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Chapters 12 and 13: DNA and the Language of Life Mendel concluded that parents pass on factors to their offspring. We now know these factors to be genes. How did scientists determine what genes were made from? Three Important Experiments 1. Griffith – 1920’s Investigating what makes people sick. Discovered two strains of pneumonia bacteria “Smooth” S-strain – forms smooth, round colonies when grown in a Petri dish. “Rough” R-strain – forms rough-edged colonies when grown in a Petri dish. A B C D A. What happened when the S strain was injected into the mouse? Mice died B. What happened when the R strain was injected into the mouse? Mice lived C. What happened when the S strain was heat-killed and then injected into the mouse? Mice lived Would a Petri dish inoculated with a sample from syringe “C” show any bacterial growth? If so, what would the colonies look like? No Growth D. What happened when the killed S strain was mixed with the R strain and injected into the mouse? Mice died Would a Petri dish inoculated with a sample from syringe “D” show any bacterial growth? If so, what would the colonies look like? What conclusion can you draw from this experiment? When heat-killed harmful bacteria were mixed with harmless, some “factor” transformed the rough, harmless bacteria into smooth, harmful bacteria. 2. Avery’s Experiment – 1940’s Wanted to determine what the transformation factor was from Griffith’s experiment. Thought the transformation factor was a macromolecule. Carbohydrate Lipids Proteins Nucleic Acids o RNA o DNA What happened when the heat-killed S strain was mixed with an enzyme to destroy RNA and then mixed with the R strain? R and S colonies visible What happened when the heat-killed S strain was mixed with an enzyme to destroy Proteins and then mixed with the R strain? R and S colonies visible What happened when the heat-killed S strain was mixed with an enzyme to destroy DNA and then mixed with the R strain? Just R colonies What happened when the heat-killed S strain was mixed with an enzyme to destroy Lipids and then mixed with the R strain? R and S colonies visible What happened when the heat-killed S strain was mixed with an enzyme to destroy Carbohydrates and then mixed with the R strain? R and S colonies visible Conclusion: No transformation when DNA is destroyed. DNA must be the transformation factor 3. Hershey and Chase’s Experiment – 1950’s Used Escherichia coli (E. coli) bacteria and bacteriophages Bacteriophages are viruses that attack bacteria Why did Hershey and Chase use bacteriophages in their experiment? Made only of Proteins and DNA What macromolecule contains sulfur? Proteins What macromolecule contains phosphorus? DNA Why were the samples agitated and placed into a centrifuge before the radiation detector was used to locate the radioactive isotopes? Separate viruses from bacteria. Viruses suspended in the liquid and bacteria in the pellet at the bottom. Where were radioactive proteins found? Where was radioactive DNA found? Liquid Pellet What conclusion was Hershey and Case able to make based on their experiment? Viruses pass DNA, not Proteins, into bacteria Why didn’t Hershey and Chase culture viruses in media containing both radioactive sulfur and phosphorus? They wouldn’t know what was actually being passed on. The Role of DNA 1. Store Information – Information is stored in the order and amount of nucleotides that make up the DNA. The sequence of DNA that codes for a particular trait is called a … Gene 2. Copy Information – During S of Interphase your cells replicate the DNA. 3. Transmitting Information – Copies of all of your genes (both sets) are passed to daughter cells at the end of Mitosis, while only 1 set is passed to daughter cells at the end of Meiosis. Building Blocks of Nucleic Acids DNA and RNA are Nucleic Acids Nucleic Acids are polymers The monomer building blocks of Nucleic Acids are Nucleotides Nucleotides are made of three subunits: 5-Carbon Sugar (Deoxyribose or Ribose) Phosphate Group Nitrogenous Base (1of 5) There are 5 different Nitrogenous Bases Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Uracil (U) Purines Pyrimidines Each base makes a different Nucleotide 4 of the 5 Nucleotides are found in DNA 4 of the 5 Nucleotides are found in RNA The sugar of one nucleotide is connected to the phosphate group of the next nucleotide by covalent bonds forming the “Sugar-Phosphate Backbone” DNA Structure Once DNA was determined to be the transformation factor, many scientists raced to discover its structure. Rosalind Franklin/Maurice Wilkins Erwin Chargaff James Watson/Francis Crick Rosalind