User:Nkuldell/mtDNA pt2

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Current revision (15:05, 1 August 2006) (view source)
 
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**nuclear K/O requires addition of 8,5-amino-levulinate to grow
**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
**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 <br>
 +
Dr Jean-Michel Camadro<br>
 +
DR1 CNRS<br>
 +
Laboratoire d'Ingénierie des Protéines et Contrôle Métabolique<br>
 +
Dpt de Biologie des Génomes<br>
 +
INSTITUT JACQUES MONOD (UMR 7592 CNRS - Universités Paris 6 & 7)<br>
 +
Couloir 43-44<br>
 +
2 Place Jussieu, F-75251 Paris Cedex 05<br>
 +
Tel : 33 (0)1 44 27 81 70; Fax : 33 (0)1 44 27 57 16 <br>
**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 <b>HA, c-myc </b> or better still (if possible) might be <b>alpha-complementing portion of b-gal.</b> . Defined as first 81 aa in [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]. 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  
**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 <b>HA, c-myc </b> or better still (if possible) might be <b>alpha-complementing portion of b-gal.</b> . Defined as first 81 aa in [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]. 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  
**heme req'd in  

Current revision

Contents

Modifications

Models for systematic re-organization of genetic systems

  • Drew's T7 paper

[[1]]
Image:Macintosh HD-Users-nkuldell-Desktop-BE109-Drew's module-rewriteT7 endy molsysbio05.pdf

  • Promoter evolution C. albicans to S. cerevisiae

[[2]]
Image:Macintosh HD-Users-nkuldell-Desktop-AlumFunds05 SynBio-chrom redesign-rewiringyeasttxn Sci05.pdf

  • Reduced E. coli genome

[[3]]
Image:Macintosh HD-Users-nkuldell-Desktop-MinimalColi Sci06.pdf

Change to make number of such changes method for generating changes comments
remove dubious ORF sequences, hypothetical proteins 11 ORFs
4781 bps total
txn' with linear DNA bearing flanking sequences?
need selectible marker to verify integration
Q0010, Q0017, Q0032, AI5_BETA, Q0092, Q0142, Q0143, Q0144, Q0182, Q0255, Q0297
remove introns 14 introns not including those deleted from deletion of hypothetical ORFs
20428 bps total
start with intronless strain from Seraphin? AI1, AI2, AI3, AI4, last 3 introns COX I, AI5_ALPHA, BI2, BI3, BI4, last 2 introns COB, SCEI
move all transcription to Watson strand 1 incident
73 bps to flip not counting promoter
flipping this may be hard. perhaps easier to swap out old and place in new, depending on overlaps with other genetic elements. tRNA-Thr
regulate promoters with alternative polymerase don't use sugar-regulated promoter has T7 RNAP been expressed in yeast? Cu or Tet?
what's left...

Summary of what's currently on mtDNA

  • 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
  • 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[[4]]
Genes in black are ??s for cutting
Seq info for intron-less mtDNA Image:Macintosh HD-Users-nkuldell-Desktop-mtDNAseq coding 55features. Downloaded from SGD 05.31.06 Phenotype info for mtDNA Image:Macintosh HD-Users-nkuldell-Desktop-phenotype mtDNA 55features. Downloaded from SGD 05.31.06

