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Molecular Genetics Part 1 – DNA & DNA Synthesis What is DNA? • • • • • • Stands for Deoxyribonucleic Acid It is an organic molecule Was known to be a chemical in cells by the end of the nineteenth century. Contains hereditary instructions. It is the chemical code for every trait. The protein “blueprint”. Can be copied and passed from generation to generation. DNA and RNA are both nucleic acids. o o They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. Hershey-Chase Experiment DNA and RNA Structure The Structure of DNA 1. Nucleotides (4 types differ in bases) a. Phosphoric Acid b. Deoxyribose sugar c. Nitrogenous bases: Adenine-Thymine Guanine-Cytosine 2. Ladder Shape 3. Double strand, helix twist Ladder Shape (Sides & Rungs) The model of DNA is like a rope ladder twisted into a spiral. Sides: • Phosphoric Acid • Sugar • Phosphoric Acid Rungs: • • • • A-T T-A G-C C-G T-A G-C C-G A-T Sugar Acid Sugar Acid Sugar Structure of DNA The Discovery of the Double Helix James Watson and Francis Crick determined that DNA is a double helix. Watson and Crick used X-ray crystallography data to reveal the basic shape of DNA. Rosalind Franklin collected the X-ray crystallography data. DNA Double Helix Chromosome DNA Code: Genes = • Segments of DNA • Code for a trait Hair Color Eye Color DNA Chromosome Code: Acid Acid Sugar-T-A-Sugar Acid Acid Sugar-G-C-Sugar Acid Acid Sugar-C-G-Sugar Triplets aka Codons • Sets of 3 Nucleotides • Code for Amino Acids, that make proteins, that ultimately code for a trait. DNA Replication – Chromosomes double in late interphase - 2n to 4n 2N – When a cell or whole organism reproduces, a complete set of genetic instructions must pass from one generation to the next. – Watson and Crick’s model for DNA suggested that DNA replicates by a template mechanism. – . DNA Replication Overview DNA Replication Review DNA Replication • Begins at specific sites on a double helix. • REPLICATION FORK • Proceeds in both directions. Leading Strand Lagging Strand Origins of Replication Steps for DNA Replication: 1. DNA untwists 2. DNA unzips - HELICASE 3. Corresponding base pairs bonded by DNA Polymerase Line up In sets of 3 nucleotides aka “triplets” or “codons” 4. DNA reforms 5. 2 strands twist into helix Identical Strand Flip to back side of “Copying DNA” Which diagram happens 1st? Check: When does DNA replication take place? Copying DNA–Replication: An Exact Copy-Practice RNA Ribonucleic Acid “DNA messenger & taxi” II. RNA Structure & Function A. What is RNA? 1. Organic Molecule 2. Stands for Ribonucleic Acid 3. Nucleic Acid 4. Three Types 1. mRNA= messenger 2. tRNA= transfer 3. rRNA= ribosomal B. Where 1. mRNA in nucleus & cytoplasm 2. tRNA only in cytoplasm 3. rRNA in the ribosomes is RNA located? rRNA mRNA mRNA tRNA What is RNA’s structure? • • • • • • • • Acid Sugar-Base Acid Sugar-Base Acid Sugar-Base Acid Sugar-Base 1. Nucleotides= a. Phosphoric Acid b. Ribose sugar c. Nitrogenous Bases: Adenine-Uracil Guanine-Cytocine 2. Single Strand 3. No Twisted helix Comparison of RNA & DNA: • • • • • • • • Acid Uracil Sugar-Base Acid Sugar-Base Acid Sugar-Base Acid Ribose Sugar-Base RNA Acid Acid Thymine Sugar-Base-Sugar Acid Acid Sugar-Base-Sugar Acid Acid DeoxySugar-Base-Sugar ribose Acid Acid DNA Ladder What are RNA’s functions: 1. mRNA= • Copies the DNA code • Deliveries message to Ribosome Why not send the original DNA code out? • DNA might be damaged! • mRNA components are reused • To copy more messages Original DNA mRNA copy RNA function continued tRNA Amino acid 2. tRNA: • in cytoplasm • Picks up an amino acid • “Taxis” the aa to the Ribosome protein factories RNA function continued 3. rRNA: • in ribosome “protein factories” • Protein synthesis III. Protein Synthesis Assembling Proteins from the DNA Instructions Protein Synthesis Transcription: 1. mRNA is copied off of DNA 2. In nucleus 3. Steps: • DNA untwists • DNA unzips • RNA codons line up Transcription Transcription: DNA Code mRNA A U T A C G G C mRNA has: • Ribose sugar • Uracil instead of thymine bases • Nuclear membrane allows it to leave! Transcription the Details The “start transcribing” signal is a nucleotide sequence called a