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Bridget, Jephte, Kristi, Matt, Teresa

Bridget, Jephte, Kristi, Matt, Teresa Set up 20 µl mix for each primer/DNA combo (ie ex/ex and in/in) on ice! 2 µl 10x F primer (1 pMol/µl = 1µM final []) 2 µl 10x R primer 1 µl DNA : use phusion amplicon for the internals if available, if not, use genomic DNA

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Bridget, Jephte, Kristi, Matt, Teresa

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  1. Bridget, Jephte, Kristi, Matt, Teresa • Set up 20 µl mix for each primer/DNA combo (ie ex/ex and in/in) on ice! • 2 µl 10x F primer (1 pMol/µl = 1µM final []) • 2 µl 10x R primer • 1 µl DNA : use phusion amplicon for the internals if available, if not, use genomic DNA • We will prepare Phusion master mix for 340 µl total volume • 68 µl 5x Phusion HF buffer • 6.8 µl 10 mM dNTP (200 µM final []) • 166.4 µl water • 3.4 µl Phusion polymerase • Add 15 µl master mix to each rxn • run on touchdown starting at 72˚ C annealing T

  2. Sequencing technologies Gene Regulation • Ion Torrent Trancriptional repressors • Illumina Circular RNA • Pyrosequencing (454) Long non-coding RNA • Solid RNA transcriptional activators • Pacific Bio miRNA • Nanopore Pol II pausing • Pol IV and Pol V • Chromatin remodeling • Digital (Droplet) PCR RNA localization • RNA degradation • RNA termination • Protein degradation • Metabolomics • Mito/Cp gene regulation • http://www.biotechniques.com/news/

  3. How to make a cell? • Must put all the right pieces in all the right places

  4. How to make a cell? • Must put all the right pieces in all the right places • Some mt & cp proteins contain subunits encoded by organelle’s genome

  5. Plastid DNA • coordination with nucleus • CP signals to nucleus: • retrograde signaling • ROS • Redox • Mg-protoporphyrin • Genome-uncoupled • (gun) mutants are • defective in retrograde • signaling

  6. Mito DNA range from 6 kb in Plasmodium to 2500 kb (muskmelons)

  7. Mito DNA • range from 6 kb in Plasmodium to 2500 kb (muskmelons) • 7 fold variation in mt genome size within cucurbit family • watermelon =330 kb, muskmelon = 2500 kb • considerable variation within same species • 5 different cytotopes in maize, vary from 540-700kb

  8. Mito DNA • range from 6 kb in Plasmodium to 2500 kb (muskmelons) • reason for large size is unknown • human mtDNA encodes 13 proteins, also rRNA & tRNA • subunits of ATP synthase, NADHdeH, CytBC1 & COX

  9. Mito DNA • human mtDNA encodes 13 proteins, also rRNA & tRNA • defects in mt DNA are nasty! • LHON (Leber's Hereditary Optic Neuropathy is due to defects in mt-encoded subunits of NADH-deH • ND1, ND4 or ND6 • mutations all have • same effect = loss of • vision, sometimes • MS-like symptoms

  10. Mito DNA • defects in mt DNA are nasty! • LHON • MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes) • ND1, ND5, TH, TL1 • & TV genes can cause it • TH,TL1 & TV encode • tRNA!

  11. Mito DNA • defects in mt DNA are nasty! • LHON • MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes) • Others: cyclic vomiting • syndrome, cox deficiency, • Deafness, ragged red fiber, • Exercise intolerance

  12. Mito DNA • defects in mt DNA are nasty! • All show maternal inheritance (used to trace human ancestry)

  13. Mito DNA • defects in mt DNA are nasty! • All show maternal inheritance • Penetrance varies depending upon proportion of defective mt

  14. Mito DNA • defects in mt DNA are nasty! • All show maternal inheritance • Penetrance varies depending upon proportion of defective mt: average ~ 5 DNA/mt, 100 mt/cell

  15. Mito DNA • defects in mt DNA are nasty! • All show maternal inheritance • Penetrance varies depending upon proportion of defective mt • Mutations increase with age! Mutate 10x faster than nDNA due to ROS

  16. Mito DNA • Mutations increase with age! Mutate 10x faster than nDNA • Defects are associated with cancer & other diseases

  17. Mito DNA • defects in mt DNA are nasty! • Mutations increase with age • Defects are associated with • cancer & other diseases

  18. Mito DNA • Oddities • Vertebrates, inverts, protists and & fungi have UGA = trpcfstop • In verts AUA = met cfisoleu • All sorts of other oddities in various groups

  19. Mito DNA • Human Oddities • 28 genes on “heavy” strand

  20. Mito DNA • Human Oddities • 28 genes on “heavy” strand • 9 on “light” strand, ND6 & 8 tRNAs

  21. Mito DNA • Human Oddities • 3 promoters: 2 on H strand, one on L • pL transcribes entire light strand; later processed into tRNA & ND6

  22. Mito DNA • Human Oddities • 3 promoters: 2 on H strand, one on L • pL transcribes entire light strand; later processed into tRNA & ND6 • pH1 transcribes • entire H strand

  23. Mito DNA • Human Oddities • 3 promoters: 2 on H strand, one on L • pL transcribes entire light strand; later processed into tRNA & ND6 • pH1 transcribes • entire H strand • pH2 may transcribe • 12S & 16S rRNA

