Molecular Genetics and Inheritance Overview

Biology 40S Unit 2: Molecular Genetics – Mechanisms of Inheritance At the end of this unit you should be able to: * Outline significant scientific contributions/discoveries that led to our understanding of the structure and function of the DNA molecule. Timeline, individual contributions, multidisciplinary collaboration, competitive environment… * Describe the structure of a DNA nucleotide, including deoxyribose sugar, phosphate group, and nitrogenous bases. * Describe the structure of the DNA molecule, including double helix, nucleotides, base-pairing and genes. * Describe the process of DNA replication, including template, semi-conservative replication, and the role of enzymes. * Compare DNA and RNA in terms of their structure, use and location in the cell. * Outline the steps involved in protein synthesis, including mRNA, codon, amino acid, transcription, tRNA, anticodon, ribosome and translation. * Relate the consequences of gene mutation to the final protein product. Examples include sickle-cell anemia * Discuss implications of gene mutation to genetic variation, including source of new alleles. * Investigate an issue related to the application of gene technology in bioresources. Include the understanding of the technology/processes involved, the economic implications, a variety of perspectives and the personal/societal and global implications. * Investigate an issue related to the application of gene technology in humans. Include the understanding of the technology/processes involved, the ethical and legal implications, a variety of perspectives and the personal/societal and global implications. UNDERSTANDING DNA: The Molecule of Life What are genes? A gene is any given segment along the DNA that encodes instructions that allow the cell to produce a specific product (typically a protein, such as an enzyme that initiates one specific action or a trait that is seen in our phenotype) How do genes work? * Although each cell contains a full complement of DNA, cells use genes selectively. * A “full compliment of DNA” is called a genome which refers to the entire set of genetic information contained within an organism’s DNA * cDNA * Some genes enable cells to make proteins needed for basic functioning (housekeeping genes) and are active in many types of cells. * Other genes, however, are inactive most of the time. * Some genes play a role in the development of an embryo and then are shutdown forever. * A normal cell activates just the genes it needs at the moment and actively suppresses the rest. NUCLEIC ACIDS In 1869, a biochemist named Fredrich Meischer (Swiss Physicist) discovers nucleic acids found in the cell’s nucleus. He isolated a non-protein substance from the nucleus of cells (pus from white blood cells on hospital bandages) and named this substance nuclein. He found that the substance was high in phosphorus. What Meischer actually isolated was nucleic acids (DNA). What is a nucleic acid? * Organisms store information about the structures of their proteins in macromolecules called nucleic acids. * A macromolecule is a molecule containing a very large number of atoms, such as protein, nucleic acid, or synthetic polymers * Organisms store and use hereditary information by coding the sequence of the amino acids of each of their proteins as a sequence of nucleotides in nucleic acids. * Amino acids are molecules that combine to form proteins * Nucleic acids are long polymers of repeating subunits (monomers) called NUCLEOTIDES. * A polymer is a large molecule made up of many smaller molecules called monomers that are linked together Nucleic acids form a code that contains the instructions a cell uses to form thousands of compounds needed for life processes. THREE important nucleic acids are: * Deoxyribonucleic acid (DNA) * Ribonucleic acid (RNA) * Adenosine Triphosphate (ATP). (We will not study this one – think back to grade 11 biology!!) What is the composition of a nucleotide? Each nucleotide is made up of three smaller units. Illustration of a simplified nucleotide: Illustration of Molecular Structures within Nucleotide: nucleotide 1. FIVE CARBON SUGAR -forms the central part of the nucleotide http://distancelearning.ksi.edu/demo/bio378/DNA_files/image004.gif TWO types of sugars: 1. ribose sugar 2. deoxyribose sugar (only difference is this sugar type has one less oxygen) http://distancelearning.ksi.edu/demo/bio378/DNA_files/image002.gif 2. PHOSPHATE GROUP -contains phosphoric acid and is located at the end of the sugar group *Note the negative charges on the phosphate group 3. NITROGEN BASE -found at the opposite end of the sugar from the phosphate group Two groups of nitrogen bases are found in nucleotides: http://www.bio.miami.edu/tom/courses/protected/MCB6/ch02/2-17.jpg i) Pyrimidine (single carbon-nitrogen ring) * two examples of pyrimidines are cytosine and thymine ii) Purine (double carbon -nitrogen ring) * two examples of purines are adenine and guanine http://www.bio.miami.edu/tom/courses/protected/MCB6/ch02/2-17.jpg Amoeba Sisters – DNA Structure and Function: (8:58) https://www.youtube.com/watch?v=_POdWsii7AI STRUCTURE OF DNA: By the 1950s it was known that DNA was a polymer of nucleotides. * The four nucleotides that make up DNA differ only in their nitrogenous bases. (adenine, guanine, cytosine, and thymine). http://www.mun.ca/biology/scarr/CG_ATGC.gif 1950: ERWIN CHARGAFF He noted that in DNA from all species tested, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine. In other words, the total abundance of purines equals the total abundance of pyrimidines, even though the actual proportions of each base vary in different species. QUESTION: Determine the amount of each nitrogen base present in a DNA molecule, if cytosine is found to be 16%. C = 16% therefore G = 16% (because they are equal) 100-16-16 = 68% (all remaining bases must be A and T) Rosalind Elsie Franklin 68/2 = 34 % (because they are also equal) A = 34 and T = 34% 1953: ROSALIND FRANKLIN (AND MAURICE WILKINS) * Franklin wanted to know what the DNA molecule looked like. She used X-ray Diffraction to find out. From the pattern that was created when the molecule was bombarded by x-rays, she suggested that the DNA is highly regular (repetitive) and uniform in shape. She also proposed that DNA was a helix of some sort. dna * produced an x-ray diffraction pattern of DNA that suggested it was in the shape of a double helix 1962: JAMES WATSON AND FRANCIS CRICK * From all the previous work on DNA, Watson and Crick knew that DNA was a linear molecule, with a sugar –phosphate backbone and the 4 nitrogenous bases were attached to the backbone * From Chargaff’s Rule they knew that every species have equal amounts of A and T, and C and G. * From Franklin’s work, they concluded that a DNA molecule consists of 2 strands attached together * They concluded that DNA is a double helix with 2 chains of nucleotides oriented in opposite directions THE MODEL OF DNA BASED ON THESE IDEAS SHAPE: DNA exists as two long, paired strands spiralled into the famous double helix. (twisted ladder) COMPOSITION: each strand is made up of millions of nucleotides. 3 COMPONENTS of a DNA nucleotide: i) SUGAR: deoxyribose ii) PHOSPHATE molecule 🡪the sugar and phosphate molecules form the backbone of the ladder 🡪Nucleotides are joined by covalent bonds to form the sides of the structure iii) NITROGEN BASES: There are FOUR different nitrogen bases in DNA that form the rungs of the ladder 1. adenine (A) 2. thymine (T) 3. cytosine (C) 4. guanine (G) The order of the bases determines the information available. (Compare to the letters of the alphabet combining to form words and sentences) * Each nitrogen base has a complimentary base pair. Adenine always bonds with Thymine Guanine always bonds with Cytosine * The nitrogen bases face inward and are connected together by WEAK HYDROGEN BONDS, like the rungs of a ladder. * DNA is composed of two chains of nucleotides lined up in opposite directions to each other. This is called ANTI-PARALLEL. * At the end of the backbone, where phosphate group is attached to the sugar at the carbon 5 position, we label it as 5’ or the “FIVE PRIME” end. sugar * The other end exposes the carbon 3 position, therefore it is labelled as the 3’ or the “THREE PRIME” end. * One strand runs 5’ to 3’, while the other runs 3’ to 5’. MODEL OF DNA http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular biology/dsDNA.jpg dna The two stands are twisted together to form a stable, DOUBLE HELIX. What is DNA and how does it work? (5:23 min) https://www.youtube.com/watch?v=zwibgNGe4aY or https://www.youtube.com/watch?v=YovwL0LwkUg (5:22 min) PRACTICE QUESTIONS SET #1: NUCLEIC ACIDS and DNA REPLICATION OF DNA REPLICATION is the process of copying DNA in the cell to produce new molecules with the same base sequence. * During replication, a single strand of nucleotides acts as a template for the formation of a complementary strand. A single strand of DNA can make a complementary strand. * Replication helps explain how one cell can divide into two identical cells, each with complete sets of DNA (genetic material) DNA replication is a SEMI-CONSERVATIVE PROCESS because half of the parental molecule is maintained (conserved) in each daughter molecule. Each molecule formed by replication consists of one new strand and one old strand conserved from the parent DNA molecule. * One strand serves as the template for the second strand. DNA replication is initiated at a region on a chromosome called an origin of replication. STEPS of DNA REPLICATION STEP 1: The enzyme DNA HELICASE unwinds or “unzips” the DNA double helix and separates the DNA into single stands by breaking hydrogen bonds between nitrogen bases. The junction between the unwound part and the open part is called a replication fork. The helicase enzyme binds to the origin and unwinds the DNA in both directions from the origin. As helicase moves along the DNA molecule, weak hydrogen bonds that hold complementary nitrogen bases together are broken, and the two edges of the ladder “unzip” separating the DNA and exposing the nucleotides. This leaves two strands of phosphates and sugars each with half the nitrogen bases. Additional single stranded DNA binding proteins (SSB) attach themselves to the individual separated strands, holding them apart and preventing them from coiling upon themselves. Illustration of Replication Fork: DNA molecule acts as a template for the synthesis of duplicate strands of DNA. STEP 2: The enzyme RNA PRIMASE adds a short length of RNA to the template strand of DNA. This acts as a primer allowing DNA Polymerase III to bind and begin replication. STEP 3: The exposed single strands act as templates for new strands. The enzyme DNA POLYMERASE III adds “free” DNA nucleotides to their complimentary bases on the DNA template. * Hydrogen bonds form between the free nucleotide and bases on the parent strand. The nucleotides (sugar and phosphate) are linked up to form the new strand. * Free nucleotides are present in large numbers around the replication fork. DNA polymerase III travels from the 3' to the 5' end of the parent strand, so adds bases 5’ to 3’ * DNA polymerase III also adds complementary nucleotides on the other side of the ladder, traveling in the opposite direction. http://bioserv.fiu.edu/%7Ewalterm/GenBio2004/chapter11_DNA/figure%2011-17.jpg NOTE: * One side is the leading strand (continuous)- it follows the helicase as it unwinds. * The other side is the lagging strand (discontinuous)– it’s moving away from the helicase * Problem: DNA polymerase III reaches the replication fork, but the helicase is moving in the opposite direction. It stops, and another DNA polymerase III binds farther down the chain. STEP 4: On the lagging strand; short lengths of DNA are formed between RNA primers. These fragments are called Okazaki Fragments. http://www.biologycorner.com/resources/DNAReplicationFork.gif The enzyme DNA POLYMERASE I removes the RNA primer and replaces it with DNA. A gap is left where the two Okazaki fragments are still unconnected. STEP 5: Final step involved, the enzyme DNA LIGASE to seal up the gap between Okazaki fragments by making a sugar-phosphate bond and making a complete DNA molecule on the lagging strand. DNA STRUCTURE & DNA REPLICATION Video: https://www.youtube.com/watch?v=8kK2zwjRV0M DNA REPLICATION VIDEO: https://www.youtube.com/watch?v=Qqe4thU-os8 RESULTS OF REPLICATION The process of replication produces two new molecules of DNA. The DAUGHTER DNA MOLECULES each rewind into a double helix. http://uoitbiology12u2014.weebly.com/uploads/2/5/8/3/25832527/5490185.png?452 NOTE: During replication, there are many points along the DNA that are synthesized at the same time. Multiple replication forks or replication “bubbles” occur. The parental DNA strands open up as daughter strands elongate on both sides of each bubble. Hundreds or thousands of bubbles can be present at once, reducing the total time needed for replication. Eventually all the bubbles fuse and form two complete daughter DNA molecules. MISTAKES IN REPLICATION * A change in the nucleotide sequence even at one location, called a mutation, may have serious effects on the cell. * The number of errors and MUTATIONS in DNA replication is reduced as enzymes proofread DNA and repair errors. Permanent damage is prevented by enzymes that act as PROOFREADERS. These enzymes called NUCLEASE run along the strands of DNA looking for mismatched pairs. Once a damaged section is detected, it can be repaired. Another enzyme snips the error from the chain, and replaces it with the correct nucleotide sequence. * Although DNA replication and proofreading prevent replication errors, some errors still do occur. * In addition DNA can be damaged by a variety of environmental factors and agents, including hazardous chemicals, and ultraviolet radiation from the sun. These factors can cause uncomplimentary nitrogen bases to become paired. PRACTICE QUESTIONS: NUCLEIC ACIDS AND DNA REPLICATION BASIC STRUCTURE OF RNA Although RNA is similar in structure to DNA, it has THREE striking differences: Description: This is a basic structure of a RNA molecule * Single stranded nucleic acid * Sugar molecule is ribose * No thymine nitrogen base but uracil nitrogen base works in its place. Cells contain THREE TYPES OF RNA, and each plays a special role in the manufacturing of proteins. 1. Messenger RNA or mRNA 2. Transfer RNA or tRNA 3. Ribosomal RNA or rRNA TYPES OF RNA Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) FUNCTION Carries the genetic information copied from DNA in the form of a series of three-base code “words,” each of which specifies a particular amino acid. i) Carries specific amino acids to mRNA to help assemble proteins. ii) Positions each amino acid at the correct place on the elongating protein chain. Ribosomal RNA (rRNA) is associated with a set of proteins to form ribosomes. Ribosomes move along an mRNA molecule, in the assembly of amino acids into protein chains. LOCATION IN THE CELL In the nucleus on DNA, copying the code and in the cytoplasm on ribosomes transferring the code In cytoplasm near or on ribosomes Inside ribosomes (in cytoplasm) which are special protein making factories. STRUCTURE A linear single strand of RNA made up of ribose sugar, phosphate and nitrogen bases (Adenine, uracil, guanine, and cytosine) Note: A sequence of 3 N-Bases that code for an amino acid on mRNA is called a CODON tRNA molecules have a characteristic cloverleaf shape with the open end carrying an amino acid and the opposite end with the exposed Nitrogen bases at one lobe is the ANTICODON. Note: Anticodon is a nucleotide triplet with a sequence of N-Bases that are complementary to the codon. Ribosomes are specialized structures in the cell that bring together the mRNA strand and tRNA carrying amino acids. rRNA found inside the ribosome is a large linear strand of RNA that always stays bound to proteins within ribosomes. Simplified Illustration A sequence of 3 N-bases found on mRNA strand are called a CODON. A sequence of 3 N-Bases found on a tRNA molecule are called an ANTICODON. rRNA is found inside Ribosome Ribosome is made up of 2 subunits (large unit and small unit that fit together to produce an active ribosome) DNA vs. RNA amoeba sisters https://www.youtube.com/watch?v=JQByjprj_mA Protein Synthesis _ The sequence of nucleotides in DNA contain information. This information is used to produce proteins. This is called the genetic code. * Proteins become important in making cell structures and regulators of cell functions. * Proteins become muscle tissue, blood cells, skin cells, nerve tissue, form enzymes, hormones, antibodies, etc... * The proteins within your body are made up of amino acids. * Only 20 different kinds of amino acids exist in your body. These 20 amino acids can be linked together in a certain order to make each different protein in our body. * The bonds linking amino acids together are called peptide bonds. A chain of amino acids forming a protein is called a polypeptide chain. This is due to the many peptides in the chain. * A simple protein might be composed of as few as eight amino acids. * A complex protein may contain in excess of 50000 amino acids. * Each different protein contains different numbers of the 20 amino acids arranged in a specific order. * DNA determines how amino acids are strung together and how proteins are made. The sequence of amino acids is determined by the sequence of nucleotides in the DNA. * By encoding the instructions for making proteins, DNA controls the cell. The sequencing of