Textbook Assignment: Chapter 15, pages 450-460 (includes end of chapter problems for all of chapter 15); Chapter 16, Pages 478-483. There is also a boxed article on pages 12-13 that should be reread at this time. Note that we are jumping around out of textbook sequence to bring related material together. The next two lectures will return to the earlier parts of Chapter 16
Major concepts
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a clever technique for the amplification of a small sample of DNA to an amount large enough to work with. It is widely used, both in basic research and in forensic studies. In extreme cases, one can start with the haploid genome from a single sperm and amplify selected sequences sufficiently for detailed analysis. Some knowledge of the DNA sequence on each side of the segment that is to be studied is needed to perform PCR.
Primers: In order to perform PCR, it is necessary to have a pair of short single-stranded oligonucleotides that are capable of priming synthesis of the sequence that is to be amplified and its reverse complement. The sequences of the two oligonucleotide primers are identical to the 5'-ends of the sense and antisense strands that will be amplified. Thus, they will hybridize to the 3'-ends of the complementary strands that serve as templates for the double stranded DNA that is to be amplified. In each case, the primer must anneal with the 3'- portion of the template strand so that it can prime DNA synthesis that proceeds 5' to 3' across the region to be amplified. The synthesis itself is carried out by a heat-stable DNA polymerase derived from bacteria that live in near-boiling water either in hot springs or in deep-ocean vents.
Temperature cycling: The original DNA, the primers, the heat-resistant DNA polymerase, and a supply of dNTPs are mixed together and placed in a temperature cycling device. The temperature is raised enough to separate the strands of the parent DNA. The temperature is then lowered enough so that the primers, which are present in excess, will anneal onto the separated DNA strands. The DNA polymerase then synthesizes a new strand that is complementary to each of the parent strands. The temperature is then raised to separate the newly synthesized strands from the original. When the temperature is lowered in the next cycle, all four strands are primed and serve as templates for synthesis of new complementary strands. In the next cycle, eight strands are primed and serve as templates, etc. By the time 30 cycles have been completed, the theoretical multiplication factor would be 230, which is roughly equivalent to 109. Even larger multiplications are possible, provided that the system does not run out of dNTPs or primers and the enzyme does not lose its activity.
Uniformity of PCR product: Each cycle is programmed with enough time at the lower temperature for synthesis to extend beyond the positions of the two primers. However, after a few cycles, most of the template strands will end at the position of the primer that initiated their synthesis. Figure 15.29 in our current textbook is somewhat misleading in that it does not show the original template DNA (red) extending beyond the primers. However, it correctly shows that all of the new strands (blue and green) start from the primers (black) and stop when their growing 3'-ends reach the ends of the primers in the previously amplified template strands. After the first few cycles of PCR, the lengths of nearly all of the strands will be the exactly the distance between the outer ends of the two primers. This high level of product uniformity makes it possible to detect the product as a sharp band in a Southern blot. If desired, the PCR product can now be cloned into a vector and further amplified. It can also be used as a sample for procedures such as DNA fingerprinting, or it can be sequenced to detect any point mutations in the region that was amplified.
Danger of contamination: Because PCR is capable of amplifying a single DNA strand, great care must be taken to avoid even the slightest contamination of the starting template or the reagents with any foreign DNA. A tiny flake of dried skin or a drop of sweat is enough to produce an amplification product that may mask or confuse the desired result. Such considerations are particularly important when PCR is used in the analysis of forensic evidence.
Eukaryotic vectors: Many eukaryotic genes cannot be expressed effectively in prokaryotic cells because of the need for removal of introns or for posttranslational modification of the protein products. Vectors that are designed for use in eukaryotic cells do not differ greatly in principle from those used in prokaryotic cells. They must be capable of entering their host cells and replicating autonomously in those cells. They must also have a site into which a foreign coding sequence can be introduced without seriously disrupting their ability to function. In many cases, the vectors are based on viruses or plasmids that occur naturally in the host cells. Frequently a bacterial origin of replication (and a means for circularization if needed) are added so that the vectors can be propagated as plasmids in bacterial cells prior to having foreign DNA cloned into them. Some so-called shuttle vectors have been designed specifically to be capable of replication both in prokaryotic and eukaryotic hosts, so that cloned gene products can be transferred readily back and forth between the two.
