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MBB262 > Barnes (protozoan genetics) > Flashcards

Barnes (protozoan genetics) Flashcards

1
Q

Are protozoa euks or proks?

A
  • euks
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2
Q

What are the characteristics of protozoa?

A
  • single-celled
  • mostly motile (diff mechanisms)
  • heterotrophs (cannot fix own C)
  • “animal-like” (not generally photosynthetic)
  • contain food vacuoles
  • massive diversity
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3
Q

Do protozoa stay at constant levels in their host after infection?

A
  • no, waves of parasitemia over weeks after time of infection
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4
Q

What is antigenic variation?

A
  • mechanism by which infectious organism, such as protozoan, bacterium or virus, alters surface proteins in order to evade host immune response
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5
Q

What is the role of abundant “immunodominant” variable surface molecules in antigenic variation?

A
  • to hide invariant surface molecules, so shieldied from IS
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6
Q

What is the strategy of antigenic variation dep on?

A
  • monoallelic expression of single surface molecule from large repertoire of structurally similar, BUT antigenically distinct molecules, on any given cell
  • periodic switching from 1 molecule to another
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7
Q

Does switching occur in response to immunological pressure?

A
  • no, spontaneously
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8
Q

How does antigenic variation occur?

A
  • DIAG*
  • single surface molecule expressed in clonal pop 1
  • surface molecule switch and one/few express diff molecule
  • original surface molecule recognised and destroyed by host
  • only cells that switched survive
  • proliferation creates clonal pop 2, w/ “new” coat expressed
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9
Q

How fast should switching occur?

A
  • should be fast enough that pop can survive, but slow enough that repertoire of surface molecules not used up too quickly
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10
Q

What does Trypanosoma brucei cause in humans?

A
  • sleeping sickness
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11
Q

What is the life cycle of Trypanosomes?

A
  • DIAG*
  • procyclic form –> metacyclic form –> long slender form –> short stumpy form –> procyclic form
  • procyclic and metacyclic in insect host, others in mammalian host
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12
Q

Which stage of Trypanosome life cycle is an eg. of antigenic variation?

A
  • long slender form –> variant surface glycoprotein (VSG)
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13
Q

What are the similarities/diffs between variant surface glycoproteins in Trypanosomes?

A
  • conserved general structure, as same job

- diffs mostly at end in exposed part

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14
Q

How many diff VSGs are there?

A
  • approx 2000
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15
Q

How many VSGs are expressed?

A
  • only one expressed on cell surface at a time
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16
Q

How does the densely packed structure of membrane mean invariant surface molecules are protected from immune response?

A
  • fit into spaces between VSGs, so tend to stick out less

- shielded by VSG coat

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17
Q

How many bloodstream expression sites are there, and where are they found?

A
  • approx 15

- in subtelomeric regions of linear chromosomes

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18
Q

What do bloodstream expression sites contain?

A
  • a diff VSG
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19
Q

What is the role of the promoter at bloodstream expression sites?

A
  • drives expression of single VSG (closest to telomere)

- drives expression of no. of expression site assoc genes (ESAGs) –> often useful for survival in mammalian host

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20
Q

What is the role of repeat seqs at bloodstream expression sites?

A
  • promote recombiantion
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21
Q

How many expression sites are expressed at a time?

A
  • only 1
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22
Q

What does RNA pol I transcribe?

A
  • generally rRNA in nucleolus

- also VSG at active expression site in trypanosomes

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23
Q

How was the role of RNA pol I showed in an experiment in Trypanosomes?

A
  • in insect stage Pol I in nucleolus transcribing rRNA

- in mammalian stage also at ESB transcribing VSG

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24
Q

How does monoallelic VSG expression occur?

A
  • expression site body is large DNAse resistant compartment, assoc w/ active expression site in bloodstream-form trypanosomes
  • control of silencing of other expression sites
  • repertoire of 2000 silent VSGs always silent (away from expression sites)
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25
Q

What is the silencing of other expression sites in Trypanosomes controlled by?

A
  • epigenetic factors –> hypoacetylation and methylation controlled by specific chromatin remodelling factors
  • specific telomere binding proteins –> assoc w/ active expression sites and silence expression sites differently
  • VEX1 protein
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26
Q

What pathways does VSG switching occur by?

