* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Chapter 12 Genetic Engineering and the Molecules of Life
Gel electrophoresis of nucleic acids wikipedia , lookup
Community fingerprinting wikipedia , lookup
Metalloprotein wikipedia , lookup
Genomic library wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Gene expression wikipedia , lookup
Proteolysis wikipedia , lookup
Two-hybrid screening wikipedia , lookup
DNA supercoil wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular cloning wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Genetic engineering wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genetic code wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Point mutation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Chapter 12 Genetic Engineering and the Molecules of Life How close to “designer babies” are we? The first draft of the human genome was completed in 2000 What have we learned from this? What are stem cells? What is recombinant DNA? The Chemistry of Heredity Deoxyribonucleic Acid (DNA) – the polymeric molecule that conveys genetic information in all species Chromosomes – in humans, there are 46 doublestranded DNA molecules, which contain all of an individual’s genetic information. These 46 chromosomes exist as 23 pairs, one set from each parent Human genome – the approximately 30,000 genes on the 46 chromosomes that code for all the proteins that convey one or more hereditary traits Gene – a section of DNA that codes for a particular protein The Chemistry of Heredity What are we made of? • All genetic information is stored in the nucleus of the millions of cells in the body. • Each nucleus contains chromosomes, 46 compact structures of intertwined molecules of DNA, and about 30,000 genes, components that convey one or more hereditary traits. • DNA is a special template written in a molecular code on a tightly coiled thread that carries all genetic information. 12.1 What makes up DNA? DNA is made of fundamental chemical units, repeated over and over. Each unit is composed of three parts: nitrogen-containing bases, the sugar deoxyribose, and phosphate groups. Adenine (A), Guanine (G), Cytosine (C), and Thymine (T) are the bases. 12.1 Nucleotides A combination of a base, phosphate group, and a deoxyribose sugar is a nucleotide. This nucleotide is an adenosine phosphate. Any of the four bases can be used to form a nucleotide. A covalent bond exists between the phosphate group and the sugar. Another covalent bond is present between the ring nitrogen of the base and a ring carbon of the sugar. 12.1 What does a segment of DNA look like? A typical DNA molecule consists of thousands of nucleotides covalently bonded in a long chain. The phosphate groups are responsible for linking each nucleotide. A phosphate group of one nucleotide reacts with an –OH group present on the deoxyribose ring of another nucleotide, forming and eliminating a H2O molecule. This –OH group reacts with the phosphate group of another nucleotide 12.1 Chargaff’s Rules Erwin Chargaff’s research showed that for all humans, the percentage of adenine in DNA is almost identical to the percentage of thymine. Similarly, the percentages of guanine and cytosine are almost equal. From this, Chargaff concluded that the bases always come in pairs; adenine is always associated with thymine and guanine is always associated with cytosine. Thus, Chargaff’s rule states: %A = %T and %G = %C 12.1 Hydrogen bond – a weak bond-like interaction that exists between a nitrogen or oxygen atom and a hydrogen atom directly bonded to a nitrogen or oxygen atom O H O R C H R R N H :N .. H H Nucleotide – combination of a base, a deoxyribose ring, and a phosphate group The Double Helix of DNA X-Ray Diffraction pattern of a hydrated DNA molecule taken in 1952. Rosalind Franklin- her data was used by Watson and Crick (below) This technique uses the fact that a molecule’s electrons diffract X-Rays at particular angles and the resulting pattern, like the one above, can be used to solve the structure of a crystal. 12.2 The Double Helix of DNA Using Rosalind Franklin’s X-ray diffraction data, Watson and Crick proposed a molecular model for DNA. This model had a double strand of repeating nucleotides. Complementary base pairing (AT, CG) is held in place by hydrogen bonds (shown in red). The nature of the base pairing required that the two strands be coiled in the shape of a double helix. 12.2 DNA Replication The process by which copies of DNA are made is called replication. The original DNA double helix partially unwinds and the two complementary portions separate. Each of the strands serves as a template for the synthesis of a complementary strand. The result is two complete and identical DNA molecules. 