The smallest bacterial plasmids are about 1. However, several very large linear DNA plasmids, up to kb long, have been found in species of Streptomyces and Nocardia. Plasmids have relatively few genes, generally less than Their genetic information is not essential to the host, and bacteria that lack them usually function normally.
Single copy plasmids produce only one copy per host cell. Multicopy plasmids may be present at concentrations of 40 or more per cell. Plasmids may be classified in terms of their mode of existence and spread. Some plasmids, conjugative plasmids, have genes for pili and can transfer copies of themselves to other bacteria during conjugation.
Plasmids were the first cloning vectors. They are easy to isolate and purify, and they can be reintroduced into a bacterium by transformation. Plasmids often bear antibiotic resistant genes, which are used to select their bacterial hosts. A recombinant plasmid containing foreign DNA often is called a chimera after the Greek mythological monster that had the head of a lion, the tail of a dragon, and the body of a goat.
One of the most widely used plasmids is pBR Plasmid pBR has both resistant genes for ampicillin and tetracycline and many restriction sites. Several of these restriction sites occur only once on the plasmid and are located within an antibiotic resistant gene. This arrangement aids detection of recombinant plasmids after transformation. For example, if foreign DNA is inserted into the ampicillin resistant gene, the plasmid will no longer confer resistant to ampicillin.
Fig: The pBR Plasmid. A map of the E. The plasmid has resistance genes for ampicillin Apr and tetracycline Tett. Many genetically engineered plasmid vectors are now available, and they offer a number of useful features that make it easier to identify host cells carrying a plasmid with an inserted DNA fragment.
One such plasmid is pUC As a vector, this plasmid has several useful properties: 1. The plasmid is small, allowing it to carry relatively large DNA inserts. In a host cell, pUC18 can replicate to form about copies per cell, producing many clones, or copies, of inserted DNA fragments.
A large number of restriction enzyme sites have been engineered into pUC18, and these are conveniently clustered in one region, called a multiple-cloning polylinker site. The pUC18 plasmid has a selection system that allows recombinant plasmids to be identified.
The pUC18 plasmid carries a fragment of the bacterial lacZ gene, and the polylinker is inserted into this fragment. When a DNA fragment is inserted into the polylinker site, the lacZ gene is inactivated, and a bacterial cell carrying pUC18 with an inserted DNA fragment will form white colonies, making them easy to identify. Fig: The plasmid pUC18 offers several advantages as a vector for cloning.
The rDNA molecules are cloned by inserting them into bacteria, using transformation or phage injection each strain reproduces to yield a population containing a single type of recombinant molecule. The overall process is outlined in the following figure.
The preparation of recombinant clone from previously isolated DNA fragments. Phage vector Both single- and double-stranded phage vectors have been employed in recombinant DNA technology. For example, lambda phage derivatives are very useful for cloning and can carry fragments about 40 kb in length.
The genes for lysogeny and integration often are nonfunctional and may be deleted to make room for the foreign DNA. The modified phage genome also contains restriction sequences in areas that will not disrupt replication. These vectors are often used to generate genomic libraries. However, this approach is less efficient than the use of complete phage particles. The process is sometimes called transfection.
Cosmids Cosmids are plasmids that contain lambda phage cos sites, necessary for packing Phage DNA into phage protein coats and can be packaged into phage capsids. The lambda genome contains a cos site at each end. When the genome is to be packaged in a capsid, it is cleaved at one cos site and the linear DNA is inserted into the capsid until the second cos site has entered. Thus any DNA between the cos sites is packaged. Cosmids typically contain several restriction sites and antibiotic resistant genes.
They can be packaged in lambda capsids for efficient injection into bacteria, but they also can exist as plasmids within a bacterial host. As much as 50kb of DNA can be carried in this way. Bacterial Artificial Chromosome: The mapping and analysis of large complex eukaryotic genomes requires cloning vectors that can accommodate very large DNA fragments.
In addition, since some human genes range from kb to over kb, vectors with large cloning capacities are useful in studying the organization of these genes. Recently, a number of vectors that use bacterial host cells have been developed. One of these vectors is based on the fertility plasmid F factor of bacteria and is called bacterial artificial chromosome BAC. Because F factors can carry fragments of the bacterial chromosome up to 1Mb in length, they have been engineered to act as vectors for eukaryotic DNA and can carry inserts of about kb.
BAC vectors carry the F factor genes for replication and copy number, and incorporate an antibiotic resistance marker and restriction enzyme sites for inserting foreign DNA to be cloned. In addition, the cloning site is flanked by promoter sites that can be used to generate RNA molecules for the expression of the cloned gene, for use as probes in chromosome walking, and for DNA sequencing of the cloned insert.
Shuttle vectors are used for experiments in which recombinant DNA is to be introduced into organisms other than E coli. For example, the yeast-E. Like E coli cloning vectors, YEp24 has an ori sequence that allows it to replicate in E.