the amino acids is regulated by the DNA. * A gene is a segment of DNA that controls the production of a protein. How does the DNA molecule provide directions for the construction of the proteins? * DNA contains the instructions for linking the amino acids together to make all the different kinds of proteins. Nitrogen bases on a DNA molecule determine the amino acids in the protein. * Although the length of DNA molecule varies from one organism to the next, the chemical composition seems identical. All organisms’ DNA is made up of nucleotides(sugar, phosphate, nitrogen base) * Each chromosome has a deoxyribose sugar backbone linked with a phosphate molecule, and only four different nitrogen bases. * The sequencing of the nitrogen bases provided the chemical code. How is the genetic code written? Description: printImage * One nucleotide alone cannot code for an amino acid, a group of three nucleotides must be used to identify the only 20 different amino acids. The table shows the full set of RNA codons and their corresponding amino acids. There are is more than one codon for the 20 amino acids. Description: aa_codon_table To read the table: Find the first letter of the RNA codon in the column titled “first letter”, then read across the rows in the column titled “second letter” to find the second letter of the codon. Finally read down the column titled “third letter” to find the last letter of the codon. This will indicate the amino acid that corresponds to that codon. EXAMPLES: 1. The RNA codon GAG codes for the amino acid glutamate 2. The RNA codon CAU codes for the amino acid histidine 3. The RNA codon CUC codes for the amino acid leucine * Three CODONS will also code for a “START” codon or a “STOP” codon which indicate the beginning and the end of protein synthesis. These codons can also be called promotors and terminators. * The only start codon is AUG called methionine * There are three stop codons: UAA,UAG, and UGA that do not code for an amino acid. * There are ONLY 20 amino acids available for a cell to use in the synthesis of proteins, BUT an amino acid can be coded for by more than one kind of triplet. (43= 64 possible combinations) * Example: Both the triplets CUC and UUG code for the amino acid leucine. NOTE: Six different triplets code for the amino acid arginine. Find them: _CGU_, __CGC__, ___CGA__, __CGG__, ___AGA__, ___AGG___. By convention, the genetic code is always presented in terms of the RNA codon rather that the nucleotide sequence of the original DNA strand. Practice Naming codons: 1. AAA Lysine 2. GCC Alanine 3. UGG Tryptophan 4. AUG Methionine 5. UAA Stop Example One: Determine the complementary strand of DNA, the mRNA codon, tRNA anticodon, and the amino acids found in the protein for the following: DNA template: TAC GAG AAT GTC GGT Complementary DNA strand: ATG CTC TTA CAG CCA mRNA strand: AUG CUC UUA CAG CCA tRNA molecules: UAC GAG AAU GUC GGU Amino acids assembled: methionine leucine, leucine, glutamine, proline Example Two: For the DNA strand given, write out the complementary DNA strand, the mRNA strand that would be produced from the original template through transcription, then complete the tRNA strand, and the amino acids found in the protein that would form. DNA template: TAC AAT GGC TGT CCC ATT Complementary strand: ATG TTA CCG ACA GGG TAA mRNA AUG UUA CCG ACA GGG UAA strand: tRNA UAC AAU GGC UGU CCC AUU molecules: Amino acids: methionine, leucine, proline, threonine, glycine, stop PRACTICE QUESTIONS SET #3: RNA & INTRODUCTION TO PROTEIN SYNTHESIS Protein synthesis amoeba sisters https://www.youtube.com/watch?v=oefAI2x2CQM PROCESS OF PROTEIN SYNTHESIS The process of making a protein involves two stages. The first stage: TRANSCRIPTION: Information from the DNA gene is copied(transcribed) onto an mRNA strand in the nucleus of the cell. DNA → mRNA The second stage: TRANSLATION: The mRNA strand arrives at the ribosome, and a protein (polypeptide chain) is synthesized (translated). mRNA → protein STAGE 1: TRANSCRIPTION The objective of transcription is to make an accurate copy of a small piece of an organism's genome. The information in DNA is copied onto an mRNA molecule. transcription Transcription of the DNA gene instruction onto mRNA strand keeps the DNA safely in the nucleus. STEPS in TRANSCRIPTION 1. RNA polymerase enzyme binds to the double helical DNA at the promoter. 2. DNA strand is unwound at promoter and the double helix is disrupted exposing the template strand and stops at the terminator. 3. mRNA is synthesized from the template strand as free RNA nucleotides join with complementary DNA nucleotides. Example DNA strand: AGC TAA CCG mRNA strand: UCG AUU GGC The base pairing during transcription is the same as when DNA replicates, except RNA has uracil instead of thymine. 4. RNA nucleotides (the phosphate molecule of one nucleotide and ribose sugar molecule of the next nucleotide) bond together with a covalent bond using the enzyme RNA polymerase. 5. RNA synthesis stops when the RNA polymerase reaches a terminator on the DNA, and the RNA strand forms, the hydrogen bonds between the complementary nitrogen bases (one from the DNA strand, and the other from the RNA strand) are broken and the molecule of messenger RNA (mRNA) is freed into the nucleoplasm. * mRNA and RNA polymerase are both released from the DNA strand. 6. After the newly transcribed RNA strand detaches from the DNA, the two strands of the original DNA then rejoin. NOTE: The base sequence in the mRNA is an exact complement of the base sequence in the DNA template strand. 7. INTRONS/EXONS: * Further modifications need to be made to the mRNA before it leaves the nucleus to code for a protein. * The initial mRNA transcripts are made up of coding regions called EXONS and noncoding regions called INTRONS. * The introns are inter-dispersed among the exons, therefore the primary mRNA transcript will contain sections of nucleotides that do not code for a part of the protein. * Introns must be removed before the mRNA can be used. Function of introns is not well understood. Some think they might play a role in the evolution of proteins, or perhaps a structural component. They are also referred to as satellite DNA. http://www.emc.maricopa.edu/faculty/farabee/biobk/exintrons.gif * The introns are removed and the exons are joined together (spliced together), producing a mature RNA with a continuous coding sequence. 8. MOVEMENT OUT OF NUCLEUS: The DNA code is now carried by the single stranded mature mRNA molecule (made of exons) out of the nucleus through a nuclear pore to the ribosomes in the cytoplasm to be translated into proteins. STAGE 2: TRANSLATION TRANSLATION: is the process by which proteins are synthesized using the DNA instruction encoded on the mRNA. (Converting information from mRNA into a protein) http://0.tqn.com/d/biology/1/0/q/translation2.gif In the process of translation, mRNA must work with tRNA (that carries the amino acids to assemble the protein). Protein synthesis occurs on the ribosomes that consist of two parts and contain rRNA that direct protein synthesis and bring mRNA and tRNA together. INITATION: 1. As translation begins, the start codon end on mRNA strand attaches itself to the ribosome, much like a ribbon tied onto a package. The two subunits (large and small) of the ribosome come together to form an active ribosome. The sequence of the amino acids is determined by the message carried from the DNA in the nucleus by the mRNA molecule. The ribosome has binding sites for both the mRNA and tRNA holding the molecules close together. 2. tRNA molecule carrying an amino acid, which is circulating within the cytoplasm, pairs up with the first exposed codon on the mRNA strand. The first codon is a “start” codon called methionine. 3. Anticodon on tRNA match up with codon on mRNA strand and the two molecules temporarily join together. Example: mRNA codon = AUG tRNA anticodon = UAC 4. A second loaded tRNA molecule arrives at the adjacent codon to the first tRNA. 5. Enzymes catalyze the formation of a PEPTIDE BOND that joins the amino acid carried by the first tRNA to the amino acid carried by the second tRNA. At the same time, the polypeptide chain is transferred to the second tRNA. 6. The tRNA from the first codon will now detach from the ribosome and search for another amino acid that fits its structure. (It is now an unloaded tRNA and is moving around the cytoplasm in search of a complimentary AA) 2. ELONGATION: 7. The ribosome moves a distance of one codon along the mRNA strand. A third tRNA molecule arrives at the exposed codon next to the second tRNA and the cycle repeats, forming a growing polypeptide chain. https://www2.estrellamountain.edu/faculty/farabee/BIOBK/ELONGATION3.gif 3. TERMINATION: 8. As the process continues, a chain of amino acids is formed until the ribosome reaches a “stop” codon or terminator codon on the mRNA, which turns off the synthesis and the ribosome stalls. No amino acid is added to the polypeptide chain. The three “stop” codons are UGA, UAG, and UAA. Since these codons do not code for an amino acid, there are no corresponding tRNA molecules. 9. The complete polypeptide chain or protein is now released from the ribosomeand exported (transported) to desired location in the body, and protein synthesis is complete. http://mol-biol4masters.masters.grkraj.org/html/Protein_Synthesis2-Prokaryotic_Mechanism_of_Synthesis_files/image002.jpg PRACTICE QUESTIONS #4: PROTEIN SYNTHESIS GENE MUTATION GENE MUTATIONS: Sometimes changes in the DNA code occur during replication or protein synthesis, and the traits or proteins in an organism change. Mutations change the sequence of the nitrogen bases in a gene, which changes the structure of a protein that the gene codes for. The mutation may produce a new trait or it may result in a protein that does not work properly resulting in structural or functional problems in cells of organisms. If a mutation is severe, an organism may not survive, or it may produce life threatening effects. In some cases gene mutations have positive effects. An organism may receive a mutation that makes it faster or stronger which increases the organisms’ chances of survival. This plays an important role in evolution, and it adds variation to a species. Some gene mutations result from a change involving many nucleotides within a gene, and some involve only one nucleotide. Mutations can be passed on to offspring Mutations can have a wide range of consequences: from no effect to lethal. EXAMPLES of mutagens 1. high radiation(X-rays, ultra violet radiation, gamma rays) 2. Radioactive material(uranium, plutonium) 3. Mustard gas, some insecticides 4. LSD (causes deletions) 5. Certain chemicals (nitrous acid) 6. Air pollutants(tobacco tars, smog) 7. viruses(HIV/AIDS) WHAT WOULD HAPPEN TO THE TRANSLATION PROCESS IF ONE NUCLEOTIDE WERE NOT TRANSCRIBED CORRECTLY? I) the mRNA is not translated because the start codon was affected II) one amino acid is incorrectly translated (point mutation) III) all amino acids after the error are affected and entirely different protein or no protein is produced(frameshift mutation) IV) there is no effect if the nucleotide affected was not between the start and stop codon, or was in an intron that was removed or was replaced with a nucleotide that still coded for the same amino acid as the original nucleotide in the sequence. 2 MAIN TYPES of GENE MUTATIONS 1. POINT MUTATIONS: Gene mutation involving a substitution of a nucleotide that affects only one nucleotide on the DNA strand, and only one amino acid in the chain. Ie: Original DNA: THE FAT CAT ATE THE RAT Mutated DNA: TWE FAT CAT ATE THE RAT 2. FRAME SHIFT MUTATIONS: Gene mutation involving addition or deletion of nucleotide that affects the entire DNA strand after mutation, including all the amino acids in the chain. Ie: Original DNA: THE FAT CAT ATE THE RAT Mutated DNA: T E FAT CAT ATE THE RAT Results: TEF ATC ATA TET HER AT POINT MUTATIONS: Change in one single nucleotide in the DNA result in changes in one codon after mutation, and therefore only one amino acid is affected. Substitution: * One nucleotide is substituted for another one * This will affect only one codon and one amino acid. * In some cases the change may not affect the amino acid since different codons may code for the same amino acid. The replacement of a single amino acid may have no affect or change the protein. FRAMESHIFT MUTATIONS: Affects all codons after mutation, and therefore all amino acids, and changes large sections of the polypeptide chain. Frameshift mutation: deletion or addition of nucleotides, so that every codon beyond the point of insertion or deletion is read incorrectly during translation. Insertion or deletion: of one nucleotide. This alters every codon from the point of the mutation on. The situation may be much more serious. PRACTICE QUESTIONS SET #5: GENE MUTATION and PRACTICE QUESTIONS SET #6: DNA MUTATIONS GENETIC ENGINEERING DNA is at the very core of what gives animals and plants their uniqueness. Genetic techniques developed in the past few decades enable scientists to explore and manipulate DNA. TECHNIQUES INCLUDE: * Cutting and pasting genes to make new organisms-Gene Transfer/Recombinant DNA * Mapping DNA-Human Genome Project * Copying DNA in a lab (PCR) * Using DNA to reveal its owner’s identity-DNA Profiling (DNA fingerprinting) * Process of Gel Electrophoresis to separate DNA fragments * Cloning animals and cells WHAT IS GENETIC ENGINEERING? * Genetic engineering involves the deliberate and controlled manipulation of the genes in an organism with the intent of making that organism better in some way.(produce a desired human effect in an organism) * This is usually done independently of the natural reproductive process. The result is a so-called genetically modified organism (GMO). * To date, most of the effort in genetic engineering has been focused on agriculture. * Genetic engineering is also known as genetic modification. * It is accomplished by adding, deleting or modifying genes to a DNA molecule in order to change the information that it contains. * By changing this information, genetic engineering changes the type or amount of proteins an organism is capable of producing. GENETICALLY MODIFIED ORGANISMS (GMO) Flavr Savr Video (8mins): https://www.nytimes.com/2013/06/24/booming/you-call-that-a-tomato.html GMO is an organism that has had an artificial genetic change using the techniques of genetic engineering such as gene transfer or recombinant DNA. The process of transferring genes is called GENETIC MODIFICATION. Genetic Modification is successful because the genetic code is universal, so genes can be transferred from one organism to another, even if they are members of different species. A gene codes for a polypeptide with the same amino acid sequence, whether it is a human cell, a bacterium, or any other cell. TRANSGENIC ORGANISMS: Transgenic Organisms are genetically engineerd organisms resulting from the insertion of “foreign” DNA from another unrelated organism. * The transgenic organism will exhibit/express the desired trait. EXAMPLES of TRANSGENIC ORGANISMS: Transgenic Bacteria: * In medicine: When a gene coding for a human protein is inserted into a bacteria, the recombinant cells may produce large amounts of the protein. * Some examples: * Human growth hormone (used for normal growth and development, treatment of dwarfism) * Insulin (treatment of diabetes) * Interferon (hormone that blocks the growth of viruses and may be used in cancer treatment) * In industry: Recombinant bacteria have been engineered to breakdown pollutants into harmless products, cleanup oil spills Transgenic Plants: * The simplest kind of GM food is one in which an undesirable gene is removed. In some cases, another more desirable gene is put in its place but in other instances, only the introduction of a new gene is needed, no DNA has to be removed. * DNA can be injected into plant cells directly or attached to plasmids of bacteria that infect the plant. * Goals of transgenic plants include increased yields, and the production of natural insecticides, become disease resistant and produce natural fertilizers. * Some examples: * Canola- genetically altered variety low in saturated fats. * Potatoes- genetically altered to have superior nutrition (high carbohydrates and proteins) with a built in resistance to potato beetles. * Fruits and Vegetables-genetically altered to have enhanced vitamin and mineral levels. Transgenic Animals: * DNA can be introduced into animal reproductive cells in a number of ways, including direct injection. * Some transgenic animals will be useful in farming and ranching because they can produce bigger and faster growing animals, more efficient with their use of feed and more resistant to disease, research to investigate human immunity. Ex: Rainbow trout used for human food are genetically engineered with cloned growth hormone gene. Growth hormone allows the trout to grow larger, and increase its commercial value, while producing more food. Glowing mice: https://www.youtube.com/watch?v=n0UzdYRnMtY IS GENETIC ENGINEERING A GOOD OR A BAD THING? Are GMO’s good or bad Video (9mins): https://www.youtube.com/watch?v=7TmcXYp8xu4 Genetic engineering raises many profound social and ethical questions. ADVANTAGES: BENEFITS, PROMISES, AND HOPES FOR THE FUTURE 1. IMPROVING FOOD PRODUCTION: * longer shelf life, slower ripening produce, growing in nutrient poor soil, resistant to extreme weather and climate, crop improvement (bigger, nutrient richer) produce a “super plant” that excels at growth and survival. 