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Genetics 301 final Exam Name:______________________________

Dr. F. Nargang I.D.#:_______________________________December 19, 2000

There are 11 questions and a total of 60 possible marks on this exam. You must do each question. Good luck!The exam booklet should contain seven pages. The exam will constitute 40% of your final grade. TIMELIMIT IS TWO HOURS.

Question 1. From the assigned paper (Jacobs et al. (2000) BioEssays 22, 564-572).

a) Suppose mitochondria from two different cell lines are each heteroplasmic for different mtDNARFLP markers represented as follows:

mtDNAs from cell line1: and

mtDNAs from cell line 2: and

These mtDNAs are introduced from their parent cell lines into one ρ0 human cell line. Assumeeach mtDNA molecule contains fully functional mitochondrial genes. Describe the mtDNAcontent of the progeny cells after several months of mitotic division if:

i) the faithful nucleoid model is true (2 mark)

Virtually all progeny should be either or

All other combination might exist in a few cells.

ii) the faithful nucleoid model is not true (2 mark)

All possible combination may be present. Also expect to see a significant number of cellshomoplasmic for each type.

b) How did the authors obtain cultured human cells with mtDNAs containing, on average, one mutationevery two kilobases? (1 mark)

Used cells that contained a "mutator" version of mt DNA polymerase.

c) Assume enucleated human cells with mitochondria capable of performing oxphos, but containingmutant mtDNAs as described above in b), were fused using cybrid transfer techniques, to a ρ0 cell with awild-type nucleus. The cells were then grown in medium containing glucose, pyrimidines, and pyruvate.After several months of growth in such medium, individual cells were assayed for their ability toperform oxidative phosphorylation. What would your assays show if the faithful nucleoid model is true?Briefly explain your answer. (2 marks)

Virtually all cells should be oxphos+ because the nucleoids contain mtDNAs with differentmutations that complement within the nucleoid. These should not readily segregate away fromeach other but be maintained as a faithful nucleoid unit.

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Question 2. Using labelled diagrams, show the behaviour of mtDNAs that you would expect to see in across of a ρ+ yeast cell with a very hypersuppressive ρ- cell. Show the mtDNAs in a zygote at time=0,time=2 hr, and time = 3 to 6 hr (ie, just prior to first bud formation). For symbols use for the ρ+

mtDNA and for the hypersuppressive ρ-. Explain what is happening at the different time pointsand describe how your model accounts for the behaviour of hypersuppressive petites. (5 marks)

Various possible answers-key is somehow the hypersensitive mtDNA must be preferentially segregated or at least move toboth sides of the zygote before bud formation so that the bud contains only or both

In further generation the might be replicated preferentially to so that has quickly aken over the culture.

Question 3. Briefly discuss a possible model to explain why mitochondrial genes have migrated to the nucleus.(2 marks)

Genome without a sexual phase can accumulate deleterious mutations since they can't be removedthrough recombination to give individual molecules without mutations.Since mitochondria have no sexual phase, the genes, originally in the mitochondria, are now safer in thenucleus.

Question 4. Multiple choice questions. There is one best answer for each question. (10 marks)

Which of the following does not occur in mitochondriaa) fatty acid oxidationb) citric acid cyclec) certain aspects of cellular iron metabolismd) glycolysise) oxidative phosphorylation

Which of the following contains the most protein subunitsa) complex Ib) complex IIc) complex IIId) complex IVe) complex V

Which of the following genes is not found in the mtDNA of all species studied thus far:a) large mitochondrial rRNA geneb) small mitochondrial rRNA genec) at least some tRNA genesd) cox1 genee) cob gene

If the sequence of a random 2 kilobase segment of mtDNA from a kinetoplast protozoan showed no ORFs, wecould conclude:a) the mtDNA was from a ρ- strainb) that the segment of mtDNA was not the gene for a mitochondrial proteinc) the mRNA from that region of the mtDNA must be editedd) the cell examined must be heteroplasmice) none of the above