promoter. – The first phase of transcription is initiation: • RNA polymerase attaches to the promoter. • RNA synthesis begins. – The second phase of transcription is elongation: • The RNA grows longer. – The third phase of transcription is termination: • RNA polymerase reaches a sequence of DNA bases called a terminator. Translation • Conversion of the message (mRNA Code) • Into a protein • By the ribosome factories Translation 1. mRNA arrives at the Ribosome (rRNA & protein) 2. tRNA picks up an amino acid (aa) 3. tRNA delivers the aa to the ribosome 4. aa are assembled into polypeptide proteins tRNA taxi A U C G U A G C mRNA code Translation: Summary I = base (U) II = mRNA III = Amino acids connected by a peptide bond IV = tRNA V = amino acid Translation: Details - 3 Phases 1. Initiation: Brings together: • The mRNA • The first amino acid with its attached tRNA • The two subunits of the ribosome Note: An mRNA molecule has a cap and tail that help it bind to the ribosome. Translation Continued 2. Elongation: Also consists of several steps • Codon recognition - The anticodon of an incoming tRNA pairs with the mRNA codon. • Peptide bond formation The ribosome catalyzes bond formation between amino acids • Translocation - A tRNA leaves the P site of the ribosome. The ribosome moves down the mRNA. • Elongation continues until the ribosome reaches a stop codon. Translation Continued 2. Termination • Stop codon is reached and translation ends. Making Proteins • How do you know what amino acids are being coded for? Summary: DNA Replication: • Make duplicate DNA • In nucleus • Copy the chromosomes • For cell division Protein Synthesis: 1. Transcription: – Make mRNA – From DNA – In nucleus 2. Translation: – – – – Make protein Off mRNA code Using amino acids In cytoplasm Part IV Genetic Changes Mutations • Any change in the DNA sequence • Mutations may result from • Internal Agents such as rrrors in: – Replication – Transcription – Cell Division • Physical or chemical agents called mutagens. – External Agents Causes of Mutations • Spontaneous Mutations • Mutagens – Agent that can cause a change in DNA – Radiation (X-rays, cosmic rays, Ultraviolet, nuclear) – Chemicals (Asbestos, benzene, Formaldahyde) – High temperatures Chromosomal Alterations • Sometimes parts of chromosomes are broken off and lost during Mitosis and Meiosis • Break and rejoin improperly during crossing over • More common in plants • Zygote usually dies, or is sterile Mutations in Reproductive Cells • Sperm or egg mutated can show up in offspring • New trait – Sometimes benefits • Protein that doesn’t work – Structural / functional problems – May not survive Mutations in Body Cells • Not passed to offspring • Cell divides, new cells also have mutation • Some mutations affect cell division – Uncontrolled cell division = Cancer Point Mutations • A change of a single base pair in DNA • THE DOG BIT THE CAT • THE DOG BIT THE CAR Frameshift Mutation • • • • A single base is added or deleted Shifts the reading of sets of 3 (codons) THE DOG BIT THE CAT THE DOB ITT HEC AT What type are these? Figure 10.21 Repairing DNA • Enzymes proofread the DNA and replace incorrect nucleotides. • Specifically the enzymes and proteins involved in replication can repair the damage. • Work well, but not perfect • More exposure to a mutagen the less likely it will be repaired Evolution Connection – Although mutations are often harmful, • They are the source of the rich diversity of genes in the living world. • They contribute to the process of evolution by natural selection. Part V Applied Genetics & Gene Regulation Question Haven’t our ancestors been able to change the plants and animals around them for thousands of years? How? & Why? How/Why did our ancestors modify their plants and animals? •cross purebreds to make hybrids… •cross hybrids to select for recessive traits… What types of traits were sought after? Before the mid-1940’s, did they know they were modifying DNA? •What if there is not any natural variation in a species to manipulate by breeding? •Is it possible to artificially create mutations in organisms and then see if those mutations can be bred? What could we do to purposely cause mutations? radiation chemicals