2. CROP IMPROVEMENT: * Crops will produce their own pest-control substances,will be beneficial to the environment because fewer chemical pesticides will be needed. Insect and pest or bacteria protection gene inserted into several crops-tomato, corn, cotton, potatoes, which give farmers new tools for integrated pest management thereby reducing pesticide use. 3. LIVESTOCK IMPROVEMENT: * Sheep, chickens, cows, pigs, trout, salmon are engineering to grow larger, produce more milk, eggs, wool, and food for growing world population * Farmers can be more in control of what crops or livestock they produce. This method is quicker than selective breeding. 4. BIOFACTORIES and VACCINE DEVELOPMENT * GMO’s produce rare proteins for medications or vaccines could be, in the long run, less costly and produce less pollution than synthesizing such proteins in laboratories. The production of drugs: pharmaceutical manufacturing of insulin, growth hormone, and interferon can be helped to save lives. * Multipurpose vaccines are made using gene technology. 5. HUMAN GENE THERAPY: * alters DNA within cells of a living organism to treat or cure a disease. New genetic therapies are being developed to treat diseases such as cystic fibrosis, AIDS and cancer. 6. ENVIRONMENTAL CLEAN-UP: * by engineering bacteria to thrive on waste products of newspaper pulp and oil. 7. ALTERNATE FUEL SUPPLIES: * Modified yeast cells or agricultural crops can produce fuel for cars. It is projected that 30% of the world’s chemical and fuel needs could be supplied by renewable resources in the first half on the next century. DISADVANTAGES: HARMFUL EFFECTS, DANGERS, AND FEARS 1. ENVIRONMENTAL IMPACT: * Efforts to keep GM plants under control in well-defined areas have failed and pollen from GM crops has escaped to neighboring fields. * Accidental release into environment and elimination of natural populations. * Genes from GM plants could be integrated into wild species giving them an unnatural advantageover other species and an ability to take over the habitat. These “super plants” out compete all other native species in nature. * Insects that are not pests could be killed by insecticide. 2. TRANSGENES: * There is a danger that genes could cross species. GMO’s may spread to unintended organisms and no one knows the consequences of genes crossing species. * spread of antibiotic resistance amongst not-target organisms. 3. UNKNOWN LONG TERM EFFECTS: * No one knows the long term effects of GMO’s in the wild. 4. MONOPOLIES * large companies or monopolies may develop, with a dependence of developing countries on companies who are seeking control of world’s commercial seed supply with “terminator seeds” (seeds that produce plants that cannot produce seeds themselves) * small businesses such as small farming operations cannot compete with large GMO operations. Critics are worried that large portions of the human food supply will be controlled by a small number of large corporations. 5. FOOD SAFETY: * health effectsof food grown from genetically engineered crops, still too new to really know effects. * There are risks for allergies, if someone is not allergic to natural tomatoes but are allergic to GM tomatoes, they will need to know which one they are eating. There is not visible difference of the fruit, and food labeling is not always clear. HUMAN GENOME PROJECT Human genome project explained Video (5 mins): https://www.youtube.com/watch?v=AhsIF-cmoQQ The human GENOME is the sum of all the DNA, carried in an organism`s cells and has been estimated to consist of between 25000 and 30000 genes distributed among our 23 pairs of chromosomes that make up our DNA. The Human Genome Project was a joint international effort of thousands of researches from laboratories worldwide that began in 1993 and ended in 2003. Their goal was to find the location of all of the genes on the human chromosomes and the base sequence of the entire DNA that makes them up. (Approximately three billion base pairs that make up the human genome) The sequencing of the entire human genome will make it easier to study how genes influence human development * Among the project`s immediate findings was the discovery that the DNA of all humans is more than 99.9% identical. This means that all differences among individuals across humanity results from variations in fewer than 1000 nucleotides in each individual`s genome. * Human genome sequencing will allow researchers to pinpoint specific nucleotide sequences that are involved in gene expression. * Over the long term, the information from the sequencing of the human genome will provide geneticists with a better understanding of the relationship between the molecular structure of human genes and the biological mechanisms of gene function. Project has identified genes that may help cure or predict diseases. * Important in detection and identification of genetic disorders early and beginning treatment before birth. * Researchers have also begun to look for genes that might predispose individuals to other medical problems such as heart disease, diabetes, and cancer. * It will allow the production of new drugs based on DNA base sequences of genes or the structure of proteins coded for by these genes. RECOMBINANT DNA: In one of the most controversial areas of genetic engineering research, scientists are able to combine genes from unrelated species. This technology is called RECOMBINANT DNA (which is used to make transgenic organisms), where genes can be extracted from the nucleus of one organism and spliced into the chromosomes of a new organism. The donated genes affect the traits of the new organism and form transgenic organisms. The Steps of Recombinant DNA Usage: Step One: Use restriction enzymes to cut DNA: Isolate the foreign DNA containing the desired gene away from their surrounding genes. This is accomplished by using a restriction enzyme. * Restriction enzymes, originally discovered in bacteria, are proteins that act like “molecular scissors” to cut foreign DNA that has invaded the cell, in specific places. * All cells have restriction enzymes which can cut strands of DNA Examples of Restriction enzymes and recognition sequences: i. EcoR1 GAATTC (sticky ends) ii. BAM HI GGATCC (sticky ends) iii. Hae III GGCC (blunt ends) iv. HIND III AAGCTT (sticky ends) * There are 100’s of restriction enzymes capable of cutting DNA * Each restriction enzyme binds to a short sequence of DNA nucleotides known as the enzyme’s recognition sequence. Some enzymes have short recognition sequences of 4, 6 or 8 nucleotides, and some have longer recognition sequences of 16 or more nucleotides. * Some restriction enzymes cut straight across the DNA strand, leaving “blunt ends” and others cut in a jagged pattern, leaving “sticky ends” of unpaired nucleotides. * These sticky ends can then be used to ligate pieces of DNA to each other using the ligase enzyme or DNA glue. The ligase enzyme is part of the normal DNA repair system. It helps restore the fragmented chromosome. Step Two: DNA RECOMBINATION USING PLASMID VECTORS Combine the desired gene from the DNA fragment with a piece of DNA from the recipient organism. * DNA fragments cannot function on their own or do not readily become part of a host organism’s chromosomes. They must become part of the genetic material of a living organism before the genes can be activated. * DNA fragments may be combined with bacterial DNA so that later it can be inserted into a bacterial cell * Plasmids are small, circular pieces of DNA from a bacteria cell that can be used as VECTORS to carry segments of cellular DNA into host cells. Two biological vectors are bacteria and viruses. * Plasmids are removed from the bacterial cell and cut with the same restriction enzyme used to produce the DNA fragments. These cuts on the plasmid and the DNA fragments will have matching “sticky ends”and can be joined together using ligase enzyme. Recombinant DNA has now been produced. Step Three: DNA INSERTION OR CLONING Insert the recombinant DNA into the host organism and many copies or clones of the recombinant DNA are made. * After the foreign DNA has been spliced into the plasmid, the plasmid is transferred by micropipette or using a gene gun and shot into a cell of the host organism. (Host organism can be bacterial cell, yeast cell, plant cell or animal cell) * When the host cell prepares to divide, it copies the recombinant DNA along with its own DNA. The process of making extra copies of recombinant DNA is a form of cloning. * CLONES are genetically identical copies. Genetic Engineering How insulin is made: https://www.youtube.com/watch?v=DIM38NlkWEo Video: https://www.youtube.com/watch?v=FAMRQz7fOaE DNA FINGERPRINTING Gel electrophoresis: * Gel electrophoresis is a laboratory technique which is used to separate fragments of DNA according to size. * Enzymes are used to cut the very long strands of DNA into varying sized smaller fragments. The DNA fragments are placed into small wells (holes) in a thin sheet of agarose gel which are aligned along one end. * The gel is then placed in a buffering solution and an electrical current is passed across the gel. (positive on one side and negative on the other) * DNA, being negatively charged(due to phosphate group), moves to the positive terminal * Smaller fragments are less impeded by the gel matrix and move faster through the gel * The fragments are thus separated according to size http://www.vce.bioninja.com.au/_Media/gel_tank_med.jpeg Lab_5_gel_1999 * In the end fragments leave a banded pattern. * Size can be calculated (in kilobases) by comparing against a known industry standard Video: Biorad overview https://www.youtube.com/watch?v=vq759wKCCUQ What wells look like (1 minute): https://www.youtube.com/watch?v=tTj8p05jAFM https://vimeo.com/85839878 Amoeba sisters: https://www.youtube.com/watch?v=ZDZUAleWX78&t=2s POLYMERASE CHAIN REACTION In the POLYMERASE CHAIN REACTION, DNA is copied again and again to produce many copies of the original molecules. Millions of copies of DNA can be produced in a few hours. This is very useful when small quantities of DNA are found and large amounts are needed for analysis. DNA from semen, blood or other tissue or even long dead specimens can be amplified using PCR. DNA PROFILING OR DNA FINGERPRINTING: DNA PROFILING (DNA FINGERPRINTING) describes the process by which individuals can be identified and compared based on their DNA sequence or DNA profile. This process requires matching an unknown sample of DNA with a known sample to see if they correspond (make a match). When DNA fragments are cut with restriction endonucleases, fragments of different lengths can be resolved using gel electrophoresis and create the DNA Fingerprint. If, after separation by gel electrophoresis, the pattern of bands formed by two samples of DNA fragments are identical, it means that both sources came from the same individual. If patterns are similar, it means that the two samples are most probably from two related people. At a crime scene, DNA is everywhere. It is present in all kinds of evidence collected at the scene, including blood, hair, skin, saliva and semen. Scientists can analyze the DNA in evidence samples to see if it matches a suspect's DNA. HOW CAN DNA PROFILING BE USED TO IDENTIFY AN INDIVIDUAL?WHY DOES DNA PROFILING/ FINGERPRINTING WORK? 99.9 %of human DNA is the same in everyone, meaning that only 0.1%of our DNA is unique! Within the human genome there are two regions found on the DNA: 1. Coding regions called GENES (99.9 %) * that contain instructions for protein synthesis 2. Non-coding regions called SATELLITE DNA (0.1%) * where its function is thought to be related to structural component of DNA SATELLITE DNA * Satellite DNA varies greatly between different individuals in the number and length of repeats called short tandem repeats (STRs) * As individuals all have a different number of repeats in a given sequence of satellite DNA, they will all generate unique fragment profiles. * These different profiles or banding patterns can be compared using gel electrophoresis Example: AATTCCG - DNA sequence may repeat several times. This is the STR's. Every individual may differ in numbers of these repeating units. http://www.vce.bioninja.com.au/_Media/dna_profiling_2_med.jpeg You may have 5 repeating STR's between two genes, whereas the person next to you may have 6 and someone else may have 33, it varies widely. Since the introduction of this technology, experts agree that information obtained from DNA testing can identify an individual with an accuracy of 99.8%, which far exceeds other testing methods such as ABO blood typing. Before PCR (3 min) https://www.youtube.com/watch?v=DOWMFvVsG0c Lab analysis before DNA https://www.youtube.com/watch?v=nPVkooi8m9I GENE THERAPY Two Methods of Gene Therapy: 1. Gene Surgery: involves removing cells from individual and growing them in culture. These cells can be transformed with a correct gene and then injected back into the individual to help cure the disorder. 2. Gene Modification: Modifying viruses so that they cannot cause disease, and then attaching DNA containing a desired gene to the viral DNA. The patient is then infected with the viruses, which carry desired gene into cells and correct the genetic disorder. Drawback to gene therapy is that the gene does not stay active for long periods of time, or the cells themselves do not have long life spans, so the treatment must be repeated often. Although the field of gene therapy is still very new, extensive debate is already underway about some of the moral and ethical issues at stake. Human Genetic Engineering is the modification of an individual's genotype with the aim of choosing the phenotype of a newborn or changing the existing phenotype of a child or adult. It holds the promise of curing genetic diseases like cystic fibrosis and increasing the immunity of people to viruses. It is speculated that genetic engineering could be used to change physical appearance, metabolism, and even improve mental faculties like memory and intelligence, although for now these uses are relegated to science fiction, but could become a reality with “designer babies”. How Crispr Works (16 mins): https://www.youtube.com/watch?v=jAhjPd4uNFY Designer babies (5 mins): https://www.youtube.com/watch?v=k1a2larfMIA CLONED ANIMALS Cloning is the technique of producing identical copies of genes, cells or organisms. It is the production of genetically identical organisms from single cells. All members of a clone and their parents have the same DNA. NATURAL CLONING: asexual reproduction in animals or plants by fragmentation, binary fission, and budding Eg: starfish growing new body if it contains part of center disc, planaria fragmented their body parts to reproduce, identical twin babies.. etc. REPRODUCTIVE CLONING: * Definition: A procedure where a full living copy of an organism is made. * It is the genetic duplication of an existing organism especially by transferring the nucleus of a somatic cell of the organism into an enucleated oocyte (egg) (surrogate mother is needed) * Cloning is very useful if an organism has a desirable combination of characteristics and more organisms with the same characteristics are wanted. THERAPEUTIC CLONING: * Definition: A procedure where damaged tissues or organs are repaired or replaced with genetically identical cells that originate from undifferentiated stem cells. * Sometimes cloning is used to produce skin, or other tissues/organs used to treat a patient. PLANT AND ANIMAL CLONING * Most plants can be cloned quite easily from pieces of roots, stem or leaves. * Animals cannot be cloned in the same way from parts of their bodies. If animal embryos are divided up at an early stage into several pieces, each piece can develop into a separate animal. (this happens naturally when identical twins are formed) ADULT MAMMAL REPRODUCTIVE CLONING Video:Story of Dolly (13 mins) https://www.youtube.com/watch?v=tELZEPcgKkE The first successful reproductive cloning of an adult with known characteristics was in 1997 in Scotland, where the first cloned adult mammal was of a sheep called “Dolly”. As scientists study how these cloned animals develop, evidence is mounting that a number of problems may be associated with animal cloning. Dolly for example showed signs of premature aging. Other cloned animals have shown problems with gene expression. THERAPEUTIC CLONING IN HUMANS Techniques are being developed to create human embryos, from which embryonic stem cells can be obtained for medical use. These stem cells have the capacity to divide and differentiate into any types of human cell. They could be used to replace tissues or even organs that have become damaged or lost in a patient. There are many ethical issues involved and research into therapeutic cloning has been banned in some countries. The use of embryonic stem cells could potentially be used for * Growing skin to repair a serious burn * Growing new heart muscle to repair an ailing heart * Growing new kidney tissue to rebuild a failing kidney Video: Ghost heart https://www.youtube.com/watch?v=j9hEFUpTVPA Ghost heart (5mins): https://www.youtube.com/watch?v=pd3TFB0wOI0 Organ transplant: https://www.youtube.com/watch?v=NMe_fOKKC24 HUMAN CLONING: * Researchers involved in human cloning distinguish between therapeutic cloning and reproductive cloning. * In all countries in the world, the cloning of human beings is illegal, this is due to ethical and religious reasons. * The potential benefits of these processes must be weighed against legal, moral and ethical issues. PRACTICE QUESTIONS SET #7: GENETIC ENGINEERING Page |

