In this article we will discuss about the Insertion of a Foreign DNA Fragment into a Vector.
Introduction to the Insertion of a Foreign DNA Fragment into a Vector:
The specific section of DNA generated by the action of restriction enzyme is spliced or incorporated into an agent called cloning vehicle. Cloning vehicle is also called vector. Cloning vehicles or vectors: short DNA molecules that can penetrate the wall of living host cells and can multiply inside the cells.
Currently small DNA molecules of bacterial plasmids, Lambda and M 13 bacteriophages of E. coli: and several animal viruses are used as cloning vectors to transfer the DNA fragment from test tube into the living host cell.
To be useful in the cloning process, a vector or cloning vehicle must have the following characteristics:
1. It must be small and well characterised molecule having means of introducing DNA into a cell.
2. It must have a replication origin enabling self-replication as well as the replication of foreign DNA segment.
3. It should be capable of selection of hybrid molecules by a straight forward assay preferably by the growth of a host cell on a solid culture medium.
The DNA fragment generated by action of restriction endonucleases has to be joined with the vector, either a plasmid or a phage, before that is cloned into a bacterium. The piasmid is separated from main chromosome by ultracentrifugation. The plasmid ring is opened by restriction enzyme and the gene is combined with the vector.
The splicing process produces a chimeric DNA or hybrid DNA molecule containing foreign DNA and the DNA of cloning vehicle. Such a DNA is also called recombinant DNA molecule. Once the foreign DNA is integrated into cloning vehicle, the chimerce DNA so formed is introduced ox transferred into a host cell. Usually the host cells used for cloning are single celled organisms such as bacteria or yeasts.
The host cell that has received recombinant DNA or chimeric DNA is then said to be transformed and the introduction of chimeric DNA into the host cell is called transformation. Gene cloning is essentially the insertion of a specific piece of foreign DNA in a cell in such a way that inserted DNA is replicated and handed on to daughter cells during cell division.
The host cell must accept the vector with foreign genes, get it incorporated its genome and start transcribing that gene.
Plasmids as Vector:
Plasmids are small, double stranded, extrachromossomal circular DNA possessing limited number of restriction sites. They are present in the bacterial cytoplasm and yeasts. The plasmid that has been used extensively in cloning is pBR 322 (Fig. 24.6) which has 4363 base pairs.
They replied independently of the bacterial chromosome and carry genes for fertility, antibiotic resistance, ability to ferment sugars and also genes for production of bacteriocins, haemolysins etc., a property d helps in the selection of transformed cells.
They differ in their size and genes contained in their DNA. Plasmids can be categorized on the basis of number of copies per cell into:
(i) Relaxed plasmids and
(ii) Stringent plasmids.
They can also be classified into conjugative and non-conjugative depending on whether or not they carry a set of transfer gene or tra genes that promote bacterial conjugation.
Generally conjugative plasmids are stringent plasmids of high molecular weight which are present in limited number (1-3 copies per cell) and the non-conjugative plasmids are relaxed type that a small with low mol. wt. and are present in multiple copies (20 – 25 copies per cell).
Plasmids that are popular in cloning contain about 6,500 base pairs and have a single site where they can be cleaved by a restriction endonuclease. Besides, there are some other plasmids which are larger enough and are difficult to handle. They are generally not used in gene cloning. Some plasmids are capable to integrate themselves into the bacterial chromosome and these are called episomes, e.g., F factors of E. coil.
Sometimes after cleavage by a restriction enzymes and during insertion of foreign DNA fragment into the plasmid, a few important genes are destroyed e.g.. Bam HI restriction enzyme destroys than ter-r gene (resistant against tetracycline).
In general, the plasmid vehicles have a different density than host DNA and thus, they can be purified easily by sedimentation. Thus to clone a piece of foreign DNA, the DNA is integrated or spliced into the plasmid DNA and the chimeric or recombinant DNA is used to transform a host bacterium such as E. coli.
The plasmid with foreign DNA then replicates within the bacterium when it grows and divides and thus the piece of foreign DNA becomes cloned.
Bacteriophages as Vectors:
Bacteriophages or their derivatives are also used as vector or cloning vehicles. Bacteriophage Lambda (λ) is one of the best understood elements in bacterial genetics.
