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Griffith’s experiments showed that hereditary material can pass from one bacterial cell to another. In 1928 Griffith was studying a bacterium called Streptococcus pneumoniae. He was trying to develop a vaccine against a virulent strain of the bacterium. Virulent – disease causing Vaccine – a substance that is prepared from killed or weakened microorganisms & introduced into the body to protect it from future infection by that microorganism. S bacteria is virulent because it has a capsule. The capsule protects it from attacks by the human immune system. It is able to survive long enough in the human body to replicate and cause disease. R bacteria is NOT virulent because it lacks a capsule. Without the capsule it is left defenseless against the human immune system. Therefore it is destroyed in the human body before it is able to cause disease. Injected mouse with live R bacteria (w/o capsule). Injected mouse with live S bacteria (w/ capsule). Killed mice Injected mouse with heat killed S bacteria. DID NOT kill mice DID NOT kill mice Injected mouse with heat killed S bacteria mixed with live R bacteria. Killed mice The R bacteria took up the DNA found in the heat killed S bacteria and became virulent. The transfer of genetic material from one cell to another cell or from one organism to another organism is called transformation. We use bacteria’s ability to do this in one form of BIOTECHNOLOGY – the use of biology for human benefit (we will study this subject in more detail at the end of the unit once you have gained an understanding of how DNA works) Spider goat video • • • Discovered that Adenine / Thymine and Cytosine / Guanine occurred in equal percentages in DNA. For Example: If you had 22 Adenine and 5 Cytosine then you would have 22 Thymine and 5 Guanine. Led to base pairing discovery (that A pairs with T and C pairs with G) • The structure of DNA was not known at this time so he did not realize that A-T / C-G had complimentary binding In 1952, two researchers, Martha Chase and Alfred Hershey, tested whether DNA or protein was the hereditary material viruses transfer when viruses enter bacteria. Used bacteriophages (viruses that infect bacteria) Remember “phage” means to eat. The viruses aren’t literally eating the bacteria (bacteria are much larger than viruses, but they are infecting/destroying them). Grew bacteriophage (with radioactive sulfur) in dish of e.coli (sulfur is only found in proteins) Grew bacteriophage (with radioactive phosphorous) in dish of e.coli (phosphorous is only found in DNA) They traced the radioactive elements that had entered the e.coli to see which one was injected into the cell. The DNA of the virus and not the proteins was what was in the e.coli causing it to produce more viral DNA. Hershey and Chase confirmed that DNA, and not protein, is the hereditary material. Watson and Crick used the information gathered by others to determine one of the most important discoveries in human history – the Double Helix. Until Hershey and Chase’s experiment, most people believed that protein was the hereditary material (because protein was involved in basically everything to do with cells and because it was believed DNA’s structure was too simple to encode the secret to life). After the Hershey and Chase experiment in 1952 proved DNA was in fact the hereditary material, the race was on to discover it’s structure to gain a better understanding of it. Watson and Crick discovered the shape of DNA(double helix) in 1953 using information gained from other scientists, mainly Wilkins / Franklin. Crick actually studied mostly proteins and Watson studied neither before 1952. Watson and Crick went to lectures from other scientists concerning DNA and compiled information they gained from them. Watson “borrowed” X-rays done by Wilkins / Franklin showing a vague picture DNA’s shape (you could tell that it was the same thickness all the way through). Watson, Crick, and Wilkins received a Nobel prize from it while Franklin (who did most of the work) died from cancer due to her exposure to X-rays. She did not receive the Nobel prize because they cannot be given posthumously (after death). Two types: DNA (deoxyribonucleic acid) RNA (ribonucleic acid) Nitrogenous base (adenine) Phosphate group Sugar The sugar is either: 1. deoxyribose (DNA) 2. ribose (RNA) The sugar in DNA (deoxyribose) has 5 carbons. Those carbons are numbered starting at the carbon attached to the base and going clockwise. This is done to tell you the direction the nucleotide is facing. (Number these on your notes) 5’ carbon 4’ carbon 3’ carbon 2’ carbon 1’ carbon The sugar and phosphate backbone (side) of DNA is the same for ALL living creatures (plants, animals, bacteria). All living