In this article we will discuss about:- 1. Meaning of Plasmids 2. Classes of Plasmid 3. Copies of Plasmids within a Cell 4. Phenotypic Effects of Plasmids 5. Origin of Plasmids and Viruses 6. Uses of Plasmids.
Meaning of Plasmids:
In bacterial cells, certain autonomously replicating circular genetic elements (DNA) are found some of which can become integrated into the bacterial chromosome, while others exist independently of the latter. These genetic elements are called plasmids and episomes depending on their integration into the bacterial chromosomes.
Jacob and Wollmann in 1958, defined episomes as DNA elements which are in addition to the normal bacterial chromosomes and that cannot arise due mutation but have to be introduced from outside; these elements are capable of integration into their host chromosomes. The self-replicating DNA molecules that exist, independently of the host chromosome are called plasmids.
However, “plasmids” and “episomes” are regarded as the same in modern terminology, and there is no sharp distinction between two since the ability to integrate into the host chromosome varies quantitatively. However, plasmids and viruses that are known to integrate into the host chromosome usually bear the adjective “episomal”.
In general, genes carried in plasmids are not essential for the host cell survival and growth, except under certain special situations.
Classes of Plasmid:
About 1000 different kinds of bacterial plasmids have been identified; they all are circular DNA molecules. Based on their size (length, molecular weight and number of base pairs), the plasmids can be divided into two major classes; large plasmids and small plasmids.
Large plasmids:
These plasmids are 60-120 kb in size and, in most cases, are conjugative, i.e., they are self-transmissible through conjugation, hence such plasmids are called conjugons. The mechanism of transfer of these plasmids to other cells by contact is coded by the plasmids themselves. Examples of such plasmids are F-plasmid (sex factor) R-factors (R determinant) and certain bacteriocinogens (Col-factors).
Small plasmids:
Small plasmids are 1.5 to 15 kb long and are non-conjugative, i.e., they lack the ability of self-transmission through conjugation. However, they can be mobilized for transfer by conjugative plasmids if present in the same cell. Examples of such plasmids are some resistance factors and some bacteriocinogens.
Apart from the above two classes, there exist certain DNA molecules that are smaller than plasmids; examples of such elements are transposable element (transposons) and IS (insertion sequences) elements. But these DNA molecules are non-replicating; therefore, they cannot be perpetuated unless they are inserted into the DNA of plasmids, temperate bacteriophages or bacterial chromosomes.
Copies of Plasmids within a Cell:
Plasmids differ considerably regarding the number of copies per cell. Large plasmids are present in only one copy per host chromosome, but small plasmids may have many copies, ranging from 10 to 20. A “replication repressor” controls the number of plasmid copies.
When the host cell protein synthesis is blocked by chloramphenicol, copies of plasmids increase and they may reach up to 1000 per cell. When the replication of different plasmids is regulated by different repressors, their characteristic number within a cell line is maintained.
Such plasmids are called compatible. But if a common repressor controls the replication of a number of different plasmids, the repressor recognizes all such different plasmids, deviation in their number is not corrected and many cells end up with one or other type of plasmid.
Thus different plasmids cannot coexist in the same cell for many cell generations. Such plasmids are called “incompatible” and they form an “incompatibility group”.
Phenotypic Effects of Plasmids:
Plasmids contain various combinations of genes governing different traits (Table 18.1). However, some very small plasmids have no known phenotypic effects.
Origin of Plasmids and Viruses:
It is believed that small DNA fragments were excised from the cell chromosome and became circularized; these fragments contained the sites for replication initiation, i.e., each fragment was a functional “replicon”.
During the course of evolution, duplications and mutations brought about changes in these DNA fragments. Some of these fragments developed symbiotic relationships with their host cells and became the present day plasmids. Some others developed a protein coat (capsid) around them to become free viral particles.
Uses of Plasmids:
Plasmids can be used as vectors in gene cloning. DNA cloning or gene cloning is the process by which DNA fragments can be amplified many folds by inserting them into a suitable plasmid or a temperate bacteriophage, and growing them in the appropriate bacterial cells/yeast cells.
Plasmids should have following characteristics for their use as vectors:
1. Plasmid should be small in size for its stability.
2. Plasmid replication should be independent of host chromosome replication, i.e., there should relaxed genetic control. This causes the accumulation of plasmids in large copy number up to even 1000 per cell.
3. There should be non-conjugative transmission of the plasmid.
A number of plasmids have been used as vectors for recombinant DNA production. One widely used plasmid vector is “pBR 322” that has been constructed from other large vectors, such as, pBR313, pBR318 and pBR320. The plasmid pBR322 contains 4362 nucleotide pairs, and the genes for resistance to ampicillin (ampR) and tertracycline (tetR.)
This plasmid also contains unique target sites for the action of a number of restriction endonucleases (Fig. 18.12).
The recombinant DNA technology or gene cloning technology involves certain specific enzymes called the restriction endonucleases; these enzymes are obtained from different organisms.
Restriction Endonucleases:
Restriction endonucleases are the enzymes that recognize specific nucleotide sequences of DNA and cut the double helix at or near their recognition sites (target sites). The target sites are small palindromic sequences having rotational symmetry; these sites generally contain 4-6 bases that are recognized by the specific restriction enzymes. For example, Eco RI endonuclease recognizes.
