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DNA the molecule of life The nucleus of every cell of your body contains DNA deoxyribonucleic acid. DNA is the only molecule known that is capable of replicating itself, thereby permitting cell division. DNA provides the directions that guide the repair of worn cell parts and the construction of new ones. Read Read pages 642-643, 646-647 DNA contains instructions that ensure continuity of life – offspring share structural similarities with those of their parents. However, not all offspring are identical to their parents. Why? New combinations of genes and mutations – a change in the DNA sequence, affect the uniqueness of descendants. Searching for the Chemical of Heredity Early 1940’s: biologists began to accept hypothesis that genetic material was found within chromosomes - long threads of genetic material found in nucleus of cells. Chromosomes are composed of relatively equal amounts of proteins and nucleic acids. Proteins (histones) - basic units are amino acids - made up of 20 different amino acids which can be arranged to make an almost infinite amount of proteins. Nucleic Acids -basic unit is the nucleotide - Nucleotides are made up of phosphates, sugar molecules and one of four different nitrogen bases: adenine, guanine, cytosine & thymine. At this point in time it was thought that the key to the genetic code lied in the proteins. This hypothesis was logical, but incorrect. 1950 – A chemist named Rosalind Franklin developed a way of using X-rays to take pictures of the DNA molecule. Her pictures showed that DNA was shaped like a spiral, or helix. 1953 – James Watson & Francis Crick developed a 3-dimensional model of the DNA molecule. This model is known as the double helix model and it resembles a twisted ladder. Watson Crick Structure of DNA The uprights of the DNA “ladder” are composed of phosphates & sugars. a. The bases form the “rungs” of the ladder b. The sugar and phosphate form the “uprights” Did you know? Human DNA is 3 billion base pairs and about 2 m in length. The two purines are adenine and guanine Double ring structures The two pyrimidines are thymine and cytosine Single ring structures Cytosine pairs with guanine Adenine pairs with thymine Nucleotides are complementary. a. A pyrimidine pairs with a purine Thymine with Adenine Cytosine with Guanine b. Bases are held together with relatively weak hydrogen bonds (They can “unzip”…) Structure of DNA Replication of DNA DNA is the only molecule that is known to be capable of duplicating itself. This process is known as replication. Replication occurs during interphase of the cell cycle. •During replication, the weak hydrogen bonds that hold the nitrogen bases together are broken. The DNA “unzips” itself into two parent strands. The enzyme helicase breaks the hydrogen bonds. DNA helicase - an enzyme that uses ATP to “unzip” the DNA molecule This process is known as the “Semi-conservative Replication Model” – Meselson & Stahl (1958). Each strand of DNA pairs with a complementary “new” strand. This produces two, “half old, half new” strands of DNA. Each “unzipped” parent strand acts as a template to which “free floating” nucleotides in the cell can attach. Enzymes called DNA polymerase bring these “free floating” nucleotides into the replication fork and pair them with their complimentary bases (A with T; C with Where G). do free floating nucleotides come from? The food we eat. Our body producing proteins. Proteins -> amino acids -> nucleotides. Enzymes called DNA ligase fuse the free nucleotides together by catalyzing the formation of the sugar-phosphate bonds between adjacent nucleotides. Steps for DNA Replication Step 1 – Unzipping and unwinding of DNA molecule. - The enzyme, Helicase, breaks the weak hydrogen bonds between the bases to form two strands Step 2 – Pairing of Nitrogenous Bases - “Free floating” nucleotides bind to the “unzipped” strands of DNA. - DNA polymerase facilitates the insertion of the nucleotides. - 2 new complementary strands formed. Step 3 – Linking of the sugar-phosphate groups - Deoxyribose sugar of one nucleotide combines with a phosphate group of an adjacent nucleotide. - DNA ligase enzyme joins the nucleotides together to form two new DNA strands. - Bases form hydrogen bonds. - Product = 2 identical strands of DNA. Complete Enzyme Function Summary Chart on page 666 Important to note: During the replication process, genetic mistakes (base pair error) can occur but these occurrences are infrequent. Environmental factors such as hazardous chemicals or radiation are one source of genetic mistakes. These factors can cause