heat What are some types of things scientists can do with DNA, today? DNA fingerprinting Identify paternity Identify innocence/guilt Cloning Convince an adult cell to grow into a whole new organism…as if it were a zygote. Genetic Engineering Taking a gene from one organism and put it in another organism DNA Fingerprinting – On November 22, 1983, • A 15-year-old girl was raped and murdered on a quiet country lane. • Three years later, another 15-year-old girl was raped and murdered. – DNA fingerprinting of DNA samples from suspects and the crime scene • Proved one man guilty and another man innocent. – DNA technology has rapidly revolutionized the field of forensics. Murder, Paternity, and Ancient DNA – DNA fingerprinting • Has become a standard criminology tool. • Has been used to identify victims of the September 11, 2001, World Trade Center attack. • Can be used in paternity cases. – DNA fingerprinting is also used in evolutionary research DNA Fingerprinting Techniques 1. The polymerase chain reaction (PCR) • • a technique by which any segment of DNA can be copied quickly and precisely. Through PCR, scientists can obtain enough DNA from even minute amounts of blood or other tissue to allow DNA fingerprinting. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Fingerprinting Techniques 3. Gel Electrophoresis • Can be used to separate the DNA fragments obtained from different sources. – The DNA fragments are visualized as “bands” on the gel. The bands of different DNA samples can then be compared. DNA Fingerprinting Techniques 2. Short tandem repeats (STRs) • Are repetitive sequences of DNA that are repeated various times in the genome. • Scientists use STR analysis to compare the number of repeats between different samples of DNA. They can prove that two samples of DNA come from the same person. • Once a set of DNA fragments is prepared, the next step in STR analysis is to determine the lengths of these fragments. Cloning – The animals in these pictures all share two unusual features. • They are all members of endangered species, and they are all clones. Cloning at the Edge of Extinction – Some scientists are now focusing their efforts on cloning members of endangered species. – The use of cloning to repopulate endangered species holds tremendous promise. Cloning Plants and Animals The Genetic Potential of Cells – Differentiated cells • All contain a complete genome. • Have the potential to express all of an organism’s genes. – Differentiated plant cells • Have the ability to develop into a whole new organism. • The somatic cells of a single plant can be used to produce hundreds of thousands of clones. Cloning of Animals – Regeneration, a similar process, • Is the regrowth of lost body parts in animals. – Reproductive Cloning in Animals – Nuclear transplantation • Involves replacing nuclei of egg cells with nuclei from differentiated cells. • Has been used to clone a variety of animals. – Scottish researchers cloned the first mammal in 1997. • Dolly, the sheep, was the product of their work. Cloning of Animals The procedure that produced Dolly is called reproductive cloning. Cloning of Animals – Other organisms have since been produced using this technique, some by the pharmaceutical industry. Therapeutic Cloning and Stem Cells – Therapeutic cloning • Produces embryonic stem cells (ES cells). – Embryonic stem cells • Can give rise to specific types of differentiated cells. – Adult stem cells • Generate replacements for nondividing differentiated cells. – Umbilical Cord Blood Banking • Are unlike embryonic stem cells, because they are partway along the road to differentiation. Human Therapeutic Cloning – In 2001, a biotechnology company announced that it had cloned the first human embryo. Genetic Engineering aka Recombinant DNA Technology If a person needs a protein, but does not have the correct gene (or if their gene for a particular protein is mutated), ……. …..What are some examples of this? “I have diabetes…I will die without insulin.” “I am a hemophiliac…how can I make my blood clot?” …then… How could they get that needed protein? 