DNA: The Molecule of Life Molecular Genetics https://www.23andme.com/ DNA and RNA Genes are segments of DNA on a chromosome that code for specific traits DNA – nucleic acid called deoxyribonucleic acid that contains the instructions necessary for a cell to build proteins from amino acids. RNA – ribonucleic acid plays a role in gene expression and protein synthesis http://www.youtube.com/watch?v=zwibgNGe 4aY What is DNA? Isolating the Material of Heredity 1869 Friedrich Miescher isolated a weakly acidic phosphorus-containing substance from the nuclei of white blood cells – Called it “nucleic acid” Early 1900’s Pheobus Levene isolated two types of nucleic acid – Called them “ribose nucleic acid” (RNA) and “deoxyribose nucleic acid” (DNA) Soon after, Thomas Hunt Morgan provided the first experimental evidence that genes are located on chromosomes (fruit flies) http://www.youtube.com/watch?v=5MQdXjRPHmQ What is a gene? Isolating the Material of Heredity In 1928 Fredrick Griffith designed an experiment to study the bacteria that were responsible for a pneumonia epidemic in London – He discovered that the dead pathogenic bacteria passed on their disease-causing properties to live, non-pathogenic bacteria – He called this the transforming principle Griffith died during world war II but several scientists built on his work In notes booklet http://www.juliantrubin.com/bigten/dnaexperiments.html Isolating the Material of Heredity In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered: – When they treated heat-killed pathogenic bacteria with a protein-destroying enzyme, transformation still occurred – When they treated heat-killed pathogenic bacteria with a DNA-destroying enzyme, transformation did not occur The results provided evidence that DNA has a role in transformation In notes booklet http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch8-1.html Isolating the Material of Heredity In 1952, Alfred Hershey and Martha Chase used radioactive labeling to show that genes are made of DNA – They used a virus that contains a protein coat surrounding a length of DNA – This virus attaches to a bacteria cell and injects genetic information into the cell – The infected cell produces new viruses and bursts which releases the new viruses to infect other cells Isolating the Material of Heredity Hershey and Chase created two batches of the virus – In one they labeled the protein coat with radioactive sulfur – In the other, they labeled the DNA with radioactive phosphorus – They found that the radioactive phosphorus was found in the bacterial cells – They concluded that DNA must direct the cell to produce new viruses – animation In notes booklet http://www.accessexcellence.org/RC/VL/GG/hershey.html Structure of DNA After isolating DNA and RNA, Levene determined that both molecules are made up of nucleotides (in long chains) Nucleotide is composed of: – 5-carbon sugar (deoxyribose in DNA, ribose in RNA) – Phosphate – Nitrogen base (4 different types) The 4 nitrogen bases belong to two chemical groups called purines and pyrimidines Purines = Adenine (A) and Guanine (G) Pyrimidines = Thymine (T) and Cytosine (C) Structure of DNA In the late 1940’s Erwin Chargaff found that nucleotides are not present in equal amounts in DNA and RNA – Nucleotides are present in varying proportions – He found that the number of adenine in DNA is equal to the number of thymine in a sample – The amount of cytosine is approximately equal to the amount of guanine This constant relationship is called Chargaff’s rule Video on Chargaff’s rule Structure of DNA In the early 1950’s, a British scientist Rosalind Franklin used x-ray photography to analyze the structure of DNA – DNA has a helical structure with two regularly repeating patterns – Nitrogenous bases are located on the inside of the structure, and the sugar-phosphate backbone is located on the outside (near the watery nucleus) – Many argue that Franklin should have shared in the Nobel Prize for discovering the structure of DNA, but she died before it was handed out NOVA program on photo 51. Structure of DNA In 1953, James Watson and Francis Crick were the first to produce a structural model for DNA Watson and Crick’s model of DNA closely resembles a twisted ladder – Deoxyribose sugar and phosphate molecules make up backbone (handrails of the ladder) – Paired nitrogen bases held together by weak hydrogen bonds make up the rungs (steps) of the ladder – The ladder twists to form