• 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

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 VChromatin remodeling

Digital (Droplet) PCR RNA localizationRNA degradationRNA terminationProtein degradationMetabolomicsMito/Cp gene regulation

http://www.biotechniques.com/news/

MetabolomicsIdentifying all the metabolites in a given tissue• GC/MS for non-polars• LC/MS for polarsAltered levels of metabolites are often earliest clues to diseasehttp://www.bbc.co.uk/news/science-environment-22013700http://www.plosone.org/article/info%3Adoi

%2F10.1371%2Fjournal.pone.0059909

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

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

Cytoplasmic inheritance1)first seen as strictly maternally inherited albino variegation• no linkage to nuclear genes• albinism strictly determined by the mother

Cytoplasmic inheritance1)first seen as strictly maternally inherited albino variegation• no linkage to nuclear genes• albinism strictly determined by the mothervariegation arises because have mix of “good”

and “bad” cp• Segregate randomly at division• eventually one form predominates

Cytoplasmic inheritanceVariegation 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

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

Plastid DNAencode ~ 100 proteins, 4 rRNA &~30 tRNA5 classes of proteins

1. ribosomal & other proteins involved in translation

2. proteins involved in transcription3. proteins involved in photosynthesis4. proteins involved in respiration• ORFs (open reading frames)sequences capable ofencoding proteins but no product has been identified

Plastid DNAencode ~ 100 proteins, 4 rRNA &~30 tRNA5 classes of proteins

in general, tend to be the more hydrophobic subunitscould have complicated exporting the gene to the nucleus

invariably also have subunits encoded by nuclear genes

Plastid DNAcpDNA encodes rubisco large subunit, nDNA encodes small subunit, holoenzyme has 8 lg & 8 small subunits

Plastid DNAcp gene expression is regulated at all levels

1) transcriptional 2) mRNA stability3) Translational: light triggers 100x increase in

some proteins but only small increase in transcription

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

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

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

Plastid DNAcoordination with nucleusCP signals to nucleus: retrograde signaling•ROS•Redox•Mg-protoporphyrin•Genome-uncoupled (gun) mutants are defective in retrogradesignaling

Plastid DNAOddities

1) many cp genes have intronsintrons 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

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

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

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!

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!

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!

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

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

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

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)

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 seenendoG in plant mt

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

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 tissueeither pollen or tapetum can blow up

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 tissueeither pollen or tapetum can blow uphave major increase in respiration and# mitochondria after meiosis40 x increase in mt/ cell in tapetum20x in sporogenous cells

CMSeither pollen or tapetum can blow uphave major increase in respiration and# mitochondria after meiosis40 x increase in mt/ cell in tapetum20x in sporogenous cellscan (usually) be overcome by nuclear "restorer" genes usually a single dominant gene