Mutants isolated by Boris Ephrussi were called “petites” anda) were unable to ferment glucoseb) were unable to grow on glycerolc) were a class of mutant with smaller than normal cellsd) included mutants of complex I

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e) none of the above

All hypersuppressive petites area) segregational mutantsb) vegetative mutantsc) found to give rise to tetrads of the type “4 wild-type: 0 petite” when crossed to wild-type cells.d) able to suppress mutations affecting cox3 translatione) found to contain the same region of mtDNA

If a wild-type Saccharomyces cerevisiae cell has a mtDNA whose size is 75 kb, then a ρ- mutant in which twothirds of the molecule has been deleted will have mtDNA molecules that area) 75 kbb) 50 kbc) 25 kbd) 12 kbe) 125 kb

Mutations in the omega gene of yeast mtDNAa) result in an inability to grow on non-fermentable carbon sourcesb) result in decreased rates of mtDNA recombinationc) have no effect on mitochondrial functiond) decrease overall oxphos ratese) result in decreased levels of Abf2 bound to mtDNA

Cybrids area) a fusion of whole cells with enucleated cells from another cell lineb) mitochondria in which fusion has led to the formation of oxphos complexes containing subunits from twodifferent cell linesc) fusions of mitochondria with ρ0 cellsd) yeast cell lines resulting from zygotes in which mitochondria have fused and mixed their contentse) cells without nuclei

The arrangement of promoters relative to the start site of transcription in yeast mtDNA is most similar toa) the arrangement in bacteria like E. colib) the arrangement in animal mtDNAc) the arrangement in phage T3d) the arrangement in the yeast nucleuse) none of the above

Question 5. You are studying various aspects of mitochondrial function in a mammalian tissue culture cell line.Make the simplest prediction of the effect of the indicated mutations by completing the following Table with“higher” or “lower” or “no change” in the appropriate box. (5 marks)

Ratio of mtDNA moleculescontaining D-loops to

molecules without D-loops

Ratio of mitochondrial rRNAs tothe number of mtDNA

molecules

Mutation in HSP that decreasesits efficiency by a factor of ten.

no change lower

Mutations in LSP that decreasesits efficiency by a factor of ten.

no change no change

Deletion of the TAS’s lower lower

Deletion of the CSB’s lower higher

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Mutations making OL ten foldless efficient

no change higher

Question 6. Why is the finding that yeast mtDNA binds to an integral membrane protein of the innermitochondrial membrane, not sufficient to explain why the mtDNA does not diffuse in a zygote? (2 marks)

Because it has been shown that inner membrane proteins diffuse in the fused mitos of the zygote.

Question 7. Explain how varying levels of the Abf2 protein affect levels of mtDNA in yeast. Give a model toexplain the data. (4 marks)

Normal Abf2 protein levels = 100% mtDNA levels2-3 fold increase= 200% mtDNA levels10-20 fold increase = severe reduction in mtDNA leading to generation of ρ0 cellsSince Abf2p is involved in mtDNA recombination a slight increase may lead to slightly morerecombination - priming of mtDNA replication may require a recombination intermediate.Therefore perhaps more DNA replication with slight increase in Abf2p but large increase could 1. trapmtDNA in recombination structure OR 2. bind mtDNA so it is inaccessible ot replication apparatus.

Question 8. In class, we examined data produced from following the behavior of a mitochondrial matrix protein(citrate synthase or CS) and mtDNA in early zygotes of Saccharomyces cerevisiae up to the point of first budformation. In the diagrams below, indicate the expected findings for each time-point considering the differentassumptions given regarding the strains used in each case. Assume you have access to all fluorescent dyes,antibodies, etc as used in the classroom discussions. Draw the zygotes with the first strain listed in the crosson the left hand side.

Symbols: Citrate synthase: mtDNA:

a) Cross 1: p+ (oliR, capS) CS- X p0, CS+. Assume that this is a normal Saccharomyces cerevisiae cross asdiscussed in class. (3 marks)

Time = 0 hours Time = just prior to first bud formation

2N 2N

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What is the expected mitochondrial genotype of a bud produced at the right hand side of the zygote at t = justprior to first bud formation.