12.2 Ribonucleic acid – the polymeric molecule consisting of phosphate, the sugar ribose, and the four bases cytosine (C), adenine (A), guanine (G), and uracil (U) Messenger RNA (mRNA) – the single-stranded RNA molecule that transcribes the genetic information of a particular gene from the DNA double helix Transfer RNA (tRNA) – the small RNA molecule that carries a particular amino acid when the genetic information in mRNA is translated into the correct amino acid sequence of a particular protein. There is a different tRNA for each of the 20 common amino acids. Cracking the Chemical Code The 3 billion base pairs in each human cell provide the blueprint for producing a human being. The specific sequence of base pairing is important in conveying the mechanism of how genetic information is expressed. The expression is seen through proteins. Through directing the synthesis of proteins, DNA can control the characteristics of an individual, including inherited illnesses. 12.3 Amino acid – the individual building blocks of proteins. There are 20 common amino acids. H R O C H2N OH Protein – a polymer of amino acids with a particular function. Proteins can be enzymes, hormones, or have other biological functions. H R1 H2N O O H H N R2 H N H R3 O OH Proteins are made of amino acids. The general formula for an amino acid includes four groups attached to a carbon atom: (1) a carboxylic acid group, -COOH; (2) an amine group, -NH2; (3) a hydrogen atom, -H; and (4) a side chain designated as R: There are 20 naturally occurring amino acids that make up proteins They differ from one another by the different R groups 12.3 Two amino acids can link together via a peptide bond: The two molecules join, expelling a molecule of water Peptide bond The process may repeat itself over and over, creating a peptide chain. Once incorporated into the peptide chain, the amino acids are known as amino acid residues. 12.3 Codons: How are they relevant in genetic expression? The order of bases in DNA determines the order of amino acids in a protein. Because there are 20 amino acids present in the proteins, the DNA code must contain 20 code “words”; each word represents a different amino acid. The genetic code is written in groupings of three DNA bases, called codons. The diagram shows possible codons, determined according to the base sequence of the nucleic acid strand. The expression of the genetic information is then seen through the specific proteins assigned. 12.3 DNA transcription and RNA translation The primary structure of a protein is its linear sequence of amino acids and the location of any disulfide (-S-S-) bridges. N-terminal carboxyl terminal The sequence is characterized by the amino terminal or "N-terminal" (NH3+) at one end; and the carboxyl terminal or "C-terminal" (COO-) at the other. Tertiary structure of the enzyme, chymotrypsin 12.4 Protein Structure Primary structure – the sequence of amino acids in a protein from the first amino acid to the last Secondary structure – the intermediate level of organization that shows helical structure and chain linkages through disulfide (–S–S– ) bonds Tertiary structure – the overall shape or conformation of the protein Most proteins contain one or more stretches of amino acids that give rise to a characteristic three dimensional structure. The most common of these are the alpha helix and the beta conformation. The telephone cord illustrates the nature of the secondary structure of the protein. Tertiary structure refers to the three-dimensional structure of the entire polypeptide. Like this tangled-up phone cord. 12.4 Active Site – the region of the enzyme where the catalytic reaction takes place Substrate – the molecule or molecules whose reaction is catalyzed by the enzyme Sickle-cell anemia is a hereditary disease, which illustrates how small changes in a protein’s primary structure can have a profoundly deleterious effect on the protein’s function. When an individual with sickle-cell anemia experiences a low oxygen concentration in the blood (e.g. during strenuous exercise), some of the red blood cells convert into a rigid, sickle or crescent-shaped form. Because these cells have lost their normal deformability, they clog tiny capillaries and cannot pass through tiny openings in the spleen and other organs. The property of sickling is caused by two mutations in the DNA sequence of the gene coding for the oxygen-transport protein, hemoglobin. Two amino acids that should be glutamic acid are replaced with valine instead. This substitution causes hemoglobin to convert to the sickled form at low oxygen concentrations. The function of a protein is dependent on its shape or threedimensional structure. Small changes in the primary structure can have dramatic effects on its properties. Sickle cell anemia is an example of a condition that develops when red blood cells take on distorted shapes due to an error in the amino acid sequence. Because these cells lose their normal shape, they cannot pass through tiny openings in the spleen and other organs. Some of the sickled cells are destroyed and anemia results. Other sickled cells can clog organs so badly that the blood supply to them is reduced. 