A bacteriophage is a virus that infects bacteria. These are commonly called phages. Bacteriophages are more complicated than plasmids. Unlike plasmids, the phage vectors are required for cloning of large DNA fragment and, therefore, gene bank or genomic libraries can be constructed.
Phage contains a head and a long tail attached to the head which are made up of proteins. Inside the proteinaceous sheath, there is a linear double stranded DNA molecule.
Phage DNA contains genes for sheath proteins and it has an origin of replication. Phages, like plasmids, lack in the machinery led to make proteins and hence they reproduce inside living bacterial cells. When a phage comes contact with a bacterial cell, the phage tail sticks to the cell wall of bacterium E. coli and it injects its linear DNA into the cytoplasm of host cell.
DNA of Lambda phage is a linear duplex molecule of about 48.5 Kb pairs possessing some 60 genes.
Functionally related genes are clustered together on the Linear map (Fig. 24.7). Gene cluster on the left of linear map code for head and tail proteins, those in the middle are involved in Lysogenic phage i.e. in recombination and the genes on the right of central region are concerned with regulation and prophage immunity to infection, DNA synthesis and lysis of host cell.
When present inside the host cell, the phage DNA may be either integrated into bacterial chromosome or it may remain in free state in the cytoplasm.
The replication cycle of phage is accomplished in two ways:
(i) Lytic process, and
(ii) Lysogenic process.
In the lytic process the linear double stranded DNA molecule injected inside the bacterial cell becomes circular through the single strand of 12 nucleotides called cos site [Fig. 24.7 (a)] which replicates by a rolling circle mechanism and produces several genomes.
In Lambda phage which replicates in lytic phase, much of the central region is not essential for phage growth and that may be deleted or substituted without impairing the infectious growth cycle. At the same time phage DNA also dried the synthesis of proteins required to produce empty heads where DNA molecules are packed.
Eventually tail is also formed and attached to the head and progeny phage particles are released out of bacterial cell (Fig. 24.8). At early stage of infection some phages produce proteins that destroy the bacterial DNA, chopping that into individual nucleotides. Lambda phage replicates in lytic phage.
In the lysogenic process the phage DNA becomes integrated into the bacterial chromoson and replicated along with the host chromosome with no detrimental effect to the host (Fig. 24.8).
The following are the advantages of phage cloning systems over plasmids:
(i) Foreign DNA can be packed in vitro into phages and transduced into bacterial host cell w better efficiency.
(ii) Long foreign DNA molecules upto 25 kPB in length can be inserted into phage DNA.
(iii) Screening and storage of recombinant DNA is more-easy in phages than in plasmids.
M-13 Phages:
M 13 Vector is a filamentous single stranded DNA phage. M-13 phage can infect male bacterial cell through pili. Once inside the host cell, phage DNA is replicated, transcribed and translated into viral gene products. The plus (+) strand of virus is used to synthesize complementary (-) strand giving rise to a double-stranded copy of M-I3 which is known as the parental replication form (RF).
The replication form serves as template from which several (100 – 200) copies of RF molecules are synthesized. Single stranded DNA phages harm the bacterial cell, instead the phage particles released continuously from the cell without any harm to it. No packaging of viral DNA is needed with this system.
M 13 particles continue to infect the surrounding male bacteria and a plaque of sic growing cells is formed. Thus with M 13 system recombinant phage can be distinguished from the non-recombinant on the basis of plaque colour.
Cosmid Vectors:
Cosmids (cos + plasmid) are artificially synthesised vectors containing plasmid DNA with a fragmeoi of λDNA including the cos site packaged in a phage particle. Cosmids consist essentially of a circular double stranded DNA with antibiotic resistant genes, several restriction sites and cos sites of λ phage.
The cosmid HC 79 for instance, consists of 1.7 kb DNA fragment consisting of cos region of λ phase inserted into any one essential region of pBR 322. Foreign DNA can be cloned into any cot the restriction sites on pBR 322 and product after ligation is packaged in vitro.
The cosmid infects the host which is selected for growth on antibiotic containing medium and the colonies formed are analysed for the presence of plasmid DNA. Recombinant cosmids, unlike vector, are maintained like plasmids in the host cell.
Recombinant cosmids are more stable and are easy to store. Larger DNA fragments can be cloned in the cosmids. Thus they combine the best i of cloning vector with maximum capabilities.