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1866- Mendel's Paper 1875- Mitosis worked out 1890's- Meiosis worked out 1902- Sutton, Boveri et. al. connect chromosomes to Meiosis. 1907- Morgans “fly room” provides support for chromosomes as the hereditary material 1928- Griffith discovers transformation 1944- Avery announces DNA is the transforming material, but no one believes him 1952 Hershey & Chase use radio-labeled bacteriophages to prove DNA must be the genetic material Review of the Experiments http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html Identify the process at the center of most biotechnology research & development Identify the scientists and describe their experiments that led understanding the Structure of DNA Identify the purpose of the Meselson Stahl experiment, explain the experimental procedures, and major conclusions Describe the 3 stages of DNA replication List the enzymes necessary for DNA replication and identify both their function and which stage they participate in Identify similarities in DNA between organisms of different species Identify differences in DNA between organisms of different species List the 3 sources of DNA Explain what a plasmid is and how they’re transferred to bacteria Explain the importance of plasmids and viruses in creating rDNA Define “virus” Explain the steps of viral gene therapy and identify diseases that may be “cured” by this therapy During the last decades of the twentieth century, a “new” biotechnology industry grew due to innovative techniques to both transfer DNA between cells and to manipulate the cells to manufacture specific proteins The manipulation of genetic information is at the center of most biotechnology research and development After the Hershey and Chase experiment, scientists were convinced that DNA contained the genetic material… But how is this information stored And how is it passed on? To answer these questions, scientists began studying the structure of DNA 1950’s arrangement of covalent bonds in nucleic acids was established, but the 3 dimensional shape was still unknown People important in elucidating the structure of DNA Erwin Chargaff Rosalind Franklin Watson & Crick In 1949, Erwin Chargaff observed that for each organism he studied, the amount of adenine always equaled the amount of thymine… A=T Likewise, the amount of guanine always equaled the amount of cytosine… C=G However, the amount of each equal pair differs between different organisms. Ultimately her diffraction pictures were used by Watson & Crick to determine the structure of DNA She determined the sugar phosphate backbones were on the outside of the DNA molecule * In 1952, she took many photographs of sections of DNA using a method called X-Ray Diffraction In the process, X-ray beams were bounced off of DNA and the rays were diffracted or scattered onto a piece of film This method is similar to shining a light on an object and analyzing its shadow X-ray crystallography is an extremely precise means of imaging the exact structure of a given molecule or macromolecule in a crystal lattice. A crystal is any regularly repeating arrangement of unit cells which range in size from less than 100 atoms — to tens of thousands is famous for being the tool first used to discover the structure of DNA. Also used to determine the structure of diamond, penicillin, numerous proteins, entire viruses In all, over 400,000 structures have been described using x-ray crystallography. Researchers using X-ray crystallography grow solid crystals of the molecules they study crystallographers aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X-rays onto an electronic detector the electronic detector today is the same type used to capture images in a digital camera. After each blast of X-rays, the researchers rotate the crystal enabling the them to capture in three dimensions how the crystal scatters, or diffracts, X-rays. The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation called a Fourier transform to calculate the position of every atom in the crystallized molecule Watson gets a copy of Rosalind Franklins x-ray diffraction of DNA and because he is familiar with the types of patterns helical molecules produce (thanks to working with Crick), he immediately knows DNA is helical He’s was also able to deduce the width of the helix and the spacing of the nitrogenous basses along it The width suggested that it was made up of 2 strands (up till now it was believed to be made of 3 strands) He also showed DNA has a uniform width the entire length of the molecule Franklin had concluded the sugar-phosphate backbones were on the outside of the helix This arrangement is appealing because it put the hydrophobic nitrogenous bases on the inside where they would be shielded from the aqueous environment They still thought like paired with like, A-A, T-T, C-C, G-G If adenine paired with adenine, and thymine paired with thymine, how would that affect the diameter of the helix? Purine-purine pairs are too wide, and pyrimidinepyrimidine pairs are too narrow Since adenine is always found in the same amount as thymine, it was determined that adenine would pair with thymine Coincidentally, adenine can only form hydrogen bonds with thymine, and cytosine can only form H-bonds with guanine Watson & Crick ended their 1 page paper on the structure of DNA by saying: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Semi-conservative: DNA strands separate each strand acts as a template to build a new strand on Each daughter DNA molecule is composed of a parent strand and a new strand Conservative: DNA strands separate each strand acts as a template to build a new strand on parent strands reassociate