Biology 40S

Unit 2:
Molecular Genetics – Mechanisms of Inheritance

At the end of this unit you should be able to:

* Outline significant scientific contributions/discoveries that led to our understanding of the structure and function of the DNA molecule.  Timeline, individual contributions, multidisciplinary collaboration, competitive environment…
* Describe the structure of a DNA nucleotide, including deoxyribose sugar, phosphate group, and nitrogenous bases.
* Describe the structure of the DNA molecule, including double helix, nucleotides, base-pairing and genes.
* Describe the process of DNA replication, including template, semi-conservative replication, and the role of enzymes.
* Compare DNA and RNA in terms of their structure, use and location in the cell.
* Outline the steps involved in protein synthesis, including mRNA, codon, amino acid, transcription, tRNA, anticodon, ribosome and translation.
* Relate the consequences of gene mutation to the final protein product.  Examples include sickle-cell anemia
* Discuss implications of gene mutation to genetic variation, including source of new alleles.
* Investigate an issue related to the application of gene technology in bioresources.  Include the understanding of the technology/processes involved, the economic implications, a variety of perspectives and the personal/societal and global implications.
* Investigate an issue related to the application of gene technology in humans.  Include the understanding of the technology/processes involved, the ethical and legal implications, a variety of perspectives and the personal/societal and global implications.

