E3

Question 1

You have measured the absorbance of a sample of DNA in solution and found that the ration A260nm/A280nm = 1.26. What can you conclude about this sample of DNA?

Answer 1

It is likely contaminated with protein.

Question 2

Which is more stable, DNA or RNA?

Answer 2

DNA

Question 3

What is the complementary sequence to CATTACGT?

Answer 3

ACGTAATG

Question 4

Which piece of DNA will have the higher Tm, one with a cytosine plus guanine content of 30% or one with a cytosine plus guanine content of 50% if both are heated under the same experimental conditions?

Answer 4

50% cytosine plus guanine will have the higher Tm.

Question 5

Chromatin interacts with the DNA backbone. Therefore, the primary structure of chromatin should have a high percentage of the following amino acids:

Answer 5

Lysine, Arginine

Question 6

Which bases pair with one another in DNA? Classify each as a purine or a pyrimidine. How many hydrogen bonds form between each of these pairs?

Answer 6

Purine (A) bonds with Pyrimidine (T) w/ double H bond. Purine (G) bonds with Pyrimidine (C) w/ triple H bond.

Question 7

Nucleobase

Answer 7

• a nitrogen-containing compound. (C)ytosine, (G)uanine, (A)denine (found in both DNA and RNA) (T)hymine (found only in DNA), and (U)racil (found only in RNA) 5 in DNA & RNA,

Question 8

Nucleoside

Answer 8

are components of nucleotides, have a nitrogenous base and a five-carbon carbohydrate group

Question 9

Nucleotides

Answer 9

are simply a nucleoside with one or more phosphate groups attached

Question 10

Nucleic acid

Answer 10

They are composed of nucleotides, monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base.

Question 11

Describe and explain how the Griffith Experiment and the Avery-MacLeod-McCarty experiments demonstrated that DNA is the genetic material

Answer 11

showed transforming substance in bacteria could change harmless strains to virulent ones. Avery-MacLeod-McCarty Experiments (1944) identified this substance as DNA by destroying proteins, RNA, or DNA in the transforming mix, revealing the destruction of DNA stopped the transformation.

Question 12

Chargaff observed that the total amount of A+G was equal to the total amount of C+T regardless of the source of DNA. Explain the structural basis for this observation

Answer 12

the structural stability and specificity of the DNA double helix. Chargaff’s rules of base pairing= transmission of genetic information.

Question 13

Describe the major feature of the DNA double helix. What force is primarily responsible for the stability of the structure?

Answer 13

DNA made of two strands that wind around each other resemble a twisted ladder, helix-like shape. Each strand has backbone made of sugar (deoxyribose) and phosphate groups. base pairing between complementary strands and stacking between adjacent bases

Question 14

Bacteria produce restriction endonucleases as a defense mechanism against phages. Explain why the enzymes do not cleave the bacteria’s genomic DNA?

Answer 14

restriction enzymes can’t cleave methylated DNA. bacterial genome is methylated, and resistant to enzymes. phage DNA is not methylated and so is cleaved by enzymes

Question 15

LacZ

Answer 15

codes for beta-galactosidase

Question 16

ApR

Answer 16

codes for beta-lactamase (ampicillin resistance)

Question 17

Ori

Answer 17

origin of replication, so the bacteria can replicate the plasmid

Question 18

Which restriction enzyme cut sites would you use to clone your blue pigment gene into pUC19 and why?

Answer 18

Any of the restriction cut sites located in the multiple cloning site (MCS), these sites only cut the plasmid once. Also the sites are in the lacZ gene and so facilitate blue/white screening.

Question 19

You digest the plasmid and your blue pigment gene with the restriction enzymes chosen in step b. Explain how the two fragments will stick together, and explain how you will stitch the phosphate backbone back up

Answer 19

fragments stick by base pairing, bc of complementary sticky ends made by digesting gene and plasmid w/ same restriction enzymes. backbone would repair w/ adding DNA
ligase

Question 20

Following transformation of the plasmid, how would you ensure that the bacteria you isolate have successfully taken up the plasmid

Answer 20

By using antibiotic selection. Plate the bacteria on agar containing antibiotic (ampicillin), only bacteria that w/ the plasmid (and antibiotic resistance) will survive.