uncomplimentary bases to become paired. Enzymes that act as “proofreaders” run along the DNA looking for genetic mistakes like these. If a damaged section is detected, it can be repaired by an endonuclease enzyme “snipping” out the error from the DNA sequence, and an enzyme called ligase, that patches the DNA back together. What do you knowSynthesis about PROTEINS? Protein Proteins in our bodies are made up of different combinations of 20 amino acids. The production of proteins is controlled by genes. Proteins are chemicals that make up the structure of cells. The “recipe” for proteins is found in the nucleus of the cell, but proteins are made in the cytoplasm. The DNA molecules do not leave the nucleus because they are too big. Instead, a messenger molecule, called mRNA, makes a copy of the DNA and takes it to the cytoplasm of the cell so that proteins can be made. The mRNA is a type of RNA (ribonucleic acid) with a structure similar to that of DNA. RNA is a single helix molecule that contains the sugar ribose. Protein Synthesis is divided into two parts: 1)Transcription 2)Translation Transcription mRNA molecules “transcribe” information from a DNA molecule. the double stranded DNA molecules in the nucleus start to “unzip” at a point where there is a gene that codes for a particular protein As the double helix uncoils, mRNA nucleotides “floating around” in the nucleus bind to the open DNA bases on one side of the DNA molecule. the mRNA transcript begins to form a long chain. Once the mRNA has been completely formed, the mRNA moves away from the DNA, and the DNA recoils back into a double helix. The single stranded mRNA molecule is small enough to pass through the nuclear pores and moves into the cytoplasm of the cell Initiation Transcription starts when the RNA polymerase enzyme binds to a segment of DNA to be transcribed It binds in front of the gene in a region called the promoter which indicated where the DNA strand should be transcribed. This is a region with a sequence of A’s and T’s ACCATAATATTACCGACCTTCG Elongation Once the RNA polymerase binds to the promoter and opens the double helix, it starts building single stranded mRNA in the 5’ to 3’ direction Similar to DNA replication, but it does not require a primer and copies only one strand The transcribed DNA strand is called the template strand It is complimentary except that in the place of of thymine there is uracil Termination Synthesis of mRNA continues until RNA polymerase reaches the end of the gene Termination sequence is at the end of the gene and signals this stop mRNA and RNA polymerase are released and go transcribe another gene Translation Translation is the process by which the mRNA strand is “translated” into an amino acid sequence (protein). The mRNA molecule attaches itself to a ribosome in the cytoplasm located in the rough endoplasmic reticulum. Every three nitrogen bases on an mRNA strand codes for one amino acid. This is called a CODON. Not all codons code for amino acids. There are 4 codes that code for initiator or terminator codons. These are codons that tell protein synthesis to start (AUG) or stop (UAA, UAG, UGA. tRNA - transfer RNA I) Carries amino acids to the mRNA ii) Clover leaf shape when the mRNA initially binds to a ribosome, the ribosome attaches to an initiator codon on the mRNA. TRNA (transfer RNA), picks up amino acids that are circulating within the cytoplasm and shuttles them to the mRNA. Each tRNA molecule has an anti-codon to each mRNA codon. This helps the tRNA find the correct amino acid in the cytoplasm The tRNA “drops off ” the amino acid (“taxi”) at the ribosome and goes to search again for more. The ribosome moves another three spaces and the next tRNA molecule binds and “drops off ” its amino acid. (The ribosome helps in the binding of mRNA and tRNA molecules). This continues until a terminator codon (“stop” codon) is reached and protein synthesis is complete. Initiation Occurs when an ribosome (carries out protein synthesis) recognizes a specific sequence on the mRNA and binds to it. Ribosomes consist of two subunits (large and small) which bind to mRNA, clamping it between them. It moves along adding new amino acids to the growing polypeptide chain each time it reads a codon Initiation The start codon is AUG If it starts reading in the wrong place all the codons will be misread. tRNA delivers the amino acids during translation At one end of the tRNA is the anticodon which is complementary to the mRNA, the other end carries the corresponding amino acid Example If mRNA has the codon UAU, the complementary base sequence of the anticodon is AUA, and the tRNA would carry the