Gene name alias mt chromosomal coordinates description
tP(UGG)Q 731 to 802 tRNA-Pro
Q0010 ORF6 3952 to 4338 Dubious mitochondrial open reading frame unlikely to encode a protein
Q0017 ORF7 4254 to 4415 Dubious mitochondrial open reading frame unlikely to encode a protein
15S_rRNA 15S_RRNA_2 6546 to 8194 Ribosomal RNA of the small mitochondrial ribosomal subunit;
tW(UCA)Q 9374 to 9447 tRNA-Trp1
Q0032 ORF8 11667 to 11957 Hypothetical protein
AI1 Q0050 13818 to 16322 Reverse transcriptase required for splicing of the COX1 pre-mRNA, encoded by a mobile group II intron within the mitochondrial COX1 gene
AI2 Q0055 13818 to 18830
CDS 13818 - 13986, and 16435 - 18830
Intron 13987 - 16434
Reverse transcriptase required for splicing of the COX1 pre-mRNA, encoded by a mobile group II intron within the mitochondrial COX1 gene
AI3 Q0060 13818 to 19996
CDS 13818 - 13986, 16435 - 16470, 18954 - 19996
Intron 13987 - 16434, 16471 - 18953
Endonuclease I-SceIII, encoded by a mobile group I intron within the mitochondrial COX1 gene
AI4 Q0065 13818 to 21935
CDS 13818 - 13986, 16435 - 16470, 18954 - 18991, 20508 - 21935
Intron 13987 - 16434, 16471 - 18953, 18992 - 20507
Endonuclease I-SceII, encoded by a mobile group I intron within the mitochondrial COX1 gene; intron is normally spliced by the BI4p maturase but AI4p can mutate to acquire the same maturase activity
AI5_ALPHA Q0070, I-SceIV 13818 to 23167
CDS 13818 - 13986, 16435 - 16470, 18954 - 18991, 20508 - 20984, 21995 - 23167
Intron 13987 - 16434, 16471 - 18953, 18992 - 20507, 20985 - 21994
Endonuclease I-SceIV, involved in intron mobility; encoded by a mobile group I intron within the mitochondrial COX1 gene
COX1 Q0045 OXI3 13818 to 26701
CDS 13818 - 13986, 16435 - 16470, 18954 - 18991, 20508 - 20984, 21995 - 22246, 23612 - 23746, 25318 - 25342, 26229 - 26701
Intron 13987 - 16434, 16471 - 18953, 18992 - 20507, 20985 - 21994, 22247 - 23611, 23747 - 25317, 25343 - 26228
Subunit I of cytochrome c oxidase, which is the terminal member of the mitochondrial inner membrane electron transport chain; one of three mitochondrially-encoded subunits
AI5_BETA Q0075 24156 to 25255
CDS 24156 - 24870, 24906 - 25255
Intron 24871 - 24905
Protein of unknown function, encoded within an intron of the mitochondrial COX1 gene; translational initiation codon is predicted to be ATA rather than ATG
ATP8 Q0080, AAP1 27666 to 27812 Subunit 8 of the F0 sector of mitochondrial inner membrane F1-F0 ATP synthase, encoded on the mitochondrial genome
ATP6 Q0085OLI2, OLI4, PHO1 28487 to 29266 Mitochondrially encoded subunit 6 of the F0 sector of mitochondrial F1F0 ATP synthase, which is a large, evolutionarily conserved enzyme complex required for ATP synthesis
Q0092 ORF5 30874 to 31014 Hypothetical protein
tE(UUC)Q 35373 to 35444 tRNA-Glu
BI2 Q0110 36540 to 38579
CDS 36540 - 36954, 37723 - 38579
Intron 36955 - 37722
Mitochondrial mRNA maturase with a role in splicing, encoded by both exon and intron sequences of partially processed COB mRNA
BI3 Q0115 36540 to 40265
CDS 36540 - 36954, 37723 - 37736, 39141 - 40265
Intron 36955 - 37722, 37737 - 39140
Mitochondrial mRNA maturase, forms a complex with Mrs1p to mediate splicing of the bI3 intron of the COB gene; encoded by both exon and intron sequences of partially processed COB mRNA
BI4 Q0120 36540 to 42251
CDS 36540 - 36954, 37723 - 37736, 39141 - 39217, 40841 - 42251
Intron 36955 - 37722, 37737 - 39140, 39218 - 40840
Mitochondrial mRNA maturase, forms a complex with Nam2p to mediate splicing of the bI4 intron of the COB gene; encoded by both exon and intron sequences of partially processed COB mRNA
COB Q0105, COB1, CYTB 36540 to 43647
CDS 36540 - 36954, 37723 - 37736, 39141 - 39217, 40841 - 41090, 42508 - 42558, 43297 - 43647
Intron 36955 - 37722, 37737 - 39140, 39218 - 40840, 41091 - 42507, 42559 - 43296