UNDERSTANDING DNA: The Molecule of Life

What are genes?
A gene is any given segment along the DNA that encodes instructions that allow the cell to produce a specific product (typically a protein, such as an enzyme that initiates one specific action or a trait that is seen in our phenotype)

How do genes work?
* Although each cell contains a full complement of DNA, cells use genes selectively.  
   * A “full compliment of DNA” is called a genome which refers to the entire set  of genetic information contained within an organism’s DNA
   * cDNA
* Some genes enable cells to make proteins needed for basic functioning (housekeeping genes) and are active in many types of cells.
* Other genes, however, are inactive most of the time.
* Some genes play a role in the development of an embryo and then are shutdown forever.
* A normal cell activates just the genes it needs at the moment and actively suppresses the rest.

NUCLEIC ACIDS
In 1869, a biochemist named Fredrich Meischer (Swiss Physicist) discovers nucleic acids found in the cell’s nucleus. He isolated a non-protein substance from the nucleus of cells (pus from white blood cells on hospital bandages) and named this substance nuclein.  He found that the substance was high in phosphorus. What Meischer actually isolated was nucleic acids (DNA).

What is a nucleic acid?
* Organisms store information about the structures of their proteins in macromolecules called nucleic acids.
   * A macromolecule is a molecule containing a very large number of atoms, such as protein, nucleic acid, or synthetic polymers
* Organisms store and use hereditary information by coding the sequence of the amino acids of each of their proteins as a sequence of nucleotides in nucleic acids.
   * Amino acids are molecules that combine to form proteins
* Nucleic acids are long polymers of repeating subunits (monomers) called NUCLEOTIDES.  
   * A polymer is a large molecule made up of many smaller molecules called monomers that are linked together
Nucleic acids form a code that contains the instructions a cell uses to form thousands of compounds needed for life processes.

THREE important nucleic acids are:
* Deoxyribonucleic acid (DNA)
* Ribonucleic acid (RNA)
* Adenosine Triphosphate (ATP). (We will not study this one – think back to grade 11 biology!!)

What is the composition of a nucleotide?
Each nucleotide is made up of three smaller units.
Illustration of a simplified nucleotide:

Illustration of Molecular Structures within Nucleotide:
 nucleotide

1.  FIVE CARBON SUGAR
        -forms the central part of the nucleotide http://distancelearning.ksi.edu/demo/bio378/DNA_files/image004.gif 
        TWO types of sugars: 
1. ribose sugar
2. deoxyribose sugar (only difference is this sugar type has one less oxygen)
 http://distancelearning.ksi.edu/demo/bio378/DNA_files/image002.gif

2. PHOSPHATE GROUP
-contains phosphoric acid and is located at the end of the sugar group
*Note the negative charges on the phosphate group

3.  NITROGEN BASE
-found at the opposite end of the sugar from the phosphate group

Two groups of nitrogen bases are found in nucleotides: http://www.bio.miami.edu/tom/courses/protected/MCB6/ch02/2-17.jpg 
i)        Pyrimidine (single carbon-nitrogen ring)
* two examples of pyrimidines are cytosine and thymine

ii)        Purine (double carbon -nitrogen ring)
* two examples of purines are adenine and guanine
 http://www.bio.miami.edu/tom/courses/protected/MCB6/ch02/2-17.jpg

Amoeba Sisters – DNA Structure and Function: (8:58)
https://www.youtube.com/watch?v=_POdWsii7AI

STRUCTURE OF DNA:
By the 1950s it was known that DNA was a polymer of nucleotides. 
* The four nucleotides that make up DNA differ only in their nitrogenous bases. (adenine, guanine, cytosine, and thymine).  http://www.mun.ca/biology/scarr/CG_ATGC.gif

1950: ERWIN CHARGAFF 
He noted that in DNA from all species tested, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine. In other words, the total abundance of purines equals the total abundance of pyrimidines, even though the actual proportions of each base vary in different species.

QUESTION: 
Determine the amount of each nitrogen base present in a DNA molecule, if cytosine is found to be 16%.

C = 16% therefore G = 16% (because they are equal)
100-16-16 = 68%              (all remaining bases must be A and T) Rosalind Elsie Franklin 
68/2 = 34 %                            (because they are also equal)
A = 34 and T = 34%

1953: ROSALIND FRANKLIN (AND MAURICE WILKINS)
   * Franklin wanted to know what the DNA molecule looked like.  She used X-ray Diffraction to find out.  From the pattern that was created when the molecule was bombarded by x-rays, she suggested that the DNA is highly regular (repetitive) and uniform in shape.  She also proposed that DNA was a helix of some sort. dna 
   * produced an x-ray diffraction pattern of DNA that suggested it was in the shape of a double helix

1962: JAMES WATSON AND FRANCIS CRICK
      * From all the previous work on DNA, Watson and Crick knew that DNA was a linear molecule, with a sugar –phosphate backbone and the 4 nitrogenous bases were attached to the backbone
      * From Chargaff’s Rule they knew that every species have equal amounts of A and T, and C and G.
      * From Franklin’s work, they concluded that a DNA molecule consists of 2 strands attached together
      * They concluded that DNA is a double helix with 2 chains of nucleotides oriented in opposite directions

THE MODEL OF DNA BASED ON THESE IDEAS
SHAPE: DNA exists as two long, paired strands spiralled into the famous double helix. (twisted ladder)
COMPOSITION: each strand is made up of millions of nucleotides.

3 COMPONENTS of a DNA nucleotide:
i)        SUGAR: deoxyribose
ii)        PHOSPHATE molecule
🡪the sugar and phosphate molecules form the backbone of the ladder 
🡪Nucleotides are joined by covalent bonds to form the sides of the structure
iii)        NITROGEN BASES: 
        There are FOUR different nitrogen bases in DNA that form the rungs of the ladder
      1. adenine (A)
      2. thymine (T)
      3. cytosine (C)
      4. guanine (G)

The order of the bases determines the information available.  (Compare to the letters of the alphabet combining to form words and sentences)
      * Each nitrogen base has a complimentary base pair.
Adenine always bonds with Thymine
Guanine always bonds with Cytosine
      * The nitrogen bases face inward and are connected together by WEAK HYDROGEN BONDS, like the rungs of a ladder.
      * DNA is composed of two chains of nucleotides lined up in opposite directions to each other.  This is called ANTI-PARALLEL.
      * At the end of the backbone, where phosphate group is attached to the sugar at the carbon 5 position, we label it as 5’ or the “FIVE PRIME” end. sugar 
      * The other end exposes the carbon 3 position, therefore it is labelled as the 3’ or the “THREE PRIME” end.
      * One strand runs 5’ to 3’, while the other runs 3’ to 5’.

MODEL OF DNA
 http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular biology/dsDNA.jpg

dna

The two stands are twisted together to form a stable, 
DOUBLE HELIX.

What is DNA and how does it work? (5:23 min)
https://www.youtube.com/watch?v=zwibgNGe4aY

https://www.youtube.com/watch?v=YovwL0LwkUg     (5:22 min)

PRACTICE QUESTIONS SET #1: NUCLEIC ACIDS and DNA

REPLICATION OF DNA
REPLICATION is the process of copying DNA in the cell to produce new molecules with the same base sequence.
         * During replication, a single strand of nucleotides acts as a template for the formation of a complementary strand.  A single strand of DNA can make a complementary strand.
         * Replication helps explain how one cell can divide into two identical cells, each with complete sets of DNA (genetic material)

DNA replication is a SEMI-CONSERVATIVE PROCESS because half of the parental molecule is maintained (conserved) in each daughter molecule. Each molecule formed by replication consists of one new strand and one old strand conserved from the parent DNA molecule.
         * One strand serves as the template for the second strand. DNA replication is initiated at a region on a chromosome called an origin of replication.

STEPS of DNA REPLICATION
STEP 1: 
The enzyme DNA HELICASE unwinds or “unzips” the DNA double helix and separates the DNA into single stands by breaking hydrogen bonds between nitrogen bases. The junction between the unwound part and the open part is called a replication fork.

The helicase enzyme binds to the origin and unwinds the DNA in both directions from the origin. As helicase moves along the DNA molecule, weak hydrogen bonds that hold complementary nitrogen bases together are broken, and the two edges of the ladder “unzip” separating the DNA and exposing the nucleotides.  This leaves two strands of phosphates and sugars each with half the nitrogen bases.

Additional single stranded DNA binding proteins (SSB) attach themselves to the individual separated strands, holding them apart and preventing them from coiling upon themselves.

Illustration of Replication Fork:

DNA molecule acts as a template for the synthesis of duplicate strands of DNA.

STEP 2: 
The enzyme RNA PRIMASE adds a short length of RNA to the template strand of DNA.

This acts as a primer allowing DNA Polymerase III to bind and begin replication.

STEP 3: 
The exposed single strands act as templates for new strands. The enzyme DNA POLYMERASE III adds “free” DNA nucleotides to their complimentary bases on the DNA template.

* Hydrogen bonds form between the free nucleotide and bases on the parent strand. The nucleotides (sugar and phosphate) are linked up to form the new strand.

* Free nucleotides are present in large numbers around the replication fork. DNA polymerase III travels from the 3' to the 5' end of the parent strand, so adds bases 5’ to 3’

* DNA polymerase III also adds complementary nucleotides on the other side of the ladder, traveling in the opposite direction. http://bioserv.fiu.edu/%7Ewalterm/GenBio2004/chapter11_DNA/figure%2011-17.jpg

NOTE: 
            * One side is the leading strand (continuous)- it follows the helicase as it unwinds.

* The other side is the lagging strand (discontinuous)– it’s moving away from the helicase

* Problem: DNA polymerase III reaches the replication fork, but the helicase is moving in the opposite direction. It stops, and another DNA polymerase III binds farther down the chain.

STEP 4:
On the lagging strand; short lengths of DNA are formed between RNA primers. These fragments are called Okazaki Fragments. http://www.biologycorner.com/resources/DNAReplicationFork.gif

The enzyme DNA POLYMERASE I removes the RNA primer and replaces it with DNA. A  gap is left where the two Okazaki fragments are still unconnected.

STEP 5:
Final step involved, the enzyme DNA LIGASE to seal up the gap between Okazaki fragments by making a sugar-phosphate bond and making a complete DNA molecule on the lagging strand.

DNA STRUCTURE & DNA REPLICATION Video:
https://www.youtube.com/watch?v=8kK2zwjRV0M 
DNA REPLICATION VIDEO:
https://www.youtube.com/watch?v=Qqe4thU-os8

RESULTS OF REPLICATION
The process of replication produces two new molecules of DNA. The DAUGHTER DNA MOLECULES each rewind into a double helix. http://uoitbiology12u2014.weebly.com/uploads/2/5/8/3/25832527/5490185.png?452

NOTE: During replication, there are many points along the DNA that are synthesized at the same time. Multiple replication forks or replication “bubbles” occur.  The parental DNA strands open up as daughter strands elongate on both sides of each bubble. Hundreds or thousands of bubbles can be present at once, reducing the total time needed for replication.  Eventually all the bubbles fuse and form two complete daughter DNA molecules.

MISTAKES IN REPLICATION
            * A change in the nucleotide sequence even at one location, called a mutation, may have serious effects on the cell.

* The number of errors and MUTATIONS in DNA replication is reduced as enzymes proofread DNA and repair errors. Permanent damage is prevented by enzymes that act as PROOFREADERS. These enzymes called NUCLEASE run along the strands of DNA looking for mismatched pairs.  Once a damaged section is detected, it can be repaired.  Another enzyme snips the error from the chain, and replaces it with the correct nucleotide sequence.

* Although DNA replication and proofreading prevent replication errors, some errors still do occur.  
            * In addition DNA can be damaged by a variety of environmental factors and agents, including hazardous chemicals, and ultraviolet radiation from the sun. These factors can cause uncomplimentary nitrogen bases to become paired.

PRACTICE QUESTIONS: NUCLEIC ACIDS AND DNA REPLICATION
BASIC STRUCTURE OF RNA
Although RNA is similar in structure to DNA, it has THREE striking differences: Description: This is a basic structure of a RNA molecule 
            * Single stranded nucleic acid
            * Sugar molecule is ribose
            * No thymine nitrogen base but uracil nitrogen base works in its place.

