User:Nkuldell/mtDNA

mtDNA (re)org

Justificiation

Given

• existing but limited techniques for tweaking mtDNA
• ability to synthesize long pieces of DNA from scratch
• defined phenotypes of S. cerevisiae with and without mtDNA
• transcription/translation of mtDNA is somewhat independent of transcription/translation of nuclear DNA

it seems like an interesting and useful effort to rewrite the mtDNA to make it more modular and editable.

To assess quality of this project idea ask want to ask
1. can it be done?
2. how?
3. why? Answer to "why" is at least in part to see what happens since I like surprises but more specific answers are needed to be more generally satisfying.

Some quick ideas as to why....
1. it will be interesting to see if different organizations of mtDNA are tolerated.
2. assuming some success, this effort could provide insights into

• mt genetics,
• mt biogenesis,
• mt gene expression,
• and coordination of mt and nuclear genes.

3. it might give rise to

• new tools for molecular manipulation and study of mt DNA.

4. this effort could also provide

• a discrete, spatially isolated, insulated platform running inside living yeast for protein engineering,
• and a compartment for implimenting logic functions or genetic manipulations separate from nuclear DNA.

5. an engineered mt genome might allow

• additional cellular functions (mitochondrial or other) to be readily added
• or existing functions to be predictably modified/improved.

6. could populate the Registry of Std Biological Parts [1] with useful inventory.
7. could provide useful teaching platform for biological engineering curriculum
8. one interesting thing to try might be to recode mtDNA, altering GC/AT balance or freeing rarely used codons for unnatural coding
9. human mitochondrial defects clinically relevant, e.g. [2] [3]

Background info

Biogenesis of mitochondria

From Pon and Schatz, Ch7 in The Molcular and Cellular Biology of the yeast Saccharomyces

• Growth phase and mt biogenesis

Growth Phase	Nutritional state	Mitochondria number	Mitochondria features
mid- and mid-late log	glucose available	few (1-2, according to Stevens in CSHLP, 1981)	poorly developed cristae
late log	glucose nearly depleted	mitochondrial mass/cell increases (from low of 3.2% of cell's volume, again according to Stevens in CSHLP, 1981)	fully developed cristae
stationary phase (early)	switch from glucose fermentation to ethanol oxidation	mitochondria arranged at periphery of cell (up to 12.6% of cell volume)	mitochondrial reticulum broken into many smaller, regular mitochondria (up to 44 in number)
stationary phase (late)	extended starvation	mitochondria degenerate	see parallel or concentric lamellae

• To a first approximation, the evolutionary history of mtDNA seems undescribed and untested.
• mtDNA is probably circular
• low GC content (about 18%)
• extreme segregation of GC- vs AT-rich regions (in Grossman et al JMB (1971) 62:565)
• islands of sequences affect melting, and long AT-rich intergenic regions are termed "spacers" but their role is not understood
• what's encoded on S. cerevisiae mtDNA:

Genes for	number	names	comments
proteins for oxidative phosphorylation	seven	apocytochrome b, subunits I-III of cytochrome oxidase, subunits 6, 8 and 9 of ATP synthase	COB is gene for cytochrome b and has six exons (B1-B6) and five introns (bI1-bI5) though some lab strains are missing first three introns, COX1 is gene for subunit I of cytochrome oxidase
protein for small subunit of mitochondrial ribosome	one	Var1p
mitochondrial tRNAs	24 or 25
tRNA maturation	one	9S
additional ORFs	about 20	within introns of large rRNA, COX1 and COB	can delete all 20 introns and strain is viable (Seraphin 1987) though deletion of individual introns can affect respiration

• yeast mtDNA is unlike other mt genomes in that it lacks subunits for NADH dehydrogenase
• yeast mtDNA has ORFs not found in other mt genomes. These ORFs thought to encode proteins that interact with nucleic acids to carry out excision of introns from mRNA, DNA transposition, DNA deletion, homologous recombination

From Fred Sherman's yeast fact sheets

in Guthrie and Fink Ch1 and [4]

