Molecular Genetics Notes PDF

Notes on Molecular Genetics 3 Molecular Genetics 1  Notes on Molecular Genetics Chapter 1: INTRODUCTION A brief history of DNA, Q and A about DNA and RNA, PROKARYOTIC AND EUKARYOTIC GENOMES. - DNA as a genetic mate...

Notes on Molecular Genetics 3 Molecular Genetics 1  Notes on Molecular Genetics Chapter 1: INTRODUCTION A brief history of DNA, Q and A about DNA and RNA, PROKARYOTIC AND EUKARYOTIC GENOMES. - DNA as a genetic material: 1- The First Piece of the Puzzle: Miescher Discovers DNA 2- Laying the Groundwork: Levene Investigates the Structure of DNA 3- Strengthening the Foundation: Chargaff Formulates His "Rules" 4- Putting the Evidence Together: Watson and Crick Propose the Double Helix 5- Why noncovalent bonds (H bonds) between the two strands? 6- What is meand by 5-3 and 3-5 directions in DNA structure? 7- Which is more inside the cell? DNA or RNA 8- Why DNA is the genetic material. - Aspects of microbial genome: 9- Anatomy of microbial and eukaryotic genomes 10- The bacterial ‘chromosome’ 11- Complications on the E. coli theme 12- The genetic organization of the prokaryotic genome 13- The genetic organization of the prokaryotic genome 14- The open reading frame 15- Operons are characteristic features of prokaryotic genomes. 16- Gene families 17- Prokaryotic genomes and the species concept 2  Notes on Molecular Genetics During your study, you will find various questions to measure your understanding. You are requested to prepare your portfolio for evaluation. The porifolio must be hand written and it will be collected at the end of the term. You can come and discuss your answers with me. “Prof. Amr M. Mowafy” 3  Notes on Molecular Genetics INTRODUCTION - Prokaryotic and Eukaryotic Living organisms have succeeded in invading and colonizing the various environments in the planet Earth, so they resisted the conditions of those environments and preserved their characteristics. These organisms have been able to live not only in normal environments (oxygen-rich, moderate in temperature and available in food), but also in environments with difficulties and dangers, such as the underground where the anaerobic environment is, near volcanoes and hot springs (where high temperatures) and in places Very cold, in desert environments, in acidic and alkaline environments, and others. These creatures have succeeded in adapting to all circumstances. Prokaryotes are the organisms most capable of adapting in such difficult environments (Fig. 1). Therefore, the more difficult the environmental conditions, the less the biodiversity of eukaryotes and the greater the predominance of prokaryotes. Figure 1: Extreme environments at which Microorganisms could survive. 4  Notes on Molecular Genetics Living organisms are divided according to their cellular structure into prokaryotes and eukaryotes. Prokaryotes include bacteria, archaea, and eukaryotes contain the rest of the groups of living organisms starting from animal protists such as amoebas, paramecium, algae, fungi, plants and various animals. The main difference between prokaryotes and eukaryotes is due to the presence of a true nucleus surrounded by a nuclear membrane in the case of eukaryotic organisms. As for prokaryotes, their genetic material spreads in the cytoplasm. Figure 2 shows the difference between protists and eukaryotes. At least 2 types of genomes are available in eukaryotic cells (nuclear and mitochondrial genomes). In case of photosynthetic eukaryotes, three types of Genomes might be available (nuclear, mitochondrial and chloroplast genomes). Both mitochondria and chloroplast have small genomes, like that of bacteria and archaea genome. By contrast, the nuclear genomes of most eucaryotes seem to have been free to enlarge. Figure 2: Left shape: The differences between Eukaryotic and prokaryotic cells, Right shape: The types of DNA inside typical eukaryotic photosynthetic organism 5  Notes on Molecular Genetics DNA AS A GENETIC MATERIAL Many people believe that American biologist James Watson and English physicist Francis Crick discovered DNA in the 1950s. In reality, this is not the case. Rather, DNA was first identified in the late 1860s by Swiss chemist Friedrich Miescher. Then, in the decades following Miescher's discovery, other scientists--notably, Phoebus Levene and Erwin Chargaff--carried out a series of research efforts that revealed additional details about the DNA molecule, including its primary chemical components and the ways in which they joined with one another. Without the scientific foundation provided by these pioneers, Watson and Crick may never have reached their groundbreaking conclusion of 1953: that the DNA molecule exists in the form of a three-dimensional double helix. - The First Piece of the Puzzle: Miescher