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BIOLOGY 207 - Dr. Locke
Lecture#23 - Cloning genes by complementation
Required readings and problems:
Reading: Open Genetics, Chapter 8
Problems: Chapter 8
Optional
Griffiths (2008) 9th Ed. Readings: pp 724--730
Problems: 9th Ed. Ch. 20: 16
Campbell (2008) 8th Ed. Readings: Concept 20.1, 20.2
Concepts:
How can we identify and select (clone) a gene of interest to us?
1. The isolation of genes proceeds via screening libraries for a gene of interest.
2. A clone containing a specific gene may be identified if it is able to complement a
host mutation (single cell organisms).
3. Unfortunately, most genes in most organisms, especially eukaryotes, cannot be
isolated by simple complementation methods.
4. Transgenes can complement host mutations and confirm which gene is mutant.
Biol207 Dr. Locke’s section
Lecture#23
Fall'11
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Genomic DNA libraries
Genomic Library: Set of clones containing genomic DNA fragments incorporated into a
vector. The sum total of the cloned DNA fragments represents the genome.
The best libraries have random set of fragments: "Shot-gunned" libraries.
However, the problem is – Which clone? of the many, has the gene of interest?
First of three methods is:
Cloning by complementation of a host mutant
 is used in single-celled organisms where many individual clones can be examined
easily, typically on a Petri dish.
 relies on the cloned gene providing a functional gene product that is absent/nonfunctional in the mutant host -> complementation.
Example: Cloning a gene for an E. coli auxotrophic mutant
- mutant in some gene called "A".
minimal media
E. coli Strain minimal media
+ supplement
auxotrophic
Agrowth
(no growth)
A+
growth
growth
Biol207 Dr. Locke’s section
Lecture#23
Fall'11
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Goal:
Clone the A+ gene, which is the mutant in the A- strain.
A genomic DNA library in a plasmid vector.
1)- Build a genomic DNA library in a plasmid vector
 Use wild type DNA (this has A+ gene and not A-) to insert the A+ gene into the
plasmid vector.
A+
E. coli
genome
Biol207 Dr. Locke’s section
A+
fragment ->
A+
clone in
Library
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2)- Use these recombinant DNA molecules containing foreign (E. coli) DNA insertion
into plasmid vector and transform the A- mutant strain E. coli.
3)- Expected results from transformation of A- strain with the library of clones:
diagrams:
AHost Cells:
Plasmid:
A-
A-
E. coli A-
E. coli A-
E. coli A-
no plasmid
+
+
plasmid
plasmid
without A+ gene with A+ gene
Plate on MM:
no growth
growth
growth
with antibiotic
with supplement
Plate on MM:
no growth
no growth
growth
with antibiotic
no supplement
Plate on MM:
no growth
no growth
growth
no antibiotic
no supplement
MM = minimal medium
Select for clone that contains a gene that will complement the A- mutant strain.
It should contain an A+ gene.
Biol207 Dr. Locke’s section
Lecture#23
Fall'11
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Numbers:
Each plasmid contains ~4.5 Kb of insert DNA and
E. coli has ~4.5 Mb DNA in the genome.
Therefore 1000 plasmid inserts = 1 E. coli genome's worth of DNA.
We need to screen the equivalent of 5000 molecules to ensure a 99% chance of
finding gene A+.
5000 bacterial clones can be produced easily and screened quickly on one Petri dish
plate.
Works well with bacterial or yeast hosts and plasmid vectors,
but what about higher organisms?
-> problems with larger, multi-cellular organisms.
Problems that prevent the use of complementation to screen for
genes in higher organisms
1)- Higher organisms have much larger genome size.
More than 5x106 plasmids would be needed to screen the human genome.
2)- Higher organisms are multi-cellular and therefore the cloned DNA has to enter all
body cells or the germline cells.
3)- Auxotrophic mutants are more difficult to find and more difficult to manipulate.
4)- Genes are often very large -> too large to fit in one vector insert.
Biol207 Dr. Locke’s section
Lecture#23
Fall'11
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Conclusion:
Complementation methods cannot be used to screen libraries for genes in higher,
multi-cellular organisms.
However, complementation can be used in higher organisms to confirm that a cloned
gene is the correct one since it should be able to "rescue a mutant".
Transgenic animals result in the rescue
(change the mutant phenotype to wild type)
of the mutant phenotype.
Transgenic animals (plants, too)
Transgene - a foreign gene that is introduced into an organism by injecting the
gene into a newly fertilized egg and incorporating it into the host's genome so it is
present in ever cell of the organism.
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Lecture#23
Fall'11
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Example Drosophila Transgenes (white+ & rosy+ genes).
white+ -> gives a red eye colour (pigmented)
white- mutant -> gives a white eye colour (unpigmented)
rosy+ -> gives a red eye colour (pigmented)
rosy- mutant -> gives a rosy eye colour (unpigmented)
Drosophila Transformation System:
Use a "P element vector system" to introduce the rosy+ gene into the germline of a
rosy- mutant strain. Fig Process:
 1) - subclone rosy+ gene into P element vector
 2) - co-inject with “helper” plasmid into the eggs of mutant rosy- strain
 3) - examine G0 and G1 progeny for rosy+ (wildtype) eyed flies
Gene rescue:The cloned rosy+ gene is incorporated into the rosy- strain genomic
DNA and the phenotype of the rosy- strain, containing the cloned rosy+ gene, is
wildtype, red eyes.
The transgene rescues (complements) the mutant strain.
This shows that the mutation is due to an absence of the cloned gene's product.
Can rosy+ rescue (complement) a white- mutation?
Test by using rosy+ transgene in white- strain.
Result:
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Lecture#23
Fall'11
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Similar transgene systems are available for most model organisms and commercially
important species.
Human Analogy - gene therapy
Theory for "gene-therapy"
There are many genetic diseases that are due to loss-of-function mutations.
If the normal functioning gene can be introduced and compensate (complement) for
the mutant genes then the effect of the disease (the mutant phenotype) can be
avoided.
Problem: need to fix somatic cells (not germline).
Drosophila rosy+ gene example above is just "gene therapy" for fruit flies.
Biol207 Dr. Locke’s section
Lecture#23
Fall'11
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