12.4 The Human Genome Project is the effort to map all the genes in the human organism. On June 26, 2000, scientists announced that a rough draft of the project to decode the genetic makeup of humans had been completed. The goal, to determine the sequence of all 3 billion base pairs in the entire genome, was completed for the approximately 30,000 genes found on the 46 human chromosomes. This information might one day help to diagnose and cure diseases, understand human development, and trace our evolutionary roots. This was a unique collaboration between government, private sector, and a philanthropic organization. 12.5 Human Genome Project – the determination of the sequence of all 3 billion base pairs in the 46 human chromosomes, including that of the approximately 30,000 genes. This project was completed in 2000. Scientists are now trying to determine the function of all the proteins encoded by the 30,000 genes. Should we be concerned that employers or insurance companies will use genetic information to discriminate against people who have or have a hereditary predisposition to certain diseases? Could this kind of information be used to try and create a race of “super” humans? Recombinant DNA Recombinant DNA is used to produce human insulin for diabetics. Insulin is a small protein (51 amino acids) that helps the body metabolize glucose (blood sugar). Plasmid – a ring of DNA that bacteria have Vector – a plasmid with a foreign gene inserted into it Clone – a collection of cells or molecules identical to an original cell or molecule Uses of Recombinant DNA Recombinant DNA is used to produce vaccines against viruses and bacteria by inserting DNA encoding for a viral or bacterial protein into a vector. The protein is expressed and then isolated and purified. This protein is then used to in a vaccine to stimulate the immune system to produce antibodies against it. A subsequent infection by the virus or bacteria leads to a rapid response by the immune system. Transgenic plants (and animals) can be created with some desirable property. Desirable properties for agricultural crops include resistance to certain pests and diseases, synthesis of specific nutrients, and resistance to particular herbicides. The latter property means that a minimal amount of herbicide can be applied per acre to clear weeds and improve crop yield. A representation of genetic engineering 12.6 The mythical creature chimera represents a combination of a lion, a goat, and a serpent. Recombinant DNA is sometimes referred to as a chimera. 12.6 Current world population (approaching 7 billion) owes much to Norman Borlaug (1914-2009; Distinguished Professor of International Agriculture at Texas A&M; winner of the 1970 Nobel Peace Prize). Borlaug is credited with saving hundreds of millions of lives by developing advanced crop breeding and agricultural practices for use in countries suffering from drought-induced famine. For example, Mexico, which imported 60% of its wheat in the 1940’s, was able to become selfsufficient by the mid-1950’s despite an ever increasing population. The green revolution of the 1950’s and 1960’s allowed the world food supply to keep pace with explosive population growth. Countries with undernourished populations The United Nations’ Food & Agriculture Organization (FAO) estimate that by 2030, it will be necessary to increase the current grain supply by 30% to feed a projected global population of 8.3 billion. A combination of rapidly increasing global population and climate change will severely challenge the world’s farmers to meet this goal. A further complication is that as per capita income climbs in China and some other middle income countries, demand for food (particularly meat) increases faster than population. Meat production means diverting some grains into animal feed. Agricultural research is focusing on developing genetically engineered drought-tolerant and disease-resistant crops to meet the challenge of increasing the global food supply. Genetic engineering allows genes from other species to be inserted into important food crops. It is a step beyond simply crossbreeding different varieties of the same plant species to develop new strains with desired characteristics. Genetically-Engineered Agriculture Transgenic Plants Virus resistant transgenic rice Frankenfood? Controversial: protestors at the 2005 WTO meeting in China 12.7 In Europe, there is widespread public apprehension towards genetically modified (GM) crops. One poll found that over 80% of Europeans view GM foods as “bad”. Even in the US, a majority (55%) disapproved of GM food products. New EU regulations require labeling and traceability of all food and animal feed containing more than 0.5% of GM ingredients. Some polls indicate that Americans would like labeling, but it has not yet become a major issue. Americans have historically placed a considerably greater degree of trust in the regulatory oversight of the US Dept. of Agriculture than