Figure 20.2 A preview of gene cloning and some uses of cloned genes
Figure 20.2 A preview of gene cloning and some uses of cloned genes
Figure 20.9 Gel electrophoresis
Figure 20.10 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β -globin gene
Figure 20.17 Can the nucleus from a differentiated animal cell direct development of an organism?
Figure 20.18 Reproductive cloning of a mammal by nuclear transplantation For the Discovery Video Cloning, go to Animation and Video Files.
Figure 20.20 Working with stem cells
Figure 20.22 Gene therapy using a retroviral vector
Figure 20.24 STR analysis used to release an innocent man from prison For the Discovery Video DNA Forensics, go to Animation and Video Files.
Figure 20.25 Using the Ti plasmid to produce transgenic plants For the Cell Biology Video Pronuclear Injection, go to Animation and Video Files. For the Discovery Video Transgenics, go to Animation and Video Files.
Overview: The DNA Toolbox <ul><li>Sequencing of the human genome was completed by 2007 </li></ul><ul><li>DNA sequencing has depended on advances in technology, starting with making recombinant DNA </li></ul><ul><li>In recombinant DNA , nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule </li></ul>
<ul><li>Methods for making recombinant DNA are central to genetic engineering , the direct manipulation of genes for practical purposes </li></ul><ul><li>DNA technology has revolutionized biotechnology , the manipulation of organisms or their genetic components to make useful products </li></ul><ul><li>To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning </li></ul>
DNA Cloning and Its Applications: A Preview <ul><li>Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids </li></ul><ul><li>Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome </li></ul><ul><li>Cloned genes are useful for making copies of a particular gene and producing a protein product </li></ul>
<ul><li>Gene cloning involves using bacteria to make multiple copies of a gene </li></ul><ul><li>Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell </li></ul><ul><li>Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA </li></ul><ul><li>This results in the production of multiple copies of a single gene </li></ul>
Fig. 20-2a DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2
Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 3
Using Restriction Enzymes to Make Recombinant DNA <ul><li>Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites </li></ul><ul><li>A restriction enzyme usually makes many cuts, yielding restriction fragments </li></ul><ul><li>The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “ sticky ends ” that bond with complementary sticky ends of other fragments </li></ul><ul><li>DNA ligase is an enzyme that seals the bonds between restriction fragments </li></ul>
DNA technology allows us to study the sequence, expression, and function of a gene <ul><li>DNA cloning allows researchers to </li></ul><ul><ul><li>Compare genes and alleles between individuals </li></ul></ul><ul><ul><li>Locate gene expression in a body </li></ul></ul><ul><ul><li>Determine the role of a gene in an organism </li></ul></ul><ul><li>Several techniques are used to analyze the DNA of genes </li></ul>
Gel Electrophoresis and Southern Blotting <ul><li>One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis </li></ul><ul><li>This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size </li></ul><ul><li>A current is applied that causes charged molecules to move through the gel </li></ul><ul><li>Molecules are sorted into “bands” by their size </li></ul>
Fig. 20-9 Mixture of DNA mol- ecules of different sizes Power source Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE RESULTS 1 2 + + – –
<ul><li>In restriction fragment analysis , DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis </li></ul><ul><li>Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene </li></ul><ul><li>The procedure is also used to prepare pure samples of individual fragments </li></ul>
Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of -globin gene Normal -globin allele Sickle-cell mutant -globin allele Dde I Large fragment Large fragment 376 bp 201 bp 175 bp Dde I Dde I Dde I Dde I Dde I Dde I
Cloning Plants: Single-Cell Cultures <ul><li>Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell </li></ul><ul><li>One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism </li></ul><ul><li>A totipotent cell is one that can generate a complete new organism </li></ul>
Cloning Animals: Nuclear Transplantation <ul><li>In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell </li></ul><ul><li>Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg </li></ul><ul><li>However, the older the donor nucleus, the lower the percentage of normally developing tadpoles </li></ul>
Fig. 20-17 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage
Reproductive Cloning of Mammals <ul><li>In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell </li></ul><ul><li>Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus </li></ul>
Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2
<ul><li>Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs </li></ul><ul><li>In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth </li></ul><ul><li>CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent” </li></ul>
Stem Cells of Animals <ul><li>A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types </li></ul><ul><li>Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells ; these are able to differentiate into all cell types </li></ul><ul><li>The adult body also has stem cells, which replace nonreproducing specialized cells </li></ul><ul><li>The aim of stem cell research is to supply cells for the repair of damaged or diseased organs </li></ul>
Fig. 20-20 Cultured stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) Different culture conditions Different types of differentiated cells Blood cells Nerve cells Liver cells Cells generating all embryonic cell types Adult stem cells Cells generating some cell types Embryonic stem cells From bone marrow in this example
