Bio 112 Lecture Slides 6 2022 PDF

Bio 112 Cell and Molecular Experiments used to elucidate: 1. Identification of the macromolecule that carries the genetic information 2. Discovering the structure of DNA 3. Discovering how DNA is duplicated DiPino Slide Set 6 Experiments that proved DNA is the genetic...

Bio 112 Cell and Molecular Experiments used to elucidate: 1. Identification of the macromolecule that carries the genetic information 2. Discovering the structure of DNA 3. Discovering how DNA is duplicated DiPino Slide Set 6 Experiments that proved DNA is the genetic material Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists The Search for the Genetic Material: Scientific Inquiry When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material The key factor in determining the genetic material was choosing appropriate experimental organisms The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them  IDENTIFICATION OF DNA AS THE GENETIC MATERIAL OF CELLS To fulfill its role, the genetic material must meet several criteria – 1. Information: It must contain the information necessary to make an entire organism – 2. Transmission: It must be passed from parent to offspring – 3. Replication: It must be copied In order to be passed from parent to offspring – 4. Variation: It must be capable of changes To account for the known phenotypic variation in each species  IDENTIFICATION OF DNA AS THE GENETIC MATERIAL OF CELLS The data of many geneticists, including Mendel, were consistent with these four properties – However, the chemical nature of the genetic material cannot be identified solely by genetic crosses Indeed, the identification of DNA as the genetic material involved a series of outstanding experimental approaches – These will be examined next Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-4 Test March 30 Frederick Griffith Experiments with Streptococcus pneumoniae Griffith studied a bacterium now known as Streptococcus pneumoniae (1928!) S. pneumoniae comes in two strains Frederick Griffith – S  Smooth Secrete a polysaccharide capsule – Protects bacterium from the immune system of animals Produce smooth colonies on solid media – R  Rough Unable to secrete a capsule Produce colonies with a rough appearance Frederick Griffith Figure 9.2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-8 In 1928, Griffith conducted experiments using two strains of S. pneumoniae: type IIIS and type IIR 1. Inject mouse with live type IIIS bacteria Mouse died Type IIIS bacteria recovered from the mouse’s blood 2. Inject mouse with live type IIR bacteria Mouse survived No living bacteria isolated from the mouse’s blood 3. Inject mouse with heat-killed type IIIS bacteria Mouse survived No living bacteria isolated from the mouse’s blood 4. Inject mouse with live type IIR + heat-killed type IIIS cells Mouse died Type IIIS bacteria recovered from the mouse’s blood Griffith concluded that something from the dead type IIIS was transforming type IIR into type IIIS  He called this process transformation The substance that allowed this to happen was termed the transformation principle  Griffith did not know what it was The nature of the transforming principle was determined using experimental approaches that incorporated various biochemical techniques  Maclyn McCarty Colin MacLeod The Experiments of Avery, MacLeod and McCarty Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material They carried out their experiments in the 1940s – At that time, it was known that DNA, RNA, proteins and carbohydrates are major constituents of living cells They prepared cell extracts from type IIIS cells containing each of these macromolecules – Only the extract that contained purified DNA was able to convert type IIR into type IIIS  In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about DNA  Evidence That Viral DNA Can Program Cells More evidence for DNA as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research Fig. 16-3 Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell  Martha Chase Chase and Hershey Alfred Hershey  In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information Hershey and Chase Experiment with Bacteriophage T2 In 1952, Alfred Hershey and Marsha Chase provided further evidence that DNA is the genetic material Figure 9.4 Inside the They studied the capsid bacteriophage T2  It is relatively simple Made up since its composed of of protein only two macromolecules DNA and protein Life cycle of the T2 bacteriophage  The Hypothesis – Only the genetic material of the phage is injected into the bacterium Isotope labeling will reveal if it is DNA or protein Testing the Hypothesis Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-15  The Hershey and Chase experiment can be summarized as such: Use precursors to DNA and precursors to