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.