Europeans do in their counterpart agencies. In 1998, the US exported $63 million worth of corn to the EU, but the exports decreased to $12.5 million in 2002 At the end of 2002, EU ministers agreed to new labeling controls for GM foods, which will have to carry a special harmless DNA sequence (a DNA bar code) identifying the origin of the crops, making is easier to spot contaminated crops and withdraw them from the food chain should problems arise. US Agricultural Department officials argue that since the US does not require labeling, Europe should not require labeling either. They claim mandatory labeling is a trade barrier since it could imply there is something wrong with GM food. Is the official US complaint to the World Trade Organization (WTO) regarding the EU ban justified? The complaint was also filed by Argentina, Canada, Egypt, Australia, New Zealand, Mexico, Chile, Colombia, El Salvador, Honduras, Peru, and Uruguay. Are individual consumers’ fears of GM foods justified? American farmers lost market share in certain countries after changing to GM crops because of skeptical consumers. Some famine-threatened African countries (e.g. Zambia, Zimbabwe, and Mozambique) have refused to accept US aid because it contains GM food. Recombinant DNA used to restore sight to children with congenital blindness A research team at the Univ. of Pennsylvania School of Medicine created a vector (a genetically engineered virus) to carry a normal version of a gene called RPE65, that is mutated in one form of Leber’s congenital amaurosis (LCA), a genetic disease that progressively damages the retinas leaving many patients totally blind in their twenties or thirties. Animal studies with mice and dogs had shown that visual improvement was age-dependent, so the research team hypothesized that younger patients would receive the greatest benefit. A clinical trial with five children and seven adults ranging in age from 8 to 44, received injections of therapeutic genes into their retinas. As expected, the greatest improvement occurred in the children, all of whom are now able to navigate a low-light obstacle course. Before they received the gene therapy, the patients had great difficulty avoiding barriers, especially in dim light. Not all the adults performed better on the obstacle course and those who did, showed more modest improvements than did the children. The clinical benefits have persisted for nearly two years after the first injections with therapeutic genes were given. Although none of the patients attained normal eyesight, six of the twelve test subjects improved enough that they are no longer classified as legally blind. Gene therapies for other retinal diseases, such as age-related macular degeneration may also be developed. These results are based on nearly twenty years of research and animal studies with mice and dogs. Is it justified? Cloning Mammals and Humans In 1996, Dolly the sheep was born – the first cloned mammal. Dolly was created by a technique called nuclear transfer. The nucleus (contains the chromosomes) from an adult cell was placed in a donor egg from another sheep whose nucleus had been removed. The nucleus and donor egg were fused with an electrical jolt. The DNA then initiated the growth of the embryo, which was then implanted into a surrogate sheep’s uterus. Since then, several other mammalian species have been successfully cloned. Dolly is an example of “reproductive cloning”, in which an embryo is transferred to a gestational carrier in the hopes that a pregnancy will result and be carried full term. Cloning Humans and Mammals 12.8 Cloning Humans and Mammals Dolly, the cloned sheep Snuppy, the cloned dog, next to his “father” 12.8 “Therapeutic cloning” refers to harvesting stem cells from 3- to 5-day-old embryos to establish stem cell lines. Scientists hope to induce these stem cells to differentiate into various specialized cells. In 2004, a team of scientists led by Woo Suk Hwang and Shin Yong Moon of Seoul National University reported that they had successfully cloned human cells to generate embryonic stem cells. In 2006, Hwang admitted the data had been fabricated and resigned from his university position. Would blastocysts created in this manner be extensions of the people whose DNA was used to create them or would they be separate, unique beings in the same way that identical twins are unique, even though they share the same genetic blueprint? Results from Stem Cell Research Scientists at the Burnham Institute for Medical Research in La Jolla, CA, have programmed embryonic stem cells into becoming nerve cells when transplanted into the brains of mice. None of the mice formed tumors, which have been a major setback in previous attempts at stem cell transplantation. This research is a first step toward developing new treatments for stroke, Alzheimer’s, and other neurological conditions. The Food & Drug Administration (FDA) approved a phase I clinical trial for the transplantation