• ~10% total cellular DNA content is mtDNA (range 5-15%) encoding mt translation machinery and 15% of mt proteins
• averages 50 copies mtDNA/cell (range 8-130), each with 70-76 kb dsDNA which is about 5X larger than human mtDNA (according to de Zamaroczy and Bernardi, 1985)
• nonMendelian inheritance, but since daughter cells receive few mitochondria find genes quickly segragate (see Guthrie and Fink Ch 5 by Bonnefory and Fox)
• wild type mtDNA is designated rho+
• cells lacking mtDNA are designated rho0 and are respiration deficient (lack cytochrome b and subunits of cytochrome oxidase and ATPase comples), retain mitochondria though they are morphologically abnormal
• cells with mutant mtDNA are described as rho- . Described mutations include cytochromes a*a3, b. These are also called "petite" mutants. They have typically lost a lot of mtDNA required for mt protein translation but have amplified remainder of genome to leave same amount of mtDNA total. Unlike rho+ cells, rho- cells do not require mt protein synthesis to replicate (as you might guess since they have lost DNA for mt protein synthesis!).
• mutant phenotypes:
  • inability to grow on Nfs (nonfermentable substrates = Nfs- ) such as ethanol or glycerol though this can arise from mutations in nuclear genes (e.g. pet1, cox4, hem1, cyc3) or single gene mutations in mtDNA , termed "mit-" "syn-" or "antR"
  • "mit-" are Nfs- from mtDNA single mutations that are respiration- but mt translation+ (e.g. cox1, cox2, cox3, cob1 or box, atp6, atp8, atp9 or pho2
  • "syn-" are Nfs- from mtDNA single mutation that results in respiration- and mt translation- (e.g. tRNAasp or M7-37)
  • "antR" is another mutant phenotype associated with mtDNA mutations. These result in antibiotic resistance e.g. rib1 for resistance to erythromycin from 21SrRNA change, rib3 for resistance to chrolampenical from 21S rRNA change, par1 for resistance to paromomycin from 16S rRNA change, and oli1 for resistance to oligomycin from ATPase subunit 9 mutation. have also described mutations in cytochrome b leading to diuron resistance.

From Bonnefoy and Fox, Ch5 in Guthrie and Fink

• replication of mtDNA poorly understood but is known to depend on mtDNA ori sequences and (for reasons unknown) on mt protein synthesis when cells are rho+.
• small bud gets minimal cytoplasmic contents (i.e. few mitochondria) leading to rapid mitotic segregation of mt DNA genotypes. Except in rare cases, heterplasmic cells rapidly give rise to homoplastic progeny (ref is in Science (1988)240:1538).
• S. c. is only species to date in which homologous recombination can be used to rewrite mtDNA
• transformable with ballistics (ref for this is 1991 Meth in Enzym)
• drug resistant phenotypes associated with "antR" mutations are only detected when strains are grown on Nfs in respiration+ strains and can arise spontaneously so they are not good transformation markers.
• nuclear auxotrophic markers such as URA3 and TRP1 are not expressed when inserted into mtDNA but (dna? rna?) can escape from mt into nucleus, readily scored on SC-ura or SC-trp.
• to express nuclear genes in mt need to rewrite in mt genetic code (ref for this is Fox in Annu Rev Genet (1987) 21:67-91). One useful example is ARG8m , which is nuclear ARG8 gene recoded for mtDNA expression. Gene is transcribed and translated in mt then diffuses to cytoplasm and complements arg- p-type. File:Macintosh HD-Users-nkuldell-Desktop-recodeformtDNA PNAS96.pdf
• Similarly GFPm has been reported in which nuclear GFP has been recoded for mt expression [5]
• yeast mating allows mitochondria of haploids to fuse, allowing homology-dependent recomb btw parental mtDNAs. In this way, mating of a haploid rho- (nonrespiring due to no mt prot synthesis) with a haploid rho+ mit- (i.e. nonrespiring due to defective cytochrome gene, e.g.) will give rise to respiring rho+ diploids which are selectible on Nfs.