proteins – Used radioisotopes to distinguish DNA from proteins 32P labels DNA specifically 35S labels protein specifically – Radioactively-labeled phages were used to infect nonradioactive Escherichia coli cells – After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells => Phage ghosts and E. coli cells were separated – Radioactivity was monitored using a scintillation counter Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-14 Fig. 16-4-1 EXPERIMENT Radioactive Phage protein Bacterial cell Batch 1: DNA radioactive sulfur (35S) Radioactive DNA Batch 2: radioactive phosphorus (32P) Fig. 16-4-2 EXPERIMENT Empty Radioactive protein Phage shell protein Bacterial cell Batch 1: DNA radioactive Phage sulfur (35S) DNA Radioactive DNA Batch 2: radioactive phosphorus (32P) Fig. 16-4-3 EXPERIMENT Empty Radioactivity Radioactive protein (phage Phage shell protein) protein in liquid Bacterial cell Batch 1: DNA radioactive Phage sulfur (35S) DNA Centrifuge Radioactive Pellet (bacterial DNA cells and contents) Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity Pellet (phage DNA) in pellet Additional Evidence That DNA Is the Genetic Material It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next This evidence of diversity made DNA a more credible candidate for the genetic material  RNA Functions as the Genetic Material in Some Viruses In 1956, A. Gierer and G. Schramm isolated RNA from the tobacco mosaic virus (TMV), a plant virus  Purified RNA caused the same lesions as intact TMV viruses Therefore, the viral genome is composed of RNA Since that time, many RNA viruses have been found  Refer to Table 9.1 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9-21 The Structure of DNA After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role  Overview: Life’s Operating Instructions In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA DNA, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DNA and reproduced in all cells of the body This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits  A Few Key Events Led to the Discovery of the Structure of DNA In 1953, James Watson and Francis Crick discovered the double helical structure of DNA The scientific framework for their breakthrough was provided by other scientists including  Linus Pauling  Rosalind Franklin and Maurice Wilkins  Erwin Chargaff  Linus Pauling In the early 1950s, he proposed that regions of protein can fold into a secondary structure  a-helix To elucidate this structure, he built ball-and-stick models  Refer to Figure 9.12b Watson and Crick raced against Pauling to determine how nucleotides fit together in DNA Building a Structural Model of DNA: Scientific Inquiry Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DNA molecule using this technique Fig. 16-6 (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA Rosalind Franklin (1920 - 1958) Rosalind Franklin was born in London on July 25, 1920. She attended St. Paul's Girls' School. When she was growing up, her parents took in two Jewish children from Nazi Germany to live in their home as part of the family. Rosalind shared her room with a woman whose father had been sent to Buchenwald. Franklin was strongly influenced by her grandfather, Arthur, who was active in social service and was so committed to Judaism that his will stipulated that only those descendants married to Jews would inherit any of his estate. Franklin’s great-uncle, Herbert Samuel, was the first High Commissioner of Palestine. Franklin studied chemistry and physics at Newnham College, Cambridge, and, in 1942, began carrying out research at the British Coal Utilization Research Association. Over the next four years she helped develop carbon fibre technology. In 1947, Franklin went to the Central Government Laboratory for Chemistry in Paris where she worked on X-ray diffraction. In 1951, she moved to King's College, London. As a woman and a Jew, Franklin felt unwelcome at King's College (the women scientists were not allowed to eat lunch in the common room where the men did). The combination of anti-Semitism and sexism also was an underlying factor in some criticism of her work. In James Watson's book, The Double Helix, Franklin is described as a difficult woman who was unwilling to share her research. She's characterized as a complainer and her appearance and clothes are criticized. Franklin worked on a DNA project that she thought was her own. When the laboratory's second-in-command, Maurice Wilkins, retruned from a vacation, however, she learned that he expected her to be his assistant rather than a colleague working as an equal. They had an uneasy relationship, complicated by the fact she was a woman in a "man's world" and their conflicting personalities. Franklin made a number of advances in x-ray diffraction techniques with DNA that allowed