Discovers DNA Although few people realize it, 1869 was a landmark year in genetic research, because it was the year in which Swiss physiological chemist Friedrich Miescher first identified what he called "nuclein" inside the nuclei of human white blood cells. (The term "nuclein" was later changed to "nucleic acid" and eventually to "deoxyribonucleic acid," or "DNA.") Miescher's plan was to isolate and characterize not the nuclein (which nobody at that time realized existed) but instead the protein components of leukocytes (white blood cells). He came across a substance from the cell nuclei that had chemical properties unlike any protein, including a much higher phosphorous content, Miescher realized that he had discovered a new substance (Dahm, 2008). - Laying the Groundwork: Levene Investigates the Structure of DNA Meanwhile, other scientists continued to investigate the chemical nature of the molecule formerly known as nuclein. One of these other scientists was Russian 6  Notes on Molecular Genetics biochemist Phoebus Levene. Levene is credited with many firsts. For instance, he was the first to discover the order of the three major components of a single nucleotide (phosphate-sugar-base); the first to discover the carbohydrate component of RNA (ribose); the first to discover the carbohydrate component of DNA (deoxyribose); and the first to correctly identify the way RNA and DNA molecules are put together. Based upon years of work using hydrolysis to break down and analyze yeast nucleic acids, Levene proposed that nucleic acids were composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen- containing bases, a sugar molecule, and a phosphate group. Indeed, many new facts and much new evidence soon emerged and caused alterations to Levene's proposal. One key discovery during this period involved the way in which nucleotides are ordered. Levene proposed what he called a tetranucleotide structure, in which the nucleotides were always linked in the same order (i.e., G-C-T-A-G-C-T-A and so on). However, scientists eventually realized that Levene's proposed tetranucleotide structure was overly simplistic and that the order of nucleotides along a stretch of DNA (or RNA) is, in fact, highly variable. Despite this realization, Levene's proposed polynucleotide structure was accurate in many regards. For example, we now know that DNA is in fact composed of a series of nucleotides and that each nucleotide has three components: a phosphate group; either a ribose (in the case of RNA) or a deoxyribose (in the case of DNA) sugar; and a single nitrogen-containing base. We also know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U) (Figure 17). 7  Notes on Molecular Genetics Figure 17: The chemical structure of a nucleotide. A single nucleotide is made up of three components: a nitrogen-containing base, a five-carbon sugar, and a phosphate group. The nitrogenous base is either a purine or a pyrimidine. The five- carbon sugar is either a ribose (in RNA) or a deoxyribose (in DNA) molecule. - Strengthening the Foundation: Chargaff Formulates His "Rules" Erwin Chargaff was one of a handful of scientists who expanded on Levene's work by uncovering additional details of the structure of DNA, thus further paving the way for Watson and Crick. As his first step in this search, Chargaff set out to see whether there were any differences in DNA among different species. First, he noted that the nucleotide 8  Notes on Molecular Genetics composition of DNA varies among species. Second, Chargaff concluded that almost all DNA--no matter what organism or tissue type it comes from--maintains certain properties, even as its composition varies. In particular, the amount of adenine (A) is usually similar to the amount of thymine (T), and the amount of guanine (G) usually approximates the amount of cytosine (C). In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually nearly equal. (This second major conclusion is now known as "Chargaff's rule.") Chargaff's research was vital to the later work of Watson and Crick, but Chargaff himself could not imagine the explanation of these relationships--specifically, that A bound to T and C bound to G within the molecular structure of DNA (Figure 18). Figure 18: What is Chargaff's rule? All DNA follows Chargaff's Rule, which states that the total number of purines in a DNA molecule is equal to the total number of pyrimidines. Table shows the relative amounts of A, T, C and G in different organisms. 