Thus it produces protruding single-stranded regions of 4 nucleotides at the 5′-ends. These ends are called “sticky” ends or “cohesive” ends and this type of cut is called “staggered cut”. Due to the presence of cohesive ends, a foreign DNA fragment containing complementary “cohesive ends can be incorporated in the DNA molecule.
Some types of restriction enzymes recognize the sequence of 4 bases, while some other types recognize the sequence of 6 bases (Table 18.3). Certain restriction enzymes such as Hindll and Hpal produce “blunt-ended” fragments, i.e., the ends are not cohesive (not protruding).
Method of Gene Cloning:
Gene cloning involves the following steps:
(a) Insertion of the foreign DNA into a cloning vector.
(b) Introduction of recombinant DNA (vector containing the foreign DNA) into bacterial cell.
(c) Selection of host cells containing recombinant DNA.
(d) Isolation of clones.
(e) Identification of specific cloned genes.
Insertion/incorporation of foreign DNA into cloning vector:
The selected plasmid is isolated from the bacterial or yeast cells. Purified plasmids are cut once with the appropriate restriction endonuclease. DNA fragments to be cloned are obtained by the action of the same restriction enzyme; these fragments are inserted into the cut vector by mixing them together allowing them to renaturate.
This permits pairing between the sticky ends of the DNA fragment with the complementary ends of the vector DNA. A plasmid containing the foreign DNA is called recombinant DNA, chimeric DNA or plasmid hybrid DNA (Fig. 18.13, 18.14).
Introduction of the recombinant DNA into bacterial cell:
The bacteria into which the plasmid hybrid DNA is introduced is called “host” and the process is known as transformation. Bacterial cells are made permeable to macromolecules by warming the cell with CaCl2 solution. The plasmid hybrid DNA is now introduced into such bacterial cells. Replication of the plasmid hybrid DNA occurs in the host cells.
In case of appropriately constructed vectors, the incorporated gene may be transcribed and translated into proteins.
Selection of cells containing recombinant DNA:
Plasmid markers, especially genes for antibiotic resistance, are used to select the cells containing recombinant DNA. Often plasmids carrying genes for resistance to two antibiotics are used as vector. One of the two resistance genes carries the target site for the restriction endonuclease that is used for cutting the plasmid as well as the DNA fragment to be cloned.
Therefore, when the DNA fragment is inserted into the plasmid, it will be located within the concerned resistance gene (which contains the target site for the restriction endonuclease used for cleaving of plasmid); this will inactivate the resistance gene.
After the insertion of the DNA fragment, two types of plasmid molecules will be produced:
(1) Plasmids having the DNA insert (in such plasmids one of the antibiotic resistance gene will be inactivated), and
(2) Plasmids without the DNA insert (both resistance genes will be active).
When this plasmid mixture is used for transformation, three types of bacterial cells will be obtained:
(i) Cells having no plasmid at all; they will be susceptible to both the antibiotics.
(ii) Cells having the non-recombinant plasmid; they will show resistance to both the antibiotics.
(iii) Cells carrying the recombinant plasmid DNA; they will be resistant to only one of the antibiotic because the other resistance gene has been inactivated due to the insertion of the” foreign DNA fragment.
Of the above three types, cells of the third type are selected since they carry the recombinant plasmid DNA (Fig. 18.14). The plasmid pBR322 is widely used as a cloning vector. It carries the genes for resistance to ampicillin (ampR) and tetracycline (tetR). Target site for the restriction enzyme Pstl is located within the ampR gene, while that for BamHl is located within the tefi gene.
If the enzyme Pstl is used for opening pBR322 and for cutting the DNA insert, the gene ampR will become inactivated due to the DNA insert being incorporated within this gene. The bacterial cells carrying such chimeric plasmids will therefore, be susceptible to ampicillin, but they will show resistance to tetracycline.
On the other hand, when BamHl is used for producing the chimeric plasmid pBR322, the cells will show resistance to ampicillin and susceptibility to tetracycline.
Isolation of cloned DNA:
Cells containing the concerned hybrid plasmid DNA are allowed to grow and proliferate. Hybrid plasmid DNA molecules are isolated and purified from these cells. The original foreign DNA fragments are excised by using the same restriction endonuclease that was used for producing them (Fig. 18.14). Thus a large number of clones of DNA fragment are produced.
Identification of cloned genes:
Purified mRNA (if available) of the cloned gene is used as a template of synthesize radioactive complementary DNA (cDNA) by the enzyme reverse transcriptase. The cDNA is used to identify the colonies containing cloned complementary sequences by the technique of colony hybridization.
One other method utilizes the sequence of amino acids in the protein produced by the gene. With the help of genetic code, the appropriate DNA sequence is identified.
Application of Molecular Cloning:
Eukaryotic genes can be transferred to prokaryotic cells and desired proteins can be “manufactured” in the prokaryote, e.g., human hormones, interferon, insulin etc. Foreign gene is incorporated into the plasmid at position adjacent to promoters of transcription. In E. coli, commonly used promoters are those for lac and trp genes.
Using this method, mammalian gene for chymosin (renin) production was incorporated in the plasmid of E. coli that produced 50,000 to 250,000 molecules of pure active mammalian chymosin per cell. Thus very rare and essential protein of mammalian cells can be produced by bacterial cells using the recombinant DNA technology.