The Basic Properties of a Cloning Vector (Genetics & Inherited Conditions)
Cloning vectors were developed in the early 1970’s from naturally occurring DNA molecules found in some cells of the bacteria Escherichia coli (E. coli). These replicating molecules, called plasmids, were first used by the American scientists Stanley Cohen and Herbert Boyer as vehicles, or vectors, to replicate other pieces of DNA (insert DNA) that were joined to them. Thus the first two essential features of cloning vectors are their ability to replicate in an appropriate host cell and their ability to join to foreign DNA sequences to make recombinant molecules. Plasmid replication requires host-cell-specified enzymes, such as DNA polymerases that act at a plasmid sequence called the “origin of replication.” Insert DNA is joined (ligated) to plasmid DNA through the use of two kinds of enzymes: restriction enzymes and DNA ligases. The plasmid DNA sequence must have unique sites for restriction enzymes to cut. Cutting the double-stranded circular DNA at more than one site would cut the plasmid into pieces and would separate important functional parts from one another. However, when a restriction enzyme cuts the circular plasmid at one unique site, it converts it to a linear molecule. Linear, insert DNA molecules, produced by cutting DNA with the same restriction enzyme as was used to cut the plasmid vector, can be joined to cut plasmid molecules using the enzyme DNA ligase. This...
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Viruses and Cloning Vectors (Genetics & Inherited Conditions)
In addition to plasmid cloning vectors, some bacteriophages (or phages) have been modified to serve as cloning vectors. Bacteriophages, like other viruses, are infectious agents that are made of a genome, either DNA or RNA, that is surrounded by a protective protein coat. Phage vectors are used similarly to the way plasmid vectors are used. The vector and insert DNAs are cut by restriction enzymes so that they subsequently can be joined by DNA ligase. The newly formed recombinant DNA molecules must enter an appropriate host cell to replicate. In order to introduce the phage DNA into cells, a whole phage particle must be built. This is referred to as “packaging” the DNA. The protein elements of the phage are mixed with the recombinant phage DNA and packaging enzymes to create an infectious phage particle. Appropriate host cells are then infected with it. The infected cells then make many copies of each recombinant molecule, along with the proteins needed to make a completed phage particle. In many cases, the final step of viral infection is the lysis of the host cell. This releases the mature phage particles to infect nearby host cells. Phage vectors have two advantages relative to plasmid vectors: First, viral delivery of recombinant DNA to host cells is much more efficient than the transformation or electroporation procedures used to introduce plasmid DNA into host cells, and second, phage vectors can be used to...
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Expression VectorsExpression vectorsPharmaceuticalscloning vectors (Genetics & Inherited Conditions)
Expression vectors are cloning vectors designed to express the gene contained in the recombinant vector. In order to accomplish this, they must also provide the appropriate regulatory signals for the transcription and translation of the foreign gene. Regulatory sequences, which direct the cellular transcription machinery, are very different in bacteria and higher organisms. Thus, unless the vector provides the appropriate host regulatory sequences, foreign genes will not normally be expressed.
Expression vectors make it possible to produce proteins encoded by eukaryotic genes (that is, genes from higher organisms) in bacterial cells. Furthermore, producing proteins in this way often results in higher production rates than in the cells from which the gene was obtained. This technology not only is of immense benefit to scientists who study proteins but also is used by industry (particularly the pharmaceutical industry) to make valuable proteins. Proteins such as human insulin, growth hormone, and clotting factors that are difficult and extremely expensive to isolate from their natural sources are readily available because they can be produced much more cheaply in bacteria. An added benefit of expression vectors is that actual human proteins are produced by bacteria and therefore do not provoke allergic reactions as frequently as insulin that is isolated from other...
