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5/2/16 Chapter 10 Molecular Biology of the Gene PowerPoint Lectures Campbell Biology: Concepts & Connections, Eighth Edition Biol REECE • TAYLOR • SIMON • DICKEY • HOGAN © 2015 Pearson Education, Inc. 1408 Dr. Doumen Lecture by Edward J. Zalisko Introduction • The 2009 H1N1 influenza (flu) virus • spread so quickly that it was declared a pandemic, • reached 207 countries, • infected more than 600,000 people, and • killed an estimated 20,000 people. • Viruses share some of the characteristics of living organisms, but are generally not considered alive because they are not cellular and cannot reproduce on their own. © 2015 Pearson Education, Inc. Introduction • So what makes up a virus ? • In general a virus is made from a • Capsid : this is the protein shell of a virus • Internal genome : usually a DNA or RNA sequence. • Some viruses have an additional viral envelope. © 2015 Pearson Education, Inc. 1 5/2/16 Introduction Examples of common viruses ( look at the size and also see next slide for comparison) © 2015 Pearson Education, Inc. Introduction © 2015 Pearson Education, Inc. Introduction • Viruses can be dangerous and combating any virus requires a detailed understanding of • molecular biology, • the study of DNA/RNA, and • its mode of replication, and how DNA serves as the basis of heredity. • The study on how viruses work has helped scientists in the discovery of the genetic material and how it works in most living cells. © 2015 Pearson Education, Inc. 2 5/2/16 SCIENTIFIC THINKING: Experiments showed that DNA is the genetic material • Early in the 20th century, the molecular basis for inheritance was a mystery. • Biologists did know that genes were located on chromosomes. But it was unknown if the genetic material was a protein based system or a nucleic acid based system. © 2015 Pearson Education, Inc. SCIENTIFIC THINKING: Experiments showed that DNA is the genetic material • Biologists finally established the role of DNA in heredity through experiments with bacteria and the viruses that infect them. • This breakthrough ushered in the field of molecular biology, the study of heredity at the molecular level. © 2015 Pearson Education, Inc. Frederick Griffith Experiment • Pneumonia was a serious cause of death following Spanish Influenza Pandemic (1918) • Frederick Griffith (1879–1941), a British bacteriologist, wanted to create a vaccine against pneumonia and started working with pneumococcus bacteria, using mice as his ‘patients’ Streptococcus pneumoniae. © 2015 Pearson Education, Inc. 3 5/2/16 Frederick Griffith Experiment • He used two strains of bacteria • S-strain (virulent): • this bacteria covers itself with a smooth capsule, protecting itself against the host’s immune system • It will kill the host • R-strain (non-virulent): • Does not have a protective capsule and gets killed by the host’s immune system. • The host will not die © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Frederick Griffith Experiment • At that time, they believed that these strains were fixed and un-changeable: the R-strain could not become the S-strain and visa versa • They also had a test to see which form was growing in a petri-dish • His experiment was to inject mice with the different strains and see what happened. © 2015 Pearson Education, Inc. 4 5/2/16 Frederick Griffith Experiment • Inject mice with living S-strain bacteria • Result: mice die • Inject mice with living R-strain bacteria • Result : mice stay alive • Inject mice with heat killed S-strain • Result : mice stay alive • Inject mice with a mix of living R-strain and heat killed S-strain • Result : mice die © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Frederick Griffith Experiment © 2015 Pearson Education, Inc. 5 5/2/16 Frederick Griffith Experiment • When they examined the blood of the dead mice from the last experiment, they found live S-strain bacteria that could be culture • All of the descendants of the transformed bacteria inherited the newly acquired ability to cause disease © 2015 Pearson Education, Inc. Frederick Griffith Experiment • Griffith concluded that some transforming factor ( he called it a “transforming principle”) present in the dead S-strain, had transformed the living Rstrain • So, the harmless strain was transformed into a deadly strain because something from the heatkilled, dead bacteria strain slipped into the harmless strain and made them virulent. © 2015 Pearson Education, Inc. Frederick Griffith Experiment • Today, we know that the "transforming principle" Griffith observed was the DNA of the