A
  • transcrip switching –> new active expression site (change in location of expression site body and epigenetic markers) and no change in DNA seqs of any expression sites
  • recombination switching (ds break repair) –> homologous recombination mechanisms: movement of VSG genes from silent repertoire, using DNA repair enzymes, into active site using small regions of homology, trigger for switching not well understood
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27
Q

What disease is Plasmodium falciparum responsible for?

A
  • causative agent of malaria
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28
Q

What is the life cycle of Plasmodium falciparum?

A
  • DIAG*

- mosquito –> human liver –> human RBCs –> mosquito

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29
Q

What gene family is PfEMP1 prod by?

A
  • var genes

- DBL multidomain-type gene family

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30
Q

Where is PfEMP1 expressed, and why does this mean it has to vary?

A
  • surface of infected RBCs

- as exposed to IS

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31
Q

How many var genes are there?

A
  • 60 per genome
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32
Q

Where is enormous diversity in var genes found?

A
  • between isolates from diff geographical locations
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33
Q

How many var genes are expressed at a time?

A
  • 1
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34
Q

How does PfEMP1 mediate the interaction of infected erythrocytes w/ human cells?

A
  • cytoadhesion to epithelium –> sticking to walls of blood vessels
  • rosetting –> infected erythrocytes form clusters w/ uninfected erythrocytes
  • platelet-mediated clumping –> infected erythrocytes form clusters w/ platelets
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35
Q

Why is necessary for PfEMP1 to mediate interaction of infected erythrocytes w/ human cells?

A
  • prevent infected erythrocytes from being recycled in spleen, which would also destroy Plasmodium cells
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36
Q

How are var genes found in P. falciparum genome?

A
  • tandem arrays
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37
Q

How do diffs in var genes lead to severe or chronic infections?

A
  • subtelomeric var genes (group A and B) tend to be expressed in severe diseases –> rapid switching rates
  • internal var clusters assoc w/ chronic infections –> slow switching rates
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38
Q

How is monoallelic expression controlled transcriptionally in P. falciparum?

A
  • inactive var genes assoc w/ repressive (closed structure) chromatin
  • eg. H3K9 acetylated at promoter of active var gne and trimethylated at silent var genes
  • long non-coding RNAs form var gene arrays (antisense compared to var genes) involved in controlling these chromatin marks
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39
Q

Where are var genes localised to, and how are they grouped together?

A
  • nuclear periphery

- grouped in bouqets

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40
Q

Why is there sub nuclear localisation of bouquet like grouping of var genes?

A
  • increases likelihood of recombination, so increased variation
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41
Q

Where does active var gene sit?

A
  • at expression site, a special location
42
Q

What is the life cycle of Giardia lamblia?

A
  • DIAG*

- faeces –> human intestine (by contaminated drinking water) –> faeces

43
Q

What does Giardia have allowing it to stick to gut epithelium, and why?

A
  • disc, that acts like sucker

- so can get nutrients from gut

44
Q

What does antigenic variation in Giardia dep on?

A
  • variant surface proteins (VSPs)
45
Q

How many VSPs are there, and at one stage is one chosen for monoallelic expressed?

A
  • around 250

- during trophozoite phase

46
Q

How freq does switching occur in Giardia?

A
  • every 6-13 gens
47
Q

What is the structure of VSPs?

A
  • DIAG*
  • 20-200 kDa extracellular domain, rich in CXXC motifs
  • conserved transmembrane domain which anchors it in pm
  • hydrophobic conserved tail –> 90% conservation w/in family
48
Q

Are VSPs toxins, and why?

A
  • similar structure

- but injection of VSP alone doesn’t cause any symptoms in animal models

49
Q

How is silencing caused by transcript degradation in Giardia?

A
  • all 200 VSP genes transcribed in nucleus to diff extents
  • transcripts for all but 1 degraded by RNA interference in cyto –> exact selection mechanism unknown
  • single VSP translated, transported to cell surface and excreted
50
Q

How is silencing caused by other mechanisms in Giardia?

A
  • and/or similar mechanism seems to trigger epigenetic silencing of all but 1 VSP gene
  • so VSPs all expressed, but not necessarily to same degree
  • Giardia has 2 nuclei w/ similar genetic info, so controlling this silencing via RNA defo seems advantageous
51
Q

Are Giardia and Tetrahymena evolutionarily related?

