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49 Cards in this Set

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  • Back
Explain how a retrovirus infects a cell.
1. virus enters cell.
2. single strand RNA is made into double strand of DNA using Reverse Transcriptase
3. DNA provirus enters cell nucleus and integrates into host DNA
4. RNA Pol II transcribes it w DNA.
5. mRNA made is spliced to make capsid, envelope and RT
6. Remaining unspliced RNA is encapsidated and buds off.
What does the "gag" code for?
capsid protein
What codes for Reverse Transcriptase?
What codes for envelope?
What are two pros to gene therapy?
1. ability to infect many tissue types
2. therapeutic gene is stably integrated which means it may be expressed long term.
What are two cons to gene therapy?
1. a cell must be actively dividing to work because a provirus cannot get through nuclear pores.

2. no control over the location of integration in the genome = could actually activate protoonco genes.
Describe the first gene therapy trial.
4 yo female c SCID (adenosine deaminase deficiency).

1. surviving T cells in pt were grown in culture and retrovirus used to implant a functional ADA gene.
2. Genetically altered T-cells were reintroduced into blood stream
3. improvement noted but necessary q3-6 month procedure to maintain.
How could the first gene therapy trail be improved upon?
The original site of gene therapy is in the T-Cell which is not long-lasting bc T-cells have a short life-span.

It would be better if they could use multipotent bone marrow cells. (Could cause cancer!!! = LMO2 gene gave pt's leukemia)
What happened to the boys in the gene therapy trial who got the new ADA gene put in at the multipotent bone marrow level as opposed to in their T-Cells?
4/9 of them got leukemia bc the altered gene inserted itself next to the LMO2 protoonco gene and activated it.
How is provirus cloned for use in gene therapy?
in a bacterial plasmid
What components are removed from viral genome for gene therapy?
The code for gag, pol and env - so the virus cannot multiply out of control.
What seals the new therapeutic gene into the viral DNA?
DNA ligase
Recombinant DNA is inserted into helper cells and grown in the culture.

What needs to be deleted from this recombinant DNA?
cis acting element for env has been deleted so that encapsidation cannot occur.

If encapsidation occurs then the virus can grow out of control.
Does the vector retrovirus genome have an intact encapsidation gene?

the HELPER retroviral pre-mRNA does NOT have encapsidation signal.
How might HSV help kill CA cells?
HSV thymidine kinases can convert gancyclovir to nucleoside 5'monophosphate analog.

If injected into human cells - human nucleotide kinases will add 2 phosphates and this will be incorporated into human DNA...but this is not a normal NTP and will stop replication = kill cells.
Do human nucleoside kinases recognize gancyclovir?

But HSV thymidine kinases do recognize this...this is taken advantage of to possibly kill CA cells.
Describe the actual trial using HSV thymidine kinases and gancyclovir.
Helper cells c HSV-tk gene were injected into a brain tumor.

The pt was then given gancyclovir BID IV X 14 days.

5 of 8 pts showed improvement. (still early in trial)
How big (# base pairs) is a simple retrovirus?
A simple retrovirus has fewer than 10,000 base pairs.
How many genes does a simple retrovirus has? What are they?
they have 3 genes: Gag, pol and env genes.
What cis acting elements does a retrovirus have?
cis-acting elements here are important for: reverse transcription, integration, proviral transcription, RNA processing, translation and encapsidation.

These are mostly located near the ends of the linear genome in LONG TERMINAL REPEATS (LTRs).
What is a provirus?
The DNA copy of the original viral genome (which is RNA).
What ends up transcribing a provirus?
Host RNA polymerase II.
How does AZT work to combat the HIV virus?
HIV RT has a high affinity for some ddNTP's - AZT has a ddTTP.

(DDI has DDInosineTP)

both stop transcription
What are two ways that translation is regulated?
1. making the mRNA less stable - giving it a shorter half-life or vice versa.

2. change the efficiency with which a ribosome initiates translation (can be modified using trans-acting factors.)
RNA editing can be regulated to regulate transcription. What is RNA editing?
RNA editing inserts, deletes or changes the identity of INDIVIDUAL NUCLEOTIDES post-transcriptionally.
What is an example of RNA editing?
Apo B mRNA is edited by APOBEC-1 enzyme. Apo B manifests differently in the liver than it does the intestine - but it their pre-mRNA's are identical.

APOBEC-1 changes CAA to UAA making it a stop codon...the intestine gets the chopped ApoB-48 version.
What does APOBEC-3G do?
It edits a viral genome in an effort to block it from viral replication. Tries defend us from viral infeciton.