Franklin and Maurice Wilkins Used X-ray crystallography to take a picture of the structure of DNA A similar picture is shown below From this picture, Watson could tell the “strandedness” of DNA. Double stranded Twisted – DNA had a uniform diameter This meant there were four options for the structure Options: 1. AA cc GG tt tt AA 2. AG ct tc GA tc AG 3. At cG tA Gc cG At 4. Ac Gt Gt cA tG Ac Further analysis allowed Watson and Crick to rule out two other options. The DNA helix had a uniform 2nm diameter. o Which two could be ruled out? Erwin Chargaff Studied the percent of each nitrogenous base found in an organism’s DNA. Nitrogenous Base Make-Up of Different Organisms’ DNA (%) Organism Mycobacterium tuberculosis Yeast Wheat Sea Urchin Marine Crab Turtle Rat Human A 15.1 G 34.9 T 14.6 C 35.4 31.3 27.3 32.8 47.3. 29.7 28.6 30.9 18.7 22.7 17.7 2.7 22.0 21.4 19.9 32.9 27.1 32.1 47.3 27.9 28.4 29.4 17.1 22.8 17.3 2.7 21.3 21.5 19.8 What observation can you make about the data? Is there a pattern? What does the data show about the make-up of DNA for different species? Based on the data, what do you think is Chargaff’s Rule? A T and G C With this data Watson and Crick “ruled-out” another option and determined the structure of DNA. Options: 1. 2. 3. 4. AA cc GG tt tt AA AG ct tc GA tc AG At cG tA Gc cG At Ac Gt Gt cA tG Ac Sugar and Phosphate Groups make-up the sides of the ladder o Sugar-Phosphate backbones run in opposite directions “Rungs” of the ladder are made of two nitrogenous bases called “Base Pairs” What makes each species unique? o Amount and sequence of “base-pairs” What type of bond connects the nucleotides in a chain together? Covalent Are they strong or weak? Strong Why important? Information is stored in nucleotide order What bonds hold the two strands together? Hydrogen Are they strong or weak? Weak Why is important? Separate the strand for replication Watson and Crick used the work from Franklin and Chargaff to determine the structure of DNA. They did determine the mechanism of replication Published their conclusions in April 1953. Won the Nobel Prize in 1962. DNA Mistakes in Movies Replication The base-pairing rule established by Chargaff provided a possible copying mechanism. Template/Semiconservative Model: When: S-phase of Interphase Where (Eukaryotic cells): In the nucleus With What: DNA, DNA nucleotides, Enzymes How: o Separate the two original strands of DNA. o Each “old” strand becomes a template to make a new complementary strand from free nucleotides Very fast: 50/sec in mammals; 500/sec in bacteria Very accurate: 1 nucleotide/billion wrong Diagrams like the ones above make it look like the parent DNA molecule is completely opened before replication occurs, but that is not true. There are many “origins of replication” along the length of the parent DNA molecule. Helicase attaches to the DNA molecule and break the hydrogen bonds holding the two strands together. o Forms replication bubbles. What is beneficial about multiple origins? One strand (Leading Strand) can be replicated in one piece. Individual nucleotides attached by DNA Polymerase Other strand (Lagging) is replicated in fragments by DNA Polymerase (attaches individual nucleotides) and then the fragments are bonded together by DNA Ligase DNA polymerases also proofread the newly replicated strand and remove incorrectly paired nucleotides DNA polymerase and DNA Ligase also repair damage done to DNA by exposure to radiation (UV and X-ray) and toxic chemicals. Replication Overview http://www.youtube.com/watch?v=zdDkiRw1PdU http://www.youtube.com/watch?v=gW3qZF9cLIA 3 Types of RNA 1. Messenger RNA (mRNA) – A single strand of RNA that is a temporary (disposable) copy of a single gene. 2. Ribosomal RNA (rRNA) – Ribosomes are made from rRNA and proteins. 3. Transfer RNA (tRNA) – Pick-up and carry amino acids to the mRNA and Ribosome to make proteins DNA to Proteins DNA - Long term storage of all information Transcription – Location: Nucleus mRNA - Short term storage of one gene’s information Translation – Location: Cytoplasm Protein One sequence of nucleotides/“gene” codes for the production of one polypeptide*. Multiple polypeptides can bond together to form more complex proteins Overview: 1. Copy part of the DNA nucleotide sequence into RNA 2. Read the mRNA 3. Attach amino acids 4. Release protein Transcription Transcription – 1 strand of DNA nucleotides (template strand) is converted into a single strand of complementary RNA nucleotides (mRNA). Language Analogy: Spoken English Written English If you break your hand and can’t write, you might need someone to transcribe (scribe) your verbal answers on a test. When: When a cell needs to produce a specific protein Where: Nucleus (Eukaryotes) With What: DNA, RNA nucleotides, RNA polymerase How: 1. RNA polymerase binds to DNA in the nucleus and separates the DNA strand for 1 gene. 2. RNA polymerase “reads” 1 strand of DNA to produce a strand of messenger RNA (mRNA). 