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Lecture SF005


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  • 1. Lecture SF005 Molecular Genetics – Protein synthesis and HGP Lecture SF005 Molecular Genetics – Genes, Proteins and the Human Genome Project R. Daniel Gietz Ph.D. References: Required Reading: Chapter 3 and Chapter 6, Genetics in Medicine, 6th Ed., Thompson and Thompson, 2001. Learning Objectives. After this lecture the student should be able to; 1. Understand the how genetic information is passed on from the DNA to the proteins. 2. Understand the structure of a human gene. 3. Understand how a gene is translated into protein. 4. Understand how DNA variation at the gene level affects the protein. 5. Understand how gene mapping can aid in cloning human disease genes. 6. Understand the goals and impact of the Human Genome project. Genetics 101 What is a Gene? The cell is the basic unit of the human body. Proteins are the molecular machines of the cells. Proteins, co-factors and RNA do everything that is needed for cellular function and is the foundation of life. The entire instruction set on how and when to produce each protein is containing with the DNA found in the nucleus and the mitochondrion. A genome is the collection of all DNA in a cell. It contains the genes that encode the proteins and the RNA products used by the cell for all its functions including cell duplication as well as cellular. A gene is a linear array nucleotides that is converted to RNA or protein products that are used by the cell. The flow of genetic information from DNA to RNA to protein is the Central dogma of Molecular Genetics. See Figure 1. Transcription DNA ↔ RNA → Protein Reverse Translation Transcription Figure 1 In human cells the DNA in organized into chromosomes. Each human chromosome contains a single piece of DNA complexed with proteins to allow the cell to make genetic transactions and manage the information stored within. (See Figure 1) There are 22 pairs of human chromosomes plus 2 sex chromosomes (X and Y). The DNA is replicated during S phase of the cell cycle just before the cells divide. This is a highly regulated process with high fidelity. Each strand is used to copy the information in a semi conservative fashion.. This is what gives DNA synthesis its high fidelity. A chromosome is how the cell organizes its DNA to allow it to manage the information stores. Normally not distinguishable in the nucleus chromosomes condense to allow the cell to manage the SF05 Page 1 of 8
  • 2. Lecture SF005 Molecular Genetics – Protein synthesis and HGP duplication and delivery of genetic information. The metaphase chromosome can be stained with a variety of agents and identified under the microscope to allow the identification of gross chromomal changes that cause certain genetic conditions such as Downs syndrome (trisomy 21). Genes are arranged on chromosomes in a linear fashion along the DNA strand. A gene is a linear array of the 4 different nucleotides and the functional unit of heredity in the cell. Human genes only make up 2-3% of the entire genome with the rest of the DNA being intergenic DNA and intervening sequences. In addition the coding portions of a human gene are not contiguous but separated by non-coding sequences called introns. There are approximately 35,000 human genes. Most genes are converted into proteins, which are the molecular machines of the cell. The protein and the gene are co-linear see Figure 2. Figure 2 Ffigure 3 The average gene being 3000 nucleotides long will translate into a protein 1000 amino acids in length. This process occurs using an RNA copy of the gene. The genetic information is in the form of nucleotide sequence and is converted using a triplet code to produce a co- linear sequence of amino acids. This process uses the universal genetic code to convert the linear array of nucleotides to a linear array of amino acids as shown in Figure 2. See Figure 3  for the list of codons. Each codon has a some redundancy except Trp. There are also 4 special codons. All proteins start with AUG Met and stop with any combination of UAA, UAG or UGA. Each protein has a primary structure, as well as a secondary, terteriary and quaternary structure. Many proteins are modified after they are produced. There are many modifications that change the structure of a protein. Once the protein is modified it can begin to do its prescribed function in the cell. Chromosome structure As mentioned in SF2 human cells have 46 chromosomes, 23 from the maternal and 23 from the paternal source. Each chromosome consists of a single DNA molecule. All the 46 DNA molecules from a single cell are approximately 1.8 meters in length. This DNA needs to be compacted into a nucleus only SF05 Page 2 of 8
  • 3. Lecture SF005 Molecular Genetics – Protein synthesis and HGP 50 µM in diameter. This is accomplished by complexing the DNA with proteins and forming chromatin. This is done by complexing DNA with histone and forming nucleosomes fibers followed by further solenoid structure formation. These solenoid structures are then further organized into loops and complexed with the nuclear matrix proteins. This allows controlled expression of genes necessary in certain cell types. Gene organization The human genome contains every gene distributed on chromosomes. This distribution is not even, as some chromosome are relatively packed with genes and others are relatively gene scarce. Genes are arranged on either DNA strand. Only about 10% of the DNA is coding, the rest being intronic or intragenic DNA. This DNA is characterized as single copy or repetitive DNA. Single copy DNA is organized into short stretches (several kilobases) and dispursed through out the genome. The repetitive DNA is characterized into a number of different categories. Satellite DNA can be grouped by size and copy number. Categories range from MegaSatellite DNA to mini to micro satellite DNA. The repetitive nature of this DNA can lead to spurious recombination and subsequent problems. Gene Cloning Molecular cloning was first established in the early 1970s after the discovery of restriction enzymes. This allowed the isolation of DNA from any source and propagation in bacteria. This allowed researchers to identify certain genes involved in human disease by cloning the DNA fragment containing the gene responsible, however the progress was a slow long involved process. With the advances in this technology over the next decades lead to the possibility of cloning and sequencing the entire human genome. With the entire human genome sequenced researcher could concentrate on gene identification rather than the technical aspect of DNA cloning. The Human Genome Project (HGP) and molecular Genetics The Human genome project (HGP) represents biology’s first big science project. The Human Genome Project initiative was put into place to acquire fundamental information needed to further our basic scientific understanding of human genetics and the role of various genes in human health and disease. The project was started in 1988 and was funded by the US department of energy (DOE). The complete DNA sequence of the human genome is only one of the goals of the HGP. Other goals include a) construction of a high resolution map of the human genome, b) Production of physical maps of all human chromosomes and other selected model organisms, c) development of capabilities for collecting, storing, distributing and analyzing data produced, d) creation of appropriate technologies necessary to achieve these goals. Genetic Mapping. Human chromosomes are mapped genetically using polymorphisms. This type of mapping relies on recombination that occurs during meiosis and is identified thru family studies. Protein polymorphisms were first used to map genes to chromosomes, however this left large gaps in the chromosome genetic map. Restriction fragment length polymorphisms where then used to used to map human chromosomes. This allowed genetic maps to be produced at a resolution of ~10 cM (centiMorgan). Microsatellite markers, also described as short tandem repeat polymorphism, were then used to reduce the resolution to one marker per 0.7 cM. This led the way to the production of the high resolution physical maps of the human chromosomes. Physical Mapping. SF05 Page 3 of 8
  • 4. Lecture SF005 Molecular Genetics – Protein synthesis and HGP There are a variety of physical maps that were constructed during the early stages of the HGP. Cytogenetic maps were produced with chromosome banding techniques to give a resolution of several megabases. Chromosome breakpoint maps using various techniques were produced to define the map to the 0.5 megabase resolution. Restriction maps using rare cutting restriction enzymes (NotI) were used to further increase the resolution to several hundred kilobases. Clone contig maps were further used to reduce the resolution to 40 kilobases. Sequence tagged site (STS) and expressed sequence tag (EST) maps also gave rise to a 40 kilobase resolution for a physical chromosomal map. Finally the DNA sequence map is the complete nucleotide sequence of each chromosome, which gives a resolution to 1 base pair. Databases The huge amounts of information generated by the HGP are stored for access in various databases. GenBank is the database that is maintained by the US National Center for Biotechnology Information (NCBI) and can be accesses by everyone with a personal computer and internet access ( There are mirror sites at the European Bioinformatics Institute (EBI) in Heidelberg ( The mapping database is found at the Genome database base established at Johns Hopkins University ( Additional genetic information on syndromes can be accessed at Online Mendelian Inheritance in Man ( db=OMIM ) Identifying Genes involved in human disease! Most genes are identified by mapping the gene location to the human genome and subsequent identification of the mutations causing the condition. This is done through studies of affected families. The mutation in the genome causing the disease is genetically mapped to a location in the human genome. This is done using genetic mapping techniques, which rely on the naturally occurring sequence variation in the human genome. A genetic disease locus sequence variation can be mapped to a specific location in the genome by looking for its association to a closely linked DNA polymorphism. These DNA polymorphisms can be restriction site length polymorphism (RFLPs), or variable number tandem repeat sequences (VNTR) as well as M HD Father X Mother other types of variable sequences such as CA repeats. A genetic linkage map is constructed by observing how frequently two DNA markers are inherited together. -Figure 4 shows that marker marker (M) and Huntingtons disease M M M (HD) loci are preferentially inherited HD HD by the offspring. The one child that inherits only marker (M) shows that * Children recombination has separated the two * Recombintaion: Frequency of this event reflects the distance markers during thethe father. of The gametes from production the between gene for the marker M and HD frequency of this event helps determine the distance between the two DNA sequences on a genetic map. Once the genetic location of the disease locus is found the positional cloning of the specific SF05 Page 4 of 8