1. Get the protein from other humans Problems: We typically don’t make more of a protein than we need…&…sharing of bodily fluids can lead to sharing diseases… example, before we tested blood for HIV many hemophiliacs got HIV from blood transfusions! 2. Use a similar protein from other animals Example: insulin from pigs Problems: Sometimes the protein is close, but not exact & can lead to rejection problems…the process can be slow and expensive 3. Make artificial proteins in the laboratory from chemicals… Problems: very expensive and time consuming…hard to make perfect… the process of translation in a cell is regulated so well that there are rarely problems…humans make lots of mistakes in comparison!!! 4. Get bacteria (or another organism) to make the human protein in large amounts. REALLY FAST! This is the thing to do…it is, NOW, easy…cheap…and very fast! This is called Recombinant DNA… to take DNA from one organism and put it into another organism in order to make a needed protein. Recombinant DNA Technology – Recombinant DNA technology is a set of techniques for combining genes from different sources into a single DNA molecule. • An organism that carries recombinant DNA is called a genetically modified (GM) organism. – Recombinant DNA technology is applied in the field of biotechnology. • Biotechnology uses various organisms to perform practical tasks. From Humulin to Genetically Modified Foods – By transferring the gene for a desired protein product into a bacterium, proteins can be produced in large quantities. – In 1982, the world’s first genetically engineered pharmaceutical product was produced. • Humulin, human insulin, was produced by genetically modified bacteria. – Humulin was the first recombinant DNA drug approved by the FDA. Genetically Modified (GM) Foods – Today, DNA technology is quickly replacing traditional plant-breeding programs. • In the United States today, roughly one-half of the corn crop and over three-quarters of the soybean and cotton crops are genetically modified. – Corn has been genetically modified to resist insect infestation. • This corn has been damaged by the European corn borer. Genetically Modified (GM) Foods – “Golden rice” has been genetically modified to contain beta-carotene. • Our bodies use beta-carotene to make vitamin A. Farm Animals and “Pharm” Animals – While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase. – These transgenic sheep carry a gene for a human blood protein. • This protein may help in the treatment of cystic fibrosis. Farm Animals and “Pharm” Animals – While transgenic animals are currently used to produce potentially useful proteins, none are yet found in our food supply. – It is possible that DNA technology will eventually replace traditional animal breeding. Recombinant DNA Techniques – Bacteria are the workhorses of modern biotechnology. – To work with genes in the laboratory, biologists often use bacterial plasmids. • Plasmids are small, circular DNA molecules that are separate from the much larger bacterial chromosome. Recombinant DNA Techniques – Plasmids can easily incorporate foreign DNA. – Plasmids are readily taken up by bacterial cells. • Plasmids then act as vectors, DNA carriers that move genes from one cell to another. – Recombinant DNA techniques can help biologists produce large quantities of a desired protein. We can Give DNA to Bacteria by… taking a piece of DNA and cutting it to have “sticky ends”, then cutting a plasmid to have sticky ends, next, sticking the DNA in the plasmid, and finally, putting the plasmid back into a bacterium. Restriction Enzymes Restriction enzymes cut the DNA so it has “sticky ends”! “Sticky ends” Restriction Enzyme What would be sticky in DNA? The Basic Steps of Bacterial Transformation 1. 2. 3. 4. 5. 6. 