a double helix From Franklin’s images, Watson and Crick knew the distance between the sugarphosphate handrails remained constant Structure of DNA The two strands that make up DNA are not identical, they are complementary to eachother – This is due to the complementary base pairs of A-T and C-G The two strands are also antiparallel – – The phosphate bridges run in opposite directions in the two strands Each end of a double stranded DNA molecule contains the 5’ end of one strand and the 3’ end of the complementary strand http://www.synapses.co.uk/genetics/tsg19.html http://www.youtube.com/watch?v= p835L4HWH68&feature=youtu.be 5' and 3' ends of DNA.mov In a segment of DNA, the number of purines equals the number of pyrimidines; this is because of the base pairing rule RULE: nitrogen bases always pair in complementary pairs – Adenine = thymine – Guanine = Cytosine Ex) if 15% of the bases were thymine, what percentage would be a) adenine b) guanine c) cytosine Example Determine the complementary strand of DNA: A I T I G I C I A I G I C I Ribonucleic Acid (RNA) compared to DNA The sugar component of RNA is ribose RNA does not have the nucleotide thymine (T), in its place is uracil (U) RNA remains single stranded There are several types of RNA – mRNA, rRNA, tRNA DNA, RNA animation DNA Replication (Synthesis) http://www.youtube.com/watch?v=8k K2zwjRV0M Crash course Replication – DNA has the ability to replicate (or duplicate) itself. This is why one cell is able to divide into two cells and each cell has identical genetic information Replication takes place during S phase of interphase DNA Replication (Synthesis) Replication is semi-conservative – Each new molecule of DNA contains one strand of the original complementary DNA and one new parent strand – Each new strand conserves half of the original molecule – Meselson-Stahl Experiment Replication takes place at several locations along the DNA molecule simultaneously – The steps are described in sequence to better understand the concept – BioFlix Replication Replication starts at a specific nucleotide sequence called the replication origin 2) During replication, weak hydrogen bonds that hold complementary nitrogen bases together are broken (This causes the two edges to “unzip”) with a special group of enzymes called helicases (gyrase breaks the hydrogen bonds) 3) This creates two y-shaped areas (replication forks) at the end of the unwound area, the unwound area is called a replication bubble 4) The parent (original) strands are conserved while two new strands created from nucleotides are formed with them (they act as a template) 1) 5) Free floating nucleotides (from diet) are attached to the exposed nitrogen bases according to the base pair rule with an enzyme called DNA polymerase – This process is called elongation – DNA polymerase attaches new nucleotides to the free 3’ end of a preexisting chain of nucleotides – Elongation can only take place in a 5’ to 3’ direction – This means that replication occurs in opposite directions along each strand of the parent DNA • • One strand is replicated continuously, it is called the leading strand The other strand is replicated in short segments, it is called the lagging strand – The short segments are called Okazaki fragments – These fragments are spliced together by an enzyme called DNA ligase 6) Since DNA polymerase cannot synthesize new fragments of DNA, and RNA primer serves as a starting point for the elongation of each new DNA strand An enzyme called primase is required to construct a primer When finished the strand, DNA polymerase removes the primer by eliminating the nucleotides in a 5’ to 3’ direction Hydrogen bonds form between the nitrogen bases 8) Special proofreading enzymes (DNA polymerase) check the new strand of DNA for mistakes. Errors are removed by cutting the mistake out and using an endonuclease and replacing it with the correct nitrogen base 7) As soon as the newly formed strands are complete, they rewind automatically into the helix structure Replication continues until the new strands are complete and the two DNA molecules separate from eachother – This is called termination This replication produces two strands of DNA from one where each strand is composed of “half-old and half-new” Replication Fork http://www.bio.davidson.edu/Courses/Molbio/ Adding Nucleotides MolStudents/spring2005/Durnbaugh/yfp.html Replication Animation Replication Review THINK WELL PART 1 AND 2 