  • 5. Lecture SF005 Molecular Genetics – Protein synthesis and HGP disease gene is accomplished. It involves the use of a low and high resolution physical map of the DNA around the disease gene. The Human Genome Project has accomplished this for the entire genome. The production of a complete high resolution physical DNA map including the complete nucleotide sequence allows the researcher to utilize this data to more easily clone the genes that may be involved in the disease studied. The completed sequencing of the human genome means that all the information on the physical map and the nucleotide sequence of all human genes has been accomplished. Once a genetic location has been established the DNA sequence of that region can be easily obtained from the database. The potential genes can be identified and subsequently screened for mutations in individuals showing the disease phenotype. This will allow researchers to more easily identify genes involved in diseases such as diabetes and heart disease as well as a host of other complex human disease conditions. Gene or DNA polymorphism Genetic Map Restriction fragments Ordered Clone library Sequence IMPACT OF HGP When the sequence in known what will it mean and was it worth the effort? It is expected that there will be many medical benefits from knowing the structure of all genes in the human genome. This will provide more comprehensive prenatal and pre-symptomatic diagnosis in individuals judged to be at risk of carrying a disease gene. In addition, information about gene structure and regulation can be used to understand the biological process in humans. It is also expected that this information will provide a framework for developing new therapies for disease, which include gene therapy approaches. While most single gene disorders are rare they are the easiest targets for developing medical therapies. The more common disorders are multifactorial, such as heart disease and adult onset diabetes, and will prove more problematic to identify the genes involved. However, ultimately the information from the HGP will be a benefit to the medical community, it just may take some time to realize them. Potential Benefits of HGP 1. Molecular medicine  Improved diagnosis of disease  Earlier detection of genetic predispositions to disease  Rational drug design  Gene therapy and control systems for drugs  Pharmacogenomics "custom drugs" The Human Genome Project is having a profound impact on biomedical research and will also affect other biological research and clinical medicine. Research associated with the HGP has played a part in the identification of genes responsible for human diseases such as myotonic SF05 Page 5 of 8
  • 6. Lecture SF005 Molecular Genetics – Protein synthesis and HGP dystrophy, fragile X syndrome, neurofibromatosis types 1 and 2, inherited colon cancer, Alzheimer's disease, and familial breast cancer. The new era of molecular medicine will be characterized less by treating symptoms and more by looking to the most fundamental causes of human disease. Specific tests will make diagnosis and earlier treatment possible for many diseases. It is possible that in the near future that the disease condition will be treated with novel therapeutic regimens based on new classes of drugs, immunotherapy techniques, avoidance of environmental conditions that may trigger disease, and possible augmentation or even replacement of defective genes through gene therapy. 2. Energy sources and environmental applications  Use microbial genomics research to create new energy sources (biofuels).  Use microbial genomics research to develop environmental monitoring techniques to detect pollutants.  Use microbial genomics research for safe, efficient environmental remediation.  Use microbial genomics research for carbon sequestration 3. Risk assessment  Assess health damage and risks caused by radiation exposure, including low-dose exposures.  Assess health damage and risks caused by exposure to mutagenic chemicals and cancer- causing toxins.  Reduce the likelihood of heritable mutations. Our ability to assess individual risk for exposure to toxic agents will improve the more we understand about the genes involved in resistance and susceptibility to them. It has been known for some time that genetic differences make some individuals more susceptible or more resistant to certain these agents. The identification of the genes involved in this variability will allow a more accurate risk assessment. This will lead to a better understand the effects of low-level exposures to radiation and other energy-related agents, especially in terms of cancer risk. 4. Bioarchaeology, anthropology, evolution, and human migration  Study evolution through germline mutations in lineages.  Study migration of different population groups based on female genetic inheritance.  Study mutations on the Y chromosome to trace lineage and migration of males.  Compare breakpoints in the evolution of mutations with ages of populations and historical events. Understanding genomics will help us understand human evolution and the common biology we share with all of life. Comparative genomics between humans and other organisms such as mice already has led to similar genes associated with diseases and traits. Further comparative studies will help determine the yet-unknown function of thousands of other genes. 5. DNA forensics (identification)  Identify potential suspects whose DNA may match evidence left at crime scenes.  Exonerate persons wrongly accused of crimes.  Identify crime and catastrophe victims.  Establish paternity and other family relationships.  Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers).  Detect bacteria and other organisms that may pollute air, water, soil, and food.  Match organ donors with recipients in transplant programs.  Determine pedigree for seed or livestock breeds. SF05 Page 6 of 8