7. Cut pieces of human DNA (genes) that code for the protein that is needed. Cut open bacterial plasmids. Stick the human gene in the plasmids. Prepare bacteria to accept the plasmids by chemically tearing small holes in the cell walls and cell membranes & freeze them so they don’t ooze out the holes! Briefly heat the bacteria to make the holes swell and allow the plasmids to sneak inside the bacteria. Cool & feed the bacteria so their cell walls and membranes can heal… Warm and feed the bacteria so they can grow…and produce the human protein. Genomics and Proteomics – Genomics is the science of studying whole genomes. • The first targets of genomics were bacteria. The Human Genome Project – In 1990, an international consortium of governmentfunded researchers began the Human Genome Project. • The goal of the project was to sequence the human genome. – Sequencing of the human genome presented a major challenge. • It is very large. • Only a small amount of our total DNA is contained in genes that code for proteins. The Human Genome Project – The Human Genome Project • Can help map specific disease genes such as Parkinson’s disease. – The Human Genome Project proceeded through several stages, • During which preliminary maps were created and refined. Genome-Mapping Techniques – The whole-genome shotgun method • Involves sequencing DNA fragments from an entire genome and reassembling them in a single stage. The Genetic Basis of Cancer – In recent years, scientists have learned more about the genetics of cancer. – As early as 1911, certain viruses were known to cause cancer. – Cancer-causing viruses often carry specific genes called oncogenes. – Proto-oncogenes • Are normal genes that can become oncogenes. • Are found in many animals. • Code for growth factors that stimulate cell division. – For a proto-oncogene to become an oncogene, a mutation must occur in the cell’s DNA. – Tumor-suppressor genes • Help prevent uncontrolled cell growth. • May be mutated, and contribute to cancer. Human Gene Therapy – Human gene therapy is a recombinant DNA procedure that seeks to treat disease by altering the genes of the afflicted person. • The mutant version of a gene is replaced or supplemented with a properly functioning one. Safety and Ethical Issues – As soon as scientists realized the power of DNA technology, they began to worry about potential dangers such as: • The creation of hazardous new pathogens • The transfer of cancer genes into infectious bacteria and viruses – Strict laboratory safety procedures have been designed to protect researchers from infection by engineered microbes. • Procedures have also been designed to prevent microbes from accidentally leaving the laboratory. The Controversy over Genetically Modified Foods – GM strains account for a significant percentage of several agricultural crops in the United States. – Advocates of a cautious approach have some concerns: • Crops carrying genes from other species might harm the environment. • GM foods could be hazardous to human health. • Transgenic plants might pass their genes to close relatives in nearby wild areas. – Negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol. • The protocol requires exporters to identify GM organisms present in bulk food shipments. – Several U.S. regulatory agencies evaluate biotechnology projects for potential risks: • • • • Department of Agriculture Food and Drug Administration Environmental Protection Agency National Institutes of Health Ethical Questions Raised by DNA Technology – Should genetically engineered human growth hormone be used to stimulate growth in HGHdeficient children? – Genetic engineering of gametes and zygotes has been accomplished in lab animals. • Should we try to eliminate genetic defects in our children? • Should we interfere with evolution in this way? – Advances in genetic fingerprinting raise privacy issues. – What about the information obtained in the Human Genome Project? • How do we prevent genetic information from being used in a discriminatory manner? Evolution Connection: Genomes Hold Clues to Evolution – Genome data has confirmed evolutionary connections, • Such as between yeast cells and human cells. – Comparisons of completed genome sequences strongly support the theory that there are three fundamental domains of life: • Bacteria • Archaea • Eukaryotes How and Why Genes Are Regulated – Four of the many different types of human cells: • They all share the same genome. • What makes them different? Gene Expression • Genes are expressed as traits • Traits are proteins or result from reactions which are regulated by proteins, such as enzymes. • Genes are expressed through protein synthesis. Gene Regulation How Does A Cell Know? Which Gene To Express & Which Gene Should Stay