her to discover crucial elements in what had become a race between competing research teams to discover the structure of DNA. Franklin produced X-ray diffraction pictures of DNA which were published in Nature in April 1953. This played an important role in establishing the structure of DNA. In fact, many scientists believe Franklin played a larger role than previously acknowledged in the research that led to the 1962 Nobel Prize that was awarded to Maurice Wilkins, Francis Crick, and James Watson for the discovery of DNA's double helix. Wilkins shared Franklin's data, without her knowledge, with Watson and Crick, at Cambridge University, and they pulled ahead in the race, ultimately publishing the proposed structure of DNA in March 1953. The difficulty in working with Wilkins and the discomfort of the environment at King's College led Franklin to leave, but the College insisted that she cease work on DNA. Franklin joined John Bernal at Birkbeck College to carry out research into the tobacco mosaic virus. Her group's findings laid the foundation for structural virology. While visiting the United States, Franklin began to experience terrible pains that she soon learned were related to ovarian cancer. She continued working up until a few weeks before her death on April 16, 1958, at age 37.  Rosalind Franklin She worked in the same laboratory as Maurice Wilkins She used X-ray diffraction to study wet fibers of DNA The diffraction pattern is interpreted (using mathematical theory) This can ultimately provide information concerning the structure of the molecule Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display  Rosalind Franklin She made marked advances in X-ray diffraction techniques with DNA The diffraction pattern she obtained suggested several structural features of DNA  Helical  More than one strand  10 base pairs per complete turn  Erwin Chargaff’s Experiment Chargaff pioneered many of the biochemical techniques for the isolation, purification and measurement of nucleic acids from living cells It was already known then that DNA contained the four bases: A, G, C and T The Data Interpreting the Data The data shown in Figure 9.14 are only a small sampling of Chargaff’s results The compelling observation was that  Percent of adenine = percent of thymine  Percent of cytosine = percent of guanine This observation became known as Chargaff’s rule  It was crucial evidence that Watson and Crick used to elucidate the structure of DNA Fig. 16-1  Watson and Crick Familiar with all of these key observations, Watson and Crick set out to solve the structure of DNA  They tried to build ball-and-stick models that incorporated all known experimental observations A critical question was how the two (or more strands) would interact  An early hypothesis proposed that the strands interact through phosphate-Mg++ crosslinks  Refer to Figure 9.15 Figure 9.15 This hypothesis was, of course, incorrect! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display  Watson and Crick They went back to the ball-and-stick units They then built models with the  Sugar-phosphate backbone on the outside  Bases projecting toward each other They first considered a structure in which bases form H bonds with identical bases in the opposite strand  ie., A to A, T to T, C to C, and G to G Model building revealed that this also was incorrect  Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior  At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data  Watson and Crick reasoned that the pairing was more specific, dictated by the base structures They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C Fig. 16-8 Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Fig. 16-7 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end (a) Key features of DNA(b) Partial chemical (c) Space-filling structure structure model  Watson and Crick Watson, Crick and Maurice Wilkins were awarded the Nobel Prize in 1962 Rosalind Franklin died in 1958, and Nobel prizes are not awarded posthumously Race to determine the structure of DNA https://www.youtube.com/watch?v=d7ET4bbkTm0 DNA Replication Experiments Concept: Many proteins work together in DNA replication and repair The relationship between structure and function is manifest in the double helix Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material The Basic Principle: Base Pairing to a Template Strand Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules Fig. 16-9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules, strands each consisting of one parental strand and one new strand  Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)  Which Model of DNA Replication is Correct? In the late 1950s, three different mechanisms were proposed for the replication of DNA – Conservative model Both parental strands stay together