Yeast artificial chromosomes: Yeast artificial chromosomes were described briefly in lecture 28 (textbook page 439). They have proven to be particularly useful vectors for large pieces of DNA, with the capacity for cloning up to one megabase in a single vector. This is enough for all but the very largest eukaryotic genes, and it often allows clusters of closely linked smaller genes to be included in a single clone. The essential elements for construction of a YAC are a piece of yeast centromere, a yeast origin of replication (autonomously replicating sequence = ARS), telomeres at the ends, a suitable insertion site for cloning, and a system for selection of YACs containing cloned sequences. The use of overlapping YACs allows long segments of chromosomal DNA to be ordered precisely for sequencing. A library of overlapping clones that covers a complete region of a chromosome is called a contig (see pages 475-477, which will be covered in lecture 32).
Animal virus vectors: Modified animal viruses can be used to introduce cloned DNA sequences into animal cells. These include retroviruses that have single-stranded RNA viral genomes, but undergo reverse transcription to form double stranded DNA, which becomes incorporated into the host genome after infection (p. 453-454). There are many examples of naturally occurring transfection involving retroviruses, such as the Rous sarcoma virus, which transfects cells with an oncogene (cancer-causing gene) known as src. The src oncogene is a cellular growth control gene (c-src) that has at some time in the past become incorporated into the virus and modified such that it causes loss of normal growth control. Modified retroviruses are widely used as vectors. A variety of other types of viruses can also be used. One example is a modified form of SV40, which is a double-stranded DNA virus that in its unmodified form can cause malignant transformation of some types of cultured cells. Highly engineered vectors that incorporate elements from more than one type of virus have also been developed (Figure 15.32).
Transfection vs. transformation: In eukaryotic cells, the word transformation is normally used only to describe a process that causes the cell to become malignant (cancerous). To avoid confusion, the introduction of a foreign gene that does not cause malignancy is referred to as transfection. In prokaryotic cells, the term transformation is commonly used to describe a genetic change caused by uptake of foreign DNA, as described in lecture 14 (pages 160-162 of textbook).
Plant vectors: The Ti (tumor-inducing) plasmid from the soil bacterium Agrobacterium tumefaciens is widely used as a plant vector. In nature, this plasmid integrates a portion of its DNA into the plant cell genome and causes a tumorous growth on the plant. Attenuated plasmids that are no longer capable of tumor formation can be used to introduce foreign genes into single plant cells, which can then be grown into complete plants by appropriate hormonal manipulation. Our textbook describes this procedure in Figure 16.20, and illustrates a tobacco leaf expressing hepatitis B antigen in Figure 16.21.
Methods of transfection: Many different methods have been developed to "force" the uptake of transfecting vectors. In some cases, direct uptake of foreign DNA can be encouraged by treatment of the cells with calcium phosphate. Another technique that sometimes works is direct microinjection of DNA into the nuclei of individual cells (Figures 15.31 and 16.19). Foreign DNA that has been encapsulated into an artificial lipid membrane called a liposome is often incorporated quite effectively by fusion of the liposome with the cellular plasma membrane. The biolistic (biological balistic) process coats microprojectiles with DNA and literally shoots them into cells or organelles. The process of electroporation uses brief exposure to high voltage electricity to punch small holes in the plasma membrane that heal after foreign DNA has entered. All of these various procedures are rather widely used, and new ones are being developed regularly.
Reporter genes: After a gene has been cloned, it is often desirable to determine both how its expression is regulated and where it is expressed in a multicellular organism. This is frequently done by attaching the promoter for the gene in question to a so-called reporter gene. To study regulation of expression, the reporter construct is then transfected into a cell type that normally expresses the gene in question. By manipulating the promoter sequence, and also by manipulating that transcription factors are expressed in the host cell, investigators can then use the reporter gene to determine how their manipulations have altered expression. Common reporters include an easily assayed enzyme called chloramphenicol acetyltransferase (CAT), beta-galactosidase (lac Z), and green fluorescent protein. To study patterns of expression, the reporter construct is transfected into an intact organism (usually by formation of a transgenic animal or plant, as described below). Whenever and wherever the gene of interest is expressed, the reporter gene will also be expressed since it is under the control of a similar promoter. When beta-galactosidase is used as the reporter, blue staining with X-gal can be used to identify sites of expression. Similarly, fluorescence can be used to determine sites of expression of green fluorescent protein, which is rapidly becoming a highly popular reporter gene. Blue/white selection of colonies of bacteria carrying recombinant vectors is another example of the use of a reporter gene (Lecture 27 and the illustration in the boxed article about reporter genes on pages 454-455).
