The Living World Fifth Edition George B. Johnson Jonathan B. Losos Chapter 14 Gene Technology Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

14.1 A Scientific Revolution genetic engineering involves moving genes from one organism to another  this process is having a major impact on medicine and agriculture

Figure 14.1 Examples of genetic engineering.

14.2 Restriction Enzymes in any genetic engineering experiment, the gene of interest must first be isolated  restriction endonucleases are special enzymes that bind to specific short sequences of DNA and make a cut these short sequences are typical 4 to 6 nucleotides long these sequences are symmetrical in that the DNA double helix has the identical sequence running in opposite directions

14.2 Restriction Enzymes the cut made by restriction enzymes is also unusual  the cut is not made in the center of the sequence but to one side this creates a break with short single strands of DNA dangling from each end these overhangs are called “sticky ends” the same sticky ends are always produced from cuts with the same restriction enzymes

14.2 Restriction Enzymes sticky ends make possible recombination of fragments generated by cut restriction enzymes  fragments from the same DNA source could pair up and be resealed by DNA ligase  fragments from different sources of DNA cut by the same restriction enzymes could also be resealed together

Figure 14.2 How restriction enzymes produce DNA fragments with sticky ends.

14.2 Restriction Enzymes where do restriction enzymes come from?  some bacteria have the ability to prevent infection by bacterial viruses these bacteria use restriction enzymes to cut up the foreign (viral) DNA hundreds of different restriction enzymes have been discovered –each kind always cuts in one kind of sequence and always makes a cut at the same place

14.3 The Four Stages of a Genetic Engineering Experiment three “ingredients” are necessary for genetic engineering 1.a source of DNA that contains the gene you want to transfer 2.a restriction enzyme to cut the DNA 3.a vehicle to carry the source DNA into the host cell

14.3 The Four Stages of a Genetic Engineering Experiment  DNA library is a collection of DNA fragments representing all of the DNA from an organism the source DNA used for genetic engineering is often obtained from a DNA library restriction enzymes are used to cut the fragment containing the gene of interest  vector is the term for the vehicle that can carry the gene of interest common vectors include both bacteria and viruses

14.3 The Four Stages of a Genetic Engineering Experiment all genetic transfer experiments share four distinct stages 1.cleaving DNA 2.producing recombinant DNA 3.cloning 4.screening

14.3 The Four Stages of a Genetic Engineering Experiment during the cleaving DNA stage, a restriction enzyme is used to cut the source DNA into fragments  many different-sized fragments will be generated  only some fragments will contain the gene of interest  the fragments can be separated from each other, based on size, by a process called electrophoresis

14.3 The Four Stages of a Genetic Engineering Experiment during electrophoresis, fragments of DNA migrate through a gel in response to an electrical current  DNA is negatively charged and moves towards the + end of the gel  smaller-sized fragments move faster than larger-sized ones  bands containing fragments of an appropriate size for the gene of interest can then be cut from the gel for use in later stages

Figure 14.4 Using restriction enzymes to cleave DNA and electrophoresis to separate the fragments.

14.3 The Four Stages of a Genetic Engineering Experiment the second stage of a genetic engineering experiment, the source DNA fragments are allowed to mix with vector DNA  the vector DNA has been cut with the identical restriction enzyme used on the source  the combination of vector and source DNA into one DNA molecule is an example of recombinant DNA

14.3 The Four Stages of a Genetic Engineering Experiment recombinant DNA is carried into a host cell using different types of vectors  plasmids are tiny circles of bacterial DNA that can replicate outside the main bacterial chromosome plasmids are introduced into a bacterial host cell by a process called transformation  viruses introduce recombinant DNA into a host cell by infection

14.3 The Four Stages of a Genetic Engineering Experiment as each host cell (normally a bacterium) reproduces, it forms clones that contain the DNA introduced by the vector the clones can be any of four types 1.a clone that has taken up a recombinant vector with the gene of interest 2.a clone that has taken up a recombinant vector without the gene of interest 3.a clone that has taken up a non-recombinant vector 4.a clone that did not take up any vector

14.3 The Four Stages of a Genetic Engineering Experiment clone library is a collection of separate clones containing fragments of source DNA cut from the entire genome of an organism genetic engineers need to identify which clone in a library contains the gene of interest  this is often the most challenging part of a genetic engineering experiment

14.3 The Four Stages of a Genetic Engineering Experiment genetic engineers first employ preliminary screens in order to eliminate  clones that do not contain any vectors  or clones that do not contain recombinant vectors

