Lecture 13-Replication Strategies and Gene Therapy (Nakai)

Question 1

In which cells are telomerases found?

Answer 1

• germ line

- cancer cells

Question 2

Why aren’t telomerases found in somatic cells?

Answer 2

• telomerase shortening is indicative of cell replications (age)
• when the cell is old the gene expression changes
• the cell is more prone to mutations when the machinery is old and so when telomeres have shortened sufficiently this is a signal for the cell to stop dividing

Question 3

Which strand is always shorter after replication and why?

Answer 3

• lagging strand
• even if you could put a primer on the very end of the strand there is nothing upstream that the DNA could bind to to replace the RNA

Question 4

Telomerase is a specialized _______

Answer 4

RT

Question 5

Describe telomerase/its activity. (3)

Answer 5

• has an RNA component
• extends TG strand of telomeres
• complement synthesized by RNA-primed DNA synthesis that is part of the telomerase

Question 6

Viruses with larger genomes generally have genetic material made out of ______ which encodes______

Answer 6

• DNA

- its own replicative machinery

Question 7

+ polarity

Answer 7

a form of genetic element in virus made of RNA–can be directly used to translate into protein. Same polarity as mRNA.

Question 8

• polarity

Answer 8

• found in viruses as a form of genetic information made of RNA that must first be transcribed into + polarity using RNA-dependent RNA polymerase

Question 9

What are the advantages to having smaller viral genomes?

Answer 9

• can replicate faster to produce large amounts of virions
• can handle errors (more plasticity) much better
• high error rate helps them thrive and increases the rate of evolution by increasing genetic diversity

Question 10

Describe HSVs genome

Answer 10

• very large

- encodes its own replicative machinery independent from host (Pol!!)

Question 11

What do E6 and E7 do?

Answer 11

• part of HPV

- bind p53 and Rb and help encourage the cell to enter into S phase

Question 12

Viruses with (-) RNA must bring what with them in the virion?

Answer 12

• RNA-dependent RNA Pol (replicase)

Question 13

The DNA intermediate of retroviruses is considered a ______

Answer 13

class of transposable element

Question 14

Ty elements

Answer 14

Included in the genomes of retroviruses

- how they discovered that retroviruses are a form of transposable elements

Question 15

Reverse transcriptase lacks ______ activity

Answer 15

proofreading

Question 16

What helps the influenza virus evolve? (2)

Answer 16

• high error rate

- segmented genome

Question 17

What is brought in a retroviral capsid?

Answer 17

• 2 copies of RNA , tRNA

Question 18

Where does reverse transcription occur and what happens afterward?

Answer 18

• in the cytoplasm

- The DNA is brought into the nucleus and integrases insert it (duplex DNA) into the host genome

Question 19

LTRs function as _____

Answer 19

promoters for transcription of 1º transcript

Question 20

Describe the features of the viral mRNA.

Answer 20

• still has cap
• polyA tail
• LTR
• gag, pol, env

Question 21

how does integrase function in retroviruses?

Answer 21

• binds LTRs to direct DNA into the host chromosome

Question 22

The LTR of a provirus includes what 3 sequences?

Answer 22

• R sequence (repeat sequence) near 5’ cap
• U5
• U3

Question 23

RnaseH

Answer 23

• degrades the RNA of an RNA-DNA duplex

Question 24

If you want to replicate a linear molecule completely you need a ______

Answer 24

primer

Question 25

Explain retroviral DNA synthesis.

Answer 25

- 5' associated tRNA acts as primer and DNA is synthesized to the end of the molecule and only making R sequence (R') - since RNA and DNA in duplex the RNA is quickly degraded - the short DNA sequence will act as a primer with it binds to the other RNA particle - DNA from whole RNA genome transcribed, RNA degraded (RnaseH) - you have LTR formation from this process

Question 26

What are the 2 most important points about how the retroviral RNA becomes DNA? What can this method lead to?

Answer 26

- RT is highly error prone and the plasticity of RNA can allow a lot of mutations to be introduced into retroviral genome - This can be a way of combining 2 retroviral strands that may code for chemotherapeutic resistance or something of the sort. RARELY if two resistant genomes from 2 different strands are incorporated onto same provirus this could lead to chemotherapeutic resistance and a highly successful strain of the virus.

Question 27

How does acyclovir work?

Answer 27

- It is a guanosine analog with a methanol group on what would be the 5' C but it lacks 2' and 3' OH - viral thymidine kinase phosphorylates the 5' methanol group making it into a 5' triphosphate so it will be incorporated into DNA - once its incorporated it stalls the machiner since there is no 2' or 3' OH

Question 28

What 2 drugs are used on HIV? What class are they?

Answer 28

- AZT, DDI | - nucleotide analogs

Question 29

How can viruses be used in gene therapy? 2 examples?

Answer 29

- to introduce needed genes - ADA into lymphocytes for SCID - TNF for cancers

Question 30

Advantages of viral vectors? (3)

Answer 30

- efficient delivery - gene stably introduced into host genes using retroviral vector - viral vectors cant replicate themselves so they don't disseminate

Question 31

psi

Answer 31

a sequence near the 5' end of viral RNA that marks it as such so it will be incorporated into DNA

Question 32

What are the disadvantages to using retroviral vectors (3)?

Answer 32

- small amount of cell types that can be infected (must be dividing) - if inserted in wrong spot can be mutagenic - they could develop the capacity to independently replicate

Question 33

How could gene therapy be used on a child with SCID?

Answer 33

- harvest ADA deficient cells - insert ADA gene into cells with neomycin resistance via retroviral vector - treat with gentimycin to select for those with gene - cells that have passed selection put back into the patient to restore function