3. Complementary RNA nucleotides pair across from the DNA nucleotides (A-U; G-C, C-G; T-A) 4. RNA polymerase links the nucleotides together. 5. The process continues until the end of the gene is reached http://www.youtube.com/watch?v=WsofH466lqk http://www.youtube.com/watch?v=Kzgnl5-8WAk (3:40 – 5:30) In eukaryotic cells, the mRNA sequence is further modified before it leaves the nucleus through nuclear envelope pores. RNA Splicing/Editing o Eukaryotic mRNA contains non-coding regions (Intervening sequence - Introns) that need to be removed before translation o Expressed sequences (Exons) are the coding regions that are maintained. The final mRNA transcript (message)is short than the original. Analogy TV Box Set or Movie Hola, mi nombre es Kevin. Translation Translation – Single stranded mRNA is used to produce a sequence of amino acids (polypeptide). Language Analogy: Spanish English Written Spanish is translated into English with the help of an interpreter. mRNA language (nucleotides) Protein language (amino acids) When: After Transcription when a cell needs to produce a specific protein Where: Cytoplasm With What: mRNA, ribosomes (rRNA + proteins), tRNA, amino acids and enzymes. How: 1. Transcribed, edited mRNA strand moves out of the nucleus and into the cytoplasm. 2. mRNA binds to a ribosome in the cytoplasm or RER. 3. A ribosome begins reading the mRNA at a start codon and continues to read the nucleotides in groups of three (codon). Why read in groups of three? How many different amino acids? 20 How many amino acids could be coded for if you read 1 nucleotide at a time? 1 4 different amino acids 4 < 20 How many amino acids could be coded for if you read 2 nucleotides at a time? 2 4 x 4 = 16 amino acids 16 < 20 How many amino acids could be coded for if you read 3 nucleotides at a time? 3 4 x 4 x 4 = 64 different amino acids 64 > 20 64 different nucleotide triplets o 61 code for amino acids (1 also “start”) o 3 code for stop THEFATCATATETHERAT THE-FAT-CAT-ATE-THE-RAT Codon Chart Redundant But Not Ambiguous Multiple codons can code for the same amino acid, but each codon can code for only1 specific amino acid. 4. Ribosomes can hold 2 codons at once in the “P” site and “A” site. 5. Transfer RNA (tRNA) with the anticodon complementary to the mRNA codon, bring specific amino acids to the ribosome/mRNA complex. 6. Peptide bonds form between two amino acids and the 1st tRNA is released. 7. mRNA moves so the 2nd tRNA and growing amino acid chain are now in the “P” site. 8. Process continues until a stop codon is reached. http://www.youtube.com/watch?v=5bLEDd-PSTQ http://www.youtube.com/watch?v=Kzgnl5-8WAk – 5:25 Overview: DNA RNA Protein Mutations Mutations – any mistake or change in the nucleotide sequence of DNA. Chromosomal mutations – large changes to regions of a chromosome. o Deletion o Duplication o Inversion o Translocation Gene (Point) mutations – change single gene Substitution Frameshift Point Mutations – mutation that involves a single nucleotide 1. Substitution – replace 1 nucleotide with another a. Silent: A mutation that changes the DNA sequence of nucleotides, but does not change the amino acid sequence of a protein. DNA: GAA – GGG – CCA RNA: CUU – CCC – GGU Amino Acids: LEU – PRO – GLY DNA: GAA – GGT – CCA RNA: CUU – CCA – GGU Amino Acids: LEU – PRO – GLY Mutation b. Expressed: A mutation that changes the DNA sequence of nucleotides and the amino acid sequence of a protein DNA: TTT – GTG – AGG RNA: AAA – CAC – UCC Amino Acids: LYS – HIS – SER DNA: TTT – GTT – AGG RNA: AAA – CAA – UCC Amino Acids: LYS – GLN – SER Mutation 2. Frameshift Mutations – changes in the DNA that can change the rest of the amino acid sequence after the mutation. a. Insertion – add a nucleotide into DNA sequence DNA: TAC – GCA – TTT RNA: AUG – CGU – AAA Amino Acids: MET – ARG – LYS DNA: TAC – GGC- ATT –T RNA: AUG – CCG – UAA – Amino Acids: MET – PRO – STOP Mutation b. Deletion – remove a nucleotide from a DNA sequence DNA: TAC – GAG – GAT – AGC RNA: AUG – CUC – CUA – UCG Amino Acids: MET – LEU – LEU – SER DNA: TAC – AGG – ATA – GC RNA: AUG – UCC – UAU – CG Amino Acids: MET – SER – TYR – Mutation Mutagenesis – the production of mutations Spontaneous - errors during DNA replication or recombination Mutagens – physical/chemical agent that causes an error. o High energy radiation (X-ray, UV) o Toxic chemicals