A
  • no, v evolutionary distinct
52
Q

What is Giardia like in the trophozoite stage?

A
  • flagellated
  • binucleate
  • both nuclei transcriptionally active
53
Q

What is Giardia like in cyst stage?

A
  • cyst wall protects
  • each contain 4 nuclei
  • dormant = downreg metabolism
54
Q

How does ploidy change throughout Giardia life cycle?

A
  • in trophozoite each nucleus cycles between diploid and tetraploid, so whole cells cycles between 4n and 8n
  • when cyst (4 nuclei) becomes trophozoite (2 nuclei) the nuclei are sorted to give 2 binucleated daughters, each w/ pair of non-identical nuclei
  • when trophozoite (2 nuclei) becomes cyst (4 nuclei) there is nuclear division but no cytokinesis
55
Q

Do the 2 nuclei have similar genetic info, and what does this suggest?

A
  • yes, v low seq heterozygosity between 2 nuclei

- suggests exchange of genetic info between them

56
Q

Why is there no evidence for a true sexual stage in Giardia, and why might this be?

A
  • conservation of 7 meiotic genes, but some important ones are not found
  • may have been meiotic ancestor at some point, but some of machinery lost
57
Q

What is the most likely explanation for the similarity between genetic info in 2 nuclei of Giardia?

A
  • diplomixis
  • fusion of nuclei in cyst following division and transfer of big chunks of DNA (or even whole chromosome exchange), followed by gene conversion
58
Q

What is Tetrahymena thermophila, where is it found and what are its diff states?

A
  • ciliated protozoa
  • freshwater ponds and hot springs
  • mostly vegetative growth, but sexual cycle in bad conditions
59
Q

What is nuclear dimorphism?

A
  • separate germline and somatic functions
60
Q

What are the only unicellular organisms to have nuclear dimorphism?

A
  • ciliated protozoans
61
Q

What are the 2 nuclei in Tetrahymena and what are their characteristics?

A
  • micronucleus (germline equivalent) –> diploid, transcriptionally silent during vegetative growth, passed on at mitosis
  • macronucleus –> polyploid (dozens of copies), actively transcribed ( hyperacetylated, open chromatin), lost during mitosis
62
Q

Why is DNA editing of somatic genome (macronucleus) req?

A
  • need strategy for silencing “junk” DNA, like repeats and transposable elements
  • other organisms deal w/ solely using heterochromatin
63
Q

What does DNA editing of somatic genome (macronucleus) involve?

A
  • transcriptionally silent germline macronucleus –> transcriptionally active somatic nucleus
  • selective loss of approx 30% genome
  • amplification of DNA seqs
  • breakage into many small chromosomes
64
Q

How is genetic material passed on during vegetative growth?

A
  • micronucleus divides mitotically
  • macronucleus divides amitotically = random distribution of expressed genome
  • so random diffs in gene expression from cell to cell –> some alleles could even be lost in some lineages (could be advantageous in some envs)
65
Q

What is the process of micronuclei being exchanged?

A
  • conjugation
  • meiosis and macronuclei disintegrate
  • 3/4 meiotic products disintegrate, leaving 1 micronucleus (n) and haploid micronuclei undergo mitosis (rep and divide)
  • haploid micronuclei exchange
  • haploid micronuclei fuse, becoming diploid (2n)
  • macronucleus reforms
66
Q

What are the 2 seqs deleted in new macronucleus genome after conjugation?

A
  • internal eliminated seq (IES) –> loss of some genetic material but ends joined back together DIAG
  • breakage eliminated seq (BES) –> breakage occurs at conserved chromosomal breakage site DIAG
67
Q

How long are internal eliminated seqs, and how many are there?

A
  • 0.5-20kb long in Tetrahymena genome

- up to 12,000

68
Q

How does excision of IESs occur, and especially what is excised?

A
  • especially transposons
  • histone mods
  • aggregation of heterochromatin
  • excision of IESs by domesticated transposase –> evo to cut DNA at these sites instead of carrying out original function
69
Q

What happens to remaining seqs after IESs excised?

