Genetics - Lecture 13, 14

Front 1

Explain how a retrovirus infects a cell.

Back 1

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.

Front 2

What does the "gag" code for?

Back 2

capsid protein

Front 3

What codes for Reverse Transcriptase?

Back 3

Pol

Front 4

What codes for envelope?

Back 4

env

Front 5

What are two pros to gene therapy?

Back 5

1. ability to infect many tissue types
2. therapeutic gene is stably integrated which means it may be expressed long term.

Front 6

What are two cons to gene therapy?

Back 6

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.

Front 7

Describe the first gene therapy trial.

Back 7

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.

Front 8

How could the first gene therapy trail be improved upon?

Back 8

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)

Front 9

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?

Back 9

4/9 of them got leukemia bc the altered gene inserted itself next to the LMO2 protoonco gene and activated it.

Front 10

How is provirus cloned for use in gene therapy?

Back 10

in a bacterial plasmid

Front 11

What components are removed from viral genome for gene therapy?

Back 11

The code for gag, pol and env - so the virus cannot multiply out of control.

Front 12

What seals the new therapeutic gene into the viral DNA?

Back 12

DNA ligase

Front 13

Recombinant DNA is inserted into helper cells and grown in the culture.

What needs to be deleted from this recombinant DNA?
Why?

Back 13

cis acting element for env has been deleted so that encapsidation cannot occur.

If encapsidation occurs then the virus can grow out of control.

Front 14

Does the vector retrovirus genome have an intact encapsidation gene?

Back 14

yes.

the HELPER retroviral pre-mRNA does NOT have encapsidation signal.

Front 15

How might HSV help kill CA cells?

Back 15

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.

Front 16

Do human nucleoside kinases recognize gancyclovir?

Back 16

No

But HSV thymidine kinases do recognize this...this is taken advantage of to possibly kill CA cells.

Front 17

Describe the actual trial using HSV thymidine kinases and gancyclovir.

Back 17

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)

Front 18

How big (# base pairs) is a simple retrovirus?

Back 18

A simple retrovirus has fewer than 10,000 base pairs.

Front 19

How many genes does a simple retrovirus has? What are they?

Back 19

they have 3 genes: Gag, pol and env genes.

Front 20

What cis acting elements does a retrovirus have?

Back 20

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).

Front 21

What is a provirus?

Back 21

The DNA copy of the original viral genome (which is RNA).

Front 22

What ends up transcribing a provirus?

Back 22

Host RNA polymerase II.

Front 23

How does AZT work to combat the HIV virus?

Back 23

HIV RT has a high affinity for some ddNTP's - AZT has a ddTTP.

(DDI has DDInosineTP)

both stop transcription

Front 24

What are two ways that translation is regulated?

Back 24

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.)

Front 25

RNA editing can be regulated to regulate transcription. What is RNA editing?

Back 25

RNA editing inserts, deletes or changes the identity of INDIVIDUAL NUCLEOTIDES post-transcriptionally.

Front 26

What is an example of RNA editing?

Back 26

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.

Front 27

What does APOBEC-3G do?

Back 27

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

Front 28

Why won't a premature stop-codon that is made by RNA editing stop translation?

Back 28

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.

Front 29

What is a common way to modulate the rate of mRNA degradation?

Back 29

change the length of its poly(A) tail.

Front 30

What is a protein that protects mRNA's poly(A) tail from being degraded?

Back 30

PABP (Poly A binding protein).

This binds 30 nucleotides of the poly(A) tail

Front 31

What does FOS protein do?

Back 31

activates the transcription of genes that promote cell growth.

Normally FOS mRNA is rapidly degraded - to guard against over-expression.

Front 32

What is required for the rapid degradation of the FOS protein?

Back 32

Removal of the poly(A) tail which requires an A/U-rich sequence in the 3'-UTR.

THIS AFFECTS THE STABILITY OF THE mRNA.

Front 33

What are microRNAs?

How do they affect stability of mRNA?

Back 33

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.

Front 34

What is the difference between a small RNA and a microRNA?

Back 34

they act similarly but small interfering RNA's (siRNA's) come from outside the cell.

Front 35

How does Fe enter the body?

Through what channel?

Back 35

absorbed through the duodenum.

Through the apical side of the enterocytes through DMTI (divalent metal ion transporter)

Front 36

What is the function of ferritin?

Back 36

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)

Front 37

What happens to the production of ferritin when the body needs iron?

What is ferroportin? Where is it found?

Back 37

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.

Front 38

How does iron get from the bloodstream into the cells?

Back 38

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.

Front 39

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?

Back 39

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.

Front 40

What is one way TfR synthesis regulated?

Back 40

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.

Front 41

How is ferritin synthesis regulated?

Back 41

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.

Front 42

Where is IRE present on mRNA of ferritin?

What does this do?

Back 42

a SINGLE IRE is found on the 5'UTR

When IRE is bound by IRP1 (iron regulatory protein 1) - Ferritin synthesis is INCREASED.

Front 43

Where is IRE found on the mRNA for TfR?

What happens when this is bound by IRP1?

Back 43

Several IRE's are found in the 3'UTR.

When IRP1 is bound to IRE, IRE is not exposed...so degradative factors don't degrade it = INCREASE IN TfR's

Front 44

What happens when IRE on ferritin mRNA is bound?

Back 44

When IRP1 is bound by IRP1 the area is NOT exposed...this means the area is blocked from translational initiation factors = DECREASE IN FERRITIN.

Front 45

What is the difference between IRE cis-acting factors for ferritin and TfR?

Back 45

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.

Front 46

When does IRP1 bind to IRE?

Back 46

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.

Front 47

What causes hereditary hyperferritinemia cataract syndrome?

What is the result?

How does this manifest?

Back 47

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.

Front 48

What mutation is the cause of hereditary hemochromotosis?

What problem does this create?

Back 48

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.

Front 49

What drug is given to patients who develop iatrogenic HH?

Back 49

deferoxamine