Silent? Patterns of Gene Expression in Differentiated Cells – In cellular differentiation: • Certain genes are turned on and off. • Cells become specialized in structure and function. – In gene expression: • A gene is turned on and transcribed into RNA. • Information flows from genes to proteins, genotype to phenotype. – The regulation of gene expression plays a central role in development from a zygote to a multi-cellular organism. Gene Regulation • When a Gene is Expressed: – It is active and is Transcribed into mRNA • When a Gene is Silent: – It is in active and is Not Transcribed Patterns of Gene Expression in Specialized Human Cells Vocabulary • RNA polymerase: RNA polymerase (RNAP or RNApol) is an enzyme that produces RNA. • Repressor: inhibits transcription of structural genes by binding to the operator • Regulatory gene: codes for the repressor Vocabulary • Promoter: area on the DNA to which the RNA polymerase attaches to begin transcription • Operator: area of the DNA to which the repressor binds; “on/off” switch • Structural genes: code for enzymes which leads to a product Gene Regulation • Expression Regulated By 1. Promoters • RNA Polymerase Binding Sites • Certain DNA Base Pair Sequences 2. Start & Stop Base Pair Sequences 3. Regulatory Sites • DNA Binding Proteins • Regulate Transcription Gene Regulation Prokaryote Gene Regulation: • What is an Operon? • Group of Genes That Operate Together • For Example: – E. coli ferments (digests) lactose • To Do That It Needs Three Enzymes (Proteins), It Makes Them All At Once! – 3 Genes Turned On & Off Together. This is known as the lac Operon (lactose Operon) Operons • Operon: made of three parts 1. Operator 2. Promoter 3. Group of genes – located together which express proteins for a similar function. Two type of operons 1. Inducible – Example: lac operon • Lac = lactose – Normally off but can be activated 2. Repressible – Example: trp operon • Trp = tryptophan – Normally on but can be inhibited Gene Regulation: lac Operon The lac Operon – – Regulates Lactose Metabolism It Turns On Only When Lactose Is Present & Glucose is Absent. Lactose is a Disaccharide – A Combination of Galactose & Glucose To Ferment Lactose E. coli Must: 1. Transport Lactose Across Cell Membrane 2. Separate The Two Sugars Gene Regulation: lac Operon Each Task Requires A Specific Protein but Proteins Not Needed If Glucose Present (why waste energy if you already have food?) so Genes Coding For Proteins Expressed Only When There Is No Glucose Present But Lactose Is Present Gene Regulation: lac Operon Gene Regulation: lac Operon ADD LACTOSE = Lactose Gene Regulation: lac Operon Gene Regulation: lac Operon Key Concept: The lac Genes Are: Turned Off By Repressors And Turned On By The Presence Of Lactose lac Gene Expression • Operon Has 2 Regulatory Regions 1. Promoter (RNA Polymerase Binding) 2. Operator (O region) Bound To A lac Repressor lac Gene Expression • lac Repressor – When Bound To O Region : Prevents Binding of RNA Polymerase To Promoter – Turns The Operon “OFF” lac Gene Expression • lac Repressor Also Binds To Lactose – Higher Affinity For Lactose • When Lactose Present lac Repressor Is Released From O Region – Allows Transcription of All Three Genes Compare and Contrast Regulation Can Be: 1. Based On Repressors 2. Based On Enhancers 3. Regulated At Protein Synthesis Eukaryotic Gene Regulation Operons Usually NOT Found In Eukaryotes Key Concept: Most Eukaryotic Genes Are Controlled Individually And Have Regulatory Sequences That Are Much More Complex Than Prokaryotic Gene Regulation Eukaryotic Gene Regulation Eukaryotic Gene Regulation • TATA Box – About 30 Base Pairs Long – Found Before Most Genes – Positions RNA Polymerase – Usually TATATA or TATAAA – Promoters Usually Occur Just Before The TATA Box Eukaryotic Promoters Enhancer Sequences – Series of Short DNA Sequences – Many Types Enormous Number Of Proteins Can Bind To Enhancer Sequences – Makes Eukaryote Enhancement Very Complex Eukaryotic Promotors • Some Enhance Transcription By Opening Up Packed Chromatin • Others Attract RNA Polymerase • Some Block Access To Genes • Key To Cell Specialization – All Cells Have Same Chromosomes – Some Liver, Skin, Muscle, etc. The End