O

3. Generate a recombinant molecule

4. Introduce recombinant molecule into new host

Figure 9.2 The Steps of a Cloning Experiment

Figure 9.3 Cloning into a High-Copy-Number Plasmid When a gene is inserted into a high-copy-number plasmid, multiple copies of that gene will be present in a single cell, resulting in the synthesis of many more molecules of the encoded protein.

High-copy-number plasmid

Gene

Gene inserted into plasmid

High-copy-number plasmid

Gene

Gene inserted into plasmid

Figure 9.3 Cloning into a High-Copy-Number Plasmid When a gene is inserted into a high-copy-number plasmid, multiple copies of that gene will be present in a single cell, resulting in the synthesis of many more molecules of the encoded protein.

Multiple copies of the gene produced

Vaccines Vaccines protect against disease by harmlessly exposing a person's immune system to a killed or weakened form of the disease-causing agent, or to a part of the agent. Although a vaccine is generally composed of whole bacterial cells or viral particles, only specific proteins, or parts of the proteins, are actually necessary to induce protection, or immunize, against the disease. The genes coding for these proteins can be cloned in yeast or bacteria so that a large amount of the pure immunizing proteins can be produced. This type of vaccine is currently used to prevent hepatitis B in humans and foot-and-mouth disease of domestic animals. ■ vaccines, p. 421

Other Commercially Valuable Proteins One of the most widely used proteins made by genetically engineered organisms is chymosin, a proteolytic enzyme used in cheese production. It is a natural component of rennin, a preparation from the stomach of calves. Chymosin causes milk to coagulate and produces desirable changes in the characteristics of cheeses as they ripen. Using genetically engineered bacteria to produce chymosin is preferable to isolating rennin from calves because the microbial product is less expensive and more reliably available. Other proteins produced by genetically engineered microbes include various restriction enzymes and bovine somatotropin, a growth hormone used to increase milk production in dairy cows.

DNA Production

In many cases, a researcher is interested in obtaining readily available supplies of certain DNA fragments. By cloning a segment of DNA into a well-characterized bacterium such as E. coli, an easy source of that sequence is available for study and further manipulation. This has been used to further our understanding in a broad range of areas, from the impact of bacteria on human health to the role of environmental bacteria in various ecological processes.

DNA for Study Genetic engineering can be used to study genomic characteristics of some of the 99% of bacteria that have not been grown in culture. This was exemplified in recent research that led to the discovery of a mechanism of energy capture in marine bacteria which had previously been described only in certain members of the Archaea. Researchers isolated random DNA fragments from ocean samples and then cloned them into E. coli. Using some of the techniques that will be discussed later, the researchers were then able to determine the nucleotide sequences of various cloned bacterial DNA. One of these was found to encode a novel gene for bacterial rhodopsin, a light-sensitive pigment that can be used to harvest energy from sunlight. The pigment can capture the energy of sunlight and use it to emit protons, generating a proton motive force; this provides the bacterium with a mechanism for phototrophy that does not require chlorophyll. Further studies showed that variations of the bacterial rhodopsin gene are widespread among marine bacteria, suggesting it might be an important mechanism for energy accumulation in ocean environments. All of this was

9.1 Applications of Genetic Engineering 223

discovered without ever growing the marine microbes in culture! ■ proton motive force, p. 55

Human genes are often cloned into bacteria to make them easier to study. A human cell contains about 30,000 genes, whereas E. coli contains only 4,500 genes; thus, a human gene cloned into the bacterium on a high-copy-number plasmid represents a much higher percentage of the total DNA in the recipient cell than in the original cell. This makes it easier to isolate the DNA as well as the gene product. In addition, since bacteria are much smaller than human cells and grow considerably faster, many more cells can be quickly grown in the same volume of growth medium. Also, the conditions for growing bacteria are much simpler than for growing human cells.

DNA for Vaccines Researchers have found that when plasmid DNA is injected into a person's tissues, the encoded proteins are expressed for a short time. This gives rise to the possibility that certain vaccines may someday be administered by injecting DNA that encodes proteins from disease-causing organisms. DNA-based vaccines are still in the experimental stage, but promising results have been obtained in animal studies.

Researching Gene Function and Regulation

Gene function and regulation can more easily be studied in E. coli because systems for manipulating its DNA have been developed. For example, regulation of gene expression can be studied by creating a gene fusion, joining the gene being studied and a reporter gene (figure 9.4). The reporter gene

Green Fluorescent Protein gene (reporter gene)

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