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Human Molecular Parasitology > Lecture 5 > Flashcards

Lecture 5 Flashcards

1
Q

Why is T. brucei the African trypanosome and T. cruzi the american trypanosome?

A

Where they act - evolutionary separated when the continents splt

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2
Q

What changes do kinetoplastids undergo as they differentiate?

A
  • Protein coat switching
  • Metabolism shift
  • Morphological transformation
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3
Q

How does the metabolism of T. brucei change?

A

From glucose metabolism in human to proline amino acid metabolism in tsetse fly midgut

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4
Q

What is key to the transitions between forms of parasite?

A

Parasite-specific gene regulation

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5
Q

Which of the kinetoplastids is the most restrained in terms of its genetics?

A

T. brucei; has fewer gene duplication events

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6
Q

Describe the genome of T. brucei

A

Nuclear; 11 chromosomes, and ~100 minichromosome (for antigenic variation, VSG proteins)
Mitochondrial (kinetoplast); ~50 maxicircles (mitochondrial function) and ~10,000 minicircles (guide RNAs for RNA editing)

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7
Q

What role do guide RNAs play?

A

Recognise mRNA and insert or delete uridine residues, to edit RNA sequence and create different proteins

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8
Q

How does Leishmania overcome gene knockouts?

A

Create a new chromosome arm to replace lost gene

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9
Q

How to Leishmania and T. cruzi increase expression of genes?

A

Gene duplications; create a new copy of the gene to be expressed

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10
Q

What are the three levels of eukaryotic gene expression?

A
  • Gene regulation (methylation, chromatin silencing and trans. factors)
  • Transcript regulation -(trans-acting factors, exosome, UBFs, siRNAs)
  • Post-translational modification (glycosylation, sumoylation, myristoylation)
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11
Q

What role does RNA pol I play in kinetoplasts?

A

Drives transcription of rRNA and surface proteins

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12
Q

How is transcription by RNA pol II different in the kinetoplasts?

A

No promoter to bind to, instead just transcribes areas lacking histones, and no introns in most genes. Activity not dependent on phosphorylation

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13
Q

What part of the mRNA is capped in kinetoplasts?

A

The 5’ end on the splice leader sequences

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14
Q

What role does RNA pol I II play in kinetoplasts?

A

Drives transcription of tRNA and splice leader RNA (in concert with Pol II)

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15
Q

What are the two surface protein genes in T. brucei?

When is each expressed?

A

VSG; for bloodstream form

Procyclin; for procyclic form

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16
Q

How is RNA pol I activity in kinetoplasts similar to in other eukaryotes?
How is it different?

A

Drives expression of rRNA and uses promoters (only RNA pol in kinetoplasts to do so)
Expresses surface proteins

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17
Q

Where are the VSG expression sites located?

A

Near the telomeres (sub-telomeric)

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18
Q

What else is expressed by VSG promoter?

A

Expression site associated genes (ESAGs)

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19
Q

What other components are found in the VSG expression site?

What are the functions?

A

70bp repeats before the VSG gene; required for recombination events
50bp before the ESAGs

20
Q

What is the main survival strategy of T. brucei?

A

Antigenic variation of the VSG coat

21
Q

What does VSG do when antibodies bind?

A

Glide to the flagella and causes digestion of the antibody

22
Q

What are the four mechanisms of VSG switching in T. brucei?

A
  • Gene conversion
  • Segmental gene conversion
  • Telomere exchange
  • Transcriptional switch
23
Q

What protein does Plasmodium spp have antigenic variation in?

A

Their PfEMP1 protein; that forms the knobs on the RBC. Encoded by Var genes.

24
Q

What are the hyper variable regions of the Var genes?

A

The DBLα and CIDR domains; can recombine within domain classes and between subclasses

25
How is splicing different in kinetoplasts? What does this cause? Outline splicing in kinetoplasts
Trans-splicing rather than cis-splicing. Polycistronic transcript unit - pre-mRNA has many genes in it. Splice leader that is already capped is added onto the 5' end on mRNA, a Poly-A tail is added to the 3' end and RNAase's cleave from upstream gene.
26
What two types of transctiption sites exist for RNA Pol II in kinetoplasts?
- Transcription start sites; expression of two poly-cistrons starts at same site but in opposite directions - Transcription termination sites; expression of two polycistrons in opposite directions ends at same site
27
What characterises transcription termination sites in kinetoplastids?
High levels of Base J; a thymidine which is hydroxylated and then glycosylated
28
How does base J work?
Physically blocks RNA PolII and is knocked off the DNA
29
Where else is base J found?
Telomeres
30
Why Leishmania express plasmids at high levels?
Lack of base J to stop transcription so plasmid continually expressed
31
Why is T. brucei used as model for kinetoplasts?
Only kinetoplast which has RNAi, so useful for knocking out genes of interest
32
When and how are kinetoplast mRNAs exported form the nucleus?
Post trans-splcining and addition of splice lead sequence, an RNA binding protein will being the UTR of mRNA and transport out of the nucleus
33
What fates exist for the mature mRNAs post-nuclear export?
- Translation - Storage - Degradation
34
Where are the mRNAs stored in the cell? | When is this storage important?
Storage granules Storage most important during transitions between stages of life cycle; due to changing their environment, e.g. starvation and heat
35
What are the components of kinetoplast mature mRNA?
Cap, splice leader, 5' UTR, ORF, 3' UTR (~90% of regulatory element) and the poly-A tail
36
In Leishmania how much crossover exists between genes expressed in each stage?
- 157 genes expressed in both promastigote and amastigote stages - 301 genes expressed only by promastigote - 51 genes expressed only by the amastigote
37
What 2 common mRNA sequences are found in Leishmania amastigote specific mRNAs?
UCUCCUUU and AAGAGAA
38
What negative regulatory elements regulate amastigote stage-specific degradation of mRNA?
SIDER elements (a transposable element) in the 3' UTR
39
What was the first characterised regulator of a surface protein in T. cruzi?
UBP1
40
What does UBP1 bind to?
Amastigote specific mRNA motifs in the 3'UTRs
41
Give an example protein that has stage specific regulation in T. brucei Where are the regulatory elements?
``` Cytochrome Oxidase (COX) proteins which are essential for metabolism of insect stage but not human stage so repressed in human In the 3'UTRs of the mRNA ```
42
How many forms of procyclin exist?
4 | EP1, 2 and 3 and GPEET
43
How is procyclin regulated?
EP1 and GPEET regulated by TbZFP3
44
Where does TbZFP3 bind?
To loop 2 (of 3) in the 3'UTR
45
Which protein completes with TbZFP3 in EP1? What is the effect? How might this be useful?
ZFP3 Ectopic expression is sufficient to cause protein coat switch to entirely EP1 If can stimulate procyclin instead of VSG in bloodstream form would kill parasite before could infect

Human Molecular Parasitology (8 decks)

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