Dr. A.K. Saha Professor Department of Zoology University of Rajshahi VECTOR Dr. A.K. Saha Professor Department of Zoology University of Rajshahi

Vector Vector is an agent that can carry a DNA fragment into a host cell. If it is used for reproducing the DNA fragment, it is called a “cloning vector.” If it is used for expressing certain gene in the DNA fragment, it is called an “expression vector”. Commonly used vectors include Plasmid, Lambda phage, Cosmid and Yeast artificial chromosome (YAC).

COSMID The cosmid can be defined as the hybrid derived from plasmids which contain cos site of phage λ (cosmid = cos site + plasmid). A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the Lambda phage.

Characteristics of Cosmid Cosmids are able to contain 37 to 52 kbp of DNA, while normal plasmids are able to carry only 1-20 kbp They can replicate as plasmids if they have a suitable origin of replication. They frequently also contain a gene for selection such as antibiotic resistance. Those cells which did not take up the cosmid would be unable to grow.

Special Features Origin of replication. A marker gene coding for antibiotic resistance. A special cleavage site for the insertion of foreign DNA and Small in size.

Polylinker pUC19 2686 bp ORI ampr Figure. A typical plasmid vector. It contains a polylinker site, an ampicillin resistance gene (ampr) and a replication origin (ORI).

Yeast Artificial Chromosome (YAC) The YAC vector is capable of carrying a large DNA fragment (up to 2 Mb), but its transformation efficiency is very low. CEN= Centromeres TEL= Telomeres ARS=Autonomous replicating sequence Figure. Cloning by the yeast artificial chromosome (YAC) vector.

Essential component of YAC vectors Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI). Procedure : 1. The target DNA is partially digested by EcoRI and the YAC vector is cleaved by EcoRI and BamHI. 2. Ligate the cleaved vector segments with a digested DNA fragment to form an artificial chromosome. 3. Transform yeast cells to make a large number of copies.

Lambda phage Phages are viruses that can infect bacteria. The major advantage of the phage vector is its high transformation efficiency, about 1000 times more efficient than the plasmid vector.

Plasmids Plasmids are extrachromosomal circular DNA molecules found in most bacterial species and in some species of eukaryotes. Many important bacterial genes are not part of the main chromosome but are on separate circles of DNA called plasmids. A plasmid is a molecule which can be stably inherited without being linked to the chromosome. Plasmids were originally defined as ‘‘extrachromosomal, hereditary determinants’’(Lederberg,1952). They are generally covalently closed circular (CCC) molecules of double-stranded deoxyribonucleic acid (DNA) that very in length from 1 kbp to 700 kbp. And occur in the majority of bacterial cells.

Fig. The structure of pBR322 showing the unique cleavage sites.

CHARACTERSTICS OF PLASMID Plasmids are important in medicine and in agriculture because they confer antibiotic resistance on pathogens of animals and man, and because they can code for toxins and other proteins which increase the virulence of these pathogens. Nitrogen Fixation: Plasmids enable species of Rhizobium to fix nitrogen in the nodules of leguminous plants. Antibiotic Production: Plasmid also code for antibiotics which can be used to control pathogenic bacteria.

Biodegradation: Plasmids genes code for a wide range of metabolic activities and enable bacteria to degrade compounds which would accumulates as pollutants. Recombinant DNA: Plasmid can be isolated quite easily from bacterial cells. New genes from other species can be inserted into isolated plasmids and the modified plasmid then can be put back into its normal host cell. Such “recombinant DNAs” are transcribed, and translated into potentially useful products.

Figure: Two super-coiled plasmid DNA molecules

TRANSFER OF PLASMID DNA The Plasmid transfer process can be divided into four stages: Formation of specific donar -recipient pairs (effective contact) Preparation for DNA transfer (mobilization) DNA transfer Formation of a replicative functional plasmid in the recipient (replication)

Figure: Plasmid transfer by conjugation between bacterial cells Figure: Plasmid transfer by conjugation between bacterial cells. The donor and recipient cells attach to each other by a pilus, an appendage present on the surface of the donor cell. A copy of the plasmid is then passed through the pilus to the recipient cell.

TYPES OF PLASMIDS Stringent or Low copy number plasmid: Which may range from 1 or 2. Relaxed or high copy number plasmids: Range from 10-100. Many types of plasmids are found in a variety of E. coli strains but three main types- F, R, and Col plasmids have been studied.

On the basis of Co-existence: F, the sex plasmid: Ability to transfer chromosomal genes (that is, genes not carried on the plasmid) and the ability to transfer F itself to a cell lacking the plasmid. R, the drug-resistance plasmid: Resistance to one or more antibiotics and often the ability to transfer the resistance to cells lacking R. Col, the colcigenic plasmid: Ability to synthesize colicins- that is, proteins capable of killing closely related bacterial strains that lack the Col plasmid. On the basis of Co-existence: Compatible plasmid: Bacteria often contain two or more different plasmid which can co-exist, and are said to be compatible Incompatible plasmid: Can not co-exist together.

Bacterial chromosome Plasmids Cell division Chromosome carrying integrated plasmid Bacterial chromosome Plasmids Cell division Chromosome carrying integrated plasmid A (a) B (b) B Figure: Replication strategies for (a) a non-integrative plasmid, and (b) an episome

Chromosomal DNA F- plasmid New Donor Old Donor Donor Recipient Pilus Donor Recipient DNA polymerase Copying of transferred strand in recipient Pilus Figure: A model for transfer of F plasmid DNA from an F+ cell by a looped rolling-circle mechanism

A new copy of F, generated by replication, is transferred to a recipient cell A copy of F remains in the donor cell cell (c) (a) (b)

A. For-high-copy number plasmids Plasmid replication Cell division and random segregation daughter cells Cell division and partitioning into plasmid daughter cells A. For-high-copy number plasmids B. For-low-copy number plasmids

Differences between chromosomal and plasmid DNA Chromosomal DNA is much larger than the DNA of plasmids. Chromosomal DNA extracted from cells is obtained as broken, linear molecules. Most plasmid DNA is extracted in a covalently closed, circular form.

Plasmids as Tools for Molecular Biology They are used as a vectors to clone DNA. A variety of different enzymes can be used to insert pieces of DNA, from animals, plants or prokaryotes, into plasmids. Circular molecules consisting partly of plasmid DNA and partly of inserted DNA can then be put back into a suitable bacterium. The plasmids replicate during bacterial growth so that many copies of the cloned DNA can easily be obtained.

The Agrobacterium Plasmid Ti This plasmid has recently become very important in plant breeding because specific genes can be inserted into Ti plasmid by recombinant DNA techniques, and sometimes these genes can become integrated into plant chromosome, thereby permanently changing the genotype and phenotype of the plant. New plant varieties having desirable and economically valuable characteristics derived from unrelated species can be developed in this way.

The Agrobacterium Plasmid Ti

Role of Ti plasmid

Role of Ti plasmid

Transgenic plant by Ti plasmid

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