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Essentials of The Living World First Edition GEORGE B. JOHNSON 10 The New Biology PowerPoint® Lectures prepared by Johnny El-Rady Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.1 Genomics The full complement of genetic information of an organism is its genome Genomics is a new field of biology concerned with the sequencing and study of genomes The first genome to be sequenced was that of the virus FX174 Frederick Sanger in 1977 obtained the sequence of this 5,375 genome The advent of automatic DNA sequencing machines has facilitated the sequencing of larger genomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Sequencing DNA DNA is first amplified The DNA fragments are then mixed with Primers + DNA polymerase A supply of the four nucleotides A smaller supply of chemically-tagged nucleotides that terminate replication The DNA is denatured into single-strands allowing DNA replication to proceed The addition of a chemically-tagged nucleotide to the growing chain halts DNA replication Thus the mixture will contain double-stranded DNA of various lengths Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Sequencing DNA The DNA mixture is separated by fragment size using gel electrophoresis Examination of the fragments from shortest to longest reveals the nucleotide sequence of the DNA Linking the sequence of the various DNA fragments will yield the sequence of the entire genome The scanning and analysis of the gel is greatly facilitated by the use of computers Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.1 How to sequence DNA One color corresponds to each nucleotide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display TABLE 10.1 EUKARYOTIC GENOMES Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display TABLE 10.1 EUKARYOTIC GENOMES Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.2 The Human Genome The sequence of the entire human genome was reported on June 26, 2000 It consists of 3.2 billion base pairs If the human genome were a book It would be 500,000 pages long It would take about 60 years to read at the rate of 8 hours a day, every day, at five bases a second Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Geography of the Genome The number of genes in humans is only about 25,000-30,000 However, there are about 4 times more mRNA molecules The genes are divided into exons and introns Thus alternative mRNA splicing can generate much more mRNA than there are genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Geography of the Genome Genes are not distributed evenly throughout the human genome Chromosome 19 is small yet packed with genes Chromosomes 4 and 8 are large yet have few genes On most chromosomes, clusters rich in genes are scattered between vast stretches of “barren” DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA That Codes for Proteins The human genome contains four different classes of protein-encoding genes 1. Single-copy genes Most genes fit in this class Silent copies, inactivated by mutation, are called pseudogenes 2. Segmental duplications Blocks of similar genes in the same order are found throughout the human genome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA That Codes for Proteins The human genome contains four different classes of protein-encoding genes 3. Multigene families Groups of related but distinctly different genes that often occur together in cluster Arose from a single ancestral sequence 4. Tandem clusters DNA sequences repeated thousands of times in tandem array The rRNA genes, for example Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Noncoding DNA Only 1-1.5% of the human genome is coding DNA There are four major types of noncoding DNA 1. Noncoding DNA within genes Together introns make up about 24% of the human genome 2. Structural DNA ~ 20% of the genome is constitutive heterochromatin Located near centromeres and telomeres Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Noncoding DNA Only 1-1.5% of the human genome is coding DNA There are four major types of noncoding DNA 3. Repeated sequences Simple sequence repeats (SSRs) Two- or three-nucleotide sequences repeated thousands of times Constitute ~ 3% of the human genome Duplicated Sequences Repeated sequences, other than SSRs Constitute ~ 7% of the human genome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Noncoding DNA Only 1-1.5% of the human genome is coding DNA There are four major types of noncoding DNA 4. Transposable elements Make up ~ 45% of the human genome They include LINEs Long interspersed elements ~ 6,000 DNA bases long Active transposons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Noncoding DNA Only 1-1.5% of the human genome is coding DNA There are four major types of noncoding DNA 4. Transposable elements Make up ~45% of the human genome They include Alu sequences ~ 300 DNA bases long Have no transposition machinery Reside within, and transpose with, LINEs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.3 A Scientific Revolution Genetic engineering is the process of moving genes from one organism to another Having a major impact on agriculture & medicine Fig. 10.3 Producing insulin Curing disease Increasing yields Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Restriction Enzymes Restriction enzymes bind to specific short sequences (usually 4- to 6- bases long) on the DNA The nucleotide sequence on both DNA strands is identical when read in opposite directions GAATTC CTTAAG Most restriction enzymes cut the DNA in a staggered fashion This generates “sticky” ends These ends can pair with any other DNA fragment generated by the same enzyme The pairing is aided by DNA ligase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Stages of a Genetic Engineering Experiment All gene transfer experiments share four distinct stages 1. 