with each other, and new strands associate with each other Dispersive: all 4 strands of DNA following replication are a mixture of old and new DNA 1st culture E. coli on a media containing a heavy isotope of nitrogen 15N Bacteria incorporate the 15N into the nitrogenous bases of their DNA Next, transfer bacteria into culture containing only 14N Any new DNA that the bacteria synthesized would be lighter than the parent DNA made in the 15N Meselson & Stahl could then distinguish DNA of different densities by centrifuging the DNA strands Heavier Strands would travel farther down then lighter strands http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf The complementary structure of DNA is also used as a basis to make exact copies of the DNA each time a cell divides Cell division allows an organism to grow, develop, and to replace old cells Enzymes, biological catalysts, are responsible for synthesizing new strands of DNA DNA replication is also called DNA synthesis Initiation Synthesis Termination ORI: origins of replication Specific sites along a DNA molecule where replication begins Bacteria have 1 circular chromosome, and 1 origin of replication Proteins can recognize the DNA sequence in the ORI, and use this sequence recognition to bind and open up a replication bubble Replication of DNA then proceeds in both directions until the entire molecule has been copied May have hundreds or even thousands of ORI’s Multiple replication bubbles form and eventually fuse, thus speeding up the replication process Replication proceeds in both directions as it does in prokaryotes At the end of each replication bubble is a replication fork, the Y-shaped region where the parental DNA is being unwound Several Kinds of proteins participate in the unwinding Helicase: enzymes that break the hydrogen bonds holding the 2 strands together Single strand binding proteins (SSBP’s): bind the now single stranded DNA to keep the 2 strands from reassociating Topoisomerases: relieves the strain caused by the untwisting of the DNA molecule by breaking the covalent bonds within the DNA backbone, swiveling, and rejoining the strands The unwound sections of DNA can now act as a template to build a new strand on Primase: an enzyme that comes in and builds an RNA primer on the parental DNA strand This step is necessary because DNA polymerase can’t initiate synthesis of a new polynucleotide, rather, it can only add nucleotides to a preexisting strand DNA Polymerase is the enzyme that builds the new DNA strand DNA Polymerases polymerize the synthesis of a new strand of DNA by adding nucleotides to a preexisting strand SSBP’s Topoisomerase The direction of DNA affects elongation DNA polymerase can only add nucleotides to a free 3’ end Thus a new DNA strand can only elongate in the 5’3’ direction Because DNA polymerase can only add to the free 3’ end of a growing polynucleotide chain, one strand of DNA will be synthesized continuously, and the other will be synthesized discontinuously The strand synthesized continuously is called the leading strand Grows in the direction towards the replication fork The strand synthesized discontinuously is called the lagging strand The lagging strand grows in the direction away from the replication fork The lagging strand is synthesized discontinuously in a series of fragments called Okazaki Fragments Okazaki Fragments must eventually be joined together to form 1 continuous DNA stand Remember, DNA polymerase can only add nucleotides to an already existing 3’end This means every time a new Okazaki fragment is started, a new RNA primer must 1st be created Before two okazaki fragments can be joined, all the RNA must be excised and replaced with DNA DNA Polymerase I is responsible for removing RNA primers Ligase: joins the sugar phosphate backbones of al Okazaki Fragments into 1 continuous strand We tend to think of DNA replication looking something akin to a train moving along a rain track This is INCORRECT in 2 important ways First: all of the before mentioned proteins involved in replication do not act individually, but rather together as one large “DNA replication Machine” Second: this DNA replication complex does not move along the DNA strands, rather the DNA moves through the complex http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html# All DNA molecules are composed of 4 nucleotide monomers, each containing 1 of the 4 nitrogenous bases Adenine Cytosine Thymine Guanine Virtually all DNA molecules form a double helix Nucleotide connect to each other through strong phosphodiester bonds between sugars and phosphate groups Hydrogen bonds between complementary nitrogenous bases hold two DNA strands together Within a double stranded segment of DNA, the 2 strands are always antiparallel to each other DNA always undergoes semiconservative replication The number of chromosomes (DNA molecules) per cell differes between organisms of different species The # of base pairs per strand can differ The number and types of genes can differ The number of noncoding sequences The shape of the chromosomes Eukaryotes = linear Prokaryotes = circular Because DNA is the molecule responsible for heredity, all living things have DNA There are 3 main categories of organisms that we get DNA from, what do you think they are? Prokaryotes Eukaryotes Viruses Prokaryotic DNA is usually contained in a single circular chromosome that is found free floating in the cytoplasm in a region of the cell known as the nucleoid This chromosome is usually supercoiled folding over on itself like a twisted rubberband Prokaryotes DO NOT have introns, which we will discuss in greater detail later Some bacteria contain extra small rings of DNA floating in