things also have the following 4 bases, but what makes us all different is the order of these bases. DNA nitrogenous bases are Think of it like reading a book. All books use the same 26 letters, but the order of those letters makes every book say something different. adenine (A) thymine (T) cytosine (C) guanine (G) RNA has A, C, and G as well, but has U (Uracil) instead of T Two DNA strands (polynucleotides) wrap around each other to form a double helix – – The two strands are connected by a hydrogen bond between complementary base pairs. A pairs with T – RNA has U instead of T (so U binds to A as well) – C pairs with G RNA is usually a single strand The 2 polynucleotide strands in a double helix run anti-parallel to each other. This means that they remain parallel, but they run in opposite directions. One side is oriented with the nucleotides going from the 5’ to 3’ direction while the other is oriented with the nucleotides running in the 3’ to 5’ direction. This occurs to keep the double helix the same thickness throughout and because of the way DNA is replicated (we will learn this tomorrow). 1. Covalent Bonds - Polynucleotides (one side of a double helix) are formed from its monomers bonding together through dehydration synthesis. Remember this means to pull out water to make strong covalent bonds. In DNA, these bonds are called phosphodiester bonds. This is because a polynucleotide sequence is never needed to be separated. 2. The phosphate group of one nucleotide bonds to the sugar of the next. The result is a repeating sugar phosphate backbone. Hydrogen bonds - As we will learn later, to use DNA you must separate the two strands; therefore, they are held together by weak, hydrogen bonds. Think of the structure like a zipper, the sides are strong but they can be separated easily These terms are often used interchangeably. Make sure you understand the differences between them. DNA – organic compound that serves as the hereditary material for all living things (the rest of these terms simply refer to different forms / amounts of DNA) Genes – a segment of DNA that codes for 1 polypeptide Chromosomes - DNA in a dividing cell (DNA is wrapped around histone proteins) Chromatin – DNA in a non-dividing cell (DNA is not wrapped around histone proteins) Genes (enough DNA to code for one protein) codes for the sequence in which the amino acids are arranged (primary structure of proteins). Genes (DNA) DO NOT code directly. Genes use an intermediary (RNA). This is because the DNA is too important to leave out in the cytoplasm where it can be damaged (so it remains in the nucleus where it is safe). If we were to damage our DNA and could not fix it, then the cell would no longer be able to function. It is okay if we damage RNA because it is just a copy of DNA, and we can make more. The DNA is transcribed into RNA, which is then translated into the amino acid sequence. Flow of information: DNA RNA Proteins DNA is read in groups of 3 bases. As stated earlier, DNA does not directly code for proteins themselves. DNA uses RNA as an intermediate. DNA is used as a template to make messenger RNA This mRNA (messenger RNA) is read by ribosomes in groups of 3 bases called codons. Each codon codes for 1 amino acid (remember that amino acids are the monomers for proteins). Both DNA and RNA are nucleic acids; therefore, they have similarities (1. both are made of nucleotides [sugar, phosphate, and base], 2. both have the same purine bases [adenine and guanine], 3. both are used in the passage of hereditary information, etc. Even so, there are 3 major differences in DNA and mRNA (there are other types of RNA that will be discussed later). DNA mRNA Pyrimidine bases C, T C, U Sugar Deoxyribose Ribose Size Double stranded Single strand Copying DNA is based on the strands of DNA being complementary (Adenine pairs with Thymine and Cytosine pairs with Guanine….or Purines pair with Pyrimidines) The two strands of the parental DNA separate and both become a template for the assembly of complementary strands of free nucleotides. Nucleotides line up one a time along the strand to create 2 new complete daughter DNA molecules. The new DNA molecules is ½ of the original and ½ new so it is considered “semi-conservative” Humans, with over 6 billion base pairs in 46 diploid chromosomes, require only a few hours to replicate. Even so, only about 1 DNA nucelotide per several billion is incorrectly paired. In other words, your body is pretty dang impressive. Remember that enzymes end in “ase” and the start of the name tells what the enzyme works on. Notice the name of each of the enzymes tells you what they do. Enzymes involved in replicated (listed in the order they are used) 1. Helicase – breaks hydrogen bond between DNA strands to “unzip” the double