Medical Applications <ul><li>One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases </li></ul><ul><li>Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele </li></ul>
Human Gene Therapy <ul><li>Gene therapy is the alteration of an afflicted individual’s genes </li></ul><ul><li>Gene therapy holds great potential for treating disorders traceable to a single defective gene </li></ul><ul><li>Vectors are used for delivery of genes into specific types of cells, for example bone marrow </li></ul><ul><li>Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations </li></ul>
Fig. 20-22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4
Forensic Evidence and Genetic Profiles <ul><li>An individual’s unique DNA sequence, or genetic profile , can be obtained by analysis of tissue or body fluids </li></ul><ul><li>Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains </li></ul><ul><li>The Innocence Project </li></ul>
Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. (a) Semen on victim Earl Washington Source of sample Kenneth Tinsley STR marker 1 STR marker 2 STR marker 3 (b) 17, 19 16, 18 17, 19 13, 16 12, 12 14, 15 11, 12 13, 16 12, 12
Environmental Cleanup <ul><li>Genetic engineering can be used to modify the metabolism of microorganisms </li></ul><ul><li>Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials </li></ul><ul><li>Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels </li></ul>
Genetic Engineering in Plants <ul><li>Agricultural scientists have endowed a number of crop plants with genes for desirable traits </li></ul><ul><li>The Ti plasmid is the most commonly used vector for introducing new genes into plant cells </li></ul><ul><li>Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops </li></ul>
Fig. 20-25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS
<ul><li>Most public concern about possible hazards centers on genetically modified (GM) organisms used as food </li></ul><ul><li>Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives </li></ul>
Genetic Engineering of Microorganisms Many medicines & useful chemicals are now produced by genetically engineered bacteria. For instance, all insulin is synthesized by transgenic E. coli that have the genes to produce human insulin . This has greatly reduced the cost and made insulin available to all diabetics who need it. Before the 1980s, all insulin was derived from the pancreases of animals (insulin from pigs differs from human insulin by only one amino acid).
The leading artificial sweetener, aspartame , is made from two amino acids, aspartic acid and phenylalanine . Both amino acids are synthesized by bacteria, and the strain that produces the phenylalanine has been genetically engineered. Genetic Engineering of Microorganisms
Vitamin A deficiency - 1 million children under 5 die per year; many go blind . Modifications to improve the nutritional value of food. Iron deficiency - 2 billion affected; 100,000 maternal deaths /yr in Africa & Asia. Severe mental impairment in women & children. Genetic engineering of rice to contain vitamin A & increased levels of iron and zinc . “Golden rice” How important is this? Fig. 38.18 Genetic engineering of corn to reduce the level of omega-6 fatty acids . http://www.nysaes.cornell.edu/comm/gmo/corn.jpg
Some crop plants have been genetically engineered to extract limiting nutrients more efficiently from the soil. Other plants have been engineered to increase the bioavailability of their essential nutrients for digestion and uptake.
Genetic engineering of crop plants to increase the quantity & quality of food that is harvested and reaches the consumer. • Shift in emphasis from vegetative body to seed/fruit. The Green Revolution was driven in part by the introduction of genes for dwarfism into cereal grains like wheat. The same genes are now engineered into other crops such as basmati rice. http://www.lsuagcenter.com/en/crops_livestock/crops/WheatOats/Diseases/Wheat+Diseases.htm
- Salty soils (77 million hectares). Plants also remove the salt from the soil! http://www.environment.gov.au/soe/2001/publications/theme-reports/water/water02-1c.html • Decreased losses due to harsh environmental conditions :
- Extreme heat or cold http://cropwatch.unl.edu/photos/cwphoto/crop07-6wheat-3.jpg http://web1.msue.msu.edu/vanburen/frstapp.htm http://boxer.senate.gov/news/photos/events/2007/01/bigfreeze/16.html • Decreased losses due to harsh environmental conditions :
Luciferase engineered in tobacco There is interest in engineering plants to signal when they are water stressed or require more nutrients through the use of reporter genes (luciferase or one of the many fluorescent proteins that can be engineered to upregulate during a particular stress). Luciferase in tobacco Fig. 17.6
• Increased shelf life for fruits and vegetables. http://www.ces.ncsu.edu/depts/cs/biotech/pics/c2h.jpg Normal Flavor saver
Other genetic improvements to the foods and beverages that humans and animals consume. To decrease the allergenicity of food . A transgenic soybean was recently engineered that does not produce a protein to which some individuals are allergic. Peanuts are currently a major target. Decaffeinated coffee - “naturally”. A Japanese group recently engineered coffee that does not synthesize caffeine. Several forage crops have been engineered to have reduced lignin content so that they are more easily digested (by sheep, cows, etc.). Efforts are also underway to engineer grains to release more carbo-hydrates, or more essential nutrients, for animal feed .
http://arabidopsis.info/students/dom/mainpage.html A number of species have been genetically engineered to more effectively clean up pollution or to detoxify specific toxins. One example is the transfer of genes from bacteria into plants, resulting in plants that can take up highly toxic methylmercury and convert it to elemental mercury. Phytoremediation