2. 3. 4. Cleaving DNA Producing recombinant DNA Cloning Screening Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.4 Genetic Engineering and Medicine Genetic engineering has been used in many medical applications 1. Production of proteins to treat illnesses 2. Creation of vaccines to combat infections 3. Replacement of defective genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Making “Magic Bullets” In diabetes, the body is unable to control levels of sugar in the blood because of lack of insulin Diabetes can be cured if the body is supplied with insulin The gene encoding insulin has been introduced into bacteria Fig. 10.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Making “Magic Bullets” Other genetically engineered drugs include Has only one extra gene: HGH Anticoagulants Used to treat heart attack patients Factor VIII Used to treat hemophilia Human growth hormone (HGH) Used to treat dwarfism Fig. 10.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Piggyback Vaccines Genetic engineering has also been used to create subunit vaccines against viruses A gene encoding a viral protein is put into the DNA of a harmless virus and injected into the body The viral protein will elicit antibody production in the animal A novel kind of vaccine was introduced in 1995 The DNA vaccine uses plasmid vectors It elicits a cellular immune response, rather than antibody production Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.7 Constructing a piggyback vaccine for the herpes simplex virus Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.5 Genetic Engineering and Agriculture Successful manipulation of the genes of crop plants has improved the quality of these plants Pest resistance Leads to a reduction in the use of pesticides Bt, a protein produced by soil bacteria, is harmful to pests but not to humans The Bt gene has been introduced into tomato plants, among others Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.5 Genetic Engineering and Agriculture Glyphosateresistant plants Herbicide resistance Crop plants have been created that are resistant to glyphosate Fig. 10.9 Glyphosatesensitive plants Petunias Herbicide resistance offers two main advantages 1. Lowers the cost of producing crops 2. Reduces plowing and conserves the top soil Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.5 Genetic Engineering and Agriculture More Nutritious Crops Worldwide, two major deficiencies are iron and vitamin A Deficiencies are especially severe in developing countries where the major staple food is rice Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden” rice to solve this problem Fig. 10.10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.10 Transgenic “golden” rice Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops The promise of genetic engineering is very much in evidence However, it has generated considerable controversy and protest Are genetic engineers “playing God” by tampering with the genetic material? Two sets of risks need to be considered 1. Are GM foods safe to eat? 2. Are GM foods safe for the environment? Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops 1. Are GM foods safe to eat? The herbicide glyphosate blocks the synthesis of aromatic amino acids Humans don’t make any aromatic amino acids, so glyphosate doesn’t hurt us However, gene modifications that render plants resistant to glyphosate may introduce novel proteins Moreover, introduced proteins may cause allergies in humans Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops 2. Are GM foods safe for the environment? Three legitimate concerns are raised 1. Harm to other organisms Will other organisms be harmed unintentionally? 2. Resistance Will pests become resistant to pesticides? 3. Gene flow What if introduced genes will pass from GM crops to their wild or weedy relatives? Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.6 Reproductive Cloning In 1938, the German embryologist Hans Spemann proposed what he called a “fantastical experiment”: Replace the nucleus of an egg cell with the nucleus of another cell Early attempts to clone animals in this way failed The breakthrough was the following insight Starvation will synchronize cells at the same point in the cell cycle Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Wilmut’s Lamb Reproductive biologist Ian Wilmut and his colleagues were able to clone the first animal in 1997 Mammary cells were removed from the udder of a six-year old sheep The nucleus was removed from an egg cell taken from another sheep Both cells were synchronized to a resting state The nucleus from the mammary cell was transferred to the enucleated egg cell An electric shock was applied to start cell division Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Wilmut’s Lamb The successful embryos (about 30 in 277 tries) were transplanted into surrogate mother sheep On July 5, 1996, “Dolly” was born Only 1 of 277 tries succeeded However, Wilmut proved that reproductive cloning is possible Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.11 Wilmut’s animal cloning experiment Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Problems with Reproductive Cloning Since Dolly, scientists have successfully cloned sheep, mice, cattle, goats