HIV produces vif - which protects it from APOBEC-3G
Why won't a premature stop-codon that is made by RNA editing stop translation?
The change occurs after the initial mRNA is made - so the premature stop doesn't make "junk" mRNA like it would if this occurred during transcripton or translation.
What is a common way to modulate the rate of mRNA degradation?
change the length of its poly(A) tail.
What is a protein that protects mRNA's poly(A) tail from being degraded?
PABP (Poly A binding protein).

This binds 30 nucleotides of the poly(A) tail
What does FOS protein do?
activates the transcription of genes that promote cell growth.

Normally FOS mRNA is rapidly degraded - to guard against over-expression.
What is required for the rapid degradation of the FOS protein?
Removal of the poly(A) tail which requires an A/U-rich sequence in the 3'-UTR.

What are microRNAs?

How do they affect stability of mRNA?
These are 22-NT long RNA's that are complementary to sequences in the 3'UTR of target mRNA's.

When micro-RNA's pair w these regions they direct cleavage and degredation of mRNA or repress translation.
What is the difference between a small RNA and a microRNA?
they act similarly but small interfering RNA's (siRNA's) come from outside the cell.
How does Fe enter the body?

Through what channel?
absorbed through the duodenum.

Through the apical side of the enterocytes through DMTI (divalent metal ion transporter)
What is the function of ferritin?
Ferritin is a storage place for Fe. It stores Fe in the enterocyte where it is supposed to be shed if the body doesn't need it.

It also stores excess cytoplasmic iron for later use (this can build-up)
What happens to the production of ferritin when the body needs iron?

What is ferroportin? Where is it found?
ferritin synthesis is repressed - and most of the iron passes through the enterocyte into the bloodstream via ferroportin.

Ferroportin is found between the enterocyte and the bloodstream.
How does iron get from the bloodstream into the cells?
Fe cannot travel in the blood alone - it is carried by tranferrin. Transferrin receptors on the cells attract the transferrin. THE TF/TfR COMPLEX IS targeted by an ENDOSOME.
What happens to an endosome containing TF/TrF complex in the cytoplasm?

What happens to the iron next? What form is iron in, Fe3+ or Fe2+ when it is stored in in ferritin or traveling with transferrin?
The endosome becomes acidic and this induces the release of Fe2+ through another DMT1 in on the endosome.

Fe is then either used by the mitochondria for heme synth or stored in ferritin. Fe3+ when it is stored/traveling with transferrin. Fe2+ when going through any DMT1.
What is one way TfR synthesis regulated?
TfR synth is regulated by changes in mRNA STABILITY

both TfR and ferritin synth are regulated by cis-acting regulatory element called IRE...but in different ways.
How is ferritin synthesis regulated?
Ferritin synthesis is regulated by changes in translational efficiency.

both TfR and ferritin synth are regulated by cis-acting regulatory element called IRE...but in different ways.
Where is IRE present on mRNA of ferritin?

What does this do?
a SINGLE IRE is found on the 5'UTR

When IRE is bound by IRP1 (iron regulatory protein 1) - Ferritin synthesis is INCREASED.
Where is IRE found on the mRNA for TfR?

What happens when this is bound by IRP1?
Several IRE's are found in the 3'UTR.

When IRP1 is bound to IRE, IRE is not degradative factors don't degrade it = INCREASE IN TfR's
What happens when IRE on ferritin mRNA is bound?
When IRP1 is bound by IRP1 the area is NOT exposed...this means the area is blocked from translational initiation factors = DECREASE IN FERRITIN.
What is the difference between IRE cis-acting factors for ferritin and TfR?
Ferritin IRE codes for initiation of translation of ferritin. When it's exposed - more ferritin is MADE.

TfR IRE codes for the degradation of TfR. When it is exposed TfR is DEGRADED.
When does IRP1 bind to IRE?
When the concentration of ferritin is LOW.

IRP1 has a site for binding Fe. When Fe is HIGH in the cell - IRP1 will bind to Fe instead of the IRE.
What causes hereditary hyperferritinemia cataract syndrome?

What is the result?

How does this manifest?
Mutations in the IRE of ferritin mRNA.

Excessive ferritin production is the result.

Ferritin builds up in the lens of the eye = cataracts.
Dz is dominant bc depression of 1/2 of ferritin mRNA molecules is enough to cause accumulation.
What mutation is the cause of hereditary hemochromotosis?

What problem does this create?
Mutation in the HFE gene - important for expression of hepcidin.

Hepcidin is a polypeptide that normally inhibits/controls ferroportin - which would control amount of Fe let into the bloodstream. If mutated - too much Fe gets in.
What drug is given to patients who develop iatrogenic HH?