Special Vectors for Cloning

Some of the special vectors are using for gene cloning purpose (recombinant DNA technology). they are

a) Ti-Plasmids

b) Shuttle Vectors

c) Expression vectors

a) Ti-Plasmid:

The DNA segment, which is transfected is called “T-DNA” and is part of a large “Ti-plasmid”(Tumor inducing), found in virulent strains of Agrobacterium tumafaciens. Similarly “Ri” (Root inducing) megaplasmids are found in the virulent strains of Agrobacterium rhizogenes. The Ti- and Ri –plasmids inducing “Crown gall disease” and “Hairy root disease”.

Structure of Ti-Plasmid:

Most Ti-plasmids have FOUR regions, which are given.

i) Region- A:

• It contains “T-DNA”, which is responsible for tumor induction so that mutations in this region lead to the production of tumors with altered morphology.

• This region transferred to plant nuclear genome, so that the region is described as “T-DNA” (Transferred DNA).

ii) Region-B:

• Responsible for Replication

iii) Region-C:

• Responsible for Conjugation

iv) Region-D:

• Responsible for “Virulence”, so that mutation in this region abolishes virulence. This region is therefore called “Virulence” (vir) region” and plays crucial role in this transfer of T-DNA into the plant nuclear genome.

T-DNA (Transfer DNA):

It contains two regions:

a) ‘onc’ region”:

It contains three genes, they are “tms1”, “tms2”, “tmr”.

tms1, tms2 Representing “Shooty locus”

“tmr” representing “Rooty locus”

These genes are responsible for the biosynthesis of two phytohormones, namely, “Auxins” and “Cytokinins”. These phytohormones in their turn alter the developmental program, leading to the formation of crown gall.

b) ‘nos’ region:

• This region responsible for the synthesis of unusual amino acid (or) sugar derivatives, which are collectively given the name “Opines”.
• Opines are derived from a variety of compounds (eg: Arginine + Pyruvate), that are found in plant cells.
• Two most common opines are “Octopine” and “Nopaline”.
• These two opines synthesis responsible enzymes coding are present in “T-DNA”.
• Outside the T-DNA, Ti-plasmid carries genes that catabolize the opines, which are utilized as a source of carbon and nitrogen.
• The T-DNA regions on all Ti and Ri-plasmids are flanked by almost perfect “25 bp direct repeat sequences”, which are essential for T-DNA transfer.

Virulence region (vir):

The vir region (~35kbp) is organized into SIX operons, namely

Vir-A, Vir-B, Vir-D, and Vir-G is  Polycistronic Vir-C, Vir-E  is Tumor formation

c) Shuttle Vectors:

• Shuttle vectors are plasmids that contain replication origin sequences for two different host species.
• The host belongs to divergent groups such as Bacteria – Yeast, Monkey – E.Coli and E.Coli – Human beings.
• The shuttle vector carries two selectable marker genes.
• It contains the Ori of both species and a number of markers each for the tow hosts.
• Shuttle vectors have been designed to replicate in cells of two different species; therefore, they contain two origins of replication, one specific for each host species, as well as those genes necessary for their replication and not provided by the host cells. 
• These vectors are created by recombinant techniques
• Some of them can be grown in two different prokaryotic species, while others can propagate in a prokaryotic species, usually E.Coli and a eukaryotic one, e.g. yeast, plants, animals.
• Since these vectors can be grown in one host and then moved into another without any extra manipulation, they are called shuttle vectors.
• Shuttle vectors are have been designed to specifically satisfy this need, i.e., the initial cloning of DNA inserts in E.coli and subsequent functional tests in the species to which the DNA inserts belong. Most of the eukaryotic vectors are, in fact, shuttle vectors.
• A shuttle vector designed to replicate in E.coli and streptomyces has been constructed as follows: 
• 1) The modules for DNA replication in streptomyces and methylenomycin A resistance are derived from a streptomyces plasmid and  2) The replication module for maintenance in E.coli and a gene for antibiotic resistance are taken from an E.coli plasmid.

d) Expression Vectors:

• Expression vectors contain expression signals that best transcribe/translate the foreign gene in a heterogeneous system.
• Sometimes these signals are Promoters and Terminators of E.Colior phage genes that are added at appropriate sites on either side of the gene to be expressed.