the cytoplasm called a plasmid Plasmids contain non-essential genes that usually give the bacteria some additional characteristic or phenotype that allows it to survive under extreme conditions R plasmids contain genes for antibiotic resistance What do you think the R stands for? Bacteria can transfer plasmids, and thus genetic information, between members of the same species, and sometimes members of different species Plasmid transfer occurs through 2 main processes Conjugation with other bacteria via the sex pilus Transformation: when a cell takes up foreign DNA from the environment The ability to transfer plasmids and thus impart new phenotypes on bacteria provides a mechanism to drive evolution and thus create “superbugs” that are resistant to antibiotics This is a leading cause of emerging infectious diseases Because plasmids are small and easily extracted from cells, they are often used as rDNA vectors Foreign DNA fragments (genes) can be cut and pasted into the plasmids, and then introduced to a new host organism Any cell can take up a plasmid, not just bacterial cells What enzyme (s) is/are necessary for cutting and pasting DNA From protists, plants, animals, and fungi Eukaryotes typically have several linear chromosomes, with each species having a characteristic chromosome number Note: every cell within a multicellular organism has the same number and type of chromosomes Eukaryotic genomes are typically larger than prokaryotes and contain more non-coding sequences of DAN (introns) than prokaryotes do Total amount of DNA per cell is NOT related to organism complexity Viruses are currently considered non-living, obligate, intracellular, parasites This means they absolutely have to (obligate) infect a cell (intracellular) and hijack its enzymes, machinery, and other organism monomers to propagate (parasite) DON’T CALL THEM ORGANISMS An organism by definition is alive, and because viruses don’t meet the characteristics of life they ar enot Call them “infectious particles” Viruses are often used in biotechnology research as vectors to carry DNA into a target cell Viruses do not have a cellular structure All viruses have a thick protein coat( capsid) surrounding a nucleic acid core (DNA or RNA, not both) Like plasmid DNA, viral DNA is small and often used create rDNA which in turn is used to creat a GMO Recombinant virus technology is one technique used to do gene therapy In this technique, a virus is used to insert corrective genes (therapy) into cells that contain defective genes Viral gene therapies are currently being researched in hopes of curing Certain cancers Some types of diabetes Cystic fibrosis The manipulation of genetic information through innovative techniques to both transfer DNA between cells and to manipulate the cells to manufacture specific proteins is at the center of most biotechnology research and development Erwin Chargaff: Adenine and thymine are always present in equal amounts, and cytosione & guanine are always present in equal amounts within a molecule of DNA Rosalind Franklin & Wilson: used X-ray crystallography to image DNA Watson & Crick: discovered that DNA is in the form of a double helix and that a purine always pairs with a pyrimidine (more specifically, Apairs with T and C with G) Purpose: to determine if the method of DNA replication was conservative, dispersive, or semiconservative Procedures: Grow bacteria in heavy N15 media Transfer bacteria to lighter N14 media for 2 rounds of replication Centrifuge and see where the bands form Conclusions: DNA replication is semiconservative Stage Enzymes Initiation Helicase: unwinds DNA Topoisomerase: relieves strain ahead of the replication fork by making cuts through one of the DNA strands and Termination Elongation SSBP’s: hold the 2 opened strands apart Primase: puts down an RNA primer DNA Polymerase III: builds the new strand of DNA DNA Polymerase I: removes RNA primers and replaces them with deoxynucleotides Ligase: catalyzes the formation of the phosphodiester bond between the sugar of one nucleotide and the phosphate group of another to connect short sequences of DNA (like okazaki fragments) All DNA molecules are composed of 4 nucleotide monomers, each containing 1 of the 4 nitrogenous bases double helix Nucleotide connect to each other through strong phosphodiester bonds between sugars and phosphate groups Hydrogen bonds between complementary nitrogenous bases hold two DNA strands together DNA is always antiparallel to each other DNA undergoes semiconservative replication The number of chromosomes (DNA molecules) per cell differes between organisms of different species The # of base pairs per strand can differ The number and types of genes can differ The number of noncoding sequences The shape of the chromosomes Eukaryotes = linear Prokaryotes = circular Most Cells Eukaryotes Plants Animals Fungi protists Prokaryotes Archeabacteria Eubacteria Viruses Small circular pieces of extrachromosomal DNA that contain non-essential genes that usually give the bacteria some additional characteristic or phenotype that allows it to survive under extreme conditions Plasmids are transferred through conjugation and transformation Foreign DNA fragments (genes) can be cut and pasted into the plasmids, and then introduced to a new host organism because plasmids are small and easy to manipulate non-living obligate intracellular parasites also called infectious particles Recombinant virus technology is a technique used to do gene therapy in which a virus is used to insert corrective genes into cells that contain defective genes Viral gene therapies are currently being researched in hopes of curing Certain cancers Some types of diabetes Cystic fibrosis