Lecture 13-Replication Strategies and Gene Therapy (Nakai) Flashcards
In which cells are telomerases found?
- germ line
- cancer cells
Why aren’t telomerases found in somatic cells?
- 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
Which strand is always shorter after replication and why?
- 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
Telomerase is a specialized _______
RT
Describe telomerase/its activity. (3)
- has an RNA component
- extends TG strand of telomeres
- complement synthesized by RNA-primed DNA synthesis that is part of the telomerase
Viruses with larger genomes generally have genetic material made out of ______ which encodes______
- DNA
- its own replicative machinery
+ polarity
a form of genetic element in virus made of RNA–can be directly used to translate into protein. Same polarity as mRNA.
- polarity
- found in viruses as a form of genetic information made of RNA that must first be transcribed into + polarity using RNA-dependent RNA polymerase
What are the advantages to having smaller viral genomes?
- 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
Describe HSVs genome
- very large
- encodes its own replicative machinery independent from host (Pol!!)
What do E6 and E7 do?
- part of HPV
- bind p53 and Rb and help encourage the cell to enter into S phase
Viruses with (-) RNA must bring what with them in the virion?
- RNA-dependent RNA Pol (replicase)
The DNA intermediate of retroviruses is considered a ______
class of transposable element
Ty elements
Included in the genomes of retroviruses
- how they discovered that retroviruses are a form of transposable elements
Reverse transcriptase lacks ______ activity
proofreading
What helps the influenza virus evolve? (2)
- high error rate
- segmented genome
What is brought in a retroviral capsid?
- 2 copies of RNA , tRNA
Where does reverse transcription occur and what happens afterward?
- in the cytoplasm
- The DNA is brought into the nucleus and integrases insert it (duplex DNA) into the host genome
LTRs function as _____
promoters for transcription of 1º transcript
Describe the features of the viral mRNA.
- still has cap
- polyA tail
- LTR
- gag, pol, env
how does integrase function in retroviruses?
- binds LTRs to direct DNA into the host chromosome
The LTR of a provirus includes what 3 sequences?
- R sequence (repeat sequence) near 5’ cap
- U5
- U3
RnaseH
- degrades the RNA of an RNA-DNA duplex
If you want to replicate a linear molecule completely you need a ______
primer
Explain retroviral DNA synthesis.
- 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
What are the 2 most important points about how the retroviral RNA becomes DNA? What can this method lead to?
- 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.
How does acyclovir work?
- 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
What 2 drugs are used on HIV? What class are they?
- AZT, DDI
- nucleotide analogs
How can viruses be used in gene therapy? 2 examples?
- to introduce needed genes
- ADA into lymphocytes for SCID
- TNF for cancers
Advantages of viral vectors? (3)
- efficient delivery
- gene stably introduced into host genes using retroviral vector
- viral vectors cant replicate themselves so they don’t disseminate
psi
a sequence near the 5’ end of viral RNA that marks it as such so it will be incorporated into DNA
What are the disadvantages to using retroviral vectors (3)?
- 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
How could gene therapy be used on a child with SCID?
- 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