14.3 The Four Stages of a Genetic Engineering Experiment clones without any vector can be identified by a simple screening process  the vector used in the experiment usually contains a gene that confers antibiotic resistance  clones are grown in a medium that contains that antibiotic  only clones that have the resistant gene will be able to grow

14.3 The Four Stages of a Genetic Engineering Experiment screening can also eliminate clones that contain recombinant vectors, but without the gene of interest  vectors are used that, in addition to the antibiotic resistance gene, contain the gene lacZ’ the lacZ’ gene encodes for the enzyme β-galactosidase β-galactosidase breaks down the sugar X-gal –metabolism of X-gal results in the formation of a blue reaction product

14.3 The Four Stages of a Genetic Engineering Experiment using a restriction enzyme whose recognition sequence lies within the lacZ’ gene, the gene will be cleaved when recombinants are formed  clones with vectors with non-recombinant DNA will appear blue  clones with vectors with recombinant DNA will appear colorless

Figure 14.5 Using antibiotic resistance and X-gal as preliminary screens of restriction fragment clones.

14.3 The Four Stages of a Genetic Engineering Experiment once the preliminary screening has occurred, the remaining clones that contain the gene of interest must be identified  hybridization is a common method that uses a probe consisting of a complementary nucleic acid sequence to that of the gene of interest

14.3 The Four Stages of a Genetic Engineering Experiment hybridization typically involves  growing previously screened clones on agar  pressing a special filter onto the colonies to create a replica of some of the cells  treating the filter with a solution to denature the DNA into single strands  washing the filter in a solution of radioactively- labeled probe

14.3 The Four Stages of a Genetic Engineering Experiment hybridization continued…  the probe only hybridizes with DNA from colonies that contain the gene of interest  the filter with hybridized DNA is overlaid on photographic film  any radioactivity will be revealed as a black spot on the film  the position of the black spot can be compared to the original master plate of colonies  the colonies containing the genes are then identified

Figure 14.6 Using hybridization to identify the gene of interest.

Figure 14.3 How a genetic engineering experiment works. Review of the four stages of a genetic engineering experiment.

14.4 Working with DNA polymerase chain reaction (PCR) is a technique to generate multiple copies of DNA  short sequences of DNA, called primers, are first synthesized  the primers sequences occur on either side of the DNA region to be amplified  the PCR technique is a way to generate a lot of DNA of interest quickly, rather than rely on bacteria to produce copies

14.4 Working with DNA there are three steps involved in PCR 1.Denaturation 2.Primer annealing 3.Primer extension

14.4 Working with DNA the DNA target sequence, primers, polymerase, and a supply of all four nucleotides are first combined together in a solution  the solution is heated to about 95°C  the polymerase used is a special heat-resistant variety call Taq polymerase  the heat causes the DNA to denature into single strands

14.4 Working with DNA as the denatured solution cools, the primers bind to their complementary sequence the polymerase then uses the primer as a starting point to move down the strand and lengthen the entire DNA fragment  because both strands behave this way, by the end of the process there are 2 copies of the original fragment the PCR process is repeated many times resulting in the desired level of amplification of DNA

Figure 14.7 How the polymerase chain reaction works.

14.4 Working with DNA recall that, in eukaryotes, genes are encoded in both translated and non-translated segments the primary mRNA transcript produced by RNA polymerase contains both the coding regions (exons) and non-coding regions (introns) the introns must be cut out from the primary transcript before the mRNA can be translated the remaining exon fragments are stitched together to form the final RNA transcript, the processed mRNA, which is eventually translated in the cytoplasm

14.4 Working with DNA it is desirable for genetic engineers to transfer DNA that is ready to be translated  one reason is that bacteria, as prokaryotes, lack the enzymes to process mRNA  to obtain DNA without introns, genetic engineers isolate first the processed mRNA corresponding to a particular gene  the enzyme reverse transcriptase produces a DNA version of this mRNA, called complementary DNA (cDNA)

Figure 14.8 cDNA: producing an intron-free version of a eukaryotic gene for genetic engineering.

14.4 Working with DNA DNA fingerprinting is a revolutionary technique used in forensic evidence  the process uses probes on DNA samples that have been cut with the same restriction endonucleases  the probes are unique DNA sequences found in non-coding regions of human DNA that are highly variable among individuals  the chances that any two individuals, other than identical twins, having the same restriction pattern for these sequences varies from 1 in 800K to 1 in 1 billion, depending on the number of probes used

14.4 Working with DNA DNA fingerprints consist of autoradiographs depicting rows of parallel bars on X-ray film  each bar represents the position of a DNA restriction endonuclease fragment that complementarily binds to a probe

Figure 14.9 Two of the DNA profiles that led to conviction.