helix 2. Primase – Adds an RNA “primer” that Polymerase can bind to so it can begin making a new strand of DNA 3. DNA Polymerase – Adds nucleotides to the 3’ end of a nucleotide build a new DNA strand. It builds a “DNA polymer” 4. DNA Ligase – Adds a few nucleotides to close the gap between Okazaki fragments on the lagging strand. It “links” DNA. Helicases Helicases are enzymes responsible for the unwinding of the DNA molecule. They unwind the DNA in both directions 37 Begins at several sites along the DNA called origins of replication Replication then proceeds in both directions creating replication bubbles Note that there is a Phosphate attached to the 5’ carbon. There is an –OH attached to the 3’ carbon that will be removed during dehydration to combine with hydrogen to make water. 5’ carbon 4’ carbon 3’ carbon 2’ carbon 1’ carbon DNA’s strands are antiparallel – they run in opposite directions. (One strand runs from 5’ to 3’ and the other runs from 3’ to 5’). VERY IMPORTANT in replication because DNA polymerases only add to the 3’ end, never to the 5’ end. In other words, daughter strands only grow 5’ to 3’. DNA always grows from 5’ to 3’ on the daughter template because DNA polymerase can only add nucleotides to the 3’ end because there is a phosphate attached to the 5’ carbon. (This means that the parent template will be read in the 3’ to 5’ direction. Remember that the new template being built will be antiparallel to the parent template). Leading Strand - Because DNA polymerases can only add nucleotides to the 3’ end, only one daughter DNA can be constructed continuously toward the replication fork. Remember that DNA strands run anti-parallel. Lagging Strand – The other daughter DNA must be constructed in segments as DNA polymerase adds nucleotides away from the replication fork to the 3’ end. This strand has to be constructed AWAY from the fork. Therefore, as the fork opens up, DNA polymerase will create a short DNA segment that will build toward the part of the daughter strand that has already been constructed. These segments are called Okazaki segments and are attached to the rest of the DNA strand by an enzyme called DNA ligase. Then, as the fork continues to open up, another segment will be added in the same manner. The Lagging Strand and Ligase This animation, shows the leading strand being synthesized followed by the lagging strand. The enzyme named ligase ties them together. 43 DNA replication ensures that every somatic cell in a multicellular organism has the same genetic information (done during Interphase before Mitosis). DNA ligase and DNA polymerase also serve a role as proof readers to quickly remove incorrectly paired base-pairs. Any change in DNA sequence is considered a mutation because changing DNA sequence will change amino acid sequence/protein (and thus the physical appearance of the organism) A change in DNA during replication results in a daughter cell that is different than its parent cell. The protein produced by the mutated gene will be different than the original, thus the trait caused will be different as well. Somatic cell mutations affect the individual but not their offspring. Gamete cell mutations do not affect the individual but do affect the offspring. Mutagenesis – production of mutations Mutagen – chemical or physical agent causing mutation 2 major kinds: 1. Base substitution – Sub 1 nucleotide for another Not as bad because only 1 codon is changed (which may mean 1 amino acid change or possibly no amino acid change at all) We will study how amino acids are coded for later 2. Insertion or deletion – insert or delete a base Alters the entire reading frame (triplet grouping) This type of mutation is very bad because it changes the entire DNA sequence and thus the entire polypeptide that is being coded for ** Note: Although mutations are almost always harmful, they are also very important. This is because mutations can on rare occasions be beneficial. Mutations provide the diversity of life that evolution can then act upon. We will discuss mutations more when we discuss exactly how DNA codes for proteins. DNA is the genetic code for all life. Even so, DNA does not directly “do” anything. Therefore, the processes of 1) transcription and 2) translation allow a cell to carry out the process of taking the code of DNA to mRNA and eventually from mRNA to protein. In other words, the flow of information in a cell goes from: DNA mRNA Protein What? DNA coding mRNA(messenger RNA) Where? Nucleus Why? DNA is double stranded and too large to get out of the nucleus through the nuclear pores. (mRNA is single stranded and can escape the nucleus). Also, DNA is too important to the cell to risk allowing it to be unprotected in the cytoplasm. DNA Methylation permanently “turns off” DNA so it cannot be transcribed. DNA that is not methylated will be transcribed, but DNA that is methylated will not. This is how all of your cells can have the same DNA but look/behave differently. The DNA that is not needed for that cell is methylated and thus turned off. 1. 