Application of Genetic Engineering (With Diagram)

Some of the most important application of genetic engineering are as follows:

For many years, the gene has been thought of as a unit of structure, its definition based on the results of recombination experiments and the analysis of mu­tant cells.

According to this older view, a gene was the smallest unit of inheritance that was not sub-divisible by recombination and the smallest unit capable of in­dependent mutation.

Today, we recognize that a nucleotide pair is the smallest structural unit that fits the recombination and mutation definitions, although it certainly is not reasonable to call a nucleotide pair a gene. Therefore, a functional definition is more ap­propriate, and the gene is better defined as a unit of inheritance that encodes one polypeptide chain or one RNA molecule.

The complete analysis of the nucleotide sequence of a gene is a substantial undertaking. Nevertheless, the primary structures of a number of genes are now known, including most of the genes encoding the vari­ous globin chains of human hemoglobin. Even small chromosomes, such as the chromo­some of the virus φX174, have been fully sequenced.

This chromosome, which consists of 5,375 nucleotides, contains nine genes whose starting and ending loca­tions are known. One of the more remarkable findings of recent years is that the nine genes do not occupy nine separate segments of the chromosome.

Instead, two of the genes are located entirely within the coding sequences of two other genes. The chromosomes of eukaryotic cells contain noncoding nucleotide sequences between and also within coding sequences. Genetic engineering may be defined as the deliber­ate manipulation or alteration of the genetic informa­tion of an organism. Among the various forms that ge­netic engineering takes is the removal of genes from one organism followed by their insertion into the genome of another.

Examples of the benefits to be de­rived through such undertakings include:

(1) The devel­opment of new strains of crop plants that are more re­sistant to adverse environmental conditions or to pests and

(2) The modification of bacteria so that they produce certain proteins (e.g., human insulin, inter­feron, etc.) and other substances of value to human­kind.

The major stages involved in genetically engineer­ing an organism for purposes such as those described above are outlined in Figure 21-19, which depicts the alteration of the genome of a bacterium so that it can produce human proteins. In the first stage of the pro­cess, human cells are disrupted and the chromosomes containing the gene to be transferred are isolated and cut into small pieces using enzymes called restriction endonucleases.

Bacterial cells are then disrupted to obtain “cloning vehicles”; the latter dually take the form of small, circular bacterial chromosomes called plasmids. Opened plasmids and pieces of the human chromosome are mixed in the presence of ligases, with the result that the human genes become spliced into the plasmid forming a chimeric or recombinant DNA molecule.

Once formed, the recombinant DNA can be trans­ferred back into the same strain of bacteria from which the original plasmids were obtained. The bacte­rial host cells are then cultured, producing a clone consisting of millions of identical cells.

During the growth of the clone, each bacterium produces a new copy of the recombinant DNA in addition to its normal chromosomal material. The result is a vast population of bacteria containing specific human genes. The ex­pression of the human genes (as well as the host’s na­tive genes) results in the production of large quanti­ties of human proteins. We will now examine each of these stages in greater detail.

1. Preparing Donor DNA:

Depending on the genes that are to be cloned, a tissue source is selected. Because nearly all cells contain a full complement of the organism’s genes, literally any tissue could be selected as a source of any gene. How­ever, the tissue that is usually chosen is the one that serves as the major source of the gene product in that organism.

Accordingly, insulin genes are isolated from pancreas tissue and interferon genes are iso­lated from white blood cells. Although hemoglobin is produced by maturing red blood cells, the genetic en­gineering studies conducted with hemoglobin have employed liver tissue as a source of the globin genes. Either fresh or previously frozen tissue may be used as a source of donor DNA.

The tissues are vigorously homogenized to free the nuclear material and a detergent solution containing specific enzymes is added to free the DNA from at­tached proteins and inactivate nucleases that might otherwise begin to degrade the DNA.

After the homogenate is deproteinated using phenol, the DNA is isolated by isopycnic density gradient centrifugation. The DNA, which forms a single band in the density gradient, is then withdrawn. The iso­lated DNA contains the genes that are to be cloned but it also contains many thousands of other genes, whose cloning may be of no interest.

