Cycle 2 BMP Workshop

Question 1

Define:

Gene expression

Answer 1

Process when a gene is turned into a function

Question 2

How does a gene become an RNA transcript?

Answer 2

Transcription

Question 3

How does an RNA transcript become a protein?

Answer 3

Translation

Question 4

Describe:

Northern Blot Technique

Answer 4

Tells us if a gene is transcribed (mRNA)

Question 5

Explain:

The acronym SNOW DROP

Answer 5

Southern Blot - DNA
Northern Blot - RNA
OOOOOOOO - OOO
Western Blot - Protein

Question 6

Describe:

Steps of Northern Blot

Answer 6

1. Extract mRNA
2. Gel Electrophoresis (separate mRNA strands by size)
3. Transfer gel to a membrane
4. Probe the membrane
5. Visualize the probes

Question 7

True or False:

In Gel Electrophoresis, the smaller mRNAs will move to negative end faster

Answer 7

False, the smaller mRnas will move to positive end faster/further than the larger mRNAs

Question 8

Describe:

Step 3 in Northern Blot

Answer 8

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

Question 9

Describe:

Step 4 of Northern Blot

Answer 9

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

Question 10

How is the fluorescent/radiactive label transferred in Northern Blot?

Answer 10

By using an x-ray film

Question 11

True or False:

Darker lines in Northern Blot means lots of transcript abundance

Answer 11

True

Question 12

Define:

Gene

Answer 12

A DNA sequence that codes for an RNA strand

Question 13

True or False:

All genes code for mRNA

Answer 13

False, also codes for rRNA and tRNA

Question 14

Which RNA codes for proteins?

Answer 14

Only mRNA

Question 15

True or False:

mRNA is the largest portion of RNA in the cell

Answer 15

False, it is the smallest portion of RNA in the cell

Question 16

How does information transfer occur?

Answer 16

Information in DNA is transribed into RNA and then mRNA is translated into proteins

Question 17

What proteins are involved in each step of information transfer?

Answer 17

Enzymes

Question 18

What shape is DNA? How is this achieved?

Answer 18

A double helix, formed by hydrogen bonding

Question 19

What shape is RNA? How does it achieve this?

Answer 19

Can be various shapes, can bond with itself using hydrogen bonding to form these shapes

Question 20

What shapes are proteins?

Answer 20

Varies; depends on function

Question 21

What is generally thought to have evolved first?

Answer 21

RNA

Question 22

Define:

Ribozyme

Answer 22

A class of catalytic RNA molecules

Question 23

What are two examples of ribozymes?

Answer 23

Ribosome: Key organelle that plays a role in the synthesis of proteins
* 2/3 rRNA and 1/3 Protein

Spliceosomes: Involved in removing introns

Question 24

Define:

Transcription

Answer 24

DNA to RNA

Question 25

# Define: Translation

Answer 25

mRNA to Protein

Question 26

What are the 2 ways to measure gene expression?

Answer 26

1. Transcript Abundance: How much mRNA corresponding to a specific gene is in the cell 2. Protein Abundance: How much of a protein is in the cell

Question 27

How is transcript abundance measured?

Answer 27

Northern Blots (RNA blot hybridization) Transcription rate and rate of mRNA breakdown

Question 28

How is protein abundance measured?

Answer 28

Western Blots Translation rate and rate of protein breakdown

Question 29

# State the similarities and differences between: DNA and RNA

Answer 29

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

Question 30

# Describe: A Nucleotide

Answer 30

5 Carbon sugar + Phosphate + Nitrogenous base

Question 31

Why is it good that RNA degrades more quickly than DNA?

Answer 31

Good temporal regulation We do not need mRNA present all the time if unnecessary as this is a waste of cellular resources

Question 32

# True or False: RNA degrades quickly, so we need to ensure than RNA samples are intact prior to experimentation

Answer 32

True

Question 33

# Define: 3 types of expression

Answer 33

1. Induced: Up-regulation in gene expression 2. Constitutive: Expression of the protein or transcript will always be the same 3. Repressed: Down-regulation in gene expression

Question 34

# State: The groups of "omics"

Answer 34

Genomics Transcriptomics Proteonomics Metabolomics

Question 35

What is the purpose of "omics"?

Answer 35

Allows us to look at whole genome, transcriptome, proteome etc.

Question 36

# In gel images: What is the RNA and how can we tell size?

Answer 36

The glowing rods are RNA, and the further down the strip the smaller it is

Question 37

How is homology proven using molecular info?

Answer 37

Look at the genes that make up each homologous structure

Question 38

# State the types of: Expression at level of transcription

Answer 38

1. Basal expression: Normal level 2. Increased expression: Transcription factors enhance 3. Decreased expression: Transcription factors inhibit

Question 39

# State the types of: Stability at level of mRNA

Answer 39

1. Basal stability 2. Increased stability, factors bind to end of mRNA (stabilizing it) 3. Decreased stability, factors bind to end of mRNA (destabilizing it)

Question 40

What is the difference between regulation at level of promoter and regulation by stability?

Answer 40

Regulation at the level of promoter controls transcription Regulation by stability is at level of mRNA, independent of transcription

Question 41

What are transcription factors?

Answer 41

Proteins, binds to DNA promoter to inhibit/promote expression

Question 42

What binds to the promoter of DNA?

Answer 42

RNA polymerase

Question 43

What is the half-life of mammalian mRNA? Protein?

Answer 43

mRNA: about 9 hrs Protein: about 46 hrs

Question 44

What is post-translational modification?

Answer 44

Modifications made after translation to make the protein functional

Question 45

What is an example of post-translational modification?

Answer 45

* 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!

Question 46

How do we link a genotype to a phenotype?

Answer 46

We mutate the gene and see what happens to the phenotype

Question 47

# Compare: Forward genetics and Reverse genetics

Answer 47

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

Question 48

How do we generate a population of mutants?

Answer 48

* Insert a gene randomly into the genomes of organisms * Use methods to get the phenotype of interest

Question 49

How does one prove a gene is responsible for a phenotype?

Answer 49

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