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Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye

Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using l H3/R1 digest and 2 log ladder as markers Use outcome to decide whether to clone or retry on Wed. Sequencing technologies Gene Regulation

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Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye

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  1. Put 2 µl loading dye in n tubes • Transfer 8 µl of each reaction to a tube containing loading dye • Load on 1% gel and run at 150 volts using l H3/R1 digest and 2 log ladder as markers • Use outcome to decide whether to clone or retry on Wed

  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. Metabolomics • Identifying all the metabolites in a given tissue • GC/MS for non-polars • LC/MS for polars • Altered levels of metabolites are often earliest clues to disease • http://www.bbc.co.uk/news/science-environment-22013700 • http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0059909

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

  5. 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

  6. Cytoplasmic inheritance • first seen as strictly maternally inherited albino variegation • no linkage to nuclear genes • albinism strictly determined by the mother

  7. Cytoplasmic inheritance • first seen as strictly maternally inherited albino variegation • no linkage to nuclear genes • albinism strictly determined by the mother • variegation arises because have mix of “good” and “bad” cp • Segregate randomly at division • eventually one form predominates

  8. Cytoplasmic inheritance • Variegation arises because have mix of “good” and “bad” cp • Segregate randomly at division • eventually one form predominates • In plants, cytoplasm comes from the egg • most pollen do not have cp or mt • can't study genetically, because no • way to mix parental organelles

  9. Plastid DNA • vary between 120 & 217 kB, according to species • most are 120-160 kB • have >20 copies/chloroplast • encode ~ 100 proteins, 4 rRNA &~30 tRNA

  10. Plastid DNA • encode ~ 100 proteins, 4 rRNA &~30 tRNA • 5 classes of proteins • ribosomal & other proteins involved in translation • proteins involved in transcription • proteins involved • in photosynthesis • proteins involved • in respiration • ORFs (open reading • frames) • sequences capable of • encoding proteins but • no product has been • identified

  11. Plastid DNA • encode ~ 100 proteins, 4 rRNA &~30 tRNA • 5 classes of proteins • in general, tend to be the more hydrophobic subunits • could have complicated exporting the gene to the nucleus • invariably also have subunits encoded by nuclear genes

  12. Plastid DNA • cpDNA encodes rubisco large subunit, nDNA encodes small subunit, holoenzyme has 8 lg & 8 small subunits

  13. Plastid DNA • cp gene expression is regulated at all levels • transcriptional • mRNA stability • 3) Translational: light triggers 100x increase in some proteins but only small increase in transcription

  14. Plastid DNA • coordination with nucleus • primarily studied during light-regulated cp development • light triggers development of proplastids • assemble thylakoids, make nearly all the proteins needed for photosynthesis

  15. Plastid DNA • coordination with nucleus • primarily studied during light-regulated cp development • nucleus controls by sending in proteins including DNA polymerases and proteases • cp degrade excess subunits

  16. Plastid DNA • coordination with nucleus • cp degrade excess subunits • when poison rbcS, rbcL is made but does not accumulate • same when poison rbcL with chloramphenicol

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

  18. Plastid DNA • Oddities • many cp genes have introns • introns are self-splicing (type II): no spliceosomes or other enzymes! • 2) mRNA editing:many cp mRNAs differ from the gene encoding them • an ACG is modified post-transcriptionally to a functional AUG start codon in several tobacco mRNAs; many other post-transcriptional changes have also been identified • editing machinery is encoded by the nucleus

  19. Mito DNA • range from 200 to 2500 kb (cf 16 kb for mammalian mito) • 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

  20. Mito DNA • range from 200 to 2500 kb (cf 16 kb for mammalian mito) • reason for large size is unknown • encodes ~ 35 proteins, also rRNA & tRNA • subunits of ATP synthase & complexes I, II, III & IV

  21. Mito DNA encodes ~ 35 proteins, also rRNA & tRNA • subunits of ATP synthase & complexes I, II, III & IV • some mRNA are trans-spliced from 2 diff transcripts!

  22. Mito DNA encodes ~ 35 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!

  23. Mito DNA encodes ~ 35 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!

  24. 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

  25. 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

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

  27. 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)

  28. 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

  29. 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

  30. 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

  31. 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 • have major increase in respiration and • # mitochondria after meiosis • 40 x increase in mt/ cell in tapetum • 20x in sporogenous cells

  32. CMS either pollen or tapetum can blow up have major increase in respiration and # mitochondria after meiosis 40 x increase in mt/ cell in tapetum 20x in sporogenous cells can (usually) be overcome by nuclear "restorer" genes usually a single dominant gene

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