ρ+ (oliR capS)

b) Cross 2: p+ (oliR, capS) CS- X p+ (oliS, capR), CS+. This cross is done in a species in which most thingsoccur as in Saccharomyces cerevisiae, but there is no tethering of mtDNA molecules. (3 marks)

Time = 0 hours Time = just prior to first bud formation

What is the expected mitochondrial genotype of a bud produced at the right hand side of the zygote at t = justprior to first bud formation?

ρ+ mixture of oliR capS and oliS capR (and some oliR capR and oliS capS - recombinants)

c) Cross 3: p+ (oliR, capS) CS- X p+ (oliS, capR), CS+. This cross is done in a species in which most thingsoccur as in Saccharomyces cerevisiae, but there is no fusion of mitochondria. (3 marks)

Time = 0 hours Time = just prior to first bud formation

What is the expected mitochondrial genotype of a bud produced at the right hand side of the zygote at t = justprior to first bud formation?

We're still assuming that tethering occurs in S. cer. so therefore ρ+ (oliS capR)

Question 9. How would the following mutations affect the removal of an intron encoding a maturase? (3marks)

i) a nonsense (ie, stop codon) mutation in the maturase coding sequence

intron not removed (maturase non functional)

ii) a nonsense mutation in the exon upstream of the maturase

intron not removed (maturase non functional)

2N 2N

2N2N

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iii) a deletion of 8 base pairs in the intron sequence following the maturase and preceding the downstream exon

intron removed (should have no effect on maturase)

Question 10. Answer the following questions about a yeast mutant that had one of its mtDNA promotersequences entirely deleted.

i) The mutant is unable to carry out oxphos. Explain why. (2 marks)

Transcripts of genes controlled by that promoter will not be made therefore some aspect of oxphos willnot be complete.

ii) Would you expect to be able to find mtDNA suppressors of the loss of ability to carry out oxphos? If youranswer “no”, explain why. If you answer “yes”, explain the nature of the suppressors you would find. (3 marks)

Yes.There would be larger deletions leading to fusion of one transcriptional unit with the one missing thepromtoer. The suppressor would have to be heteroplasmic.Also containing some the the promoter deleted mtDNA (simialr to pet494 suppressors)

Question 11. Short answer questions.

a) Circle the scenario that is more suggestive of a small bottleneck for mtDNA inheritance? (1 mark)

i) Mother with no detectable mt DNA mutations in her somatic tissues↓

Daughter with 30% mutant mtDNA↓

Severely affected child with mtDNA disease

ii) Mother with no detectable mt DNA mutations in her somatic tissues↓

Daughter with low level of mutant mtDNA↓

Daughter with 10% mutant mtDNA↓

Daughter with 40% mutant mtDNA↓

Severely affected child with mtDNA disease

b) In which mitochondrial compartment are each of the following proteins located? (2 marks)

i) citrate synthase matrix

ii) Tom6p outer membrane

iii) Yta10p inner membrane

iv) F0 ATP synthase inner membrane

c) What is a chondriolite (1 mark)

A unit of mtDNA inheritance/transmission containing more than one mtDNA molecule ie. a nucleoid withmore than one mtDNA

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d) Why do mutations in the mgt1 gene affect the segregation or transmission of different ρ- mtDNAs to adifferent extent? (2 marks)

The ρ- mtDNAs with larger deletions (therefore more repetitive DNA) will be affected to the greatestextent because they will have more recombination junctions formed. These will not be resolved due tothe lack of mgt1 protein.

e) What evidence exists to suggest that mtDNA mutations in an individual increase with age? (2 marks)

Eg. Individuals sampled at different ages show increase in mtDNA mutations as they get older.52 years - no mutations in mtDNA71 years - mutations detected in mtDNA (same individual)

(1.5 marks for example of mt disease patients getting symptoms as they age.)