  • 7. Lecture SF005 Molecular Genetics – Protein synthesis and HGP  Authenticate consumables such as caviar and wine. DNA typing can be used to identify any organism or even individual. As DNA sequencing technologies become more amenable to automation and high through put modalities the identification of individuals will become more precise and direct through genome sequencing. Currently the identification of individuals is done by scanning 10 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (DNA fingerprint). With this technology there is a very small chance that any two individuals are identical. 6. Agriculture, livestock breeding, and bioprocessing  Disease-, insect-, and drought-resistant crops.  Healthier, more productive, disease-resistant farm animals.  More nutritious produce .  Biopesticides.  Edible vaccines incorporated into food products.  New environmental cleanup uses for plants like tobacco. Ethical Legal Social Issues of the HGP The Human Genome Project was completed in 2003. One of the key research areas was ethical, legal, and social issues research (ELSI). The U.S. Department of Energy (DOE) and the National Institutes of Health (NIH) devoted 3% to 5% of their annual Human Genome Program budgets toward studying the ethical, legal, and social issues (ELSI) surrounding availability of genetic information. This represents the world's largest bioethics program. It has become a model for ELSI programs around the world (including Canada). Rapid advances in the science of genetics and its applications have presented new and complex ethical and policy issues for individuals and society. ELSI programs that identify and address these implications have been an integral part of the U.S. HGP since its inception. These programs have resulted in a body of work that promotes education and helps guide the conduct of genetic research and the development of related medical and public policies. A continuing challenge is to safeguard the privacy of individuals and groups who contribute DNA samples for large-scale sequence-variation studies. Other concerns have been to anticipate how the resulting data may affect concepts of race and ethnicity; identify potential uses (or misuses) of genetic data in workplaces, schools, and courts; identify commercial uses; and foresee impacts of genetic advances on the concepts of humanity and personal responsibility. Societal Concerns Arising from the New Genetics 1. Fairness in the use of genetic information by insurers, employers, courts, schools, adoption agencies, and the military, among others. Who should have access to personal genetic information, and how will it be used? 2. Privacy and confidentiality of genetic information. Who owns and controls genetic information? 3. Psychological impact and stigmatization due to an individual's genetic differences. How does personal genetic information affect an individual and society's perceptions of that individual? How does genomic information affect members of minority communities? 4. Reproductive issues including adequate informed consent for complex and potentially controversial procedures, use of genetic information in reproductive decision making, and reproductive rights. Do healthcare personnel properly counsel parents about the risks and limitations of genetic SF05 Page 7 of 8
  • 8. Lecture SF005 Molecular Genetics – Protein synthesis and HGP technology? How reliable and useful is fetal genetic testing? What are the larger societal issues raised by new reproductive technologies? 5. Clinical issues including the education of doctors and other health service providers, patients, and the general public in genetic capabilities, scientific limitations, and social risks; and implementation of standards and quality-control measures in testing procedures. How will genetic tests be evaluated and regulated for accuracy, reliability, and utility? (Currently, there is little regulation at the federal level.) How do we prepare healthcare professionals for the new genetics? How do we prepare the public to make informed choices? How do we as a society balance current scientific limitations and social risk with long-term benefits? 6. Uncertainties associated with gene tests for susceptibilities and complex conditions (e.g., heart disease) linked to multiple genes and gene-environment interactions. Should testing be performed when no treatment is available? Should parents have the right to have their minor children tested for adult-onset diseases? Are genetic tests reliable and interpretable by the medical community? 7. Conceptual and philosophical implications regarding human responsibility, free will vs genetic determinism, and concepts of health and disease. Do people's genes make them behave in a particular way? Can people always control their behavior? What is considered acceptable diversity? Where is the line between medical treatment and enhancement? 8. Health and environmental issues concerning genetically modified foods (GM) and microbes. Are GM foods and other products safe to humans and the environment? How will these technologies affect developing nations' dependence on the West? 9. Commercialization of products including property rights (patents, copyrights, and trade secrets) and accessibility of data and materials. Who owns genes and other pieces of DNA? Will patenting DNA sequences limit their accessibility and development into useful products? Additional Reading: For further information on the Human Genome Project SF05 Page 8 of 8