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Lecture 20

BIOL 5060 Lecture 20: 4:6_RecombinantDNATech_Hoffman

7 Pages
11 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
20

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4/6_RecombinantDNATech_Hoffman 4/6/2017 2:58:00 PM
Yeast
Autonomously-Replicating plasmids
Developed to introduce DNA into cells and ALSO in the reverse
fashion (benefit over most other plasmids that cannot come back
out of the gene once they’ve been inserted)
o This tells if the plasmid is responsible for the behavior of the
cell
o Or you can lose the original one, insert a new one and tell the
biological affect of the new one
Budding yeast- high copy and low copy number plasmids
o Centromere is only about 125 bp
o Micro centromere allows the low copy number of plasmids-
o Without a centromere, it can have a large copy number
Fission yeast- only high copy number plasmids
o Centromeres, depending on the chromosome, are about 50-
110 kb
o Huge centromeres (close to the size of mammals)—we don’t
have the ability to do low copy number of plasmids
Selectable markers- allow us to know which cells received a plasmid
o Often a gene of the biosynthetic pathway
o Nomenclature (genotypic and phenotypic)
Budding Yeast Nomenclature (genotype)
Underline or Italics= wild-type gene
Lowercase- recessive mutant allele
Leu2 ^(R37A)- means arginine at amino acid 37
is now adenine
LEU2-7- dominant mutant allele
Mutant because of allele number
Dominant because of caps
Leu2p- represents the protein (not underlined,
over the first letter is capitalized)
Leu2-3p- mutant protein
Fission Yeast (genotype)
ura4+- wild-type genes
we don’t use uppercase in fission yeast
nomenclature
find more resources at oneclass.com
find more resources at oneclass.com
ura4-297- mutant allele
since we don’t use uppercase there’s no
way of indicating a dominant vs. recessive
allele
ura4() and…
ura4-D18= deletion alleles
Phenotype nomenclature
Ura+/ Ura- - no numbers because we aren’t
referring to a gene; we are referring to the
behavior of a cell
- = cell will not grow unless we give it Ura
to do so
o ** USEFUL ON NEXT PROBLEM SET
o Common selectable markers
Budding yeast
LEU2, URA3, HIS3, TRP1
Fission yeast (pombe)
LEU2 (from S.cerevisiae; used in fission yeast to
complement a Leu1 mutation)
ura4+
his7
lys2+
URA3+ and ura4+ both encode OMP decarboxylase
(part of uracil biosynthetic pathway)
By introducing these two genes to a URA3, or
ura4 mutant cell…?
These are both selectable and counter-selectable
Selectable marker- presence is required for
growth in medium lacking uracil
Counter-selection- absence is required for growth
in media containing 5-fluoro-orotic acid
The cell will take something up and die as it
is trying to make 5-fluoro-orotic acid into
uracil
5FOA kills the cells with the URA3 or ura4
plasmid to produce a collection of cells
find more resources at oneclass.com
find more resources at oneclass.com

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Description
4/6_RecombinantDNATech_Hoffman 4/6/2017 2:58:00 PM Yeast Autonomously-Replicating plasmids • Developed to introduce DNA into cells and ALSO in the reverse fashion (benefit over most other plasmids that cannot come back out of the gene once they’ve been inserted) o This tells if the plasmid is responsible for the behavior of the cell o Or you can lose the original one, insert a new one and tell the biological affect of the new one • Budding yeast- high copy and low copy number plasmids o Centromere is only about 125 bp o Micro centromere allows the low copy number of plasmids- o Without a centromere, it can have a large copy number • Fission yeast- only high copy number plasmids o Centromeres, depending on the chromosome, are about 50- 110 kb o Huge centromeres (close to the size of mammals)—we don’t have the ability to do low copy number of plasmids • Selectable markers- allow us to know which cells received a plasmid o Often a gene of the biosynthetic pathway o Nomenclature (genotypic and phenotypic) ▪ Budding Yeast Nomenclature (genotype)  Underline or Italics= wild-type gene  Lowercase- recessive mutant allele  Leu2 ^(R37A)- means arginine at amino acid 37 is now adenine  LEU2-7- dominant mutant allele • Mutant because of allele number • Dominant because of caps  Leu2p- represents the protein (not underlined, over the first letter is capitalized)  Leu2-3p- mutant protein ▪ Fission Yeast (genotype)  ura4+- wild-type genes • we don’t use uppercase in fission yeast nomenclature  ura4-297- mutant allele • since we don’t use uppercase there’s no way of indicating a dominant vs. recessive allele  ura4(∆) and…  ura4-D18= deletion alleles ▪ Phenotype nomenclature  Ura+/ Ura- - no numbers because we aren’t referring to a gene; we are referring to the behavior of a cell • - = cell will not grow unless we give it Ura to do so o ** USEFUL ON NEXT PROBLEM SET o Common selectable markers ▪ Budding yeast  LEU2, URA3, HIS3, TRP1 ▪ Fission yeast (pombe)  LEU2 (from S.cerevisiae; used in fission yeast to complement a Leu1 mutation)  ura4+  his7  lys2+ ▪ URA3+ and ura4+ both encode OMP decarboxylase (part of uracil biosynthetic pathway)  By introducing these two genes to a URA3, or ura4 mutant cell…?  These are both selectable and counter-selectable  Selectable marker- presence is required for growth in medium lacking uracil  Counter-selection- absence is required for growth in media containing 5-fluoro-orotic acid • The cell will take something up and die as it is trying to make 5-fluoro-orotic acid into uracil • 5FOA kills the cells with the URA3 or ura4 plasmid to produce a collection of cells lacking plasmid- doesn’t cause the cell to lose the plasmid!! It just identifies the cells that have lost the plasmid • passive process in which we select for the cells that have lost the plasmid • You can mess up your 5FOA medium by putting to much uracil in it—if there is too much uracil, the cells will turn off there uracil biosynthetic pathway and become resistant to 5FOA • Autonomously replicating plasmids are readily lost by yeast (as compared to E.
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