interuniversity programme of molecular biology...
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Eyong eyong christopherVrije Universiteit Brussel
Interuniversity Programme of Molecular Biology
Institute of Molecular Biology and Biotechnology
MSc. Molecular Biology
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Definition(s)Reasons Types
Advantages and disadvantages
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Cloning; Using specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough materials for further study
A vector; DNA molecule for transferring foreign genetic material into another cell
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Stability
Self-replication
Molecular weight
Isolation
Single cut sites
Detection
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Cloning in yeast has been due to the desire to cloning to control or improve synthesis of an important metabolic product (e.g. a hormone )
Yeast Sacchriomycete cereviciae is one of the most important organism in biotecnology.
Cloning vectors for yeast has been effective due to the presence of a plasmide called the 2µm plasmid.
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2µm plasmid
2µm plasmid is an excellent base .
For cloning vector due to;
-Size of 6kb ideal for cloning.
_Existence of an origin of repliction.
_The REP1 and REP2 assisitin replication by coding for proteins .
_FLP codes for proteintsthat converts this form in to a form in which the geneorder has been rearranged.
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Selection is based on nutirion instead of drugs.
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The different types of yeast vectors include.
Yeast episomal plasmid(YEps).
Yeast intergrative plasmids(YIps).
Yeast replicating plasmids(YRps)
Yeast artificial chromosomes(YAC)
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SHUTTLE VECTOR
YEp13 constitute a recombination of E. coli cloning vector with the naturally occuringyeast 2µm plasmid.
LEU2 functions as the selection maker
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Often, much of the cloning is done in prokaryotic systems (E coli) and then the recombinant vectors are grown in eukaryotic (yeast).
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Yeast intergrative
chromosome (YIp5)
>YIp are basicallybacterial plasmidscarrying the yeast gene..
>URA3 is a gene codes for orotidine-5¢-phosphate
decarboxylase (an enzyme that catalyses one of the steps in the biosynthesis
pathway for pyrimidinenucleotides)
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Yeast replicating
plasmid (YRp7)
>YRp has the applity to raplicateindependently.
>TRP1 is the selectable marker.
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Yeast artificial
chromosomes (YAC)
YAC is made of
>2 selectablemarkers (URA3 and TRP1) with the TRP1 and ori extentto include CEN4
>2 TEL ends
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Cloning strategy
with pYAC3
>Restriction enzyme BamH1 and SnaB1.
>2 fragments bound edby TEL ends and SnaB1 site.
>DNA to be clone isligated between the twofragments.
>Protoplasttransformation is usedto introduce YAC in to S. cerevisiae
>SUP4 is the selectablemarker.
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Factors such as
Trasformation frequency.
Copy values.
Stability of recombinants.
Unique advantage of YAC
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Principles of Gene Manipulation and GenomicsS.B. Primrose and R.M. Twyman (seventh edition)
www.freepatentsonline.com
www.blackwellpublishing.com/geneclonin
http://dbb.urmc.rochester.edu/labs/sherman_f/yeast
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An investment in knowledge always pays
the best interest.