2. mRNA(messenger RNA)- takes DNA message to ribosomes Promoter sequence – a nucleotide sequence on DNA that signals for transcription to begin at this area 1. 3. 4. 5. This is the site for RNA Polymerase binding and determines which of the two strands of DNA is to be transcribed Terminator sequence – sequence of DNA that signals the end of transcription and the end of the gene Template strand – strand of DNA used to construct mRNA Noncoding/Nontemplate strand- strand of DNA not used to make mRNA 6. Helicase – transcription enzyme that breaks the Hydrogen bonds between DNA bases so that transcription can begin 7. RNA Polymerase – transcription enzyme that adds RNA nucleotides to the DNA template by helping to form Hydrogen bonds between the bases of DNA and mRNA 1) Initiation – RNA polymerase binds to promoter DNA on the coding strand after Helicase has separated the strands 2) RNA elongation – RNA polymerase “slides” down DNA coding strand creating mRNA as it goes by adding RNA nucleotides by correct base pairing rules (A to U and C to G) As RNA synthesis continues, the RNA strand peels away from its DNA template and the two DNA strands come back together 3) Termination - RNA polymerase reaches terminator DNA and the polymerase detaches from the RNA and the gene (section of DNA that has just be transcribed) Changes 1) G-Cap and Poly-A tail – A single Guanine base is added to one end of the mRNA and long tail of 50 to 250 Adenine nucleotides to the other end These help to export mRNA from nucleus, protect mRNA, and help ribosome bind to mRNA Neither of these are translated into the protein 2) RNA Splicing DNA sequences that code for polypeptides are not continuous Intron – internal noncoding regions Exon – coding regions of DNA that are the parts of a gene that are to be expressed as amino acids Introns are “cut” out of the mRNA and the exons are “pasted” together tRNA (transfer RNA) – transfers amino acids from cytoplasm to ribosomes Has a site on top for amino acid attachment The bottom of the tRNA is known as an anticodon Acts as the “interpreter” when translating “nucleic acid language” to protein “language” rRNA (ribosomal RNA) – a type of RNA that, along with proteins, make up the 2 subunits of ribosomes What? mRNA is read by ribosomes (made of rRNA and proteins) and proteins are built from these instructions Where? Ribosomes in the cytoplasm Why? To create proteins to carry out basically every function in the body Codon – mRNA is read by the ribosome in groups of 3 bases. Each codon (3 mRNA bases) codes for 1 amino acid Amino acid – monomer (building block) of protein Anticodon – 3 bases on the bottom of tRNA that are complementary (opposite) to the codons on mRNA. Anticodons on the bottom of tRNA ensure that each codon codes for only 1 amino acid Ribosome – Reads mRNA codons and sends out signal to tRNA to bring in appropriate amino acid (by matching codon of mRNA to anticodon of tRNA) tRNA – type of RNA that transfers amino acids from cytoplasm to ribosomes 1) Initiation – binding of mRNA to ribosome mRNA binds to small ribosomal subunit tRNA then binds to the start codon (which is AUG) to bring in first amino acid – MET Large ribosomal subunit binds to the small one, creating a functional ribosome Ribosome now has 2 binding sites P site = holds tRNA with growing polypeptide A site = vacant site where next amino-acid bearing tRNA will bind 2) Elongation – Amino acids are added one by one to first amino acid. Occurs in 3 step process. Codon recognition – Anticodon of incoming tRNA molecule, carrying its amino acid, pairs with mRNA codon in A site Peptide bond formation - Polypeptide separates from tRNA in P site and attaches by a peptide bond to amino acid carried by tRNA in A site Translocation - P site tRNA now leaves the ribosome, and ribosome translocates (moves) the tRNA in the A site, with its attached polypeptide, to the P site. The codon and anticodon remain bonded so tRNA and mRNA move as a unit. This opens the A site for the next amino acid to be brought in by a tRNA 3) Termination – Elongation continues until a stop codon reaches the A site Ribosome then breaks apart and finished polypeptide is released from tRNA where it was growing mRNA(messenger RNA)- takes DNA message to ribosomes where it is gives the code for constructing proteins to rRNA rRNA (ribosomal RNA) – rRNA and proteins combine to make ribosomes. Ribosomes construct proteins. tRNA (transfer RNA) – transfers amino acids to ribosomes so protein can be built