methods for re-writing mtDNA

1. yeast transformation

From Guthrie and Fink Ch5

• DNA prepecipitated on metal particles, such as tungsten particles from BioRad, cat #165-2265 for 0.4 um or cat#165-2266 for 0.7 um particles or 0.6 um gold particles cat#165-2262. Helium shock from BioRad instrument (PDS-1000/He system) used to rupture membranes (nuclear and mitochondrial) and accelerate metal particles into cells. DNA on particles introduced to nucleus and mitochondria in this way.
• DNA on particles has genetic marker for selection via complementation of nuclear DNA phenotype. Colonies that recover from this selection are checked for desired mtDNA phenotype, e.g. rho+ if starter strain was rho-, marker rescue expressed in trans.
• std strains from ATCC, derived from DBY947, which is S288c type strain
  • ATCC 201440 = MCC109rh0 MATalpha ade2-101, ura3-52, kar1-1 (rho0)
  • ATCC 201442 = MCC123rhoO MAT a ade2-101, ura3-52, kar1-1 (rho0)
• kar1-1 allele is karyogamy-defective mutation, allowing mitochondria to fuse efficiently but reducing nuclear fusion during mating rxn, allowing haploid mitochodrial cytoductants to be isolated.
• transformation of rho0 strains is often ~10 to 20 x more efficient than isogenic rho+ with small mtDNA deletion
• successful transformation of linear DNA with as little as 260 bp of homologous seq flanking deletion mutation in rho+ recipient. This result may be key for strain construction success.

2. yeast mating

placeholder

methods for measuring respiration

Methods used to measure respiration and reactive oxygen species (ROS) in File:Macintosh HD-Users-nkuldell-Desktop-mtDNA re-org-RPO41muts MCB06.pdf

1. changes in chronological life span
2. balanced mt translation
3. respiration in stationary-phase glucose cultures
4. changes in ROS production
5. sensitivity to oxidative stress

what's known and what isn't about mtDNA replication

• catalytic subunit for mtDNA transcription RPO41 is also required for mtDNA maintainence, suggesting it acts as a primase for replication.
• MIP1 is a nuclear gene, mutation of which leads to loss of mtDNA but does not affect nuclear DNA synthesis.
• replication of mtDNA in rho+ cells requires protein synthesis. Observation is that inhibition of protein synthesis (e.g. with chloramphenicol or by mutation of mitochondrial protein synthesis machiner) leads to mtDNA instability and loss.
• replication of mtDNA in rho- cells defective for mitochondrial protein synthesis does not require protein synthesis (obviously). This difference in rho+ and rho- requirements suggests that rho+ and rho- mtDNA replicate by different mechanisms or that inhibition of translation induces rho+ mtDNA instabilty.

what's known and what isn't about mtDNA transcription

• mtDNA transcription by nucleus-encoded RNAP made from two non-identical subunits
  • RPO41 , 145 catalytic subunit [6] and
  • MTF1 , specificity factor to allow mt promoter recognition [7] (43 kD according to Schinkel 1987 or 70 kD according to Ticho and Getz 1988)
• synthesis of both subunits is glucose repressed
• RNAP for mtDNA transcription is most like T3 or T7 RNAP rather than bacterial
• mt "promoter" 5' tataagta
• T7 RNAP concensus promoter BBa_R0085 5'taatacgactcactata ggg aga, where green is polymerase recognition seq and red is transcription initiation site.
• introns can be deleted from large rRNA (normally has 1 intron), COB (normally has up to 5 introns) and COXI (normally up to 10 introns) . Strain lacking all introns is viable and have normal oxidative phosphorylation (Seraphin PNAS 1987 File:Macintosh HD-Users-nkuldell-Desktop-intronlessmtDNA PNAS87.pdf)

what's known and what isn't about mt translation

• alternative genetic code:

[[8]] [[9]]

• multiple nuclear-encoded gene products targetted to mt inner membrane to enable mt translation by binding to 5'UTR of mt-encoded transcripts.

[[10]]

Placeholder: tools for studying/re-organizing mtDNA

• b-gal alpha complementation with alpha fragment on incoming DNA and omega in residence on mt genome. Can also use complementation to study prot localization to mt.
• 5' and 3' truncations of nuclear marker (eg ura) vs marker on mt genome (eg his)
• alpha-galactosidase vs beta-galactosidase reporters. Two proteins have different pH optima (4 vs 7) so require different assay conditions. Former is encoded by yeast MEL1 that is not transcribed in S288c bkgd. This enzyme is cell wall associated but is also secreted into media in stationary phase cells so can be assayed on plates but do not get full activity if using liquid assays. Colorimetric assay for alpha-galactosidase uses X-alpha-gal or alpha-PNPG vs beta-galactosidase uses X-gal or ONPG.
• fluorescence to follow mt prot localization/biogenesis
• HO endonuclease inducible in mt?
• measuring respiration?

other detailed yeast mt reviews

• Butow et al in Methods in Enzymology (1996) 264:265
• Perlman et al in Methods in Enzymology (1979) 56:139
• Fox et al in Methods in Enzymology (1991) 194:149
• B. Dujon in The Molecular Biology of S.c.: Life cycle and inheritance" CSHLP (1981)