http://www.youtube.com/watch?v=aSI LNKbhNLg&feature=related http://www.youtube.com/watch?v=CR http://www.biosci.ohio-state.edu/~mgonzalez/Micro521/04.html http://distancelearning.ksi.edu/demo/bio378/lecture.htm Genetic Engineering and Recombinant DNA Genetic Engineering – refers to the alteration of an organism’s genome (complete set of genes) by selectively removing, adding, or modifying DNA Recombinant DNA – the process of cutting out DNA from one genome and placing the DNA into another genome resulting in a transgenic organism Examples of transgenic organisms: – Genetically modified bacteria for use in medicine and bioremediation (environmental clean-up) – Transgenic plants to improve crop yield and resistance to environmental effects – Cloned animals (livestock) and organs for human use Recombinant DNA – How do they do it? Use restriction enzymes (endonucleases) to cut strands of DNA within their interior (at specific sequences) – animation Then ligase (enzyme that fuses segments of DNA) is used as a biological glue Production of human insulin Gene in the human genome that codes for insulin is cut out using restriction enzymes The plasmid of an E-coli bacteria is cut using restriction enzymes so that the gene for insulin can be inserted using a ligase Bacteria can read the DNA and produce insulin for us to later harvest and use Another example is the insertion of the gene that codes for growth hormone into animals so that they grow faster Note: Biotechnology refers to the use of organisms or biological products for commercial and/or industrial processes - video What are the Issues? – Costs/where money is spent – Motivation for the product – Biological characteristics of the product – Heath effects – Environmental effects – Freedom of Information/Privacy – Who Owns the technology/patents – Issues Animation Gel Electrophoresis 1) 2) Technique used to separate DNA fragments by size for the purpose of identification in paternal or criminal suits (animation) Sample of DNA is cut using restriction enzymes from hair, blood, skin, etc. This produces a number of DNA segments of different lengths. The different pieces of DNA (referred to as restriction-fragment-length-polymorphisms or RFLP) are tagged with a radioactive isotope 3) Using an agarose gel that contains holes or wells along one side, the samples of DNA are inserted into the wells. A known sample is loaded with it as a comparison 4) Electric current is run through the gel, causing the movement of the negatively charged DNA fragments. The shortest strands move the farthest (lowest weight) and the longer strands (heavier) will not move as far. 5) This causes separation of the DNA into bands. The gel is left to set 6) When combined with staining or X-ray film, the patterns are used to determine the presence or absence of particular DNA or proteins DNA Fingerprinting Developed in 1985 – used to identify whether or not a sample of DNA comes from a specific person People have similar DNA, however every human (with the exception of identical twins, triplets, etc.) have some unique noncoding segments of DNA called introns; exons are segments of DNA that actually code for proteins Sample of DNA is placed through gel electrophoresis as well as samples from individuals who are suspected as “owners” of the sample Because of introns, each individual will have a different number of sites where the restriction enzyme will cut This results in a unique number and length of fragments which produces a unique banding pattern (fingerprint) when x-rayed Fingerprints are used to identify criminals, paternity or kinship Animation Lane A: DNA from crime scene cut with Enzyme 1 Lane B: DNA from crime scene cut with Enzyme 2 Lane C: DNA from Suspect 1 cut with Enzyme 1 Lane D: DNA from Suspect 1 cut with Enzyme 2 Lane E: DNA from Suspect 2 cut with Enzyme 1 Lane F: DNA from Suspect 2 cut with Enzyme 2 Protein Synthesis http://www.youtube.com/watch?v=itsb2 SqR-R0 Crash course Genetic code is determined by the arrangement of nitrogen bases within the strands of DNA Each gene codes for the production of a specific protein transcription – DNA RNA translation protein Proteins Proteins are composed of 20 different amino acids that are strung together in endless combinations Compose cell membranes, cell organelles, muscle filaments, hair, hair color, enzymes (regulate speed of chemical reactions in cells), antibodies (disease-control agents), hormones Genetic Code It takes the code of 