They are carriers or vehicles of desired DNA fragments (passenger DNA) which can undergo independent replication to increase copies of desired genes, e.g., plasmids, bacteriophages. Number of plasmids in some bacterial cells can be 15-100 (even higher), through the common number is 1-2.
Similarly, a number of bacteriophages mature in a bacterium. Therefore, if we are able to recombine a piece of DNA with bacteriophage genome or a plasmid and introduce the same in a plasmid free bacterium, the vector would multiply, equal to number of plasmids or bacteriophages that normally develop in it.
Context
- Features Required in Cloning Vector
- Insertional Inactivation
1. Features Required in Cloning Vector.
A cloning vector must have its own origin of replication (ori), selectable marker (for identifying recombinant from nonrecombinant) and preferably a single recognition site for cloning. Cloning vectors are of four types-plasmids, viruses, cosmids and artificial chromosomes. Plasmid vector for plants are different from others.
Plasmids.
They are small extranuclear circular DNA which carry extrachromosomal genes in bacteria and some fungi. They replicate independently. The best known vectors, which are available commercially were PBR322 and pUC-18. Both of them have been modified from natural plasmids of Escherichia coli. pUC series of plasmid vectors are named so because they were initially developed at the University of California. Some common pUC plasmids are pUE-7, pUE-8, pUC-12, pUC-13, pUC-18, pUC-19. They have been derived from pBR-322. The Plasmid are about 2700 bp long. They have the following Features:
(i) Origin of Replication (ori).
It is DNA sequence which is specialised to initiate replication. The sequence also possesses nearby replication control which determines the number of copies it would form. Therefore, the selected plasmid should have an origin of replication that supports high copy number.
It is DNA sequence which is specialised to initiate replication. The sequence also possesses nearby replication control which determines the number of copies it would form. Therefore, the selected plasmid should have an origin of replication that supports high copy number.
(ii) Selectable Marker.
The plasmid has a selectable marker. Normally genes encoding for antibiotic resistance (e.g., ampicillin, chloramphenicol, tetracycline, kamamycin) are considered useful as selectable marker for Escherichia coli. pBR 322 (after Bolivar and Rodriguez, 1977) has two resistance genes, ampicillin resistance and tetracycline resistance while pUC-18 has only ampicillin resistance gene. Selectable marker is meant for distinguishing a recombinant from nonrecombinant DNA. It also helps in eliminating the nontransformed si bacterial cells from transformed bacterial cells. Transformation is the passage of foreign DNA or plasmid containing the passenger DNA into a host cell.
The plasmid has a selectable marker. Normally genes encoding for antibiotic resistance (e.g., ampicillin, chloramphenicol, tetracycline, kamamycin) are considered useful as selectable marker for Escherichia coli. pBR 322 (after Bolivar and Rodriguez, 1977) has two resistance genes, ampicillin resistance and tetracycline resistance while pUC-18 has only ampicillin resistance gene. Selectable marker is meant for distinguishing a recombinant from nonrecombinant DNA. It also helps in eliminating the nontransformed si bacterial cells from transformed bacterial cells. Transformation is the passage of foreign DNA or plasmid containing the passenger DNA into a host cell.
(iii) Cloning or Unique Restriction Sites.
Recognition site or site where alien DNA can be inserted should preferably be single and responsive to commonly used restriction enzyme. Occurrence of more than one recognition site can cause DNA fragmentation and disturbs gene cloning. In PBR322 alien DNA is ligated generally in the area of BamHI site of tetracycline resistance gene. The recombinant plasmid does not possess tetracycline resistance but continues to have ampicillin resistance. The plasmids are passed into bacteria during transformation. The non-recombinants can flourish in medium having both ampicillin and tetracycline.
Recombinants can grow only in ampicillin containing medium but will die out in tetracycline containing medium.
2. Insertional Inactivation
Inactivation of gene, due to insertion of alien, DNA is known for insertional inactivation. Plasmids with antibiotic resistance genes as selectable markers require to follow a cumbersome procedure requiring antibiotic containing culture plates. Therefore, alternate selectable markers have been produced. They use chromogenic substrate, Xgal. The common recognition or cloning site is lac gene Z that codes for enzyme galactosidase. Insertion of alien DNA into it inactivates the gene and prevents synthesis of enzyme galactosidase. Chromogenic substrate produces blue-coloured colonies if the plasmids do not have inserts and white coloured colonies if the recombinant plasmids are present.
2. Cloning Vectors for Plants.
Tumour inducing plasmid (Ti) of soil bacterium Agrobacterium tumefaciens* has been made nonvirulent by deleting the part that is responsible for its virulence.
However, the modified plasmids does contains the tumour that inducing gene T-DNA. It is, however, employed as recognition or cloning site. A foreign gene is inserted in T-DNA region of the plasmid.
The T-gene becomes inactive. The recombined or cloning vector is now passed into plasmid free bacterium that is allowed to infect the plant or single cells in tissue culture. The plasmid with the desired inserted gene passes into various parts. The inserted gene expresses its effect. Agrobacterium tumefaciens normally infects only dicot plants but the one with modified non virulent plasmid can also cause genetic changes in monocots.
Natural Genetic Engineer. Agrobacterium tumefaciens is called natural genetic engineer because part of its Ti plasmid, called T-DNA, is transferred and incorporated into host cell DNA where it is expressed alongwith host DNA. And genes contained in T-DNA, therefore, becomes part of host genetic system.
3. Disarmed Viruses As Vectors for Animals and Others.
Bacterial viruses or bacteriophages (eg., lambda 1 virus) are used as clones for introducing desired genes into bacteria. Retroviruses which have the ability to transform normal animal cells into cancerous cells (e.g., Simian Virus 40) can be used for similar purpose in animals. Herpes virus and Adenovirus are common vectors used in human beings.
Cauliflower Mosaic Virus is used in case of plants. Virus that carrying a foreign gene becomes harmless and therefore, proves to be a good vector.
4. Cosmids.
These are plasmids in which the phage lambda cos sites have been inserted. A cosmid can be packed in the phage coat. It is useful in carrying large number of DNA fragment. A similar cloning vector is phagemid. It is formed by combining phage DNA with plasmid.5. Artificial Chromosomes.
They are used for cloning large DNA segments bigger than 100 kb.
Bacterial artificial chromosomes (BACs) have been prepared from F-factor of Escherichia coli by combining it with chloramphenicol resistance gene and a segment having cloning site. YAC (Yeast artificial chromosomes), BACs (mammalian artificial chromosomes) and HACs (human artificial chromosomes) have been constructed by using centromeric and telomeric sequence alongwith high molecular weight and autonomous replicating sequences.
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