Cytochrome b, mitochondrially encoded subunit of the ubiquinol-cytochrome c reductase complex which includes Cobp, Rip1p, Cyt1p, Cor1p, Qcr2p, Qcr6p, Qcr7p, Qcr8p, Qcr9p, and Qcr10p
OLI1 Q0130, ATP9, OLI3 46723 to 46953 F0-ATP synthase subunit 9 (ATPase-associated proteolipid), encoded on the mitochondrial genome; mutation confers oligomycin resistance; expression is specifically dependent on the nuclear genes AEP1 and AEP2
tS(UGA)Q2 48201 to 48290 tRNA-Ser
VAR1 Q0140, VARI 48901 to 50097 Mitochondrial ribosomal protein of the small subunit, mitochondrially-encoded; polymorphic in different strains due to variation in number of AAT (asparagine) codons; translated near the mitochondrial inner membrane
Q0142 ORF9 51052 to 51228 Hypothetical protein
Q0143 ORF10 51277 to 51429 Hypothetical protein
Q0144 54109 to 54438 Hypothetical protein
21S_rRNA 21S_rRNA_3, 21S_rRNA_4 58009 to 62447
Intron 60725 - 61867
Noncoding_exon 58009 - 60724, 61868 - 62447
Mitochondrial 21S rRNA; intron encodes the I-SceI DNA endonuclease
SCEI Q0160OMEGA, I-SceI 61022 to 61729 I-SceI DNA endonuclease, encoded by the mitochondrial group I intron of the 21S_rRNA gene; mediates gene conversion that propagates the intron into intron-less copies of the 21S_rRNA gene
tT(UGU)Q1 63862 to 63937 tRNA-Thr
tC(GCA)Q 64415 to 64490 tRNA-Cys
tH(GUG)Q 64596 to 64670 tRNA-His
Q0182 ORF11 65770 to 66174 Hypothetical protein
tL(UAA)Q 66095 to 66179 tRNA-Leu
tQ(UUG)Q 66210 to 66285 tRNA-Gln
tK(UUU)Q 67061 to 67134 tRNA-Lys
tR(UCU)Q1 67309 to 67381 tRNA-Arg
tG(UCC)Q 67468 to 67542 tRNA-Gly
tD(GUC)Q 68322 to 68396 tRNA-Asp
tS(GCU)Q1 69203 to 69288 tRNA-Ser
tR(ACG)Q2 69289 to 69362 tRNA-Arg
tA(UGC)Q 69846 to 69921 tRNA-Ala
tI(GAU)Q 70162 to 70237 tRNA-Ile
tY(GUA)Q 70824 to 70907 tRNA-Tyr
tN(GUU)Q 71433 to 71503 tRNA-Asn
tM(CAU)Q1 72630 to 72705 tRNA-Met
COX2 Q0250, OXI1, OXII 73758 to 74513 Subunit II of cytochrome c oxidase, which is the terminal member of the mitochondrial inner membrane electron transport chain; one of three mitochondrially-encoded subunits
Q0255 ORF1, RF1 74495 to 75984
CDS 74495 - 75622, 75663 - 75872, 75904 - 75984
Intron 75623 - 75662, 75873 - 75903
Hypothetical protein
tF(GAA)Q 77431 to 77505 tRNA-Phe
tT(XXX)Q2 78162 to 78089 Note: this feature is encoded on the Crick strand. tRNA-Thr
tV(UAC)Q 78533 to 78608 tRNA-Val
COX3 Q0275, OXI2 79213 to 80022 Subunit III of cytochrome c oxidase, which is the terminal member of the mitochondrial inner membrane electron transport chain; one of three mitochondrially-encoded subunits
tM(CAU)Q2 85035 to 85112 tRNA-fMet
RPM1 85295 to 85777 RNA component of mitochondrial RNase P, which also contains the protein subunit Rpm2p; Rnase P removes 5' extensions from tRNA precursors
Q0297 ORF12 85554 to 85709 Hypothetical protein

Reporters

Available

  • ARG8m [5]
  • GFPm [6]
  • BARSTAR [7]
  • RIPm (not really a reporter but nuclear gene recoded and moved to mito) [8]

To make?

  • 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 [[9]] (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

DNA sequence =1115 bp [[10]] Prot sequence = ~372 aa [[11]]

  • Could try PUT1 [12]
    • 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 [13]
    • 5-aminolevulinate synthase, catalyzes the first step in the heme biosynthetic pathway [14]; 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 [15], protein is 549 aa [16]
    • 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

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

    • 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 [17]. 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[18]
  2. fatty acid biosynthesis pathway [19]] , and
  3. methionine biosynthesis pathway [20]
    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 [[21]]
  • 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 [[22]]
  • 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

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