3 nucleotides (a codon) to code for one amino acid Humans can make 12 of the 20 amino acids, we must consume the other 8 essential amino acids Simple protein = 8 amino acids Complex protein = 50 000 amino acids Sequencing amino acids is determined by DNA Replacement of a single amino acid can change a protein Genetic Code The genetic code has three important characteristics 1. The genetic code is redundant (more than one codon can code for the same amino acid) 2. The genetic code is continuous (reads as a series of three letter codons without spaces, punctuation or overlap) 3. The genetic code is nearly universal (almost all organisms use the same code – this is good for genetic engineering and biotechnology) Role of DNA in protein synthesis DNA is in nucleus, but protein synthesis occurs on the ribosomes in the cytoplasm Carrier molecule (mRNA – messenger RNA) is responsible for reading the information from the DNA (transcription) and carry it to the ribosomal RNA (rRNA) in the cytoplasm where it will be translated into an amino acid sequence by transfer RNA (tRNA) RNA (Ribonucleic Acid) 1) 2) 3) 4) 5) 6) Different from DNA in that: It’s single stranded It contains the sugar ribose It is located throughout the cell (DNA is only in the nucleus with some also in the mitochondria) It contains the base uracil (U) instead of thymine (T) There are three types: mRNA, rRNA, tRNA It’s shorter (no introns) Transcription Protein synthesis begins with transcription (RNA synthesis) of DNA DNA never leaves the nucleus (protected) Steps of Transcription 1) DNA molecule unzips (like in replication), however, RNA nucleotides are now added to the necessary areas (exons) by RNA Polymerases For each gene, only one strand of the DNA is transcribed, this is called the sense strand. The other is called the anti-sense strand. 2) 3) 4) 5) 6) As double helix uncoils, nucleotides from the mRNA find the appropriate pair by using single DNA strand as a template (5’ to 3’ direction) Uracil binds to exposed adenine bases and cytosine binds to exposed guanine bases mRNA joined and fused in a long chain mRNA move away from DNA and the DNA strands rejoin again mRNA leave the nucleus in search of the ribosomes http://fig.cox.miami.edu/~cmallery/150/gene/mol_gen.htm _____________________________DNA I I I I I I I I A G C T T A T C U C G A A U A G I I I I I I I I _____________________________RNA mRNA reads code from DNA RNA codes for amino acids Some codes in mRNA are not for amino acids but are terminators and initiators Terminators – end protein synthesis (stop codon) Initiators – turning protein synthesis on (start codon). Also called promoter site, starts RNA transcription Transcription Animation http://www.coolschool.ca/lor/BI12/unit6/U06L01.htm Example Original DNA sequence: AAT GCC AGT GGT TCG CAC AAA a) Write the complementary DNA sequence b) Write the mRNA sequence c) How many amino acids are there? d) What is the amino acid sequence? Do the same for the following DNA sequence: TAC CAC GTG GAC TGA GGA CTC CTC ATC ATA Translation Translation is the next stage of protein synthesis Involves translating codons found on the mRNA into a chain of amino acids (to form a protein) Transfer RNA, tRNA is made up of a single strand of RNA that folds into a clover-leaf shape – One lobe contains the anticodon, three nucleotides that are complementary to the mRNA codon – At the opposite end is a binding site for the amino acid that corresponds to the codon Ribosomal RNA, rRNA is a linear strand of RNA that remains associated with the ribosomes More on tRNA The exposed bases are called the anticodon Each kind of tRNA molecule has a specific anticodon Ex) Glutamate carried by a tRNA molecule that carries either the CUU or the CUC anticodon (opposite to mRNA codons) Ex) Valine carried by a tRNA molecule that has the CAA anticodon Steps of translation 1) 2) 3) 4) 5) 6) Translation is activated when an mRNA molecule binds to an active ribosome complex in such a way that two codons are exposed The first tRNA molecule carrying the amino acid methionine, base pairs with the start codon on the mRNA, (AUG) Another tRNA molecule arrives at the codon next to the first tRNA, and the first tRNA passes its amino acid on to the second tRNA Enzymes catalyze the formation of a peptide bond between the two amino acids The ribosome moves along the mRNA strand one codon at a time The first tRNA molecule detaches from the mRNA and picks up another amino