2. Preparing the Cloning Vehicle:

The Plasmid Method Cloning vehicles may take the form of small circular bacterial chromosomes called plasmids (as described in the example presented ear­lier) or phage DNA. Although plasmids account for only a small percentage of the total DNA of a bacte­rium, they contain a number of vital genes including those that confer antibiotic resistance to the cell (e.g., the gene for penicillinase). The presence of these genes is essential in the subsequent search for cells containing the donor DNA. Because of their small size, plasmids are readily separated from the main chromosomes of disrupted bacteria by centrifugation.

Isolated donor DNA is treated with one or more of the restriction endonucleases (Table 21-3) that cleave the DNA into specific fragments. Restriction endonucleases possess site specificity, cleaving the DNA wherever certain nucleotide sequences, called restriction sites, occur. Some restriction endonu­cleases make “staggered cuts” in the DNA so that the exposed cut ends have a number of unpaired bases (Fig. 21-20).

Because the nucleotide sequences that are recognized by the restriction endonucleases are palindromes (i.e., base sequences that read the same in the forward direction along one chain and the back­ward direction along the other), the single-stranded ends are complementary.

When treated with the same restriction enzymes, plasmids also produce molecules having complemen­tary, single-stranded ends. The donor DNA fragments and the plasmid DNA are then mixed in the presence of DNA ligase under conditions that permit the DNAs to be spliced together.

The recombinant DNA that results has variable composition depending on which donor DNA fragments combined with the plasmid DNA. (Remember, the donor DNA consists of a mix­ture of DNA fragments only some of which contain the genes to be cloned!) Recombinant DNA can be taken up by bacterial cells that are made transiently permeable to such macromolecules. Not all of the bacteria mixed with re­combinant DNA will incorporate the modified plas­mids but those that do can be identified if the bacteria used are antibiotic-sensitive.

This is because incorpo­ration of the recombinant DNA simultaneously con­fers antibiotic resistance to the bacteria. Therefore, only these bacteria will proliferate when cultured in the presence of antibiotic. (Those that do not take up the recombinant DNA, and therefore do not simulta­neously acquire the antibiotic genes, die). Once incor­porated by the bacteria, the recombinant DNA is rep­licated along with the remaining cellular DNA during growth.

Identification of the particular bacterial cells that contain the specific genes to be cloned demands specific assay procedures. The procedure (which is some­times referred to as the “shotgun” approach) may be illustrated using as an example the gene that encodes an enzyme involved in the synthesis of the amino acid histidine (i.e., the his gene).

The various forms of re­combinant DNA produced by the procedure described above would be introduced into a mutant variety of E. coli cells that are unable to synthesize histidine. After transfer of the recombinant DNA to the cells, they would be cultured using a medium that lacked histi­dine.

In this way, the cells that had not incorporated recombinant DNA containing his gene would be unable to produce the necessary enzyme for histidine synthesis and would not survive in the histidine-free medium. Only the cells acquiring his gene would survive; thus, the appropriate cells are selected and the gene is cloned as these cells grow and divide. A different selection method is used for each gene to be cloned.

The Bacteriophage Method:

A bacteriophage (or phage) is a virus that infects bacteria. The initial stage of the bacteriophage method involves the isola­tion of a small number of phage from a bacterial cul­ture. Bacteria can be cultured on the surface of a shal­low dish containing nutrient medium (e.g., an agar plate), and on such a medium the bacteria grow until they cover the dish’s surface. Because phage-infected bacteria eventually lyse, releasing new viruses that begin another cycle of infection, clear areas contain­ing lysed cells and freed bacteriophage are soon seen on the surface of the nutrient medium; these areas are called plaques.

Phage particles removed from a plaque are mixed with a large culture of bacteria whose infection quickly yields much more phage. Phage and bacteria are separated by centrifugation and the phage is treated with enzymes that remove the protein coat; the phage DNA is then purified by density gradient centrifugation. Isolated phage DNA and donor DNA are then treated in a manner similar to that described earlier for plasmids with the result that the donor DNA is integrated into the phage DNA, thereby form­ing a cloning vehicle.

3. Gene Cloning Methods Using Reverse Transcription:

In some instances it is possible to isolate from donor cells the mRNA for specific proteins. For example, globin chains mRNA can be isolated from reticulo­cytes, albumin mRNA can be isolated from chicken oviduct cells, proinsulin mRNA can be isolated from pancreas cells, and interferon mRNA can be isolated from white blood cells. The isolated mRNAs can then be used as template for the synthesis of complemen­tary DNA strands (called cDNA) using reverse transcriptase, an enzyme encoded in the genome of certain RNA viruses (Fig. 21-21).