Question 21

How would you know which bacteria have a plasmid with your gene inserted?

Answer 21

Blue/white screening. Place X-gal in agar, if bacteria produce lacZ colonies will be blue, if not the colonies will be white. Since the gene in the middle of lacZ, that will make the protein inactive, so the white colonies will have the correct gene insert.

Question 22

You are now interested in inserting your blue pigment gene into a tomatoes plant. You assume that people would love to eat blue tomatoes. Explain how you might go about inserting this gene into the plant

Answer 22

You would use the Ti plasmid from Agrobacterium tumefaciens. remove tumor causing genes from plasmid and replace with blue pigment gene. plasmid would be reintroduced back into the bacteria, that would be used to infect the plants. plasmid would go into the plant’s chromosome changing it with the gene.

Question 23

What is Taq polymerase? Where was it isolated and from which organism? Why is it crucial for the polymerase chain reaction?

Answer 23

Taq polymerase is a heat stable DNA polymerase. If was isolated from the bacteria Thermus aquaticus in a hotspring in Yellowstone National park. The enzyme is important in PCR because it does not denature at high temperature, so new enzyme does not have to be added at each cycle of the reaction

Question 24

Describe using a diagram how the polymerase chain reaction works and results in exponential amplification of DNA. What steps are involved, what temperatures are required, and what components are needed for the reaction to occur.

Answer 24

1. Denaturation: 94 °C, DNA strands separate
2. Annealing: Cool to ~55 °C, synthtetic DNA primers form duplex with the separated DNA strands.
3. Extension: 72 °C, stable DNA polymerase copies the DNA chain Repeat steps 1-3 25-30 times

To perform the reaction you will need -heat stable DNA polymerse (Taq or equivalent)
-dNTP (deoxy nucleotide triphosphates)
-template
-primers
-Mg2+

Question 25

You wish to make a single nucleotide change in your gene for the blue pigment. Describe how you could accomplish this

Answer 25

use site-directed mutagenesis. Perform a PCR using primers that have desired mutation. The two primers **must** anneal to the same place on the gene, but to both Strands of DNA. You will end up with a mixture of parental DNA and new mutated DNA. The parental DNA is methylated while the new mutated DNA is not. Use DpnI to digest the parental (non-mutated) DNA. Transform bacteria

Question 26

Explain how the CRISPR-Cas system works as a bacterial immune system

Answer 26

short palindromic repeats of DNA coded in genome. Invading DNA from viruses incorporated in-between CRISPR sequences.

Question 27

Briefly outline one of two strategies for recreating extinct species.

Answer 27

CRISPR - Sequence the passenger pigeon genome See how it is different from existing pigeon species Use CRISPR technology to change existing pigeon DNA to be passenger pigeon DNA The other approach would be somatic nuclear transfer (see you lecture notes)

Question 28

You grow a culture of E. coli in media containing 15NH4Cl, you then switch the bacteria to media containing 14NH4Cl for three generations (an eightfold increase in population). What would be the ratio of hybrid DNA (15N-14N) to light DNA (14N-14N) at this point?

Answer 28

After 3 generation there would be a ratio of 2 to 6

Question 29

On the diagram draw arrows indicating the electron flow that occurs in the formation of the new phosphodiester bond. In words describe what is happening. What are the products of the reaction?

Answer 29

DNA polymerase catalyzes the formation of a new phosphodiester bond. The 3’OH group on the end of the growing chain is deprotonated to become a nucleophile (O-). It attacks the 5’- phosphate on the incoming deoxy nucleotide triphosphate forming a new phosphodiester bond, releasing pyrophosphate

Question 30

Why is DNA synthesis discontinuous on the lagging strand?

Answer 30

The DNA polymerase has only 1 catalytic activity, the formation of a new DNA chain in the 5’ to 3’ direction. The enzyme can only attach nucleotides to a free 3’OH group. Since the parental DNA is anti-parallel, the two strands go in opposite directions (one is 5’ to 3’, the other is 3’ to 5' This means when the two strands are being copies, only the 3’ to 5’ strand will be continuously copied, as its new complementary strand would be the correct 5’ to 3’ direction.

Question 31

How are the discontinuous fragments joined to make a continuous strand of DNA?

