• 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
• Ion Torrent
• Illumina
• Pyrosequencing (454)
• Solid
activators
• Pacific Bio
• Nanopore
Digital (Droplet) PCR
Gene Regulation
Trancriptional repressors
Circular RNA
Long non-coding RNA
RNA transcriptional
miRNA
Pol II pausing
Pol IV and Pol V
Chromatin remodeling
RNA localization
RNA degradation
RNA termination
Protein degradation
Metabolomics
Mito/Cp gene regulation
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
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 places
Some mt & cp proteins contain subunits encoded by
organelle’s genome
Cytoplasmic inheritance
1)first seen as strictly maternally inherited albino
variegation
• no linkage to nuclear genes
• albinism strictly determined by the mother
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
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
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
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
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
Plastid DNA
cpDNA encodes rubisco large subunit, nDNA encodes
small subunit, holoenzyme has 8 lg & 8 small subunits
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
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
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
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
Plastid DNA
coordination with nucleus
CP signals to nucleus:
retrograde signaling
•ROS
•Redox
•Mg-protoporphyrin
•Genome-uncoupled
(gun) mutants are
defective in retrograde
signaling
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
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 seen
endoG 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 tissue
either 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 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
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