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 H3/R1 digest and 2 log ladder as markers Use outcome to decide whether to clone or retry on Wed

2. Sequencing technologiesGene Regulation Ion TorrentTrancriptional repressors IlluminaCircular RNA Pyrosequencing (454) Long non-coding RNA SolidRNA transcriptional activators Pacific Bio miRNA NanoporePol II pausing Pol IV and Pol V Chromatin remodeling Digital (Droplet) PCRRNA 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%2Fj ournal.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 1)first seen as strictly maternally inherited albino variegation no linkage to nuclear genes albinism strictly determined by the mother

7. Cytoplasmic inheritance 1)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 1.ribosomal & other proteins involved in translation 2.proteins involved in transcription 3.proteins involved in photosynthesis 4.proteins involved in respiration 5.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 1)transcriptional 2)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 1)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