S-strain bacteria • That DNA survived the heating process and was taken up by the R-strain, providing the genes to make the protective capsule • The exact nature of the transforming principle (the DNA) was verified by later experiments (eg. The Hershey Chase experiment) © 2015 Pearson Education, Inc. 6 5/2/16 Hershey – Chase Experiments • In 1952, Alfred Hershey and Martha Chase used bacteriophages to show that DNA is the genetic material • Bacteriophages (or phages for short) are viruses that infect bacterial cells. • They used a bacteriophage called of T2, a virus that infects the bacterium Escherichia coli (E. coli). © 2015 Pearson Education, Inc. A bacteriophage : a virus that infects bacteria © 2015 Pearson Education, Inc. Reproductive Cycle of a Bacteriophage • The phage attaches to the cell wall of a bacterium • It injects its DNA into the bacterial cell • The viral DNA directs the destruction of the host DNA • Viral genes instruct the making of replicate viral DNA 1 A phage attaches itself to a bacterial cell. 2 The phage injects its DNA into the bacterium. © 2015 Pearson Education, Inc. 7 5/2/16 Reproductive Cycle of a Bacteriophage • The viral DNA takes charge of the protein production line of the bacteria (eg. Ribosomes). • Viral genes direct the protein machinery to make new viral proteins and assemble new viruses • Bacterium lyses and new viruses spill out 3 The phage DNA directs the host cell to make more phage DNA and proteins; new phages assemble. 4 The cell lyses and releases the new phages. © 2015 Pearson Education, Inc. Figure 10.1c-0 Reproductive Cycle of a Bacteriophage 1 A phage attaches itself to a bacterial cell. 2 The phage injects its DNA into the bacterium. 3 The phage DNA directs the host cell to make more phage DNA and proteins; new phages assemble. The cell lyses and releases the new phages. © 2015 Pearson Education, Inc. Hershey-Chase Experiments • Hershey and Chase knew their biology and biochemistry very well to realize that sulphur is an element found in certain amino acids • On the other hand, phosphorus is an element mainly found in nucleic acids such as RNA and DNA • They came up with a brilliant idea to figure out weather protein or nucleic acids were the biomolecule that carries the genetic information. © 2015 Pearson Education, Inc. 8 5/2/16 Hershey-Chase Experiments Grow viruses in a media with radioactive sulfur Sulfur will become incorporated into protein Thus the coat/shell of the viruses will be radioactive Grow viruses in a media with radioactive phosphorus Phosphorus will become incorporated into DNA/RNA Radioactive material will now be inside the virus © 2015 Pearson Education, Inc. Hershey-Chase Experiments • They infected bacteria • with radioactive sulfur labeled viruses ( so the radioactive element is in the protein coat) Or • with radioactive phosphorus labeled viruses ( so the radioactive element is inside in the DNA) • After a while, they used a simple blender to shake off all the viruses from the bacteria and centrifuged it all down ( bacteria would pellet down – viruses remain in upper suspension liquid) © 2015 Pearson Education, Inc. • In bacteria infected with radioactive sulfur, most radioactivity remained in suspension liquid, none in the bacterial pellet. • The new viruses that eventually came out of the bacterial pellet had no radioactive sulfur • In bacteria infected with radioactive phosphorus, most radioactivity was found within the bacterial pellet • The new viruses that eventually came out of the bacterial pellet contained radioactive phosphorus © 2015 Pearson Education, Inc. 9 5/2/16 Figure 10.1b-0 Phage Bacterium Radioactive protein Empty protein shell The radioactivity is in the liquid. Phage DNA DNA Centrifuge Pellet Batch 1: Radioactive protein labeled in yellow Radioactive DNA Centrifuge Pellet The radioactivity is in the pellet. Batch 2: Radioactive DNA labeled in green © 2015 Pearson Education, Inc. Hershey-Chase Experiments The Hershey and Chase experiment strongly supported DNA as the hereditary material while it also showed protein was NOT the hereditary material. © 2015 Pearson Education, Inc. The search for the DNA structure • After the 1952 Hershey-Chase experiment convinced most biologists that DNA was the material that stored genetic information, a race was on to determine how the structure of this molecule could account for its role in heredity. • Researchers focused on discovering the threedimensional shape of DNA. © 2015 Pearson Education, Inc. 10 5/2/16 Watson and Crick • American James D. Watson journeyed to Cambridge University in England, where the