Cells contain THREE TYPES OF RNA, and each plays a special role in the manufacturing of proteins.
            1. Messenger RNA or  mRNA
            2. Transfer RNA or  tRNA
            3. Ribosomal RNA or  rRNA

TYPES OF RNA
	Messenger RNA (mRNA)
	Transfer RNA 
(tRNA)
	Ribosomal RNA (rRNA)
	FUNCTION

Carries the genetic information copied from DNA
in the form of a series of three-base code “words,” each of which specifies a particular amino acid.
	i) Carries specific amino acids to mRNA to help assemble proteins.

ii) Positions each amino acid at the correct place on the elongating protein chain.

Ribosomal RNA (rRNA) is associated with a set of proteins to form ribosomes.

Ribosomes move along an mRNA molecule, in the assembly of amino acids into protein chains.
	LOCATION IN THE CELL
	In the nucleus on DNA, copying the code and in the cytoplasm on ribosomes transferring the code
	In cytoplasm near or on ribosomes

Inside ribosomes (in cytoplasm) which are special protein making factories.
	STRUCTURE
	A linear single strand of RNA made up of ribose sugar, phosphate and nitrogen bases (Adenine, uracil, guanine, and cytosine)
Note: A sequence of 3 N-Bases that code for an amino acid on mRNA is called a CODON

tRNA molecules have a characteristic cloverleaf shape with the open end carrying an amino acid and the opposite end with the exposed Nitrogen bases at one lobe is the ANTICODON.
Note: Anticodon is a nucleotide triplet with a sequence of N-Bases that are complementary to the codon.
	Ribosomes are specialized structures in the cell that bring together the mRNA strand and tRNA carrying amino acids.

rRNA found inside the ribosome is a large linear strand of RNA that always stays bound to proteins within ribosomes. 
	Simplified
Illustration

A sequence of 3 N-bases found on mRNA strand are called a CODON.

A sequence of 3 N-Bases found on a tRNA molecule are called an ANTICODON.

rRNA is found inside Ribosome

Ribosome is made up of 2 subunits
(large unit and small unit that fit together to produce an active ribosome)
	DNA vs. RNA amoeba sisters https://www.youtube.com/watch?v=JQByjprj_mA
Protein Synthesis                                                                  _
The sequence of nucleotides in DNA contain information.  This information is used to produce proteins.  This is called the genetic code.
            * Proteins become important in making cell structures and regulators of cell functions. 
            * Proteins become muscle tissue, blood cells, skin cells, nerve tissue, form enzymes, hormones, antibodies, etc...
            * The proteins within your body are made up of amino acids.
            * Only 20 different kinds of amino acids exist in your body.  These 20 amino acids can be linked together in a certain order to make each different protein in our body.  
            * The bonds linking amino acids together are called peptide bonds.  A chain of amino acids forming a protein is called a polypeptide chain. This is due to the many peptides in the chain.
            * A simple protein might be composed of as few as eight amino acids.  
            * A complex protein may contain in excess of 50000 amino acids.  
            * Each different protein contains different numbers of the 20 amino acids arranged in a specific order.
            * DNA determines how amino acids are strung together and how proteins are made.  The sequence of amino acids is determined by the sequence of nucleotides in the DNA.
            * By encoding the instructions for making proteins, DNA controls the cell.  The sequencing of the amino acids is regulated by the DNA.
            * A gene is a segment of DNA that controls the production of a protein.

How does the DNA molecule provide directions for the construction of the proteins?

* DNA contains the instructions for linking the amino acids together to make all the different kinds of proteins.  Nitrogen bases on a DNA molecule determine the amino acids in the protein.
            * Although the length of DNA molecule varies from one organism to the next, the chemical composition seems identical.  All organisms’ DNA is made up of nucleotides(sugar, phosphate, nitrogen base)
            * Each chromosome has a deoxyribose sugar backbone linked with a phosphate molecule, and only four different nitrogen bases.
            * The sequencing of the nitrogen bases provided the chemical code.

How is the genetic code written? Description: printImage 
            * One nucleotide alone cannot code for an amino acid, a group of three nucleotides must be used to identify the only 20 different amino acids.

The table shows the full set of RNA codons and their corresponding amino acids.  There are is more than one codon for the 20 amino acids. 
 Description: aa_codon_table

To read the table:
Find the first letter of the RNA codon in the column titled “first letter”, then read across the rows in the column titled “second letter” to find the second letter of the codon.  Finally read down the column titled “third letter” to find the last letter of the codon.
This will indicate the amino acid that corresponds to that codon.

EXAMPLES:
1. The RNA codon GAG codes for the amino acid glutamate
2. The RNA codon CAU codes for the amino acid histidine
3.  The RNA codon CUC codes for the amino acid leucine
            * Three CODONS will also code for a “START” codon or a “STOP” codon which indicate the beginning and the end of protein synthesis.  These codons can also be called promotors and terminators.
            * The only start codon is AUG called methionine
            * There are three stop codons: UAA,UAG, and UGA that do not code for an amino acid.
            * There are ONLY 20 amino acids available for a cell to use in the synthesis of proteins, BUT an amino acid can be coded for by more than one kind of triplet. (43= 64 possible combinations)  
            * Example: Both the triplets CUC and UUG code for the amino acid leucine.

NOTE: Six different triplets code for the amino acid arginine.
Find them: _CGU_, __CGC__, ___CGA__, __CGG__, ___AGA__, ___AGG___.

By convention, the genetic code is always presented in terms of the RNA codon rather that the nucleotide sequence of the original DNA strand.
Practice Naming codons:

1. AAA           Lysine

2.        GCC        Alanine

3.        UGG        Tryptophan

4.        AUG        Methionine

5.        UAA                Stop

Example One:
Determine the complementary strand of DNA, the mRNA codon, tRNA anticodon, and the amino acids found in the protein for the following:

DNA template:                 TAC        GAG   AAT   GTC   GGT

Complementary DNA strand: ATG        CTC    TTA     CAG    CCA

mRNA strand:                        AUG        CUC    UUA    CAG   CCA

tRNA molecules:                UAC        GAG   AAU    GUC   GGU

Amino acids assembled:        methionine        leucine,   leucine,   glutamine,   proline

Example Two:
For the DNA strand given, write out the complementary DNA strand, the mRNA strand that would be produced from the original template through transcription, then complete the tRNA strand, and the amino acids found in the protein that would form.

DNA template: TAC         AAT        GGC        TGT        CCC         ATT

Complementary 
strand:                  ATG         TTA        CCG            ACA           GGG          TAA

mRNA                    AUG          UUA     CCG           ACA            GGG         UAA
strand:

tRNA                UAC          AAU            GGC            UGU            CCC          AUU
molecules:

Amino acids:        methionine, leucine,         proline, threonine, glycine, stop

PRACTICE QUESTIONS SET #3: RNA & INTRODUCTION TO PROTEIN SYNTHESIS

Protein synthesis amoeba sisters https://www.youtube.com/watch?v=oefAI2x2CQM

PROCESS OF PROTEIN SYNTHESIS
The process of making a protein involves two stages.

The first stage:
TRANSCRIPTION: Information from the DNA gene is copied(transcribed) onto an mRNA strand in the nucleus of the cell.

DNA → mRNA

The second stage:
TRANSLATION: The mRNA strand arrives at the ribosome, and a protein (polypeptide chain) is synthesized (translated).

mRNA → protein

STAGE 1: TRANSCRIPTION
The objective of transcription is to make an accurate copy of a small piece of an organism's genome. The information in DNA is copied onto an mRNA molecule.  transcription

Transcription of the DNA gene instruction onto mRNA strand keeps the DNA safely in the nucleus.

STEPS in TRANSCRIPTION
            1. RNA polymerase enzyme binds to the double helical DNA at the promoter.
            2. DNA strand is unwound at promoter and the double helix is disrupted exposing the template strand and stops at the terminator. 
            3. mRNA is synthesized from the template strand as free RNA nucleotides join with complementary DNA nucleotides.

Example         DNA strand:                 AGC  TAA  CCG
                mRNA strand:                UCG  AUU  GGC

The base pairing during transcription is the same as when DNA replicates, except RNA has uracil instead of thymine.

4. RNA nucleotides (the phosphate molecule of one nucleotide and ribose sugar molecule of the next nucleotide) bond together with a covalent bond using the enzyme RNA polymerase.

5. RNA synthesis stops when the RNA polymerase reaches a terminator on the DNA, and the RNA strand forms, the hydrogen bonds between the complementary nitrogen bases (one from the DNA strand, and the other from the RNA strand) are broken and the molecule of messenger RNA (mRNA) is freed into the nucleoplasm.

* mRNA and RNA polymerase are both released from the DNA strand.

6. After the newly transcribed RNA strand detaches from the DNA, the two strands of the original DNA then rejoin.

NOTE: The base sequence in the mRNA is an exact complement of the base sequence in the DNA template strand.

7. INTRONS/EXONS:
            * Further modifications need to be made to the mRNA before it leaves the nucleus to code for a protein. 
            * The initial mRNA transcripts are made up of coding regions called EXONS and noncoding regions called INTRONS.
            * The introns are inter-dispersed among the exons, therefore the primary mRNA transcript will contain sections of nucleotides that do not code for a part of the protein.  
            * Introns must be removed before the mRNA can be used.  Function of introns is not well understood. Some think they might play a role in the evolution of proteins, or perhaps a structural component.  They are also referred to as satellite DNA. http://www.emc.maricopa.edu/faculty/farabee/biobk/exintrons.gif 
            * The introns are removed and the exons are joined together (spliced together), producing a mature RNA with a continuous coding sequence.

8. MOVEMENT OUT OF NUCLEUS:

The DNA code is now carried by the single stranded mature mRNA molecule (made of exons) out of the nucleus through a nuclear pore to the ribosomes in the cytoplasm to be translated into proteins.

STAGE 2: TRANSLATION

TRANSLATION: is the process by which proteins are synthesized using the DNA instruction encoded on the mRNA. (Converting information from mRNA into a protein) http://0.tqn.com/d/biology/1/0/q/translation2.gif

In the process of translation, mRNA must work with tRNA (that carries the amino acids to assemble the protein).  Protein synthesis occurs on the ribosomes that consist of two parts and contain rRNA that direct protein synthesis and bring mRNA and tRNA together.

INITATION:

1. As translation begins, the start codon end on mRNA strand attaches itself to the ribosome, much like a ribbon tied onto a package.  The two subunits (large and small) of the ribosome come together to form an active ribosome.

The sequence of the amino acids is determined by the message carried from the DNA in the nucleus by the mRNA molecule.
The ribosome has binding sites for both the mRNA and tRNA holding the molecules close together.

2. tRNA molecule carrying  an amino acid, which is circulating within the cytoplasm, pairs up with the first exposed codon on the mRNA strand.  The first codon is a “start” codon called methionine.

3. Anticodon on tRNA match up with codon on mRNA strand and the two molecules temporarily join together.

Example:                        mRNA                                codon                 =                 AUG 
  tRNA                            anticodon                 =                UAC

4. A second loaded tRNA molecule arrives at the adjacent codon to the first tRNA.

5. Enzymes catalyze the formation of a PEPTIDE BOND that joins the amino acid carried by the first tRNA to the amino acid carried by the second tRNA. At the same time, the polypeptide chain is transferred to the second tRNA.

6. The tRNA from the first codon will now detach from the ribosome and search for another amino acid that fits its structure.  
(It is now an unloaded tRNA and is moving around the cytoplasm in search of a complimentary AA)

2. ELONGATION:

7. The ribosome moves a distance of one codon along the mRNA strand.  A third tRNA molecule arrives at the exposed codon next to the second tRNA and the cycle repeats, forming a growing polypeptide chain.

https://www2.estrellamountain.edu/faculty/farabee/BIOBK/ELONGATION3.gif

3. TERMINATION:

8. As the process continues, a chain of amino acids is formed until the ribosome reaches a “stop” codon or terminator codon on the mRNA, which turns off the synthesis and the ribosome stalls. No amino acid is added to the polypeptide chain.

The three “stop” codons are UGA, UAG, and UAA.  Since these codons do not code for an amino acid, there are no corresponding tRNA molecules.

