Vehicles for Gene Cloning

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A DNA molecule needs to display several features to be able to act as a vehicle for gene cloning. A cloning vehicle also needs to be-
• Most importantly it must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells.
• Relatively small,
• Ideally less than 10kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate.
Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: plasmids and bacteriophage chromosomes. Besides these two, other DNA molecules are also identify to serve the purpose of cloning vehicles. The list is as follow-
1. Plasmids – (up to 10kb DNA can be cloned)
2. Bacteriophages - (up to 20-40kb DNA can be cloned)
3. Cosmid - (up to 40kb DNA can be cloned)
4. Phagemid
5. YACs - (up to 2MB)
6. BACs - ( up to 300kb DNA can be cloned)
7. Viruses as vectors.
Plasmids
Basic features of plasmids
Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell (Figure). Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium.
Marker Gene for antibiotic resistance: The ability to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence in the bacterium of a plasmid carrying antibiotic resistance genes. In the laboratory antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid (Figure).
Origin of replication: All plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell quite independently of the main bacterial chromosome also called non-integrative plasmids (Figure(a)). The smaller plasmids make use of the host cell's own DNA replicative enzymes in order to make copies of themselves, whereas some of the
larger ones carry genes that code for special enzymes that are specific for plasmid replication.
A few types of plasmid are also able to replicate by inserting themselves into the bacterial chromosome (Figure (b )). These integrative plasmids or episomes may be stably maintained in this form through numerous cell divisions, but will at some stage exist as independent elements.
Size and copy number: The size and copy number of a plasmid are particularly important as far as cloning is concerned. Plasmids range from about 1.0 kb for the smallest to over 250 kb for the largest plasmids, so only a few are useful for cloning purposes.
The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. The factors that control copy number are not well understood, but each plasmid has a characteristic value that may be as low as one (especially for the large molecules) or as many as 50 or more. Generally speaking, a useful cloning vehicle needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained.
Conjugation and compatibility
Plasmids fall into two groups: conjugative and non-conjugative.
Conjugative plasmids are characterized by the ability to promote sexual conjugation between bacterial cells (Figure), a process that can result in a conjugative plasmid spreading from one cell to all the other cells in a bacterial culture. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes, which are present on conjugative plasmids but absent from the non-conjugative type. However, a non-conjugative plasmid may, under some circumstances, be cotransferred along with a conjugative plasmid when both are present in the same cell.
Compatible and incompatible plasmids: Several different kinds of plasmid may be found in a single cell, including more than one different conjugative plasmid at anyone time. In fact, cells of E. coli have been known to contain up to seven different plasmids at once. To be able to coexist in the same cell, different plasmids must be compatible. If two plasmids are incompatible then one or the other will be quite rapidly lost from the cell. Different types of plasmid can therefore be assigned to different incompatibility groups on the basis of whether or not they can coexist, and plasmids from a single incompatibility group are often related to each other in various ways. The basis of incompatibility is not well understood, but events during plasmid replication are thought to underlie the phenomenon.
Plasmid classification / Types
The most useful classification of naturally occurring plasmids is based on the main characteristic coded by the plasmid genes. The five main types of plasmid according to this classification are as follows:
(1) Fertility or 'F' plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids, e.g. F plasmid of E. coli.
(2) Resistance or 'R' plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections, e.g. RP4, commonly found in Pseudomonas, but also occurring in many other bacteria.
(3) Col plasmids ability to synthesised colicins, proteins that kill closelt related strains that lack ‘col’ plasmid, e.g. ColE 1 of E. coli.
(4) Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, e.g. TOL of Pseudomonas putida.
(5) Virulence plasmids confer pathogenicity on the host bacterium; e.g. Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants.
Commonly used plasmids in Rec-DNA Technology are pBR322, pUC, pSP64 and pSP65, pGEM3 etc
The useful properties of pBR322
The genetic and physical map of pBR322 (Figure) gives an indication of why this plasmid has been such a popular cloning vector.
The first useful feature of pBR322 is its size. it was stated that a cloning vector ought to be less than 10 kb in size, to avoid problems such as DNA breakdown during purification. pBR322 is 4363 bp, which means that not only can the vector itself be purified with ease, but so can recombinant DNA molecules constructed with it. Even with 6 kb of additional DNA, a recombinant pBR322 molecule is still a manageable size.
The second feature of pBR322 is that, it carries two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells containing the plasmid, and each marker gene includes unique restriction sites that can be used in cloning experiments. Insertion of new DNA into pBR322 that has been restricted with Pst1, Pvu1 or ScaI inactivates the ampR gene, and insertion using any one of eight restriction endonucleases (notably BamHI and HindIII) inactivates tetracycline resistance. This great variety of restriction sites that can be used for insertional inactivation means that pBR322 can be used to clone DNA fragments with any of several kinds of sticky end.
A third advantage of pBR322 is that it has a reasonably high copy number. Generally there are about 15 molecules present in a transformed E. coli cell, but this number can be increased, up to 1000-3000, by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol (p. 44). An E. coli culture therefore provides a good yield of recombinant pBR322 molecules.
Plasmids in organisms other than bacteria
Although plasmids are widespread in bacteria they are by no means as common in other organisms. The best characterized eukaryotic plasmid is the 2um circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The discovery of the 2um plasmid was very important as it has allowed the construction of vectors for cloning genes with this very important industrial organism as the host. Other yeast plasmids so far identified are as follow-
Yip or yeast integrative plasmid
YEp or yeast episomal plasmid
YRp or yeast replicative plasmid
Yep or yeast centromere containing plasmid
All the above cited plasmids can be used as shuttle vectors which allow the gene sequences to be transferred back and forth between Yeast and E.coli. These shuttle vectors have both E.coli and Yeast replication gene. Some plasmids can retrive the gene from yeast chromosomes. These are called retriever vectors
However, the search for plasmids in other eukaryotes (e.g. filamentous fungi, plants and animals) has proved disappointing, and it is suspected that many higher organisms simply do not harbour plasmids within their cells.
Bacteriophages as vector:
Basic features of bacteriophages
Bacteriophages, or phages as they are commonly known, are viruses that specifically infect bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or occasionally ribonucleic acid RNA molecule ) carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules (Figure).
The general pattern of infection, which is the same for all types of phage, is a three-step process (Figure).
(1) The phage particle attaches to the outside of the bacterium and injects its DNA chromosome into the cell.
(2) The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on the phage chromosome.
(3) Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium.

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