User:Nkuldell/mtDNA pt2

Modifications
 Models for systematic re-organization of genetic systems  [] [] []
 * Drew's T7 paper
 * Promoter evolution C. albicans to S. cerevisiae
 * Reduced E. coli genome

Summary of what's currently on mtDNA

 * what's encoded on S. cerevisiae mtDNA:


 * 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
 * S. cerevisiae mt genome is ~ 5X the size of the human one, which has 16,568 base pairs with 37 genes (13 proteins, 22 tRNAs and two rRNAs) according to wikipedia mtDNA entry.

mt genetic material to keep / to cut
Info from SGD 05.31.06[] Genes in black are ??s for cutting Seq info for intron-less mtDNA. Downloaded from SGD 05.31.06 Phenotype info for mtDNA. Downloaded from SGD 05.31.06

Available

 * ARG8m
 * GFPm
 * BARSTAR
 * RIPm (not really a reporter but nuclear gene recoded and moved to mito)

To make?
DNA sequence =1115 bp [] Prot sequence = ~372 aa [] Dr Jean-Michel Camadro DR1 CNRS Laboratoire d'Ingénierie des Protéines et Contrôle Métabolique Dpt de Biologie des Génomes INSTITUT JACQUES MONOD (UMR 7592 CNRS - Universités Paris 6 & 7) Couloir 43-44 2 Place Jussieu, F-75251 Paris Cedex 05 Tel : 33 (0)1 44 27 81 70; Fax : 33 (0)1 44 27 57 16
 * Could try MEL1, B-gal for assay-able enzymes (latter is very big and MEL1 p-type of common strain is not known)
 * Could try xFPs though GFP is done and is not very bright (according to E. Sia)
 * Could try LYS12 [] (aka LYS10,LYS11)
 * Homo-isocitrate dehydrogenase, an NAD-linked mitochondrial enzyme required for the fourth step in the biosynthesis of lysine, in which homo-isocitrate is oxidatively decarboxylated to alpha-ketoadipate
 * Could try PUT1
 * Proline oxidase, nuclear-encoded mitochondrial protein involved in utilization of proline as sole nitrogen source; PUT1 transcription is induced by Put3p in the presence of proline and the absence of a preferred nitrogen source
 * Could try HEM1
 * 5-aminolevulinate synthase, catalyzes the first step in the heme biosynthetic pathway ; an N-terminal signal sequence is required for localization to the mitochondrial matrix; expression is regulated by Hap2p-Hap3p
 * nuclear gene is 1.647 kb, protein is 549 aa
 * SGD reports systematic deletion is inviable but also reports null is viable, heme and methionine auxotroph
 * Mark has nuclear K/O with KanMX
 * nuclear K/O requires addition of 8,5-amino-levulinate to grow
 * Mark believes there is a UV or spec method for measuring heme levels
 * antiserum to HEM1 described in 1986 paper and may be available from
 * alternatively could epitope tag at C-terminus (N-terminus is used for mt import and probably shouldn't mess with this section for fusion). Possible tags are HA, c-myc  or better still (if possible) might be alpha-complementing portion of b-gal. . Defined as first 81 aa in . Could then select for mitochondrial expression of HEM1 in nuclear mutant by selection on -heme or -met plates and measure expression levels with Western if alpha-specific antibody is available, and assay on X-gal plates or liquid assay in strain expressing mito-targetted omega-portion. If fusion was directed to COX2 (instead of other regions of mito genome that will eventually want to clean up) then can select for integrants as respiration defective, and don't need to worry about
 * heme req'd in
 * 1) ergosterol biosynthesis pathway
 * 2) fatty acid biosynthesis pathway ], and
 * 3) methionine biosynthesis pathway though no step directly requiring heme seen from cursory look at biochemical pathways.

Strain Info
 Most strains are S273-10B background which has low freq of spont petite formation relative to other strains such as S288c  (according to E. Sia)

 DSF160 (common transformation recipient)
 * Genotype: MATalpha ade2-101 leu2del ura3-52 arg8del::URA3 kar1-1 [rho0]
 * ref is Steele et al (1996) PNAS 93(11):5253-7 []
 * Note: biolistically transform with pRS415 and mito-targetted material, select for LEU+, replica to TF236 tester strain to find mito transformants

 TF236  (tester for mitochondrial transformation)
 * Genotype: ino1::HIS3 arg8::hisG pet9 ura3-52 lys2 cox3::arg8m-1
 * ref is Bonnefoy and Fox (2000) MGG 262:1036 []
 * Note: hisG is scar from URA3 disruption a la Kleckner method
 *  Note  (important): pet9, aka op1, is synthetic lethal in combination with rho0. From SGD: PET9 is major ADP/ATP carrier of the mitochondrial inner membrane, exchanges cytosolic ADP for mitochondrially synthesized ATP; required for viability in many common lab strains carrying a mutation in the polymorphic SAL1 gene