after DNA replication – Semiconservative model The double-stranded DNA contains one parental and one daughter strand following replication – Dispersive model Parental and daughter DNA are interspersed in both strands following replication Fig. 16-10 First Second Parent cell replication replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models They found a way to experimentally distinguish between daughter and parental strands They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope The first replication produced a band of hybrid DNA, eliminating the conservative model A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Their experiment can be summarized as such – Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations The population of cells had heavy-labeled DNA – Switch E. coli to medium containing only 14N (a light isotope of Nitrogen) – Collect sample of cells after various times – Analyze the density of the DNA by centrifugation using a CsCl gradient Fig. 16-11a EXPERIMENT 1 Bacteria 2 Bacteria cultured in transferred to medium medium containing containing 14N 15 N RESULTS 3 DNA sample 4 DNA sample Less centrifuged centrifuged dense after 20 min after 20 min (after first (after second More application) replication) dense Fig. 16-11b CONCLUSION First replication Second replication Conservative model Semiconservative model Dispersive model  Interpreting the Data After ~ two generations, DNA is of two After one generation, DNA is types: “light” and “half-heavy” “half-heavy” This is consistent with only the This is consistent with both semi- semi-conservative model conservative and dispersive models  conclusion The first replication produced a band of hybrid DNA, eliminating the conservative model A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model In vitro Bacterial Replication and Eukaryotic DNA Replication DNA Replication Can Be Studied In Vitro The in vitro study of DNA replication was pioneered by Arthur Kornberg in the 1950s – He received a Nobel Prize for his efforts in 1959 Kornberg hypothesized that deoxynucleoside triphosphates are the precursors of DNA synthesis He also knew that these nucleotides are soluble in an acidic solution while long DNA strands are not  Arthur Kornberg Roger Kornberg Me and a couple of my favorite scientists Arthur Frank Christian DNA Replication Can Be Studied In Vitro In this experiment, Kornberg mixed the following – An extract of proteins from E. coli – Template DNA – Radiolabeled nucleotides These were incubated for sufficient time to allow the synthesis of new DNA strands – Addition of acid will precipitate these DNA strands – Centrifugation will separate them from the radioactive nucleotides  The Hypothesis – DNA synthesis can occur in vitro if all the necessary components are present Testing the Hypothesis – Refer to Figure 11.17  EUKARYOTIC DNA REPLICATION Eukaryotic DNA replication is not as well understood as bacterial replication – The two processes do have extensive similarities, The bacterial enzymes described in Table 1.1 have also been found in eukaryotes – Nevertheless, DNA replication in eukaryotes is more complex Large linear chromosomes Tight packaging within nucleosomes More complicated cell cycle regulation  Multiple Origins of Replication Eukaryotes have long linear chromosomes – They therefore require multiple origins of replication To ensure that the DNA can be replicated in a reasonable time In 1968, Huberman (“Bobometer”) and Riggs provided evidence for the multiple origins of replication DNA replication proceeds bidirectionally from many origins of replication  Bidrectional DNA synthesis Replication forks will merge Part (b) shows a micrograph of a replicating DNA chromosome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display  Multiple Origins of Replication The origins of replication found in eukaryotes have some similarities to those of bacteria – Origins of replication in Saccharomyces cerevisiae are termed ARS elements (Autonomously Replicating Sequence) They are 100-150 bp in length They have a high percentage of A and T They have three or four copies of a specific sequence – Similar to the bacterial DnaA boxes  Multiple Origins of Replication Origin recognition complex (ORC) – A six-subunit complex that acts as the initiator of eukaryotic DNA replication It appears to be found in all eukaryotes – Requires ATP to bind ARS elements – Single-stranded DNA stimulates ORC to hydrolyze ATP  Eukaryotes Contain Several Different DNA Polymerases Mammalian cells contain well over a dozen different DNA polymerases Four: alpha (a), delta (d), epsilon (e) and gamma (g) have the primary function of replicating DNA – a, d and e : Nuclear DNA – g : Mitochondrial DNA  Trends Biochem Sci. (6):212-7. Eukaryotic DNA replication: yeast bares its ARSs. Campbell J.