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Lecture 25

BIOL 5060 Lecture 25: Final Lecture Review

6 Pages
44 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
25

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5/2_RecDNATech_Hoffman 5/2/2017 4:13:00 PM
Scaling Up (is the theme)
1) Synthetic Lethality (something that predates recombinant DNA/ a genetic
interaction between mutations in two different genes)
Not a phenotype seen in either defect on its own, but when the two
are put together it is seen
Mut1- mut2+x crossed with mut1+mut2-
o Having mutations in opposite genes
o We did a screen for a genotype and genes that are
functionally related came up (where we got these two from)
o Both different from wild-type because we screened them from
a synthetic screen
So… what is the double mutant phenotype?
o Cross them and look at progeny
o 2/3 of progeny= 1-2-, 1-2+, 1+2-, 1+2+
tetrad: in the most common type f tetrad, you should
have one double mutant
one is wild-type, two are mutant and we can infer that
the double mutant is dead
o 1/6 Mut1- mut2+x
o 1/6 mut1+mut2-
2) Synthetic lethal Screen (previously discussed)- a way of starting with
mut1- and looking for mut1-mut2-
3) in order to cover everything, move on to a Synthetic Gene Array (SGA)
you can cross a. your deletion or mutant strain with b. all viable
haploid deletion strains
put 2 different markers in two mutants (deletions) and score all
double mutants BUT diploid cells will also have the 2 recombinant
markers as will the haploid targets
need a trick to not see growth of diploid and only see growth of
haploid that has deletion of each selectable marker
o need to insert a gene that is controlled by the mating type of
the strain into a haploid where its not expressed
o so we look for strains that have both the deletion selectable
markers and the last, magic marker, that marks for haploid
only; only the double mutant recombinant progeny will grow
find more resources at oneclass.com
find more resources at oneclass.com
magic marker eliminates other strains that can grow
(most importantly diploid- that will have both deletion
markers)
Global Two-hybrid Screen- systematic fusion of Gal4 activation domain
(GAD) to 6000 ORF; it’ll be fused to ever ORF in budding yeast
If we have our GOI, we can make a PCR product that has homology
with a selection marker that can recombine in to select for GF1A
resistance
o Contains GOI without stop codon and then gets reframed with
activating strain by transfusion
Caveat: possible that the full length construct will not interact
where a fragment construct will
o Cannot replace 2-hybrid screen but allows for a global
assessment, exhaustive search but potentially with false
negatives (when full length protein can’t get into the nucleus
whereas a shorter fragment could get a hit)
puts GAD on everything
Systematic GFP fusions- global localization of protein
Allows for Quantitation- how many molecules of protein per cell
under various conditions
Systematic TAP Tags- allows for affinity capture (purification) of all proteins
to identify members of protein complexes
Pass it over a column to bind, then pass protease over it to elute
those that don’t bind (that don’t contain probe) and do another
round of affinity purification for a different probe- elute it again-
mass spec to identify mass of peptides
Review
LacO array- marks LacO sequence (place in a chromosome); allows you to
identify particular place because LacI protein binds it, which is fused to GFP
When you do microscopy, you see and intense small dot in the
nucleus that represents this sequence
If sister chromatids aren’t cohesive, you will see two spots
find more resources at oneclass.com
find more resources at oneclass.com

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Description
5/2_RecDNATech_Hoffman 5/2/2017 4:13:00 PM Scaling Up (is the theme) 1) Synthetic Lethality (something that predates recombinant DNA/ a genetic interaction between mutations in two different genes) • Not a phenotype seen in either defect on its own, but when the two are put together it is seen • Mut1- mut2+x crossed with mut1+mut2- o Having mutations in opposite genes o We did a screen for a genotype and genes that are functionally related came up (where we got these two from) o Both different from wild-type because we screened them from a synthetic screen • So… what is the double mutant phenotype? o Cross them and look at progeny o 2/3 of progeny= 1-2-, 1-2+, 1+2-, 1+2+ ▪ tetrad: in the most common type f tetrad, you should have one double mutant ▪ one is wild-type, two are mutant and we can infer that the double mutant is dead o 1/6 Mut1- mut2+x o 1/6 mut1+mut2-  2) Synthetic lethal Screen (previously discussed)- a way of starting with mut1-  and looking for mut1-mut2-  3) in order to cover everything, move on to a Synthetic Gene Array (SGA) • you can cross a. your deletion or mutant strain with b. all viable haploid deletion strains • put 2 different markers in two mutants (deletions) and score all double mutants BUT diploid cells will also have the 2 recombinant markers as will the haploid targets • need a trick to not see growth of diploid and only see growth of haploid that has deletion of each selectable marker o need to insert a gene that is controlled by the mating type of the strain into a haploid where its not expressed o so we look for strains that have both the deletion selectable markers and the last, magic marker, that marks for haploid only; only the double mutant recombinant progeny will grow ▪ magic marker eliminates other strains that can grow (most importantly diploid- that will have both deletion markers) Global Two-hybrid Screen- systematic fusion of Gal4 activation domain (GAD) to 6000 ORF; it’ll be fused to ever ORF in budding yeast • If we have our GOI, we can make a PCR product that has homology with a selection marker that can recombine in to select for GF1A resistance o Contains GOI without stop codon and then gets reframed with activating strain by transfusion • Caveat: possible that the full length construct will not interact where a fragment construct will o Cannot replace 2-hybrid screen but allows for a global assessment, exhaustive search but potentially with false negatives (when full length protein can’t get into the nucleus whereas a shorter fragment could get a hit) •  puts GAD on everything Systematic GFP fusions- global localization of protein • Allows for Quantitation- how many molecules of protein per cell under various conditions Systematic TAP Tags- allows for affinity capture (purification) of all proteins to identify members of protein complexes • Pass it over a column to bind, then pass protease over it to elute those that don’t b
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