9. The complete polypeptide chain or protein is now released from the ribosomeand exported (transported) to desired location in the body, and protein synthesis is complete.

http://mol-biol4masters.masters.grkraj.org/html/Protein_Synthesis2-Prokaryotic_Mechanism_of_Synthesis_files/image002.jpg

PRACTICE QUESTIONS #4: PROTEIN SYNTHESIS
GENE MUTATION
GENE MUTATIONS: Sometimes changes in the DNA code occur during replication or protein synthesis, and the traits or proteins in an organism change.

Mutations change the sequence of the nitrogen bases in a gene, which changes the structure of a protein that the gene codes for.  The mutation may produce a new trait or it may result in a protein that does not work properly resulting in structural or functional problems in cells of organisms.  If a mutation is severe, an organism may not survive, or it may produce life threatening effects.  In some cases gene mutations have positive effects.  An organism may receive a mutation that makes it faster or stronger which increases the organisms’ chances of survival.  This plays an important role in evolution, and it adds variation to a species.

Some gene mutations result from a change involving many nucleotides within a gene, and some involve only one nucleotide.
Mutations can be passed on to offspring
Mutations can have a wide range of consequences: from no effect to lethal.

EXAMPLES of mutagens 
               1. high radiation(X-rays, ultra violet radiation, gamma rays) 
               2. Radioactive material(uranium, plutonium)
               3. Mustard gas, some insecticides 
               4. LSD (causes deletions)
               5. Certain chemicals (nitrous acid)
               6. Air pollutants(tobacco tars, smog)
               7. viruses(HIV/AIDS)

WHAT WOULD HAPPEN TO THE TRANSLATION PROCESS IF ONE NUCLEOTIDE WERE NOT TRANSCRIBED CORRECTLY?
I)        the mRNA is not translated because the start codon was affected
II)        one amino acid is incorrectly translated (point mutation)
III)        all amino acids after the error are affected and entirely different protein or no protein is produced(frameshift mutation)
IV)        there is no effect if the nucleotide affected was not between the start and stop codon, or was in an intron that was removed or was replaced with a nucleotide that still coded for the same amino acid as the original nucleotide in the sequence.

2 MAIN TYPES of GENE MUTATIONS

1. POINT MUTATIONS:

Gene mutation involving a substitution of a nucleotide that affects only one nucleotide on the DNA strand, and only one amino acid in the chain.

Ie:        Original DNA:                 THE FAT CAT ATE THE RAT
                Mutated DNA:         TWE FAT CAT ATE THE RAT

2. FRAME SHIFT MUTATIONS:

Gene mutation involving addition or deletion of nucleotide that affects the entire DNA strand after mutation, including all the amino acids in the chain.

Ie:        Original DNA:                 THE FAT CAT ATE THE RAT
                Mutated DNA:         T   E FAT CAT ATE THE RAT
                        Results:                 TEF  ATC ATA TET HER AT

POINT MUTATIONS: 
Change in one single nucleotide in the DNA result in changes in one codon after mutation, and therefore only one amino acid is affected.

Substitution: 
               * One nucleotide is substituted for another one 
               * This will affect only one codon and one amino acid.  
               * In some cases the change may not affect the amino acid since different codons may code for the same amino acid. The replacement of a single amino acid may have no affect or change the protein.

FRAMESHIFT MUTATIONS: 
Affects all codons after mutation, and therefore all amino acids, and changes large sections of the polypeptide chain.

Frameshift mutation: deletion or addition of nucleotides, so that every codon beyond the point of insertion or deletion is read incorrectly during translation.

Insertion or deletion: of one nucleotide. This alters every codon from the point of the mutation on. The situation may be much more serious.

PRACTICE QUESTIONS SET #5: GENE MUTATION   and     PRACTICE QUESTIONS SET #6: DNA MUTATIONS

GENETIC ENGINEERING
DNA is at the very core of what gives animals and plants their uniqueness. Genetic techniques developed in the past few decades enable scientists to explore and manipulate DNA.

TECHNIQUES INCLUDE:
               * Cutting and pasting genes to make new organisms-Gene Transfer/Recombinant DNA
               * Mapping DNA-Human Genome Project
               * Copying DNA in a lab (PCR)
               * Using DNA to reveal its owner’s identity-DNA Profiling (DNA fingerprinting)
               *  Process of Gel Electrophoresis to separate DNA fragments
               * Cloning animals and cells

WHAT IS GENETIC ENGINEERING?
               * Genetic engineering involves the deliberate and controlled manipulation of the genes in an organism with the intent of making that organism better in some way.(produce a desired human effect in an organism)
               * This is usually done independently of the natural reproductive process. The result is a so-called genetically modified organism (GMO). 
               * To date, most of the effort in genetic engineering has been focused on agriculture.
               * Genetic engineering is also known as genetic modification.
               * It is accomplished by adding, deleting or modifying genes to a DNA molecule in order to change the information that it contains.
               * By changing this information, genetic engineering changes the type or amount of proteins an organism is capable of producing.

GENETICALLY MODIFIED ORGANISMS (GMO)
Flavr Savr Video (8mins): https://www.nytimes.com/2013/06/24/booming/you-call-that-a-tomato.html

GMO is an organism that has had an artificial genetic change using the techniques of genetic engineering such as gene transfer or recombinant DNA.   The process of transferring genes is called GENETIC MODIFICATION.

Genetic Modification is successful because the genetic code is universal, so genes can be transferred from one organism to another, even if they are members of different species.  A gene codes for a polypeptide with the same amino acid sequence, whether it is a human cell, a bacterium, or any other cell.

TRANSGENIC ORGANISMS:
Transgenic Organisms are genetically engineerd organisms resulting from the insertion of “foreign” DNA from another unrelated organism.

* The transgenic organism will exhibit/express the desired trait.

EXAMPLES of TRANSGENIC ORGANISMS:  
Transgenic Bacteria:
               * In medicine: When a gene coding for a human protein is inserted into a bacteria, the recombinant cells may produce large amounts of the protein.  
               * Some examples:
               * Human growth hormone (used for normal growth and development, treatment of dwarfism) 
               * Insulin (treatment of diabetes)
               * Interferon (hormone that blocks the growth of viruses and may be used in cancer treatment)
               * In industry: Recombinant bacteria have been engineered to breakdown pollutants into harmless products, cleanup oil spills

Transgenic Plants:
               * The simplest kind of GM food is one in which an undesirable gene is removed.  In some cases, another more desirable gene is put in its place but in other instances, only the introduction of a new gene is needed, no DNA has to be removed.
               * DNA can be injected into plant cells directly or attached to plasmids of bacteria that infect the plant.
               * Goals of transgenic plants include increased yields, and the production of natural insecticides, become disease resistant and produce natural fertilizers.
               * Some examples:
               * Canola- genetically altered variety low in saturated fats.
               * Potatoes- genetically altered to have superior nutrition (high carbohydrates and proteins) with a built in resistance to potato beetles.
               * Fruits and Vegetables-genetically altered to have enhanced vitamin and mineral levels.

Transgenic Animals:
               * DNA can be introduced into animal reproductive cells in a number of ways, including direct injection.
               * Some transgenic animals will be useful in farming and ranching because they can produce bigger and faster growing animals, more efficient with their use of feed and more resistant to disease, research to investigate human immunity.

Ex: Rainbow trout used for human food are genetically engineered with cloned growth hormone gene.  Growth hormone allows the trout to grow larger, and increase its commercial value, while producing more food.

Glowing mice: https://www.youtube.com/watch?v=n0UzdYRnMtY

IS GENETIC ENGINEERING A GOOD OR A BAD THING?
Are GMO’s good or bad Video (9mins): https://www.youtube.com/watch?v=7TmcXYp8xu4

Genetic engineering raises many profound social and ethical questions.

ADVANTAGES: BENEFITS, PROMISES, AND HOPES FOR THE FUTURE
                  1. IMPROVING FOOD PRODUCTION:
                  * longer shelf life, slower ripening produce, growing in nutrient poor soil, resistant to extreme weather and climate, crop improvement (bigger, nutrient richer) produce a “super plant” that excels at growth and survival.

2. CROP IMPROVEMENT: 
                  * Crops will produce their own pest-control substances,will be beneficial to the environment because fewer chemical pesticides will be needed.  Insect and pest or bacteria protection gene inserted into several crops-tomato, corn, cotton, potatoes, which give farmers new tools for integrated pest management thereby reducing pesticide use.

3. LIVESTOCK IMPROVEMENT: 
                  * Sheep, chickens, cows, pigs, trout, salmon are engineering to grow larger, produce more milk, eggs, wool, and food for growing world population
                  * Farmers can be more in control of what crops or livestock they produce.  This method is quicker than selective breeding.

4. BIOFACTORIES and VACCINE DEVELOPMENT
                  * GMO’s produce rare proteins for medications or vaccines could be, in the long run, less costly and produce less pollution than synthesizing such proteins in laboratories.  The production of drugs: pharmaceutical manufacturing of insulin, growth hormone, and interferon can be helped to save lives.
                  * Multipurpose vaccines are made using gene technology.

5. HUMAN GENE THERAPY: 
                  * alters DNA within cells of a living organism to treat or cure a disease.  New genetic therapies are being developed to treat diseases such as cystic fibrosis, AIDS and cancer.

6. ENVIRONMENTAL CLEAN-UP: 
                  * by engineering bacteria to thrive on waste products of newspaper pulp and oil.

7. ALTERNATE FUEL SUPPLIES: 
                  * Modified yeast cells or agricultural crops can produce fuel for cars. It is projected that 30% of the world’s chemical and fuel needs could be supplied by renewable resources in the first half on the next century.

DISADVANTAGES:  HARMFUL EFFECTS, DANGERS, AND FEARS
                  1. ENVIRONMENTAL IMPACT:
                  * Efforts to keep GM plants under control in well-defined areas have failed and pollen from GM crops has escaped to neighboring fields.  
                  * Accidental release into environment and elimination of natural populations. 
                  * Genes from GM plants could be integrated into wild species giving them an unnatural advantageover other species and an ability to take over the habitat.  These  “super plants” out compete all other native species in nature.
                  * Insects that are not pests could be killed by insecticide.

2. TRANSGENES: 
                  * There is a danger that genes could cross species.   GMO’s may spread to unintended organisms and no one knows the consequences of genes crossing species.
                  * spread of antibiotic resistance amongst not-target organisms.

3. UNKNOWN LONG TERM EFFECTS:
                  * No one knows the long term effects of GMO’s in the wild.

4. MONOPOLIES
                  * large companies or monopolies may develop, with a dependence of developing countries on companies who are seeking control of world’s commercial seed supply with “terminator seeds” (seeds that produce plants that cannot produce seeds themselves)
                  * small businesses such as small farming operations cannot compete with large GMO operations. Critics are worried that large portions of the human food supply will be controlled by a small number of large corporations.

5. FOOD SAFETY:
                  * health effectsof food grown from genetically engineered crops, still too new to really know effects.
                  * There are risks for allergies, if someone is not allergic to natural tomatoes but are allergic to GM tomatoes, they will need to know which one they are eating.  There is not visible difference of the fruit, and food labeling is not always clear.

HUMAN GENOME PROJECT
Human genome project explained Video (5 mins): https://www.youtube.com/watch?v=AhsIF-cmoQQ

The human GENOME is the sum of all the DNA, carried in an organism`s cells and has been estimated to consist of between 25000 and 30000 genes distributed among our 23 pairs of chromosomes that make up our DNA.

The Human Genome Project was a joint international effort of thousands of researches from laboratories worldwide that began in 1993 and ended in 2003.  Their goal was to find the location of all of the genes on the human chromosomes and the base sequence of the entire DNA that makes them up.  (Approximately three billion base pairs that make up the human genome)

The sequencing of the entire human genome will make it easier to study how genes influence human development 
                  * Among the project`s immediate findings was the discovery that the DNA of all humans is more than 99.9% identical.  This means that all differences among individuals across humanity results from variations in fewer than 1000 nucleotides in each individual`s genome.
                  * Human genome sequencing will allow researchers to pinpoint specific nucleotide sequences that are involved in gene expression. 
                  * Over the long term, the information from the sequencing of the human genome will provide geneticists with a better understanding of the relationship between the molecular structure of human genes and the biological mechanisms of gene function. Project has identified genes that may help cure or predict diseases.