Gene knockouts: Gene knockout experiments in mice and other species are currently providing major insight into the functions of many different genes. Embryonal stem cells are cultured cells that have retained totipotent developmental potential and can become any part of the animal, including the germ cells, when injected into a sufficiently early embryo. Techniques have been developed for permanent inactivation of a specific gene in cultured embryonal stem cells of mice. When injected into early embryos, some of these cells become germ cells, such that some of the gametes that are procuded when the animals mature contain only the inactivated gene. These gametes produce heterozygous progeny in crosses with wild-type mice. Crosses of two of the heterozygous progeny can produce homozygous embryos that do not have any functional copies of the knockout gene. This procedure is outlined on pages 469-470 and in figure 16.11 of our textbook.
Developmental roles of specific genes: Studies on knockout mice are providing valuable information about the functions of specific genes, including their roles during early embryonic development. They are also revealing a surprising degree of redundancy in some control systems. For example, the c-src gene can become a cancer-causing oncogene when it is excessively activated by loss of a regulatory function, and it also plays an important role in normal cellular multiplication when properly regulated. However, total inactivation of this gene in knockout mice had only minor effects of development to term and live birth of relatively normal animals. Ultimately it was shown that two other genes with similar functions could adequately replace nearly all of the functions of the c-src gene. When all three of these genes were knocked out, cellular multiplication could not occur normally, causing early embryonic lethality. Many similar cases are known with other gene families.
Transgenic animals: Methods have been developed that allow cloned genes to be incorporated into germ line cells of a number of animal species. Most of the pioneering work has been done with mice, but transgenic pigs, sheep, goats and cows have also been developed. The first step is to transfect the gene in question into an embryonal stem cell (ES cell), which is then injected into an early embryo. The resulting chimeric animals are then bred with wild type mice, creating heterozygotes for the transgene. Mating of two of the heterozygotes can then generate 1/4 of the offspring that are homozygous for the transgene. The process for producing transgenic mice that express a foreign gene is virtually identical to the procedure for gene knockout (figure 16.11), expect that a functional gene of foreign origin is added to the ES cell, in most cases with all of its normal genes left unaltered.
Growth hormone: Transgenic animal research is a rapidly expanding area of research that is already producing a lot of useful data. In one early study, a transgenic mouse with multiple copies of a rat growth hormone gene grew much larger than a normal mouse (see page 272 of our textbook). However, attempts to produce larger pigs with human growth hormone were less successful.
Use of milk gene promoters: One very interesting application of transgenic animals has been to link genes coding for desired proteins to promoters for milk protein genes in transgenic animals. This results in the desired protein being produced in the milk of the animal, sometimes in quite large amounts. These techniques are beginning to be used, particularly in sheep and goats, to generate rather large amounts of foreign proteins in the milk. One of the advantages is that correct glycosylation of the proteins occurs in these systems. The textbook describes the production of alpha-1-antitrypsin in amounts up to 35 grams per liter in milk (pages 479-480).
MCDB transgenic mouse facility: A facility for research on transgenic mice has been constructed in the new part of the MCDB building and studies on a variety of transgenic and knockout mice are currently being undertaken by several members of the MCDB faculty.
Transgenic plants Transgenic approaches are beginning to be widely used with plants also. Thus, for example, crop plants have been developed that have transgenic resistance to glycophosphate, the active herbicidal ingredient of the weed killer, Roundup. Glycophosphate inhibits an enzyme needed for synthesis of amino acids, without which plants cannot grow. A bacterial gene that confers resistance to glycophosphate has been transfected into crop plants, making them resistant. A Ti plasmid vector was used for transfection of cultured tobacco cells, which were then used to regenerate plants (Figure 16.20. These plants will survive selectively, while weeds will not, in a field sprayed with glycophosphate.:w!