A
  • amplification
  • on av 68 copies of most seqs, but amplified to diff degrees
  • some much more, eg. rRNA genes 200 fold
70
Q

How is DNA detection in macronucleus guided by scnRNAs?

A
  • dsRNA gen in meiotic nuclei
  • chopped into chunks around 28nt long, matching sections of genome to be detected (don’t know how chosen) = scanning RNAs
  • scnRNAs bind to complementary seq in genome of new macronucleus
  • scnRNAs attract proteins to create specific heterochromatin structures
  • this chromatin structure guides cleavage by “domesticated” transposase-like enzyme, ds breaks then repaired
71
Q

What are breakage eliminated seqs and what do they involve?

A
  • breakage and de novo telomere addition
  • at specific 15bp CBS site (assoc w/ repetitive seqs)
  • gen up to 300 chromosomes from 5 micronuclear chromosomes (av 600kb in size)
  • enzymes involved not known –> poss directed by Pot2, a protein related to telomere binding protein Pot1
72
Q

What is a summary of macronucleus formation?

A
  • elimination of IESs –> guided by scnRNAs
  • breakage at BESs –> chromosome fragmentation guided by CBSs
  • de novo telomere formation
  • genome amplification
73
Q

Why is Tetrahymena a good model organism for telomeres?

A
  • macronucleus contains 100s of chromosomes, so high proportion of telomere seq
74
Q

What has Tetrahymena helped to study?

A
  • Elizabeth Blackburn used it in late 1970s before mol cloning techniques of today existed (eg. PCR), to investigate end rep probelm and telomeres, as needed large quantities of DNA
  • telomerase enzyme discovered in Tetrahymena
75
Q

What are 3 kinetoplastid organisms, what disease do they cause and where?

A
  • Trypanosoma brucei –> Human Africa Trypanosomiasis
  • Trypanosoma cruzi –> Chagas disease, S America
  • Leishmania major –> Leishmaniasis, various hot places
76
Q

What did seq of kinetoplastid organisms allow and show?

A
  • allowed comparison between species

- lots of similarities between genomes, even though look and act quite diff

77
Q

What are the characteristics of mito?

A
  • organelle in most euk cells
  • devoted to energy prod –> gen of ATP by OP
  • contain circular genomes, w/ genes for OP and protein prod
  • double membrane and circular genome related to proteobacteria –> likely originated from endosymbiosis
  • prokaryote style translation, transport of proteins in and out of organelle
78
Q

What is the trypanosome kinetoplast and what is its structure?

A
  • single mito per cell

- disk shaped structure containing complex DNA network (kDNA) –> 10% total DNA

79
Q

How have kDNA networks evolved?

A
  • to be more complicated in diff branches of kinetoplastid phylogenetic tree
80
Q

How can kDNA be visualised in trypanosome cell, and what do you see?

A
  • using DAPI to stain DNA

- see separate smaller clump of DNA away from main nuclear DNA

81
Q

What does the kinetoplast house which is useful for its function?

A
  • houses membrane ETC for OP
82
Q

What are the diffs in mito in diff life cycle stages of trypanosome?

A
  • in mammalian bloodstream –> lots of glucose, so OP not needed, high levels of glycolysis, TCA and OP proteins not expressed
  • in insect host –> lower glucose, so OP needed and mito more active, TCA and OP proteins expressed
83
Q

What are the 2 types of kinetoplast DNA and what do they code for?

A
  • maxicircles –> dozens per cell, ≈23kb, encode rRNAs and protein for energy transduction (normal mito functions, eg. ETC)
  • minicircles –> 100s per cell, ≈60 diff types, ≈1kb, encode gRNAs for RNA editing, all essential for expression of maxicircle genes
84
Q

What shape are both types of kinetoplast DNA?

A
  • usually circular
85
Q

What is the structure of DNA molecules in kinetoplast DNA?

A
  • interlocked in single network
  • chainmail structure overall, forming disk
  • minicircles stretched out side by side
86
Q

Why does kinetoplast RNA undergo extensive pan-editing?

A
  • insertion and deletion of many uridine residues –> completely unrecognisable from RNA transcribed from maxicircle
87
Q

How does gene editing of kinetoplast RNA occur?