amino acid tyrosine Elongation The ribosome has two sites for tRNA to attach: the aminoacyl (A) site and the peptidyl (P) site The anticodon (UAC) complimentary to the start codon (AUG) enters the P site The next tRNA carrying the required amino acid enters the A site A peptide bond forms between the two and a polypeptide chain is forming The ribosome has shifted over one so the second tRNA is now in the P site, allowing the A site to be open. This continues until the entire code of mRNA has be translated and the ribosome reaches a stop codon Termination Eventually, the ribosome reaches one of the three stop codons. A protein known as a release factor recognizes the ribosome has stalled and helps release the polypeptide chain from the ribosome The protein has been synthesized In Summary mRNA is formed in the nucleus (transcription) mRNA travels to ribosome which serves as the framework for binding the mRNA and tRNA molecules Cell cytoplasm contains a pool of amino acids. Each can only link to one specific tRNA. This linkage requires energy (ATP) and a specific enzyme for each amino acid 5. There are some key differences between DNA and RNA. DNA RNA deoxyribose ribose thymine (T) uracil (U) dble strand single strand long short 1 type 3 types nucleus only nucleus, cytoplasm Bonding A and T form double bonds C and G form triple bonds eg.) Predict the mRNA and tRNA base sequences, as well as the amino acid sequence, for the following DNA molecule: DNA: T A C G C A T T G C C A T A T C C G A C T mRNA: AUG CG U A A C G G U A U A G G C U GA UACG C A U UG C C A U A U CC G A C U tRNA: amino acid: Methionine/Start; Arg;enine Aspartate; Glycine; Isoline; Glycine; stop Gene Recombinations 1) Recombinant DNA An application of genetic engineering in which genetic information from one organism is spliced into the chromosome of another organism. Genetic transformation- introduction and expression of foreign DNA in a living organism DNA Sequencing Before a DNA sequence can be used to make recombinant DNA, an isolated piece of DNA containing that sequence must be identified This DNA sequencing was used during the Human Genome Project Enzymes and Recombinant DNA Restriction endonucleases (restriction enzymes) are like molecular scissors than can cut DNA at a specific base-pair sequence (its recognition site) Most recognition sites are 4 to 8 base pairs long and are usually palindromic Ex. 5’ GAATTC 3’ 3’ CTTAAG 5’ Restriction Enzymes See Table 2 on page 680 When one strand is longer than the other and has exposed nucleotides that lack complimentary bases it is called a sticky end When the DNA fragments are fully base paired it is called a blunt end It is more useful to have sticky ends because they can be more easily joined together Methylases Enzymes than can modify a restriction enzyme recognition site by adding a methyl (-CH3) group to one of its base sites. They protect a gene fragment from being cut in an undesired location First identified in bacteria cells and were used to protect the DNA from digestion by its own restriction enzymes DNA Ligase Joins segments of DNA together Hydrogen bonds will form between the complimentary bases The Process Polymerase Chain Reaction PCR allows scientists to make billions of copies of pieces of DNA from extremely small quantities of DNA This depends on Taq polymerase which synthesizes DNA during replication Enzymes have an optimum temperature range that they function, and Taq polymerase is stable at much higher temperatures than DNA polymerase Each PCR cycle doubles the number of DNA Polymerase Chain Reaction A technique used to amplify a specific gene in a DNA sample. Basically a molecular photocopier. A common gene is selected (i.e. insulin, myosin) and then amplified exponentially. (1 strand; 2; 4; 8; 16, etc.) Performed prior to gel electrophoresis. DNA is pipetted into these tubes, nucleotides and DNA polymerase are added. The mixture is heated to separate DNA, and let the polymerase work! Other uses for PCR: Cloning a gene so it can be inserted into various samples. Genetic diagnosis: sickle cell anemia or cystic fibrosis Transformation Any process by which foreign DNA is incorporated into the genome of a cell A vector is the delivery system that is used to move the foreign DNA into the cell An organism that has foreign DNA is said to be transgenic Transformation of Bacteria Bacteria are the most commonly transformed organism Transgenic bacteria have been engineered to produce hGH 1) Identify and isolate the DNA fragment that is being transferred 2) DNA fragment is introduced into the vector Plasmids are small, circular, double-stranded DNA molecules that are commonly used as vectors. It contains genes and is replicated and expressed. 