more senior Francis Crick was studying protein structure with a technique called X-ray crystallography. • While visiting the laboratory of Maurice Wilkins at King’s College in London, Watson saw an X-ray image of DNA produced by Wilkins’s colleague, Rosalind Franklin. © 2015 Pearson Education, Inc. Watson and Crick Rosalind Franklin and her important X-ray diffraction analysis of DNA © 2015 Pearson Education, Inc. DNA is a double-stranded helix • Watson deduced the basic shape of DNA to be a helix (spiral) with a uniform diameter and the nitrogenous bases located above one another like a stack of dinner plates. • The thickness of the helix suggested that it was made up of two polynucleotide strands. © 2015 Pearson Education, Inc. 11 5/2/16 DNA is a double-stranded helix • Watson and Crick realized that DNA consisted of two polynucleotide strands wrapped into a double helix. • The sugar-phosphate backbone is on the outside. • The nitrogenous bases are perpendicular to the backbone in the interior. • Specific pairs of bases give the helix a uniform shape. • A pairs with T, forming two hydrogen bonds, and • G pairs with C, forming three hydrogen bonds. © 2015 Pearson Education, Inc. Hydrogen bond (dotted lines) C G T Sugarphosphate backbone A T A C Sugarphosphate backbone G Pairs of nitrogenous bases linked with hydrogen bonds © 2015 Pearson Education, Inc. Figure 10.3c Pairs of nitrogenous bases linked with hydrogen bonds Sugar-phosphate backbone Twist © 2015 Pearson Education, Inc. 12 5/2/16 Watson and Crick © 2015 Pearson Education, Inc. Figure 10.3d-0 C C G G G Hydrogen bond C G C G A C Base pair A T T T C A G A T A T C G C C G C A A T A G G T T T A Ribbon model Partial chemical structure Computer model © 2015 Pearson Education, Inc. Watson and Crick • In 1962, the Nobel Prize was awarded to James D. Watson, Francis Crick, and Maurice Wilkins. • Rosalind Franklin probably would have received the prize as well but for her death from cancer in 1958. • Nobel Prizes are never awarded posthumously. • The Watson-Crick model gave new meaning to the words genes and chromosomes. The genetic information in a chromosome is encoded in the nucleotide sequence of DNA. • It still wasn’t clear how it was encoded…. © 2015 Pearson Education, Inc. 13 5/2/16 DNA and RNA are polymers of nucleotides • DNA and RNA are nucleic acids consisting of long chains (polymers) of chemical units (monomers) called nucleotides. • A DNA nucleotide is composed of a • nitrogenous base, • five-carbon sugar, called deoxyribose • phosphate group. • The nucleotides are joined to one another by a sugarphosphate backbone. © 2015 Pearson Education, Inc. Figure 10.2a-3 A DNA nucleotide Nitrogenous base (can be A, G, C, or T) Thymine (T) Phosphate group Sugar (deoxyribose) DNA nucleotide © 2015 Pearson Education, Inc. Sugar-phosphate backbone A DNA poly-nucleotide A A • A DNA nucleotide can have 4 different nitrogencontaining base: • adenine (A), • cytosine (C), • thymine (T), and • guanine (G). • A DNA polynucleotide will have those 4 bases represented in a certain order Covalent bond joining nucleotides C C DNA nucleotide T Phosphate group Nitrogenous base Sugar T G G G G Two representations of a DNA polynucleotide © 2015 Pearson Education, Inc. 14 5/2/16 The nitrogen-containing bases in DNA Thymine (T) Cytosine (C) Adenine (A) Guanine (G) Purines Pyrimidines • • • • Pyrimidines have a 6 ring structure and Purines are a combination of a 5 ring with a 6 ring Each base is part of a nucleotide. They are important in the DNA structure as they form the “spokes” of the ladder, the connections what holds the double helix together. T on one polynucleotide strand backbone always pairs with an A on the other strand G on one polynucleotide strand backbone always pairs with an C on the other strand © 2015 Pearson Education, Inc. A complete DNA Is a double helix A T • A complete DNA is a double helix of two polynucleotides, twisted around each other as shown earlier T G C A C G T A C G G T A • Notice the base pairing between the two strands C G T T A A C G T A A DNA double helix © 2015 Pearson Education, Inc. 10.2 DNA and RNA are polymers of nucleotides • The full name for DNA is deoxyribonucleic acid, • RNA (ribonucleic acid) is unlike DNA in that it • Has nucleotides made from the the sugar ribose (instead of deoxyribose in DNA) and • The nitrogenous bases are similar except that uracil (U) is used instead of thymine (T). © 2015 Pearson Education, Inc. 15 5/2/16 An RNA nucleotide Nitrogenous base (can be A, G, C, or U) Phosphate group Uracil (U) Sugar (ribose) © 2015 Pearson Education, Inc. 16