and pigs However, problems and complications arise, leading to premature death Dolly died in 2002, having lived only half a normal sheep life span Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Importance of Genomic Imprinting During gamete development, the DNA undergoes a process termed genomic imprinting The process involves the methylation (addition of –CH3 groups) to cytosine residues in the DNA This locks genes in either the “on” or “off” position Normal animal development requires chemical reprogramming of the DNA Takes months to years in adult reproductive tissues Cloning fails because there is not enough time for the re-programming to be done properly Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.7 Embryonic Stem Cells The blastocyst, an early embryo, consists of A protective outer layer that will form the placenta Inner cell mass that will form the embryo The inner cell mass consists of embryonic stem cells These are pluripotent Capable of forming the entire organism As development proceeds, cells lose their pluripotency They become committed to one type of tissue They are then called adult stem cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.7 Embryonic Stem Cells Embryonic stem cells could be used to restore tissues lost or damaged due to accident or disease Experiments have already been tried successfully in mice Damaged spinal neurons have been partially repaired The course of development is broadly similar in all mammals Therefore, the experiments in mice are very promising Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.7 Embryonic Stem Cells The research in human embryonic stem cells is associated with two serious problems 1. Finding a source Harvesting them from discarded embryos raises ethical issues Fig. 10.13 Human embryonic stem cells 2. Immunological rejection Implanted stem cells will likely be rejected by the immune system of the individual Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.7 Embryonic Stem Cells Therapeutic cloning follows this basic approach: A cell is obtained from an individual who lost a tissue function due to an accident or disease It is cloned to produce an embryo Embryonic stem cells are harvested and grown in tissue culture The stem cells are then injected back into the same individual There, they divide and ultimately differentiate into healthy tissue Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.15 How human embryos might be used for therapeutic cloning Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.15 How human embryos might be used for therapeutic cloning Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.7 Embryonic Stem Cells Therapeutic cloning solves the problem of immune rejection Cells are cloned from the individual’s own tissues, Therefore, they pass the immune system’s “self” identity check However, the process is still controversial Some fear that the cloned embryo might be brought to term by inserting it into a human uterus Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.8 Gene Therapy Gene therapy involves the introduction of “healthy” genes into cells that lack them It was first used successfully in 1990 Two girls were cured of a rare blood disorder caused by a defective adenosine deaminase gene The girls stayed healthy Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.8 Gene Therapy Researchers then set out to apply gene therapy to cystic fibrosis In 1994, the technique was first tried on mice A normal copy of the gene, cf, was added to the vector adenovirus The virus was then squirted into the lungs of mice that carried a defective cf gene The mice had their immune systems disabled The “healthy” gene was thus introduced into lung cells And the mice were successfully cured! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10.8 Gene Therapy Researchers then targeted humans in 1995 The same basic approach was used as was used with mice For eight weeks, the gene therapy seemed successful However, the gene modified-cells in the patients’ lungs came under attack by the immune system The healthy genes were lost, and with them the chances for a cure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A comprehensive 1995 review of human gene therapy trials revealed three problems 1. The adenovirus elicits a strong immune response It causes the common cold, so antibodies were formed due to previous colds 2. In rare cases, the immune response can be very severe If many patients are treated, a few may die 3. The adenovirus inserts its DNA randomly in human chromosomes This will cause mutations and potentially cancers Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display More Promising Vectors Within a few years, researchers had a much more promising vector A tiny virus called adeno-associated virus (AAV) AAV only has two genes and thus needs adenovirus to replicate AAV has several advantages over adenovirus 1. It inserts genes into human DNA less frequently 2. It does not elicit a strong immune response Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Success with the AAV Vector In 1999, AAV successfully cured anemia in rhesus monkeys AAV was also used to cure dogs of a hereditary disorder leading to retinal degeneration & blindness And human trials are under way again! In 2000, scientists performed the first gene therapy experiment for muscular dystrophy Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 10.16 Using gene therapy to cure a retinal degenerative disease in dogs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display