9  Notes on Molecular Genetics - Putting the Evidence Together: Watson and Crick Propose the Double Helix Chargaff's realization that A = T and C = G, combined with some crucially important X-ray crystallography work by English researchers Rosalind Franklin and Maurice Wilkins, contributed to Watson and Crick's derivation of the three-dimensional, double-helical model for the structure of DNA. Watson and Crick's discovery was also made possible by recent advances in model building, or the assembly of possible three-dimensional structures based upon known molecular distances and bond angles, a technique advanced by American biochemist Linus Pauling. In fact, Watson and Crick were worried that they would be "astonished" by Pauling, who proposed a different model for the three-dimensional structure of DNA just months before they did. In the end, however, Pauling's prediction was incorrect. Using cardboard cutouts representing the individual chemical components of the four bases and other nucleotide subunits, Watson and Crick shifted molecules around 10  Notes on Molecular Genetics on their desktops, as though putting together a puzzle. They were misled for a while by an erroneous understanding of how the different elements in thymine and guanine (specifically, the carbon, nitrogen, hydrogen, and oxygen rings) were configured. Only upon the suggestion of American scientist Jerry Donohue did Watson decide to make new cardboard cutouts of the two bases, to see if perhaps a different atomic configuration would make a difference. It did. Not only did the complementary bases now fit together perfectly (i.e., A with T and C with G), with each pair held together by hydrogen bonds, but the structure also reflected Chargaff's rule (Figure 19). Figure 19: The double-helical structure of DNA. The 3-dimensional double helix structure of DNA, correctly elucidated by James Watson and Francis Crick. Complementary bases are held together as a pair by hydrogen bonds. 11  Notes on Molecular Genetics Although scientists have made some minor changes to the Watson and Crick model, or have elaborated upon it, since its inception in 1953, the model's four major features remain the same yet today. These features are as follows: DNA is a double-stranded helix, with the two strands connected by hydrogen bonds. A bases are always paired with Ts, and Cs are always paired with Gs, which is consistent with and accounts for Chargaff's rule. Most DNA double helices are right-handed; that is, if you were to hold your right hand out, with your thumb pointed up and your fingers curled around your thumb, your thumb would represent the axis of the helix and your fingers would represent the sugar-phosphate backbone. Only one type of DNA, called Z-DNA, is left-handed. The DNA double helix is anti-parallel, which means that the 5' end of one strand is paired with the 3' end of its complementary strand (and vice versa). As shown in Figure 4, nucleotides are linked to each other by their phosphate groups, which bind the 3' end of one sugar to the 5' end of the next sugar. Not only are the DNA base pairs connected via hydrogen bonding, but the outer edges of the nitrogen-containing bases are exposed and available for potential hydrogen bonding as well. These hydrogen bonds provide easy access to the DNA for other molecules, including the proteins that play vital roles in the replication and expression of DNA (Figure 20). 12  Notes on Molecular Genetics Figure 20: Base pairing in DNA. Two hydrogen bonds connect T to A; three hydrogen bonds connect G to C. The sugar-phosphate backbones (grey) run anti-parallel to each other, so that the 3’ and 5’ ends of the two strands are aligned. One of the ways that scientists have elaborated on Watson and Crick's model is through the identification of three different conformations of the DNA double helix. In other words, the precise geometries and dimensions of the double helix can vary. The most common conformation in most living cells (which is the one depicted in most diagrams of the double helix, and the one proposed by Watson and Crick) is known as B-DNA. There are also two other conformations: A-DNA, a shorter and wider form that has been found in dehydrated samples of DNA and rarely under normal physiological circumstances; and Z-DNA, a left-handed conformation. Z- DNA is a transient form of DNA, only occasionally existing in response to certain types of biological activity (Figure 21). Z-DNA was first discovered in 1979, but its existence was largely ignored until recently. Scientists have since discovered that 13  Notes on Molecular Genetics certain proteins bind very strongly to Z-DNA, suggesting that Z-DNA plays an important biological role in protection against viral disease (Rich & Zhang, 2003). Figure 21: Three different conformations of the DNA double helix. (A) A-DNA is a short, wide, right-handed helix. (B) B-DNA, the structure proposed by Watson and Crick, is the most common conformation in most living cells. (C) Z-DNA, unlike A- and B- DNA, is a left-handed helix. 14  Notes on Molecular Genetics Compare between DNA and RNA 15