14.4 Working with DNA DNA fingerprints have been used in courts of law since 1987  while an individual DNA fingerprint is not 100% accurate, it is as reliable as traditional fingerprinting used in evidence when multiple probes are used  any source of DNA (i.e., a hair, a speck of blood, or semen) can be used in DNA fingerprinting to convict or to clear a suspect  however, laboratory analyses of DNA must be carried out properly to ensure accuracy

14.5 Genetic Engineering and Medicine much of the promise of genetic engineering lies in improving medicine  specifically, to aid in curing and preventing illnesses one such application comes in the form of “magic bullets”  many genetic disorders, such as diabetes, involve a failure to make a critical protein  genetic engineering can supply persons suffering from the disease with the protein they lack by engineering another organism, usually a bacterium, to do it  the donated protein is like a “magic bullet” to the disorder

Table 14.1 Genetically Engineered Drugs

Figure Genetically engineered human growth hormone.

14.5 Genetic Engineering and Medicine recombinant viruses are produced by genetic engineering against common viruses, such as herpes and hepatitis  genes encoding part of the protein coat of these viruses are spliced into a harmless fragment of the vaccinia (cowpox) virus  the vaccinia virus acts as a vector for introducing the viral genes and, after translation, proteins into a human  the human develops immunity against the viruses prior to exposure to the true form  the utilization of one vaccine to introduce genes from another virus is called a piggyback vaccine

Figure Constructing a subunit, or piggyback, vaccine for the herpes simplex virus.

14.6 Genetic Engineering of Farm Animals gene technology is having a major impact on the breeding and rearing of agricultural animals  use of genetically engineered hormones recombinant bovine somatotropin (BST) has been used in cows to increase milk production  use of transgenic animals these animals have been engineered to have specific desirable genes this precludes having to wait through several generations of selective breeding

Figure The production of bovine somatropin (BST) through genetic engineering.

14.7 Genetic Engineering of Crop Plants genes in crop plants have been successfully manipulated through genetic engineering in order to  make plants more resistant to diseases caused by insects  make plants resistant to herbicides  improve their nutritional balance and protein content  make plants hardier against environmental stress

14.7 Genetic Engineering of Crop Plants engineering crops to be resistant to insect pests can reduce pesticide use  Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein that is toxic when eaten by crop pests  inserting the gene producing Bt protein into crop plant chromosomes makes the crop pest resistant

14.7 Genetic Engineering of Crop Plants a big success in genetic engineering has been the creation of crop plants that are resistant to the herbicide glyphosphate  glyphosphate is used in orchards and agricultural fields to control weeds  plants cannot make aromatic amino acids needed for protein production  genetic engineers found a gene in bacteria that made aromatic amino acids in the presence of glyphosphate  this gene was then inserted into plant genomes using a DNA particle gun, or gene gun

Figure Shooting gene into cells.

Figure Genetically engineered herbicide resistance.

14.7 Genetic Engineering of Crop Plants genetically modified (GM) crops are commonly cultivated in the United States  some of the benefits of GM crops include increased soil preservation and reduced pesticide usage  these benefits translate into reduced prices for consumers because cultivating GM crops is cheaper and more efficient

14.7 Genetic Engineering of Crop Plants the real promise of plant genetic engineering is to prevent GM plants with desirable traits that directly benefit the consumer  “golden” rice is a solution from genetic engineering to nutrient deficiencies common to regions of the world where the major staple food is rice  rice eaters often are deficient in two major micronutrients, iron and vitamin A

14.7 Genetic Engineering of Crop Plants the development of transgenic, “golden,” rice involved the splicing of genes from different sources Figure Transgenic “golden” rice.

Table 14.2 Genetically Modified Crops

14.7 Genetic Engineering of Crop Plants are there any risks associated with genetic engineering of crops?  the potential for risks have alarmed many activists and scientists  two sets of risks need to be considered is eating genetically modified food dangerous? are GM crops actually harmful to the environment?

Inquiry & Analysis Does the gene conferring resistance to herbicide pass to other plants of this species, A. stolonifera? What general statement can be made about the effect of distance on the likelihood that the herbicide resistance gene will pass to another plant? p. 258, graph of frequency of GM Sentinel plants versus distance