understand this.

Answer 19

• 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.

Question 20

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.

Answer 20

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

Question 21

Three: Determining the site of insertion (pretty easy…although I don’t tell you how).

Answer 21

Gene isolated using polymerase chain reaction (PCR), since insertional mutagen genetic code is known and can be used to determine sequence of neighbouring DNA.

Question 22

Four: Rescuing the mutant phenotype…(also called GENETIC COMPLEMENTATION)…why is this important.

Answer 22

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!

Question 23

What’s a ribozyme and the two examples

Answer 23

Ribozymes are a class of RNA molecules that are catalytic like enzymes.

Example 1: RIBOSOMAL RNA, the centre of protein synthesis is a ribozyme. 2/3 of ribosome is rRNA.

Example 2: SPLICEOSOME, splices introns driven by a ribozyme (RNA!)

Question 24

Gene expression: transcription, translation, mRNA and protein breakdown (decay)

Answer 24

Gene expression is highly regulated at many levels and is highly conserved across all forms of life. It constitutes the FLOW OF INFORMATION from gene to transcript to protein, or from DNA which undergoes TRANSCRIPTION to form an mRNA pool which is then TRANSLATED into protein.

Over time, mRNA and proteins decay!

Applies to mRNA as well as rRNA!!

Question 25

What processes determine the amount of a particular mRNA or protein that is in a cell at a specific time?

Answer 25

Transcript abundance, or the amount of mRNA in a cell at a given time, depends on the rate of mRNA TRANSCRIPTION AND the rate of mRNA degradation.
Protein abundance depends on the rate of TRANSLATION AND the rate of protein degradation.

Question 26

Structural differences between DNA and RNA

Answer 26

DNA - double helix, deoxyribose (deoxy means lack of oxygen, carbon 2)
RNA - single strand, ribose

Question 27

Two reasons RNA is less stable than DNA…be able to explain “more reactive” with DNA vs RNA structural differences.

Answer 27

1) RNA is more REACTIVE since it is composed of ribose instead of deoxyribose, such that it has a hydroxyl group attached to its 2nd carbon instead of DNA’s hydrogen atom. This increases its susceptibility to hydrolysis. If it bonds to the P, it cuts the RNA strand and renders it useless - breaks sugar-phosphate backbone.
2) RNA may be degraded by ribonucleases (enzyme)

Question 28

Why is it a good thing that mRNA degrades rapidly.

Answer 28

mRNA’s rapid degradation allows for TEMPORAL REGULATION OF GENE EXPRESSION, which is important becuase not all genes need to be expressed at all times (e.g. stress-induced genes)

Question 29

What distinguishes intact versus degraded RNA…do you understand the gel picture.

Answer 29

Intact RNA shows up as thick bands of glowing white, while degraded RNA doesn’t show up at all.

(rRNA is larger than mRNA)

Question 30

What is heat shock and what does HSP do.

Answer 30

Heat shock is a cellular protective mechanism activated when an organism is exposed to high temperatures.
HSP stands for Heat Shock Protein and is a molecular “chaperone” protein that helps other proteins stay functional at high temperatures.

Question 31

Chlamy heat shock response. Understand the transcript abundance and protein abundance blots.

Answer 31

1) Extract sample from room temperature Chlamy
2) Heat the sample to 40deg
3) Extract samples every few hours

Question 32

Can you link the changes in expression seen on the blots with processes outlined in Outcome 7. IMPORTANT MIDTERM QUESTION

Answer 32

RNA blotting (transcript abundance): At RT, there’s very minimal hsp1 RNA, which increases up until 3h at 40deg and decreases.

As T increases, the rate of hsp1 transcription increases. As T returns to normal, hsp1 mRNA is broken down.

Western blotting (protein abundance): HSP protein abundance increases up to 24h. Actin abundance does not increase.

As T increases, the rate of HSP translation increases. As T returns to normal, HSP is broken down.

Question 33

Distinction between constitutive, induced and repressed gene/protein expression.

Answer 33

Constitutive: Gene expression doesn’t change.
Induced: Gene expression increases.
Repressed: Gene expression decreases.

Question 34

What are house-keeping genes/proteins and why do they show constitutive expression.

Answer 34

House-keeping proteins are proteins required for the normal day-to-day functioning of the cell and so are always translated and degraded at the same rate, resulting in a constant level of abundance.

Question 35

Genomics, transcriptomics, proteomics, metabolomics….what are these and what is the usefulness of each…(in broad terms).

Answer 35

-omics: characterization of groups of molecules, helps to understand biological processes

Genomics: looking at an entire genome (of a specimen with a certain phenotype)
Transcriptome: look at all the genes that are actually expressed
Proteomics: look at all the proteins corresponding to the gene
Metablomics: look at the metabolic profile (products/growth factors)

Question 36

Why is it that, in many cases, proteomics and metabolomics provide more “meaningful” data to a researcher that genomics or transcriptomics.

Answer 36

Proteomics and metabolomics are more indicative of gene function and actual impact on than genomics and transcriptomics. However, they tend to be more expensive and difficult to do.

Question 37

Transcriptome profile of Chlamydomonas during the standard growth curve..why do you think expression changes. (you don’t need to memorize the figure legend).

Answer 37

Suites of genes in Chlamy change in different phases (as a function of cell growth, nutrient environment, interactions with other cells) - in stationary and growth phases. Down-regulated genes early in the growth phase are the genes involved in DNA replication, RNA stability, respiration, photosynthesis, transport, etc.

Question 38

What characteristic would two sequences have (DNA or PROTEIN) that would suggest to you that they are evolutionarily related (descended from the same ancestor)…homologous.

Answer 38

Some characteristics that suggest that two sequences are homologous are:
- sequence similarity in their primary structure (amino acid sequence for proteins, nucleotide sequence for DNA)
- structural homology or structural similarities in the proteins