User:Nkuldell/mtDNA

mtDNA (re)org

Given

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

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

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,
• 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 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

Biogenesis of mitochondria

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

• To a first approximation, the evolutionary history of mtDNA seems undescribed and untested.

From Fred Sherman fact sheet

in Guthrie and Fink Ch1 and [2]

• ~10% total cellular DNA content is mtDNA encoding mt translation machinery and 15% of mt proteins
• averages 50 copies mtDNA/cell (range 8-130), each with 70-76 kb dsDNA
• 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 [3]
• 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

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.

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)