acid as another tRNA attaches to the mRNA. NOTE: The process begins with the presence of an initiator (start codon) AUG and ends with the presence of a stop (terminator codon) UAA, UGA, or UAG on the mRNA. Remember that the sequence of amino acids was originally derived from the message carried by mRNA from the nucleus (DNA) Translation animation 1 Translation animation 2 Translation 3 Protein Synthesis Process http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Translation.html http://www.scq.ubc.ca/?p=263 Example Write a tRNA and amino acid sequence for the following DNA sequence: TAC CAC TGA GGA CTC CTC CAT CAT Protein synthesis animation http://www.youtube.com/watch?v=983lhh20rGY Analogy of protein synthesis and RNAi http://www.pbs.org/wgbh/nova/body/rnai.html DNA song http://www.youtube.com/watch?v=FUA6_Ucw3i4 Make a Recipe Card for Protein Synthesis Use your notes and the 4 by 6 index card I gave to you to write your own “recipe card”. Mutations http://www.youtube.com/watch?v=ecZbhf96W9k A mutation is a permanent change in the DNA sequence caused by mutagens (mutagenic agents) Mutations – are inheritable – Arise from mistakes in DNA replication when one nitrogen base is substituted for another – Creates a new genetic code that gives new instructions to make amino acids (causes a different protein to be made) Mutagenic agents Physical: – Pushing or tugging chromosomes Chemical: – Carcinogens, mustard gas, poor nutrition Medications: – Some antibiotics Radiation: – X-ray, ultraviolet radiation, cosmic rays Replication mistakes: – Natural mistakes occur during mitosis or meiosis Nutritional: – Lacking certain nucleotides in diet means you are unable to provide the proper free nucleotide base and this causes a mismatch Biological: – Most viruses use genetic material of chromosomes to reproduce. They join existing DNA to cause permanent damage Mutation Animation http://www.youtube.com/watch?v=efstlgoynlk&feature=fvw If a mutation is present in the gametes, it will be passed on to further generations. This is why it is particularly dangerous for pregnant women Mutations can be grouped under 2 categories: 1) Chromosomal mutations 2) Point mutations Chromosomal mutations Large mutations that visibly effect the structure or number of chromosomes – Ex) nondisjunction, fragile-X-syndrome – http://www.youtube.com/watch?v=y1FOEteaM9Q&feature=related Point Mutations Alter a single gene. There are several types: 1) Base substitution – a foreign base replaces the normal base in each strand of DNA. This could result in one amino acid being different animation – ACGCCA becomes CCGCCA – Ex) Sickle cell anemia – substitution of one nitrogen base causes an inability to carry sufficient oxygen 2) Insertion: A base is added into the normal DNA sequence – ACGCCA becomes AACGCCA 3) Deletion: a base is removed from the normal DNA sequence – ACGCCA becomes CGCCA – Ex) Cystic fibrosis – deletion of 7th, 8th, and 9th nitrogen bases causes an inability to produce protein that regulates chloride channels – animation NOTE: both insertion and deletion result in a frame-shift mutation because every amino acid after the point of mutation may be affected 4) Translocation: a sequence of nitrogen bases is removed from one area and placed in another – ABCDEFGHIJ becomes ABFGHIJCDE 5) Inversion: reversal of a sequence of nitrogen bases – ABCDEFGHIJ becomes ABEDCFGHIJ 6) Duplication: duplicating a set or sequence of nitrogen bases twice in one location – ABCDEFGHIJ becomes ABCABCDEFGHIJ 7) Silent mutations: no phenotypic effect because certain amino acids have more than one code – GTA (CAU) and GTG (CAC) both code for histidine NOTE: The body can repair some mutations, but not all Human Genome Project Human Genome Project – animation Oncogenes and Cancer In normal cells, protein synthesis is carried out by structural genes only when required Because protein synthesis is not always required, a regulator gene produces a repressor protein which switches off protein synthesis and reducing the rate of cell division P53 animation http://www.youtube.com/watch?v=WZdErhuL Ctc Uncontrolled cell division is cancer. Cancer is caused by a mutation due to an environmental factor or carcinogen Mutation could be a base substitution that prevents the production of the repressor protein, or the movement of a gene from one part of the chromosome to another If the structural gene is separated from its regulator gene, it cannot be turned off (cancer forms) – These genes that cannot be turned off are called oncogenes Most common oncogene = ras Found in 50% of colon cancers and 30% of lung cancers Ras makes a protein that acts as an “on” switch for cell division. Once a sufficient number of cells are produced, it should shut off Cancer-causing oncogene produces a protein that blocks the “off” switch (causes cell division to continue at an accelerated rate) Epigenetics Epigenetics Explanation – Tale of Two Mice Diagnosing Genetic Disorders Amniocentesis and Chorionic Villus Sampling can take cell samples from a developing fetus or embryo – This sample can be screened for genetic markers for certain disorders • Uses a DNA probe which identifies problem genes Steps in Cloning a Gene http://www.learnalberta.ca/content/seb3 0/html/interactiveLauncher.html?interact ive=StepsInCloningAGene.swf Gene Therapy Targets genetic causes of diseases rather than their symptoms A DNA vector carries foreign DNA into target cells of the patient – The vector is usually a virus that has been genetically altered to carry a desired gene – The virus will eventually transfer the new gene into the cell’s genome – Sickle Cell Anemia -- Hope from Gene Therapy – http://www.youtube.com/watch?v=ujf72mjy0Bg&feature=rela ted Gene Therapy Somatic Gene Therapy – correcting genetic disorders in somatic cells – Mutations can still be passed on to offspring Germ-line Therapy – correcting the genetic information in sperm and egg – Could have many negative effects on future generations – Currently banned in Canada Ames Test Technology to determine quickly, cheaply, and accurately if a chemical is mutagenic. Any chemical that is mutagenic has potential to be carcinogenic We must test products for their cancercausing agents http://diverge.hunter.cuny.edu/~weigang/Images/08-22_amestest_1.jpg Performed on bacteria that have been mutated so they cannot produce histadine on their own (we must supply them with it in order for them to survive) Plate this bacteria on a petri dish with no histidine and the chemical being tested (expect no growth) If bacteria are found, conclusion can be made that microbe has been mutated and is now producing its own histidine » The more colonies that are found, the higher the strength of mutagenic the chemical is which indicates it is highly carcinogenic Often chemical is added to liver enzymes because chemicals are often harmless to an individual until they are broken down in the liver into toxic metabolites NOTE: some chemicals can cause cancer in some individuals and not in others (because of different nitrogen base sequences in each individual) Biological Warfare Most disease-causing agents can be exploited for biological weaponry Microbe or toxin produced by microbe may be harmful to livestock, grains, bacteria in soil, or humans Fortunately, few organisms are suited for mass destruction Examples AIDS – not transmitted by air Clostridium botulinum – deadly in water (not air)…one kg of toxin in water reservoir kills ~50 000 people (60% of population dies in 24 hrs) Research/Testing Stations Britain = Porton Down USA = Camp Detrick in Maryland Canada = Suffield in Alberta Preferred microbe was anthrax bacillus (affects cattle and humans). Anthrax Deadly spores (rod-shaped bacterium) live long periods of time, are highly contagious, and resistant to many environmental factors We currently have the ability to create the “superbug” through merging genes for rapid reproduction and environmental resistance…what do you think would happen then? http://www.youtube.com/watch?v=CmtLYQKT21I&feature=related Mitochondrial DNA Mitochondria = responsible for cellular respiration 1960’s – discovered that mitochondria contains its own DNA (mtDNA) They have an amount of control over what they do (not completely controlled by nuclear DNA) Endosymbiotic Hypothesis Mitochondria once were free living bacteria that were engulfed by other cells. The two cells developed mutualistic relationship (mitochondria had protection and food, engulfing cell had a source of energy and oxygen) Evidence to support theory MtDNA resembles the loops of DNA found in bacteria and viruses 2) The mtDNA is tiny compared to nuclear DNA 3) Mitochondria divide and replicate independently of the cell itself * the same theory is used to explain how photosynthetic cells gained chloroplasts 1) NOTE: The mtDNA in our bodies is maternal because sperm’s mitochodria are lost when their tail falls off.