of a human embryonic stem cell-derived cell population into spinal cord-injured individuals on January 23, 2009. Eight to ten paraplegics who had had their injuries no longer than two weeks before the trial begins, will be selected since the neural stem cells must be injected before scar tissue forms. These first trials are mainly to test for the safety of the procedures. Based on earlier results with mice, researchers say the restoration of myelin sheaths (insulation around nerve cells) and an increase in mobility is probable. The injections are not expected to fully restore mobility. In November 2010, the first patient, a recent paraplegic, was injected with two million embryonic stem cells in the injured spinal cord region with the goal of regenerating spinal cord tissue. The cells had been induced to become specialized nerve cells. The embryonic stem cells came from a leftover embryo from a fertility treatment, which would have been otherwise discarded. Embryonic stem cells are valued by researchers for their ability to be transformed into any type of cell. There are some restrictions tied to federally funded research involving embryonic stem cell lines. The company developing this treatment has spent ~$175 million thus far with no federal funding. Animal model studies shown movement in previously paralyzed rodents, but the results in humans are not expected to be that dramatic. Human embryonic stem cells could be used as models for human genetic diseases. The relative inaccessibility of human tissue is an obstacle to research in these areas. This approach could be very valuable in studying cystic fibrosis or fragile-X syndrome or other genetic diseases where no reliable animal model exists. Embryos with a genetic disease could be identified by prenatal genetic diagnosis (PGD) and used to establish a stem cell line featuring the genetic disorder. The National Institutes of Health (NIH) announced the approval of thirteen new human embryonic stem cell lines for NIH funding on Dec. 2. 2009. Where do we go from here? Is saving a human life worth the cost of a potential human life? 12.8 What We Should Know from Ch. 10 and 12 Be able to write structural formulas and lineangle drawings. Be able to draw isomers. Recognize functional groups. Recognize enantiomers and chiral carbons. Understand the structure of DNA and how it is translated into a protein sequence. Understand how the base pairs are held together by hydrogen bonds. Understand what recombinant DNA is and how it works. Be able to give examples of recombinant DNA technology. Understand the polymerase chain reaction and be able to give examples of this method. Understand cloning via nuclear transfer. RNA Codon Table Ala (Alanine) Leu (Leucine) Arg (Arginine) Lys (Lysine) Asn (Asparagine) Met (Methionine) Asp (Aspartic acid) Phe (Phenylalanine) Cys (Cysteine) Pro (Proline) Gln (Glutamine) Ser (Serine) Glu (Glutamic acid) Thr (Threonine) Gly (Glycine) Trp (Tryptophan) His (Histadine) Tyr (Tyrosine) Ile (Isoleucine) Val (Valine) START STOP GCU, GCC, GCA, GCG UUA, UUG, CUU, CUC, CUA, CUG CGU, CGC, CGA, CGG, AGA, AGG AAA, AAG AAU, AAC AUG GAU, GAC UUU, UUC UGU, UGC CCU, CCC, CCA, CC G CAA, CAG UCU, UCC, UCA, UC G, AGU, AGC GAA, GAG ACU, ACC, ACA, AC G GGU, GGC, GGA, GGG UGG CAU, CAC UAU, UAC AUU, AUC, AUA GUU, GUC, GUA, GUG AUG UAA, UGA, UAG AUG serves as the codon fo r the amino acid methionine and also as the “start” site on messenger RNA (mRNA). The first AUG codon on mRNA is where translation into protein beg ins. DNA makes up the chromosomes (23 pairs in humans) in cells’ nuclei. There are four bases A, C , G and T . A and T form base pairs as do G and C. RNA use s the base uracil (U) in place of thymine (T) as well as A, C and G. When a gene in a chromosome is transcribed, the DNA strands are pulled apart and a complementary RNA strand is formed from the DNA strand containing the gene. The DNA double helix then reforms and the mRNA travels from the cell’s nucleus to the cytosol. The mRNA is then translated into protein. Below is shown a small section of a DNA strand. What would the base sequence o f the complementary strand be after one round of DNA replication? TACAGA CCACATAAAACCGAA ATGTCTGGTGTATTTTGGCTT What would the base sequence o f the mRNA be after transcription of this section of DNA? Remember RNA uses U in place of T. AUGUCUGGUGUAUUUUGGCUU What would be the amino acid sequence of the protein formed from translation of this section of mRNA? You may use the three letter abbreviations for the amino acids. Met-Ser-Gly-Val-Phe-Trp-Leu How does chang ing the original DNA sequence to TACAGACCACATAAAACAGA A chang e the amino acid sequence of the protein that is produced? Met-Ser-Gly-Val-Phe-Cys-Leu Draw the hyd rogen bond s between the AT base pair. H O H3C H N H H Thymine N O H N N N dR N N H Adenine dR Circle the chiral carbons in the molecules shown below. Chiral carbons have four different substituents. O CH3 CH3 O H3C H O O H3CO Na