3) Plasmid vector and DNA are cut by the same restriction enzyme(s) 4) Plasmid and DNA fragment are mixed together and incubated with DNA ligase 5) This produces recombinant plasmids that contain the foreign DNA fragment 6) Plasmid is introduced into bacterial cell 7) Once the bacterium has been transformed, it makes many copies of the recombinant plasmid This is often called gene cloning For transformation to be successful, the plasmid must only have one recognition site for the restriction enzyme or else it would be cut into a bunch of useless pieces Naturally occurring plasmids do not always have a single site, so engineered plasmids are used They contain multiple cloning sites, which is a single region that contains unique recognition sites for an assortment of restriction enzymes When are these technique used? a) Medical Applications Scientists have already placed a number of human genes into bacteria. The gene that produces human insulin has been spliced into E.coli bacteria. The human DNA directs the bacteria to produce human insulin, a hormone essential for regulating blood sugar. Diabetes Mellitus Insufficient/lack of production of insulin. People with this disorder regulate their blood sugar levels by taking insulin injections. Traditionally, insulin has been extracted from pig and cow pancreases. Downsides of this are that the insulin differs from human insulin and some people have allergic reactions to it. Human insulin produced by E. coli was first marketed in Canada in 1983. b) Agricultural Genetic engineering is also being used to produce strains of wheat, cotton, and soybeans that carry a bacterial gene that makes the plants resistant to the herbicides used by many farmers to control weeds. This gene would make it easier to grow crops while still ensuring that the weeds are destroyed. The first gene-spliced fruit that was approved for human consumption were tomatoes engineered with “anti-sense” genes that retard spoilage. Crop plants are also being engineered to resist certain pathogens and pest insects. Tomato and tobacco plants, for example, have been engineered to carry and express certain genes of viruses that normally infect and damage plants. By having the viral gene, these plants are “vaccinated” against attack by the viruses themselves. 2) DNA Fingerprinting If enough body tissue or semen is left at a crime scene, forensic laboratories can perform tests to determine the blood type or tissue type. DNA Fingerprinting This DNA fingerprint can then be used to compare against other fingerprints (possible suspects, DNA found at a crime scene or family member DNA). Prior to 1993, these tests had limitations - they required LARGE amounts of DNA (not what you would see on CSI). DNA Fingerprinting Now because of a technology called polymerase chain reaction (PCR), a small DNA sample can be copied/amplified which means multiple copies of it can be made to provide enough DNA to produce a fingerprint. Such tests have limitations. First, they require fairly fresh tissue in significant amounts. Second, because there are so many people with the same blood type, this approach can only exclude a subject; it is not evidence of guilt. DNA testing CAN identify the guilty individual with a higher degree of certainty, since the DNA VNTR (variable number tandem repeat) nucleotide sequence of everyone is unique (except identical twins). Two humans will have the vast majority of their DNA sequence in common. Genetic fingerprinting exploits highly variable repeating sequences called microsatellites. Think of DNA as a train. All trains you see have different numbers of cars on them. This is just like different people having different numbers of VNTR sequences on their DNA. The probability of two people having the same number of VNTR’s for one gene is 1/900 000! How do they do this? The technology used to produce a DNA fingerprint is called gel electrophoresis. Steps involved: The amplified DNA sample is placed in a well in the agarose gel (analogous to a lane in a swimming pool). –The VNTR fragments (of differing sizes) in the DNA are separated when electricity is applied to the gel. DNA fragments have a negative charge and are therefore pulled to the other side of the gel due to their attraction to the opposite positive side. The distance that the fragments travel in the gel are based on the size of the fragments. Segments of DNA from PCR Who would move through the agarose “gel forest” faster???? The smallest fragments travel the furthest. After a period of time, the fragments finish