YEp24 also contains the selectable marker URA3 a wild type yeast gene for an enzyme required for uracil biosysthesis. This marker enable yeast ura3 mutant host cells containing YEp24 to be identified.
YEp24 also carries a yeast-specific sequence, the two-micron circle 2u , that allows it to replicate autonomously in a yeast cell. Thus, YEp24 is able to replicate in both yeast and E. Not all shuttle vectors have the ability to replicate in the nonbacterial host. A cloned gene is not always expressed in the host cell without further modification of the recombinant vector. To be transcribed, the recombinant gene must have a promoter that is recognized by the host RNA polymerase.
Translation of its mRNA depends on the presence of leader sequences and mRNA modifications that allow proper ribosome binding. These are quite different in eukaryotes and prokaryotes, a prokaryotic leader must be provided to synthesize eukaryotic proteins in bacterium.
Finally, introns in the eukaryotic genes must be removed because the prokaryotic host will not excise them after trascription of mRNA; a eukaryotic protein is not functional without intron removal prior to translation. Thus, before a specific RNA or protein product can be produced in a particular host cell, a suitable DNA construct must be prepared.
The expression system is composed of an expression vector and a specific host cell. Expression vectors usually consist of small, circular plasmids specifically designed with several key features that allow a foreign gene inserted into the plasmid to be expressed in the host cell. Important elements of the plasmid include: 1. The most widely used expression vectors for E. A typical prokaryotic expression vector is represented in the following diagram: These vectors are often derivatives of plasmid pBR and contain the necessary transcription and translation start signals.
Some expression vectors contain portions of the lac operon and can effectively regulate the expression of the cloned genes in the same manner as the operon. Somatostatin, the residue hypothalamic polypeptide hormone that helps regulate human growth, provides an example of useful cloning and protein production. Besides the 42 bases coding for somatostatin, the polynucleotide contain a codon for methionine at the 5-prime end the N-terminal end of the polypeptide and two stop codons at the opposite end.
To aid insertion into the plasmid vector, the 5-prime ends of the synthetic gene were extended to form single stranded sticky ends complementary to those formed by EcoRI and BamHI restriction enzymes. The synthetic gene was then spliced into the vector by taking advantage of its cohesive ends. Finally, a fragment containing the initial part of the lac operon including the promoter, operator, ribosome binding site, and much of the beta-galactosidase gene was inserted next to the somatostatin gene.
The plasmid now contained the somatostatin gene fused in the proper orientation to the remaining portion of the beta-galactosidase gene. After introduction of the chiremic plasmid into E. Tranaslation foremed a protein consisting of the total hormone polypeptide attached to the beta-galactosidase fragment by a methionine residue. Cyanogen bromide cleaves peptide bonds at methinine residues and released the hormone. Once free, the polypeptide was able to fold properly become active.
A similar approach was used to manufacture human insulin. A set of DNA clones derived from a single individual represents a library. Cloned libraries can represent an entire genome, a single chromosome, or a set of genes that are actively transcribed in a single cell type.
Ideally, a genomic library is a collection of clones that contains at least one copy of all the sequences represented in the genome. One approach to obtaining a clone of a gene is to isolate it from a genomic library through the use of a specific probe.
There are three ways to produce genomic libraries: 1. Genomic DNA is completely digested by a restriction enzyme, and the resulting DNA fragments are then cloned in a cloning vector.
This technique does have a drawback. If the specific gene the researchers want to study contains restriction sites for the enzyme, the gene will be split into two or more fragments when the DNA is digested by the restriction enzyme. In this case, the gene would then be cloned in two or more fragments. Thus, an entire library would need to contain a very large number of recombinant DNA molecules, and screening for the specific gene would be very laborious.
The problems of genes split into fragments and the large number of recombinant DNA molecules can be minimized by cloning longer DNA fragments. For example, the passage of the syringe needle will produce a population of overlapping DNA fragments. However, since the ends of the resulting DNA fragments have not been generated by cutting with restriction enzymes, additional enzymatic manipulations are necessary to add appropriate ends to the molecules for insertion into vector cloning sites.
Another approach for producing DNA fragments of appropriate size for constructing a genomic library is to perform a partial digestion of the DNA with restriction enzymes that recognize frequently occurring four-base —pair recognition sequences. Partial digestion means that only a portion of the available restriction sites is actually cut with the enzyme.
The ideal result of partial digestion is a population of overlapping fragments representing the entire genome. To facilitate the study of genes, they can be isolated and amplified. One method of isolation and amplification of a gene of interest is to clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells.
The recombinant DNA molecule is placed in a host cell, either prokaryotic or eukaryotic. The host cell then replicates producing a clone , and the vector with its foreign piece of DNA also replicates. The foreign DNA thus becomes amplified in number, and following its amplification can be purified for further analysis. Restriction endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere, depending on the type of enzyme.