Question 32

the structure of the anti-retroviral drug AZT. What nucleotide does the drug resemble?

Answer 32

nucleotide Thymidine

Question 33

Describe a plausible mechanism for how this drug AZT would inhibit the growth of HIV.

Answer 33

The drug lacks a 3’OH group (azide group in its place), which means it cannot have additional nucleotides, attaches to it. If the drug becomes phosphorylated, and incorporated into the virus’ growing DNA chain, the reverse transcriptase will be unable to add new nucleotides, thus terminated DNA replication.

Question 34

Given what you know about DNA replication, why does the drug kill the virus but not people?

Answer 34

The reverse transcriptase has no proofreading ability so cannot remove the drug form its growing DNA chain. DNA pol III has proofreading ability so it can remove the drug from the growing DNA chain, making it less toxic to humans than to the virus

Question 35

You have discovered a new class of DNA polymerase in bacteria from Mars. The polymerase lacks 3’ to 5’ exonuclease activity, would you expect it to be more or less accurate that E. coli DNA polymerase III? Explain your answer

Answer 35

You would expect this new polymerase to be less accurate than DNA polymerase III. The 3’ to 5’ exonuclease activity is the proofreading activity of the enzymes that allows them to remove nucleotides that have been incorrectly added to the chain

Question 36

Turnover number

Answer 36

is the speed that the polymerase can copy DNA

Question 37

The Processivity

Answer 37

is how many nucleotides can be added to the new chain of DNA before the polymerase dissociates from the chain

Question 38

5’ to 3’ polymerization activity

Answer 38

is the ability to make new DNA

Question 39

5’ to 3’ exonuclease activity

Answer 39

is the repair activity

Question 39

What would you expect the major function(s) of each of the DNA polymerases to be in DNA replication and why?

Answer 39

Pol A = major polymerase involved in DNA replication. high turnover number = rapid copying of genome. high processivity to copy a large portion of the genome before falling off. has polymerization activity and proofreading activity. Pol B = proofreading and repair. small turnover number and processivity unlikely to copy whole genome. has both proofreading and repair activity.

Question 40

the 3’ to 5’ exonuclease activity

Answer 40

is the proofreading activity.

Question 41

Consider the following DNA duplex: 5’-ATGCCAGGTCAAGTTAGTAA-3’ 3’TACGGTCCAGTTCAATCATT-5' What is the sequence of the template strand (5’ to 3’)?

Answer 41

5’-TTACTTAACTTGACCTGGCAT-3’

Question 42

Consider the following DNA duplex: 5’-ATGCCAGGTCAAGTTAGTAA-3’ 3’-TACGGTCCAGTTCAATCATT-5’ What is the sequence of the coding strand (5’ to 3’)?

Answer 42

5’-ATGCCAGGTCAAGTTAGTAA-3’

Question 43

Consider the following DNA duplex: 5’-ATGCCAGGTCAAGTTAGTAA-3’ 3’-TACGGTCCAGTTCAATCATT-5’ What is the sequence of the mRNA produced from the DNA (5’ to 3”)?

Answer 43

5’-AUGCCAGGUCAAGUUAGUAA-3’

Question 44

Provide a definition for a promoter. What are the roles of Pribnow box, the -35 region and the TSS?

Answer 44

The promoter region of DNA that is not transcribed into mRNA, serves a role in gene regulation. The Pribnow box is a AT rich sequence where the DNA duplex unwinds and the transcription bubble forms. The -35 region is where the sigma factor bind and initiate transcription. The TSS is the transcription start site, it is the first nucleotide from the DNA that gets transcribed.

Question 45

What are sigma factors? Provide an example of how different sigma factors can be used to control gene expression.

Answer 45

Sigma factors are a subunit of RNA polymerase that bind the promoter region to initiate transcription. The sigma factors only bind specific sequences in the promoter, thus provide a way of controlling gene expression by producing different sigma factors at different times. An example is the control of early, middle and late genes by different sigma factors. The early genes are transcribed by the early sigma factor, which also produces the middle genes sigma factor. This will then turn on the middle genes, which produces the late genes sigma factor. This mechanism ensures sequential control of gene expression and different periods of the bacterial life cycle