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Recombination Up CloseBIO. "Biotechnology in Perspective." Washington, D.C.: Biotechnology Industry Organization, 1990.Manipulating the GeneThe attractions of genetically-engineered products are clear. A wide range of materials for use in medicine, agriculture, and industry that are currently in short supply could be produced in much greater quantities if the genes regulating their production by higher plants and animal cells could be incorporated into the microbial genome. Growing bacteria in bioreactors under conditions that favor the synthesis of the material is relatively straightforward. Industries such as baking and brewing have a wealth of experience in just these areas. Why not simply grow the cells of the higher organisms that make the product in the first place? That is clearly a potential alternative solution, but in practice many such cells cannot readily be grown in vitro.By contrast, very effective methods are available for growing micro-organisms rapidly and at low cost. If we examine the case of insulin, for example, we find that cells of the human pancreas - the source material - cannot be grown easily in isolation, but genetic engineering can result in cells that will readily manufacture a precursor of insulin, just as our own bodies do, and then enzymatically remove a polypeptide to give recombinant human insulin. Although certain ground rules for genetic engineering have been established, after over a decade of intensive effort it is still the case that few industrial problems in this area can be solved by applying a standard formula. The method adopted in a given case will depend on the gene in question and the microbe which is to receive it. Nevertheless, four main stages can be recognized. The first of these involves obtaining the gene which codes for the product. This isolated gene must then be inserted into the microorganism, often a bacterium. In many ways these are the simplest stages to achieve. Conditions must be established under which the microbes will synthesize the required product in appropriate amounts. This may require particular ingenuity, and the same is true at the final stage in which the product is collected, isolated, and purified.
Isolation of GenesHuman cells contain thousands of genes, so the key initial step is to isolate the gene. Rather than search through the mass of DNA in the human genome (only a fraction of which has been sequenced to date), genetic engineers have turned to gene expression as a way to isolate specific genes. Using this method to isolate the insulin gene, for example, human pancreas cells are examined to find the messenger RNA molecules that have been copied from that gene as part of the process of protein synthesis. There will be other kinds of RNA as well as that coding for insulin, but already the scale of the problem is greatly reduced. Until recently, there was no way to connect the genetic information in mRNA back to DNA. It was then discovered that some viruses can do precisely this. This was something of a surprise, since it involved a reversal of the "central dogma" of molecular biology (DNA makes RNA makes protein).The enzyme that achieves this is known as reverse transcriptase. Viruses consist simply of a core of genetic material wrapped in a protein coat, and are much smaller than bacterial cells. They can reproduce themselves only when they invade cells. Some viruses employ RNA as their genetic material, and in the case of retroviruses, reverse transcriptase inside the virus is used to generate DNA. In this way, retroviruses convert their RNA 'genes' into a DNA form, and because of the universality of the genetic code, the host cell is deceived into replicating new viruses. Biotechnologists can adopt a similar strategy by mixing reverse transcriptase with human mRNAs in vitro, thereby producing the complementary or copy (cDNA) sequence for the required gene, e. g., insulin.
Plasmids Help to Transfer Genes between OrganismsThe insertion of genes is facilitated by bacterial plasmids (small circles of DNA), which are smaller than the bacterial chromosome. Some plasmids can pass readily from one cell to another, even when the cells are clearly from different species far apart on the evolutionary scale. By inserting the human cDNA gene into the plasmid ring, it can be readily introduced into its microbial host for a biotechnological application. Plasmids used in this way are known as vectors and certain viruses can perform the same role. The stitching in place of, say, a human gene, is achieved by a family of remarkable 'cut-and-paste' enzymes, the restriction endonucleases and ligating enzymes.
Gene CloningWith the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate themselves. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen replicas. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for, say, insulin) will in a relatively short time produce many millions of similar cells (clones) containing the same human gene.
Recombinant DNA TechnologyRecombinant DNA technology requires DNA extraction, purification, and fragmentation. This molecular dissection is achieved using specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene. This portion of the DNA is then coupled to a carrier molecule and the hybrid DNA is introduced into a chosen cell for reproduction and synthesis. Modern laboratories even have access to a DNA synthesizer (gene machine), which provides a desired DNA sequence. Recombinant DNA allows the genome to be manipulated much more readily than by classical breeding methods and can therefore circumvent incompatibility between species. Already human genes have been incorporated into bacteria, permitting large-scale synthesis of rare biochemicals, such as the hormone insulin and the natural anti-viral agent interferon.
Recombinant DNA Technology Produces Human ProductsFifteen or more years ago if you posed the question "which is the most difficult cellular component to analyze?," any biochemist would have undoubtedly answered "DNA." Its enormous size and its chemical monotony (only four repeated nucleotides) meant that its sequence could be approached only by indirect procedures of protein or RNA sequencing; genetic analysis was an alternative approach, but was time consuming and inefficient.When the same question is posed today, a very different answer is forthcoming. DNA is by now one of the simplest molecules in the cell to examine, and specific regions can be obtained in unlimited quantities. Sequencing of DNA can now be achieved at a rate of several hundred nucleotides per day.
Recombinant DNA TechniquesThat such a transformation has been achieved is a direct result of recombinant DNA technology. Such has been the progress in this field that this approach has superseded conventional methods for determining the amino acid sequence of proteins. It is this technology that is offering industry the possibility of large-scale production of protein hormones and vaccines at economically viable costs. Recombinant DNA technology is not a single technique - it is a combination of methods, some quite new, others borrowed from established microbial genetic procedures.Recombinant DNA techniques allow even the minor proteins in a cell to be studied. This is a major advance over conventional methods, which require several hundred grams of cells to purify even a major protein (i.e., one that makes up 1% or more of the total cell protein). From such source material, conventional procedures (chromatographic and electrophoretic methods) would yield perhaps negligible quantities of pure protein. Since many of the chemicals that would be of great commercial value are present in small quantities in cells, it is instructive to follow the sequence of steps that enable purification using recombinant DNA technology.
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