Cut one strand at non specific sites,bp 2 subunit,methylas e and endonuclease are separate. Restriction endonucleases are named for the organism in which they were discovered, using a system of letters and numbers.
The Hin comes from the first letter of the genus name and the first two letters of the species name; d is for the strain type; and III is for the third enzyme of that type. Over type II restriction endonucleases have been isolated and characterized to date. Approximately are available commercially for use by molecular biologists. Each orthodox type II restriction endonuclease is composed of two identical polypeptide subunits that join together to form a homodimer.
These homodimers recognize short symmetric DNA sequences of 48 bp. Six base pair cutters are the most commonly used in molecular biology research. Usually, the sequence read in the 5 3 direction on one strand is the same as the sequence read in the 5 3 direction on the complementary strand. Sequences that read the same in both directions are called palindromes from the Greek word palindromos for run back Some enzymes, such as EcoR1, generate a staggered cut, in which the single-stranded complementary tails are called sticky or cohesive ends because they can hydrogen bond to the singlestranded complementary tails of other DNA fragments.
DNA ligases catalyze formation of a phosphodiester bond between the 5phosphate of a nucleotide on one fragment of DNA and the 3-hydroxyl of another. This joining of linear DNA fragments together with covalent bonds is called ligation. The basic procedure of molecular cloning involves a series of steps. First, the DNA fragments to be cloned are generated by using restriction endonucleases, Second, the fragments produced by digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors.
Vectors can replicate autonomously independent of host genome replication in host cells and facilitate the manipulation of the newly created recombinant DNA molecule. Third, the recombinant DNA molecule is transferred to a host cell. Within this cell, the recombinant DNA molecule replicates, producing dozens of identical copies known as clones. As the host cells replicate, the recombinant DNA is passed on to all progeny cells, creating a population of identical cells, all carrying the cloned sequence.
Finally, the cloned DNA segments can be recovered from the host cell, purified, and analyzed in various ways. Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are that they: i can independently replicate themselves and the foreign DNA segments they carry; ii contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector; iii carry a selectable marker usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell to distinguish host cells that carry vectors from host cells that do not contain a vector; and iv are relatively easy to recover from the host cell.
There are many possible choices of vector depending on the purpose of cloning. The greatest variety of cloning vectors has been developed for use in the bacterial host E. Thus, the first practical skill generally required by a molecular biologist is the ability to grow pure cultures of bacteria.
Plasmids are naturally occurring extrachromosomal double stranded circular DNA molecules that carry an origin of replication and replicate autonomously within bacterial cells. The plasmid vector pBR, constructed in , was one of the first genetically engineered plasmids to be used in recombinant DNA. Plasmids are named with a system of uppercase letters and numbers, where the lowercas p stands for plasmid.
In the case of pBR, the BR identifies the original constructors of the vector Bolivar and Rodriquez , and is the identification number of the specific plasmid. These early vectors were often of low copy number, meaning that they replicate to yield only one or two copies in each cell. Plasmid vectors are modified to contain a specific 1.
Cutting the circular plasmid vector with one of these enzymes results in a single cut, creating a linear plasmid. A foreign DNA molecule, referred to as the insert, cut with the same enzyme, can then be joined to the vector in a ligation reaction. The degree of self-ligation can be reduced by treatment of the vector with the enzyme phosphatase, which removes the terminal 5-phosphate When the 5-phosphate is removed from the plasmid it cannot be recircularized by ligase, since there is nothing with which to make a phosphodiester bond.
But, if the vector is joined with a foreign insert, the 5phosphate is provided by the foreign DNA. The ligation reaction mixture of recombinant and nonrecombinant DNA is introduced into bacterial cells in a process called transformation. The traditional method is to incubate the cells in a concentrated calcium salt solution to make their membranes leaky. Alternatively, a process called electroporation can be used that drives DNA into cells by a strong electric current.
Successfully transformed bacteria will carry either recombinant or nonrecombinant plasmid DNA. Multiplication of the plasmid DNA occurs within each transformed bacterium.
A single bacterial cell placed on a solid surface agar plate containing nutrients can multiply to form a visible colony made of millions of identical cells. As the host cell divides, the plasmid vectors are passed on to progeny, where they continue to replicate.
Isolation and genotypic characterization of different yeast strains from Nepalese mixed culture Murcha. The unusual origin of the polymerase chain reaction. Scientific American. Replication and transcription of eukaryotic DNA in Escherichia coli. Molecular cell. Construction of a novel human artificial chromosome vector for gene delivery.
Biochemical and biophysical research communications. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nature Genetics. Chromosome-based vectors for gene therapy. Structure and function of type II restriction endonucleases. Nucleic acids research. The Yale Journal of Biology and Medicine.
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