* Important in detection and identification of genetic disorders early and beginning treatment before birth.
                  * Researchers have also begun to look for genes that might predispose individuals to other medical problems such as heart disease, diabetes, and cancer.
                  * It will allow the production of new drugs based on DNA base sequences of genes or the structure of proteins coded for by these genes.

RECOMBINANT DNA: 
In one of the most controversial areas of genetic engineering research, scientists are able to combine genes from unrelated species.  This technology is called RECOMBINANT DNA (which is used to make transgenic organisms), where genes can be extracted from the nucleus of one organism and spliced into the chromosomes of a new organism.  The donated genes affect the traits of the new organism and form transgenic organisms.

The Steps of Recombinant DNA Usage: 
Step One:  Use restriction enzymes to cut DNA: 
Isolate the foreign DNA containing the desired gene away from their surrounding genes.  This is accomplished by using a restriction enzyme.
                  * Restriction enzymes, originally discovered in bacteria, are proteins that act like “molecular scissors” to cut foreign DNA that has invaded the cell, in specific places. 
                  * All cells have restriction enzymes which can cut strands of DNA

Examples of Restriction enzymes and recognition sequences:         
i.        EcoR1                GAATTC (sticky ends)        
ii.        BAM HI                GGATCC (sticky ends)
iii.        Hae III                GGCC (blunt ends)                
iv.        HIND III                AAGCTT (sticky ends)

* There are 100’s of restriction enzymes capable of cutting DNA 
                  * Each restriction enzyme binds to a short sequence of DNA nucleotides known as the enzyme’s recognition sequence. Some enzymes have short recognition sequences of 4, 6 or 8 nucleotides, and some have longer recognition sequences of 16 or more nucleotides.   
                  * Some restriction enzymes cut straight across the DNA strand, leaving “blunt ends” and others cut in a jagged pattern, leaving “sticky ends” of unpaired nucleotides.
                     * These sticky ends can then be used to ligate pieces of DNA to each other using the ligase enzyme or DNA glue. The ligase enzyme is part of the normal DNA repair system.  It helps restore the fragmented chromosome.

Step Two: DNA RECOMBINATION USING PLASMID VECTORS
Combine the desired gene from the DNA fragment with a piece of DNA from the recipient organism.

* DNA fragments cannot function on their own or do not readily become part of a host organism’s chromosomes.  They must become part of the genetic material of a living organism before the genes can be activated.
                        * DNA fragments may be combined with bacterial DNA so that later it can be inserted into a bacterial cell
                        * Plasmids are small, circular pieces of DNA from a bacteria cell that can be used as VECTORS to carry segments of cellular DNA into host cells.  Two biological vectors are bacteria and viruses.
                        * Plasmids are removed from the bacterial cell and cut with the same restriction enzyme used to produce the DNA fragments.  These cuts on the plasmid and the DNA fragments will have matching “sticky ends”and can be joined together using ligase enzyme.  Recombinant DNA has now been produced.

Step Three:
DNA INSERTION OR CLONING
Insert the recombinant DNA into the host organism and many copies or clones of the recombinant DNA are made.

* After the foreign DNA has been spliced into the plasmid, the plasmid is transferred by micropipette or using a gene gun and shot into a cell of the host organism. (Host organism can be bacterial cell, yeast cell, plant cell or animal cell)
                        * When the host cell prepares to divide, it copies the recombinant DNA along with its own DNA.  The process of making extra copies of recombinant DNA is a form of cloning.
                        * CLONES are genetically identical copies.

Genetic Engineering

How insulin is made: https://www.youtube.com/watch?v=DIM38NlkWEo 
Video: https://www.youtube.com/watch?v=FAMRQz7fOaE

DNA FINGERPRINTING
Gel electrophoresis:
                        * Gel electrophoresis is a laboratory technique which is used to separate fragments of DNA according to size.
                        * Enzymes are used to cut the very long strands of DNA into varying sized smaller fragments. The DNA fragments are placed into small wells (holes) in a thin sheet of agarose gel which are aligned along one end. 
                        * The gel is then placed in a buffering solution and an electrical current is passed across the gel. (positive on one side and negative on the other)
                        * DNA, being negatively charged(due to phosphate group), moves to the positive terminal
                        * Smaller fragments are less impeded by the gel matrix and move faster through the gel
                        * The fragments are thus separated according to size

http://www.vce.bioninja.com.au/_Media/gel_tank_med.jpeg

Lab_5_gel_1999

* In the end fragments leave a banded pattern.
                        * Size can be calculated (in kilobases) by comparing against a known industry standard
Video: Biorad overview https://www.youtube.com/watch?v=vq759wKCCUQ 
What wells look like (1 minute): https://www.youtube.com/watch?v=tTj8p05jAFM 
https://vimeo.com/85839878 
Amoeba sisters: https://www.youtube.com/watch?v=ZDZUAleWX78&t=2s

POLYMERASE CHAIN REACTION
In the POLYMERASE CHAIN REACTION, DNA is copied again and again to produce many copies of the original molecules.  Millions of copies of DNA can be produced in a few hours.  This is very useful when small quantities of DNA are found and large amounts are needed for analysis.  DNA from semen, blood or other tissue or even long dead specimens can be amplified using PCR.

DNA PROFILING OR DNA FINGERPRINTING:
DNA PROFILING (DNA FINGERPRINTING) describes the process by which individuals can be identified and compared based on their DNA sequence or DNA profile. This process requires matching an unknown sample of DNA with a known sample to see if they correspond (make a match).

When DNA fragments are cut with restriction endonucleases, fragments of different lengths can be resolved using gel electrophoresis and create the DNA Fingerprint.  If, after separation by gel electrophoresis, the pattern of bands formed by two samples of DNA fragments are identical, it means that both sources came from the same individual.  If patterns are similar, it means that the two samples are most probably from two related people.

At a crime scene, DNA is everywhere. It is present in all kinds of evidence collected at the scene, including blood, hair, skin, saliva and semen. Scientists can analyze the DNA in evidence samples to see if it matches a suspect's DNA.

HOW CAN DNA PROFILING BE USED TO IDENTIFY AN INDIVIDUAL?WHY DOES DNA PROFILING/ FINGERPRINTING WORK?
99.9 %of human DNA is the same in everyone, meaning that only 0.1%of our DNA is unique!

Within the human genome there are two regions found on the DNA: 
                        1. Coding regions called GENES (99.9 %)
                        * that contain instructions for protein synthesis
                        2. Non-coding regions called SATELLITE DNA (0.1%)
                        * where its function is thought to be related to structural component of DNA

SATELLITE DNA

* Satellite DNA varies greatly between different individuals in the number and length of repeats called short tandem repeats (STRs)
                        * As individuals all have a different number of repeats in a given sequence of satellite DNA, they will all generate unique fragment profiles.
                        * These different profiles or banding patterns can be compared using gel electrophoresis

Example:
AATTCCG  - DNA sequence may repeat several times. This is the STR's.  Every individual may differ in numbers of these repeating units.   http://www.vce.bioninja.com.au/_Media/dna_profiling_2_med.jpeg

You may have 5 repeating STR's between two genes, whereas the person next to you may have 6 and someone else may have 33, it varies widely.

Since the introduction of this technology, experts agree that information obtained from DNA testing can identify an individual with an accuracy of 99.8%, which far exceeds other testing methods such as ABO blood typing.

Before PCR (3 min) https://www.youtube.com/watch?v=DOWMFvVsG0c
Lab analysis before DNA
https://www.youtube.com/watch?v=nPVkooi8m9I

GENE THERAPY
Two Methods of Gene Therapy:
                        1. Gene Surgery: involves removing cells from individual and growing them in culture.  These cells can be transformed with a correct gene and then injected back into the individual to help cure the disorder.
                        2. Gene Modification: Modifying viruses so that they cannot cause disease, and then attaching DNA containing a desired gene to the viral DNA.  The patient is then infected with the viruses, which carry desired gene into cells and correct the genetic disorder.

Drawback to gene therapy is that the gene does not stay active for long periods of time, or the cells themselves do not have long life spans, so the treatment must be repeated often.
Although the field of gene therapy is still very new, extensive debate is already underway about some of the moral and ethical issues at stake.

Human Genetic Engineering is the modification of an individual's genotype with the aim of choosing the phenotype of a newborn or changing the existing phenotype of a child or adult.

It holds the promise of curing genetic diseases like cystic fibrosis and increasing the immunity of people to viruses.

It is speculated that genetic engineering could be used to change physical appearance, metabolism, and even improve mental faculties like memory and intelligence, although for now these uses are relegated to science fiction, but could become a reality with “designer babies”.

How Crispr Works (16 mins): https://www.youtube.com/watch?v=jAhjPd4uNFY 
Designer babies (5 mins): https://www.youtube.com/watch?v=k1a2larfMIA 
CLONED ANIMALS
Cloning is the technique of producing identical copies of genes, cells or organisms. It is the production of genetically identical organisms from single cells.  All members of a clone and their parents have the same DNA.

NATURAL CLONING: asexual reproduction in animals or plants by fragmentation, binary fission, and budding 
Eg: starfish growing new body if it contains part of center disc, planaria fragmented their body parts to reproduce, identical twin babies.. etc.

REPRODUCTIVE CLONING:
                        * Definition: A procedure where a full living copy of an organism is made.
                        * It is the genetic duplication of an existing organism especially by transferring the nucleus of a somatic cell of the organism into an enucleated oocyte (egg) (surrogate mother is needed)
                        * Cloning is very useful if an organism has a desirable combination of characteristics and more organisms with the same characteristics are wanted.

THERAPEUTIC CLONING:
                        * Definition: A procedure where damaged tissues or organs are repaired or replaced with genetically identical cells that originate from undifferentiated stem cells.
                        * Sometimes cloning is used to produce skin, or other tissues/organs used to treat a patient.

PLANT AND ANIMAL CLONING
                        * Most plants can be cloned quite easily from pieces of roots, stem or leaves.
                        * Animals cannot be cloned in the same way from parts of their bodies.  If animal embryos are divided up at an early stage into several pieces, each piece can develop into a separate animal.  (this happens naturally when identical twins are formed)

ADULT MAMMAL REPRODUCTIVE CLONING
Video:Story of Dolly (13 mins) https://www.youtube.com/watch?v=tELZEPcgKkE 
The first successful reproductive cloning of an adult with known characteristics was in 1997 in Scotland, where the first cloned adult mammal was of a sheep called “Dolly”.

As scientists study how these cloned animals develop, evidence is mounting that a number of problems may be associated with animal cloning.

Dolly for example showed signs of premature aging.  Other cloned animals have shown problems with gene expression.

THERAPEUTIC CLONING IN HUMANS
Techniques are being developed to create human embryos, from which embryonic stem cells can be obtained for medical use.  These stem cells have the capacity to divide and differentiate into any types of human cell.  They could be used to replace tissues or even organs that have become damaged or lost in a patient.  There are many ethical issues involved and research into therapeutic cloning has been banned in some countries.

The use of embryonic stem cells could potentially be used for 
                        * Growing skin to repair a serious burn
                        * Growing new heart muscle to repair an ailing heart
                        * Growing new kidney tissue to rebuild a failing kidney

Video: Ghost heart https://www.youtube.com/watch?v=j9hEFUpTVPA 
Ghost heart (5mins): https://www.youtube.com/watch?v=pd3TFB0wOI0 
Organ transplant: https://www.youtube.com/watch?v=NMe_fOKKC24

HUMAN CLONING:
                        * Researchers involved in human cloning distinguish between therapeutic cloning and reproductive cloning.
                        * In all countries in the world, the cloning of human beings is illegal, this is due to ethical and religious reasons.
                        * The potential benefits of these processes must be weighed against legal, moral and ethical issues.
PRACTICE QUESTIONS SET #7: GENETIC ENGINEERING
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Transcript for:Molecular Genetics and Inheritance Overview

Transcript for:
Molecular Genetics and Inheritance Overview