Single-stranded DNA produced by reverse tran­scription can be used as template for the synthesis of a complementary DNA strand. The resulting double- stranded DNA can then be inserted into an opened plasmid or phage DNA and cloned in bacteria as de­scribed above. Because the recombinant DNA used in this approach is specific (not random recombinant DNA molecules as are produced in the shotgun method), the necessity of using special E. coli mutants for cell selection is precluded.

The incorporation of DNA molecules produced by reverse transcription into plasmids requires the use of enzymes called ter­minal transferases instead of restriction endonucleases, because reverse transcription does not pro­duce DNA molecules that have offset complementary nucleotide chains at their ends (i.e., the enzymes do not produce staggered cuts). Terminal transferases add single-stranded tails to the 3′ ends of the DNA strands.

4. Genetic Engineering of Mammalian Cells:

Genes from a number of different eukaryotic cells have been excised and then cloned in bacterial cells us­ing the restriction endonuclease and reverse tran­scription techniques. The bacteria containing the re­combinant DNA are thus able to produce a number of eukaryotic proteins.

The hormone proinsulin was the first such eukaryotic protein whose production was achieved in cultures of bacteria transformed using re­combinant DNA. The gene encoding this protein was synthesized using reverse transcriptase and then transferred to the bacterial cells. Human interferons (proteins that act as antiviral agents) are also being made by applying the recombinant DNA technology to bacterial cells. In this instance, interferon mRNA is isolated from human white blood cells.

Interferon:

The production of human interferon using recombinant DNA methods has attracted a great deal of public and scientific attention in recent years be­cause interferon has also been found to attenuate the growth of transformed human cells. In a number of specific instances, cancerous tumors have been shown to go into remission after patients have been treated with interferon.

Interferon was discovered in the 1950s by A. Isaacs and J. Lindenmann, who were studying a phenomenon called “virus interference” in which cells treated with one type of virus resisted in­fection by other types. Isaacs and Lindenmann found that virus interference was not due to one virus block­ing the growth of another rather, the infection by one virus was shown to induce a cell to produce and secrete a substance that conferred viral resistance to other cells. Because the substance interfered with the ca­pacity for viruses to produce an infection, they called the substance “interferon.”

Human interferons are now known to be a class of glycoproteins produced by a number of different cells including lymphocytes and fibroblasts. Synthesis of the interferons by these cells is triggered by the derepression of the interferon genes of the cell’s genome.

Once released from the cells, the in­terferons react with receptors in the plasma mem­branes of other cells and in some yet uncertain way block viral infection of these cells. Because interferon is produced and secreted in very small quantities, in­tensive study of the action of interferon has been ham­pered for decades as a result of the difficulty of isolat­ing sufficient quantities of this material.

Because certain cancers are believed to be trans­mitted by viruses, and interferons have been shown to diminish the growth of tumors, it is tempting to sug­gest that interferon administration could prevent or cure virus-mediated cancers. However, it has not been shown that the effects of interferon on tumor growth result directly from antiviral activity.

Rather, the in­terferon may act indirectly by stimulating other body defense mechanisms. Application of the recombinant DNA technology for large-scale interferon production should soon provide the scientific community with enough of this most interesting material to allow de­finitive tests of its clinical value. At the present we are seeing only the “tip of the iceberg” regarding the ben­efits that may be derived from the use of bacteria for cloning human genes and producing human proteins.

The genes of eukaryotic cells can also be cloned in other eukaryotic cells, but this is a much more difficult task. This is because the membranes of mammalian cells are not so readily made permeable to recombi­nant DNA. One method for transferring genes to mammalian cells that has had some success is the use of a whole virus as a vector for such transfer.

The gene to be transferred is first linked to the viral DNA and the recombinant DNA-containing virus is used to infect the animal cells. The gene encoding the beta chain of rabbit hemoglobin has been successfully in­corporated into the genome of the Simian virus (SV- 40).

When these viruses are then allowed to infect mammalian cells, the beta globin chain gene is trans­ferred along with the native viral genes and rabbit beta globin chains are subsequently synthesized in these cells. Some success has also been achieved using mechanical microinjection procedures to transfer re­combinant DNA-containing plasmids directly into the nuclei of mammalian cells.