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Chargaff’s Rules Erwin Chargaff became interested in DNA in 1944; he showed that for all human DNA, the percentages of guanine and cytosine are nearly identical, 19.1 and 18.4%, respectively, and the percentages of adenine and thymine, 31.0 and 31.5%, respectively, likewise are almost identical. From this remarkable observation, Erwin Chargaff concluded that the bases always come in pairs; adenine always associated with thymine and guanine always associated with cytosine. Earlier, scientists had held the view that DNA was composed of a large number of repeats of GACT and had explained the deviation of the percentages from 25% as an experimental error. Chargaff’s Rules In addition to his first rule: %G = %C and %A = %T Chargaff also postulated that different species have DNA of different composition, suggesting DNA as the carrier of genetic information. Pyrimidines Purines The Double Helix of DNA X-ray diffraction pattern of a hydrated DNA molecule taken in 1952. This technique is based on the fact that the electrons of a molecule diffract Xrays at particular angles; the resulting pattern (e.g., that above) can be used to solve the structure of a crystal. Rosalind Franklin – her X-ray data led Watson and Crick (below) to the double helix 12.3 The Double Helix of DNA Based on 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. 12.3 Structure of DNA Nucleic acids form a double helix: DNA is a double helix consisting of two strands of polynucleotides. There are three types of structural features: 1) phosphoric acid and deoxyribose form a condensation co-polymer; 2) within the polymer strand the bases are held together by pi-stacking; 3) two strands, running in opposite directions, are connected to each other through highly specific hydrogen-bonds between their organic bases. H Bonding Compounds like alcohols, ethers, and amines, which contain lone pairs and hydrogens on an electronegative atom (O and N), that is N–H and O–H functions, can form hydrogen bonds The O atoms of ketones also can form hydrogen bonds with a suitable H-donor Hydrogen Bonds Structure Alcohols form hydrogen bonds (cf. H2O) Alcohols are both H-bond donor and H-bond acceptor This type of association raises the boiling point considerably (unbelievably), e.g., H3C–CH3 –88° CH3OH 65° HDR-F-2012 DNA bases contain lone pairs on N and C=O functions as well as N–H groups; therefore they can form hydrogen bonds Pyrimidines Purines HDR-F-2012 Place cytosine to the right of guanine and rotate cytosine by -30° and guanine by 30° HDR-F-2012 Place thymine to the right of adenine and rotate thymine by -30° and adenine by 30° HDR-F-2012 HDR-F-2012 Component Bases of DNA Structure of DNA Nucleic acids form base pairs The individual polynucleotide strands are hydrogenbonded to each other through their organic bases; the strands run in opposite directions. Structure of DNA If a piece of DNA in one strand has the sequence –AGCTACGATC-, the complementary strand has the sequence –TCGATGCTAG- 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. A complete set of genetic information is packaged into chromosomes packed into the cell nucleus. 12.3 Replication of DNA DNA replicates by unwinding and assembling new complementary strands. The double helical nature of DNA suggested to Watson and Crick a mechanism for the replication of DNA during cell division. Each of the two strands in a parent DNA molecule serve as a template for the assembly of a new complementary strand in a new DNA molecule. This process occurs with an error frequency of less than 1 in 10 billion base pairs during DNA replication. The nearly 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 enacted through proteins. DNA directs the synthesis of proteins and, thereby, controls the characteristics of an individual, including inherited diseases. Let’s review Proteins 12.4 Proteins 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 There are 20 naturally occurring amino acids that make up proteins. They differ from one another by the different R groups 12.4 Proteins Two amino acids can link together via a peptide bond: amino acid residue The two molecules join, expelling a molecule of water. Peptide bond As the process repeats itself again and again, it creates a peptide chain. Once incorporated into the peptide chain, the amino acids are known as amino acid residues. 12.4 The Genetic Code The order of amino acids in a protein, the genetic code, is determined by the order of bases in DNA (Marshal Nirenberg, Nobel Prize, Phys. & Med. 1968). Because human protein contains 20 amino acids, the DNA code must contain 20 code “words”, each representing a specific amino acid. The four DNA bases can be combined to a sufficient number of three DNA base groups, called codons, to represent each amino group uniquely; this sequence of codons is the genetic code. The diagram shows three codons, GTA, AAA, and GGC, which for the incorporation of histidine, phenylalanine, and proline, respectively. Some amino acid residues can be produced by different codes AAA lysine CCC proline GGG glycine Protein Structure The primary structure of a protein is its linear sequence of amino acids, including the location of any disulfide (–S–S–) bridges. 12.5 Protein Structure The secondary structure of a protein is the folding pattern within a segment of the protein chain. 12.5 Protein Structure 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 12.5 Protein Function and Structure 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 of their irregular shape these cells 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. 12.5 Genetic Engineering • When a species is genetically engineered, the DNA in the cell is modified. • When the genes are changed, the proteins synthesized by the genes are modified. • When the cell grows and develops, a plant with new characteristics from the different DNA is generated. • Before genetic engineering, when humans selected for plants with certain characteristics or crossbred different strains, genes were manipulated. The genetic traits for modern corn were selected over time, starting with an early ancestor, teosinte (bottom). 12.6 Genetic Engineering 12.6 Genetic Engineering • Genetic engineering is transgenic – where an organism is created by the transfer of genes across species. • Genetic engineering can also be used to do the same thing as crossbreeding, just more efficiently and faster. Transgenic rice with virus-resistance 12.6 Reasons for Genetic Engineering • Make crops more resistant to disease, tolerant of stresses (salt, heat, or drought) • Develop soybeans that produce high yields of biofuel per acre • Use of enzymes to create new drugs • Develop vaccines that grow in edible products Developing strains of algae for new biofuels 12.7 Genetically-Engineered Agriculture Transgenic Plants Virus resistant transgenic rice Frankenfood? Greenpeace activists dumping papaya during a Bangkok protest. 12.8 Genetic Engineering and Sight 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. Genetic Engineering 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. Genetic Engineering 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. Genetic Engineering 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. 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 (containing 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. Cloning Mammals and Humans 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 Mammals and Humans Cloning Humans and Mammals Cloning Mammals and Humans Cloning Mammals and Humans Cloning Humans and Mammals Cloning Humans and Mammals Dolly, the cloned sheep Dolly, the cloned sheep Snuppy, the cloned dog, next to his “father Dolly, a cloned sheep, Snuppy, a next cloned dog, Snuppy, the cloned dog, to his “father” next to his “father” Therapeutic Cloning “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. Therapeutic Cloning 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? 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. Stem Cell Research The Food & Drug Administration (FDA) approved a phase I clinical trial for the transplantation of a human embryonic stem cellderived cell population into spinal cord-injured individuals on January 23, 2009. Eight to ten paraplegics who were injured less than two weeks before the trial begins, will be selected, because the neural stem cells must be injected before scar tissue forms. These first trials are mainly to test for the safety of the procedures. Stem Cell Research 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. Stem Cell Research 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 without federal funding. Animal model studies have shown movement in previously paralyzed rodents, but the results in humans are not expected to be that dramatic. Stem Cell Research 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. Stem Cell Research Human embryonic stem cells are grown typically on a mouse feeder layer; they thrive on it Stem Cell Research Stem Cell Research The Busch administration did not allow the National Institutes of Health (NIH) to provide any funding for introduction of new human embryonic stem cell lines. On Dec. 2, 2009, the NIH announced the approval of thirteen new human embryonic stem cell lines for NIH funding, still a strictly limited number. Genetic Engineering Where do we go from here? Is saving a human life worth the cost of a potential human life? 12.8