Lecture 8a

Question 1

In E. coli, what is the origin of replication called?

Answer 1

oriC

This stands for ‘origin of Chromosomal replication’

Question 2

What direction does synthesis of DNA proceed around the bacterial chromosome?

Answer 2

Bidirectionally

Question 3

Where do the replication forks eventually meet?

Answer 3

They eventually meet at the opposite side of the bacterial chromosome.

Question 4

How is bacterial DNA replication initiated?

Answer 4

It is initiated by the binding of DnaA proteins to the DnaA box sequences.

Question 5

What does the binding of DnaA proteins stimulate?

Answer 5

It stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex.

Question 6

What does the large DnaA protein complex cause?

Answer 6

The DnaA box region wraps around the DnaA protein complex and separates the AT-rich region.

Question 7

Label the AT-rich region and the DnaA boxes.

Answer 7

Question 8

What separates DNA bidirectionally, creating 2 replication forks?

Answer 8

DNA helicase

Question 9

After the AT-rich region has been separated, what occurs next?

Answer 9

DNA helicase loads onto the single strands.

Question 10

What is DNA helicase and what does it do?

Answer 10

It is composed of 6 subunits and travels along the DNA in the 5’ to 3’ direction using energy from ATP to ‘unzip’ the strands.

Question 11

From the single origin of replication, what do we get in bacteria?

Answer 11

Synthesis of leading and lagging strands.

Question 12

How many RNA primers are needed for leading strand synthesis?

Answer 12

One RNA primer is made at the origin of replication by Primase.

Question 13

What does Primase do?

Answer 13

Loads the RNA primers onto a single strand.

Question 14

What does DNA polymerase III do?

Answer 14

Attaches nucleotides in a 5’ to 3’ direction.

Question 15

Does leading and lagging strand synthesis take place in the 5’ to 3’ or 3’ to 5’ direction?

Answer 15

In the 5’ to 3’ direction.

Question 16

What differs directionally about the leading and lagging strand?

Answer 16

The leading strand goes towards the replication fork, whereas, the lagging strand goes away from the replication fork.

Question 17

How many primers are required for lagging strand synthesis?

Answer 17

Many RNA primers are required because many small DNA fragments are built.

Question 18

What are Okazaki fragments?

Answer 18

Small DNA fragments from lagging strand synthesis.

Question 19

What does DNA polymerase I do?

Answer 19

Removes the RNA primers using its 5’ to 3’ exonuclease activity. Then, it replaces the RNA with DNA.

Question 20

What does DNA ligase do?

Answer 20

Covalently attaches the Okazaki fragments.

Question 21

T/F: Eukaryotes also have a single origin of replication.

Answer 21

False! Eukaryotes have multiple origins of replication.

Question 22

Why do eukaryotes require multiple origins of replication?

Answer 22

Eukaryotes have long linear chromosomes, so we need multiple origins of replication to ensure that the DNA can be replicated in a reasonable time.

Question 23

Who provided evidence for the multiple origins of replication in 1968?

Answer 23

Huberman and Riggs

Question 24

What did Huberman and Riggs do to provide evidence for multiple origins of replication?

Answer 24

They took growing eukaryotic cells and fed them radioactive deoxythymidine that got incorporated into DNA. Then, they exposed the DNA to film.

Question 25

Where are LoxP sites located? What do we use them for?

Answer 25

On each side of a conditional gene. If we add cre recombinase, this will delete out the gene.

Question 26

T/F: In a conditional knockout, the gene is deleted from 100% of cells.

Answer 26

False! It is unlikely that the gene is deleted from 100% of cells.

Question 27

How do we only reduce gene expression in certain tissues?

Answer 27

We make sure to only put the gene for cre recombinase expression in that tissue.

Question 28

What do MicroRNA genes do?

Answer 28

They encode short hairpin RNAs (shRNAs) that are then processed to produce miRNAs.

Question 29

What do shRNAs do?

Answer 29

Reduce gene expression by binding to a complementary mRNA strand and cleaving it.

Question 30

How do we measure the amount of mRNA remaining after reducing gene expression?

Answer 30

We use ‘Taqman’

Question 31

What does Taqman contain?

Answer 31

An inactive reporter and a quencher.

Question 32

What does the inactive reporter do?

Answer 32

When it becomes active, it will fluoresce. This enables us to visualize cDNA.

Question 33

What does the quencher do?

Answer 33

It prevents the reporter from fluorescing.

Question 34

Describe the process of ‘Taqman’ to measure the amount of mRNA remaining.

Answer 34

1. Reverse transcriptase produces a single-stranded cDNA from the mRNA.
2. A PCR primer, DNA polymerase, and Taqman are annealed to the single-stranded cDNA.
3. DNA polymerase adds nucleotides to extend from the primer. The polymerase has a 5’ to 3’ exonuclease activity, so it will separate the quencher from the reporter.
4. The reporter will then fluoresce.

Question 35

What do we need Real-Time (RT) PCR for?

Answer 35

We need to quantitate the DNA or RNA produced from the Taqman probe. Initially, so little fluorescence is produced that nothing is detected, so we need to PCR the products to exponentially increase them.

Question 36

What does RT PCR do?

Answer 36

It is carried out in a thermocycler that can measure changes in fluorescence emitted by detector molecules

Question 37

Describe the RT PCR process.

Answer 37

1. Initially, so little fluorescence is produced that nothing is detected.
2. Next, when Taqman probe, DNA polymerase, and nucleotides are not limiting, product doubles with every cycle.
3. Then, the reaction falls as reagents become limiting and plateaus when one or more are used up.

Question 38

How does initial DNA concentration relate to PCR cycles needed to detect fluorescence?

Answer 38

The higher the initial DNA concentration, the fewer PCR cycles that are needed to detect fluorescence. This minimal concentration needed to detect fluorescence is called the Cycle Threshold (Ct).

Question 39

Describe the process of using internal standards for RT PCR.

Answer 39

We can take known lower and higher concentration standards for the fluorescence and quantitate them on the same graph as our unknown sample. This allows us to obtain precise quantitation by seeing where our sample falls compared to the known standards.