separating. The bands can be viewed when a dye is applied and a pattern of bands can be seen. This pattern is known as a DNA fingerprint. The banding patterns on one person’s DNA fingerprint can then be compared to the banding patterns of another DNA fingerprint. Who is Guilty? Suspect 2! How do you know? The bands match Other uses for DNA fingerprinting: Identification of catastrophe victims Identify endangered species (poaching) Prevent sports memorabilia fraud Superbowl footballs 2000 Summer Olympics memorabilia DNA and Mutations Mutations are inheritable changes in the genetic material They can arise from mistakes in DNA replication when one nitrogen base is substituted for another X-rays, UV rays, and chemicals that can alter DNA and are called mutagenic agents If DNA is altered, mRNA will also be affected. A change in a single amino acid could result in a completely different protein being produced There are three main types of mutations: Substitution – one base replaces another Insertion – a base is inserted between two existing bases Deletion – a base is deleted from the DNA sequence These types of mutations can cause a shift in the DNA sequence, which would result in a miscoded protein. If the protein is a vital one, the cell may die. When mutations occur, they are repeated each time the cell undergoes mitosis (or meiosis) Point Mutations Changes to one single base pair of DNA Silent mutation No effect on the operation of the cell because they occur in the non-coding region. ACA ACU Missense mutation Leads to a different amino acid being placed in the polypeptide Nonsense mutation A stop codon replaces a codon specifying an amino acid Is often lethal to cells Sickle cell anemia Sickle cell anemia is a genetic disease caused by the incorrect codon of one amino acid in a protein Valine replaces glutamine as the sixth amino acid in one of the protein changes coded for by DNA Such a small change has a serious impact on the formation of red blood cells, causing them to have a sickle shape, which reduces their oxygen carrying capacity. Gene Mutations Changes the coding for the amino acid Deletion When one or more nucleotide is removed, which can lead to significant changes to the protein Insertion Inserting an extra nucleotide will cause a shift in the codons causing different amino acids to be inserted Translocation is the relocation of groups of base pairs from one part of the genome to another Usually occurs between two non-homologous chromosomes, disrupting the normal structures of genes. Leukemia Inversion is a section of the chromosome that has reversed its orientation in the chromosome No gain or loss of genetic material, but a gene may be disrupted Oncogenes: Cancer Cancer can be caused by nitrogen base substitution, or the movement of genetic material from one part of the chromosome to another. There are many cancer causing agents (carcinogens): X rays, UV radiation & mutagenic chemicals, that promote the alteration of normal genes into cancer causing genes. (oncogenes) Every cell in your body has identical DNA, but each cell is specific to its purpose. In a skin cell, the “skin genes” are expressed, while the “muscle genes” are not expressed. How does a skin cell turn on certain genes while turning off others? - by having “regulator genes.” – act like switches to turn “on” or “off ” segments of the DNA molecule. Regulator genes produce proteins that have the ability to turn “on” and “off ” the process of cell division. Oncogenes can either turn ON cell division (leave the switch regulating cell division OPEN ) or INHIBIT an “off ” switch. Both situations could lead to cancer. Studies have indicated that cancer-causing oncogenes are found in normal strands of DNA. The question is if oncogenes are found in normal cells, than why do normal cells not become cancerous? Current theory is that the cancer gene, (oncogene), has been transposed to another gene site. Transposon- a DNA section that can change positions within a genome Such transpositions may have been brought about by environmental factors or mutagenic chemicals. The movement of the oncogene away from it’s regulator gene may have caused the problem. Causes of Genetic Mutations Spontaneous mutations are caused by an error of the genetic machinery Mutagenic agents can cause induced mutations due to exposure Phylogeny is the proposed evolutionary history of a group of organisms Species that are closely related will share very similar DNA sequences Mitochondrial DNA can be used to trace inheritance down the maternal line in mammals, as the egg is the only source of the mitochondria passed on to new offspring