Commercial production: A variety of transgenic plants are in varying stages of experimentation or commercial production. For example, frost-resistant and salt-tolerant plants that can be grown in otherwise hostile environments are being pursued vigorously. In some cases, unexpected new problems are encountered with genetically engineered crops. One such case is the Flavr Savr tomato, which remains firm for a much longer time after ripening because the activity of the enzyme polygalacturonase has been greatly reduced (see boxed article on pages 12-13 of textbook). This allows vine ripened tomatoes to remain in good condition for a long enough time to be shipped to stores and displayed without rapidly going bad. However, the methods that were used for packing and shipping tomatoes that were harvested green and ripened artificially later proved to be too rough for ripe tomatoes. This resulted in an unexpected market failure of the first major commercial crop, and a requirement for developing new methods to handle and ship the ripe tomatoes.
Environmental concerns: In many cases, there are also concerns that the transgenes could get into other types of plants. This could, for example generate herbicide resistant weeds. In addition, many of the selective procedures involved in generating recombinant vectors involve the use of antibiotic resistance. There is concern that plasmids carrying antibiotic resistance could be released into the environment causing various types of pathogens to become antibiotic resistant.
Biotechnology: The use of expression vectors to produce commercial quantitites of proteins that cannot readily be obtained in adequate amounts from natural sources has captured the imagination of investors and resulted in rapid growth of biotechnology companies worldwide. Although a number of companies now have products on the market and a few like Genentech have become quite successful, most are still in early growth and development stages.
Cost effectiveness: One of the limitations of this type of manufacture is that production of proteins through recombinant DNA technology is relatively expensive. The only proteins that are reasonable candidates for commercial production in this manner are those that are needed in relatively small amounts and can be sold at a price high enough to be profitable. Examples would include hormones and other types of regulatory proteins that are used clinically in relatively small amounts, as well as a variety of types of proteins that are needed in small amounts for basic research in molecular biology, such as cellular growth factors.
Post-translational modification: Another limitation is that many proteins must undergo various types of post-translational modification before they are biologically active. This often includes the addition of complex polysaccharides in a process known as glycosylation. Eukaryotic proteins produced in bacterial expression vector systems are not adequately glycosylated, thus limiting the use of prokaryotic systems to those proteins that do not require glycosylation to function. A variety of eukaryotic expression vector systems have been developed to overcome this problem, including use of yeast, cultured insect cells, and cultured mammalian cells to generate the desired products. In addition, transgenic dairy animals are also beginning to be used to produce large amounts of desired proteins in their milk, as described above.
Hormones: Examples of pharmaceutical products that are now being produced commercially by recombinant DNA methods include human insulin and human growth hormone. The use of expression systems allows synthesis of human forms of these hormones, thus avoiding any possibility of allergic reactions caused by hormones from animal sources, which tend to have slightly different amino acid sequences. Another advantage is avoiding possible transmission of disease from use of human blood products or cadaver material as a source. Prior to the availablity of recvombinant DNA products, there were known cases of transmission of AIDS virus in human factor VIII preparations and Creutzfeld-Jacob disease (caused by prions) in human growth hormone preparations. Hormone-like growth factors used for serum-free cell culture are also mostly produced through use of recombinant DNA technology.
Vaccines: Another area of major interest is the possible use of proteins generated from cloned genes as antigens for the production of vaccines that carry no risk of accidental transmission of the disease. Our textbook describes a licensed project in which a surface protein from the hepatitis B virus in being produced in yeast cells in commercial quantities (pages 480-481). There is also a description of the experimental production of the same antigen in tobacco plants. There is also the interesting possibility of producing edible vaccines in transgenic plants (see boxed article on pages 12-13).
Treatment of human genetic diseases Most human genetic diseases are caused by mutations that cause an enzyme or some other type of protein to become non-functional. In theory, production of the missing protein from an expression vector that would function in the patient's cells could cure such diseases. In practice, the procedure is not that simple, largely because most proteins must be expressed selectively in specific tissues and because the amounts that are produced must be carefully controlled. Nevertheless, there have been a few successful treatments. The textbook briefly describes successful treatment of a severe combined immunodeficiency caused by absence of adenosine deaminase activity (pages 471-473 and figure 16.12). A recent followup report stated that the two original patients in this project had sustained a long-term alleviation of the symptoms resulting from insertion of a functional adenosine deaminase gene into some of their white blood cells and infusion of the cells back into their bodies. Similar possible treatments for a variety of other inherited diseases are being studied extensively.