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Artificial Chromosomes (Genetics & Inherited Conditions)
In 1987, a new type of cloning vector was developed by David Burke, Maynard Olson, and their colleagues. These new vectors, artificial chromosomes, filled the need created by the Human Genome Project (HGP) to clone very large insert DNAs (hundreds of thousands to millions of base pairs in length). One of the goals of the HGP—to map and ultimately sequence all the chromosomes of humans, as well as a number of other “model” organisms’ genomic sequences—required a vector capable of propagating much larger DNA fragments than plasmid or phage vectors could propagate. The first artificial chromosome vector was developed in the yeast Saccharomyces cerevisiae. All the critical DNA sequence elements of a yeast chromosome were identified and isolated, and these were put together to create a yeast artificial chromosome (YAC). The elements of a YAC vector are an origin of replication, a centromere, telomeres, and a selectable marker suitable for yeast cells. A yeast origin of replication (similar to the origin of replication of bacterial plasmids) is a short DNA sequence that the host’s replicative enzymes, such as DNA polymerase, recognize as a site to initiate DNA replication. In addition to replicating, the new copies of a chromosome must be faithfully partitioned into daughter cells during mitosis. The centromere sequence mediates the partitioning of the chromosomes during cell division because it serves as the...
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Impact and Applications (Genetics & Inherited Conditions)
Cloning vectors are one of the key tools of recombinant DNA technology. Cloning vectors make it possible to isolate particular DNA sequences from an organism and make many identical copies of this one sequence in order to study the structure and function of that sequence apart from all other DNA sequences. Until the development of the polymerase chain reaction (PCR), cloning vectors and their host cells were the only means to collect many copies of one particular DNA sequence. For long DNA sequences (those over approximately ten thousand base pairs), cloning vectors are still the only means to do this.
Gene therapy is a new approach to treating and perhaps curing genetic disease. Many common diseases are the result of defective genes. Gene therapy aims to replace or supplement the defective gene with a normal, therapeutic gene. One of the difficulties faced in gene therapy is the delivery of the therapeutic gene to the appropriate cells. Viruses have evolved to enter cells, sometimes only a very specific subset of cells, and deliver their DNA or RNA genome into the cell for expression. Thus viruses make attractive vectors for gene therapy. An ideal vector for gene therapy would replace viral genes associated with pathogenesis with therapeutic genes; the viral vector would then target the therapeutic genes to just the right cells. One of the concerns related to the use of viral vectors for gene therapy is the random...
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Further Reading (Genetics & Inherited Conditions)
Anderson, W. French. “Gene Therapy.” Scientific American 273, no. 3 (September, 1995): 124. Provides a good review of the promises and problems of gene therapy.
Brown, T. A. “Vectors for Gene Cloning: Plasmids and Bacteriophages.” In Gene Cloning and DNA Analysis: An Introduction. 5th ed. Malden, Mass.: Blackwell, 2006. Describes the principles and methods of gene cloning and DNA analysis for readers with little knowledge of these subjects. Contains more than 250 two-color illustrations.
Cohen, Philip. “Creators of the Forty-seventh Chromosome.” New Scientist 148, no. 2003 (November 11, 1995): 34. Describes the efforts to develop human artificial chromosomes.
Friedmann, Theodore. “Overcoming the Obstacles to Gene Therapy.” Scientific American 276, no. 6 (June, 1997): 96. Elaborates on the relative merits of different delivery systems for gene therapy.
Hassett, Daniel E., and J. Lindsay Whitton. “DNA Immunization.” Trends in Microbiology 4, no. 8 (August, 1996): 307-312. Reviews the process of DNA immunization and compares it to traditional vaccination strategies.
Jones, P., and D. Ramji. Vectors: Cloning Applications and Essential Techniques. New York: J. Wiley, 1998. A laboratory manual that allows quick and easy access to the key protocols required by those working with vectors.
Lodge, Julia, Peter A. Lund, and Steve...
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Web Sites of Interest (Genetics & Inherited Conditions)
Molecular Biology, Cloning Vectors. http://www.web-books.com/MoBio/Free/Ch9A4.htm. This page in an online book about molecular biology uses text and illustrations to describe cloning vectors.
Waksman Student Scholars, Genetic Engineering Vectors. http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/cloningvectors.html. The Waksman Student Scholars site was designed by professors at Rutgers University to be a resource about molecular biology for high school students and teachers. The site includes a page providing information about cloning vectors.
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