  24. Mito DNA • Human Oddities • 3 promoters: 2 on H strand, one on L • pL transcribes entire light strand; later processed into tRNA & ND6 • pH1 transcribes • entire H strand • pH2 may transcribe • 12S & 16S rRNA • In vitro only need • TFAM & TFB2M to • transcribe pL & pH1

  25. Mito DNA • Human Oddities • 3 promoters: 2 on H strand, one on L • pL transcribes entire light strand; later processed into tRNA & ND6 • pH1 transcribes • entire H strand • pH2 may transcribe • 12S & 16S rRNA • In vitro only need • TFAM & TFB2M to • transcribe pL & pH1 • Uncertain if pH2 is used

  26. Mito DNA • Human Oddities • DNA replication: controlled by nuclear genes

  27. Mito DNA • Human Oddities • DNA replication: controlled by nuclear genes • Separate origins for H and L strands!

  28. DNA replication: controlled by nuclear genes • Separate origins for H and L strands! • Replicates in D-loop manner: starts at OH & heads towards OL displacing opposite strand until hits OL & new fork starts replicating in opposite direction.

  29. Mito DNA • range from 6 kb in Plasmodium to 2500 kb (muskmelons) • 7 fold variation in mt genome size within cucurbit family • watermelon =330 kb, muskmelon = 2500 kb • considerable variation within same species • 5 different cytotopes in maize, vary from 540-700kb

  30. Plant Mito DNA encodes ~13 proteins, also rRNA & tRNA • subunits of ATP synthase & complexes I, II, III & IV • some mRNA are trans-spliced from 2 diff transcripts!

  31. Mito DNA encodes encodes ~13 proteins, also rRNA & tRNA • subunits of ATP synthase & complexes I, II, III & IV • some mRNA are trans-spliced from 2 diff transcripts! • some mRNA are edited: bases changed after synthesis!

  32. Mito DNA encodes encodes ~13 proteins, also rRNA & tRNA • subunits of ATP synthase & complexes I, II, III & IV • some mRNA are trans-spliced from 2 diff transcripts! • some mRNA are edited: bases changed after synthesis! • Mech to prevent nucleus from stealing genes? • Find cp & nuc genes in mtDNA!

  33. Mitochondrial DNA • some mRNA are trans-spliced from 2 diff transcripts! • some mRNA are edited: bases changed after synthesis! • Mech to prevent nucleus from stealing genes? • mtDNA recombines to form new genes: see many smaller molecules cf one big circle

  34. Mitochondrial DNA • mtDNA recombines to form new genes: see many smaller molecules cf one big circle: some poison pollen development to create cytoplasmic male sterility

  35. Mitochondrial DNA • mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility • Pollen don't transmit mito!

  36. Mitochondrial DNA • mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility • Pollen don't transmit mito! • May be due to PCD (apoptosis)

  37. Mitochondrial DNA • mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility • Pollen don't transmit mito! • May be due to PCD (apoptosis) • Only have seen • endoG in plant mt

  38. Mitochondrial DNA • mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility • Pollen don't transmit mito! • Widely used in plant breeding • Eg hybrid corn

  39. CMS • mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility • described in over 150 different spp. • can affect either sporophytic or gametophytic tissue • either pollen or tapetum can blow up

  40. CMS either pollen or tapetum can blow up have major increase in respiration and # mt after meiosis 40 x increase in mt/ cell in tapetum 20x in sporogenous cells

  41. CMS can (usually) be overcome by nuclear "restorer" genes usually a single dominant gene

  42. CMS can (usually) be overcome by nuclear "restorer" genes usually a single dominant gene mtDNA recombines to form new defective proteins, Nucleus fixes them

  43. Apoptosis (programmed cell death) • Occurs as normal part of development • Is also triggered by many kinds of damage • Especially to DNA

  44. Cell death vs necrosis • Necrosis: • Passive • Indiscriminate • Often follows irreversible injury • Characterized by progressive loss of membrane integrity swelling of cytoplasm, release of cell constituents • PCD • Active • Orderly process mediated by intracellular death programs • May or may not be due to an external factor • Nuclear condensation • Condensation of PM

  45. Programmed cell death (PCD) Dev’l cell death • Cell plays active role in its demise • Genetically controlled • Pathways • Apoptosis • Autophagy • Plant PCD (Scott & Logan, 2008, Plant Signaling & Behavior)

  46. PCD Mammalian apoptosis • e.g. patterning of hands/feet Phases • Induction (perception) • Effector (commitment) • Degradation (dismantling of cell contents) syndactyly

  47. PCD : role of mitochondrion Mitochondria --sensor of death signals &initiator of biochem processes leading to cell death http://bifi.unizar.es/research/pro_pro_inter_elec_transfer/research.php

  48. Programmed cell death (PCD) • Autophagy • Intracellular recycling process – lysosomes (animals);vacuoles (plants) -- hydrolases • Can be used to prevent premature cell death • Upregulated  PCD Apoptosis Autophagy (Scott & Logan, 2008, Plant Signaling & Behavior)

  49. Programmed cell death (PCD) • Plant PCD • Changes in shape and position of mitochondria (Mitochondrial morphology transition, MMT) • Nuclear condensation • Condensation of PM from cell wall • Deregulated: dev’l defects, lethality MMT (Scott & Logan, 2008, Plant Signaling & Behavior)

  50. Apoptosis (programmed cell death) • Occurs as normal part of development • Is also triggered by many kinds of damage • Especially to DNA • Many cancer cells do not commit apoptosis

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