Interuniversity Programme of Molecular Biology...
Transcript of Interuniversity Programme of Molecular Biology...
Eyong eyong christopherVrije Universiteit Brussel
Interuniversity Programme of Molecular Biology
Institute of Molecular Biology and Biotechnology
MSc. Molecular Biology
Definition(s)Reasons Types
Advantages and disadvantages
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
Stability
Self-replication
Molecular weight
Isolation
Single cut sites
Detection
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.
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.
Selection is based on nutirion instead of drugs.
The different types of yeast vectors include.
Yeast episomal plasmid(YEps).
Yeast intergrative plasmids(YIps).
Yeast replicating plasmids(YRps)
Yeast artificial chromosomes(YAC)
SHUTTLE VECTOR
YEp13 constitute a recombination of E. coli cloning vector with the naturally occuringyeast 2µm plasmid.
LEU2 functions as the selection maker
Often, much of the cloning is done in prokaryotic systems (E coli) and then the recombinant vectors are grown in eukaryotic (yeast).
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)
Yeast replicating
plasmid (YRp7)
>YRp has the applity to raplicateindependently.
>TRP1 is the selectable marker.
Yeast artificial
chromosomes (YAC)
YAC is made of
>2 selectablemarkers (URA3 and TRP1) with the TRP1 and ori extentto include CEN4
>2 TEL ends
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.
Factors such as
Trasformation frequency.
Copy values.
Stability of recombinants.
Unique advantage of YAC
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
An investment in knowledge always pays
the best interest.