A
  • based on guide RNA templates
  • acted on by endonucleases
  • either makes nick = INSERTION EDITING, terminal uridyl transferases (TUTases) added and end ligated back together
  • or exoUases remove new residues and ends ligated back together = DELETION EDITING
88
Q

Why does the chainmail network exist in kinetoplast DNA?

A
  • so minicircles not lost at cell division
89
Q

How is kinetoplast DNA rep?

A
  • minicircles released from network to be rep
  • topoisomerase II essential for this process and alters supercoiling of DNA molecules –> so promising trypanocidal drug target
  • each molecule remains nicked until all circles copied, to keep track of which ones have been rep
90
Q

What are the 2 types of members of apicomplexa?

A
  • parasitic

- free-living (non-parasitic)

91
Q

What are the characteristics of chloroplasts?

A
  • organelle in plants and algae
  • thylakoid membranes contain machinery for ps
  • contains circular DNA
  • genes for protein synthesis and ps machinery
  • prokarytoic style translation, plus transport of proteins in and out of organelle
  • double membrane and circular genome related to cyanobacteria –> likely originates from endosymbiosis
92
Q

Why is the apicoplast genome not sufficient for function?

A
  • also req trafficking of add proteins to apicoplast –> controlled by specific signal seqs
93
Q

What is apicoplast genome similar to?

A
  • seq similar to algal chloroplasts
94
Q

What is the structure of the apicoplast genome, and what does it code for?

A
  • 35kb circular genome
  • ≈50 mostly housekeeping genes –> ribosome subunits, tRNAs, RNA pol, chaperone for protein import
  • encodes many genes for translation that closely resemble ribosomes etc. from bacteria
95
Q

What is Chromera velia and what did it provide?

A
  • free-living, photosynthetic relative to apicomplexans

- missing link between free-living (photosynthetic) and parasitic (non-photosynthetic) members of family

96
Q

How did Chromera velia provide the missing link in apicomplexa evo?

A
  • plastid surrounded by 4 membranes (2 endosymbiosis events)
  • plastid photosynthetic genes similar to red algae and plastid housekeeping genes similar to apicomplexa species
  • therefore points to endosymbiotic origin of apicoplast from red algae
97
Q

What suggests the apicoplast is essential?

A
  • lots invested in maintaining it

- 5-10% of Plasmodium nuclear genome has apicoplast import signals

98
Q

If malaria doesn’t carry out ps, then why is the apicoplast essential?

A
  • has biosynthetic roles –> inc FAs, isopentenyl pyrophosphate (IPP) , haem
  • many of these energetically expensive to make so made sense to do this at site of energy prod
  • FAs and haem can be scavenged, but IPP synthesis essential
99
Q

What is IPP, and how is it gen?

A
  • important intermediate in prod of many isoprenoid molecules, inc cholesterol
  • 2 distinct biosynthetic pathways to gen IPP, w/ diff intermediates
  • -> mevalonate (used by animals and fungi)
  • -> DOXP (used by bacteria and plants)
100
Q

How can apicoplasts be a good drug target?

A
  • fosmidomycin and clindamycin are anti-malarial drugs w/ diff modes of action
  • fosmidomycin blocks DOXP pathway for IPP biosynthesis (pathway used in apicoplast)
  • clindamycin originally dev to treat bacterial infections –> inhibits bacterial ribosome
  • translation inhibitors, eg. chloramphenicol and clindamycin, are drugs against apicomplexan parasites
  • dev against bacteria, but also block translation of apicoplast genes, as they have bacteria like translation machinery
101
Q

How have host cells come to rely on apicoplast biosynthetic pathways over evolutionary time?

A
  • ancestral ps organism –> plastid needed for ps function
  • cytoplasmic biosynthetic pathways for IPP (by mevalonate pathway) were lost and IPP exported from apicoplast into Plasmodium cell
  • present day apicomplexans don’t need ps, but still need plastid IPP prod (by DOXP pathway)
102
Q

How can the need for an apicoplast be avoided?

A
  • clindamycin lethal to parasite (blocks apicoplast translation)
  • but adding high levels IPP to culture media during clindamycin treatment relieves effect and cells survive
  • shows, at least in culture, it is IPP biosynthetic role of apicoplasts that is essential

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