Biochemical Genetics PDF - Lecture Notes

‫بسم هللا الرحمن‬ ‫الرحيم‬ ‫‪1‬‬ Biochemical Genetics 2  Biochemical Genetics  Prof. Dr. Eman Mahmoud Fahmy  Dr. Marwa Mahmoud 3 DNA Structure and Analysis 4 It is concern about structure a...

‫بسم هللا الرحمن‬ ‫الرحيم‬ ‫‪1‬‬ Biochemical Genetics 2  Biochemical Genetics  Prof. Dr. Eman Mahmoud Fahmy  Dr. Marwa Mahmoud 3 DNA Structure and Analysis 4 It is concern about structure and the function of genetic material 5  Chapter Concepts Except in some viruses, DNA serves as the genetic material in all living organisms on Earth. According to the Watson–Crick model, DNA exists in the form of a right-handed double helix. The strands of the double helix are antiparallel and are held together by hydrogen bonding between complementary nitrogenous bases. 6  The structure of DNA provides the means of storing and expressing genetic information. RNA has many similarities to DNA but exists mostly as a single-stranded molecule. Insome viruses, RNA serves as the genetic material. Many techniques have been developed that facilitate the analysis of nucleic acids, most based on detection of the complementarity of nitrogenous bases. 7  The Genetic Material Must Exhibit Four Characteristics  Replication  Storage of information  Expression of information  Mutation 8 Cell Cycle 9 10  Information Flow Central Dogma of Molecular Genetics 11 Replication 12 Central Dogma 14 Until 1944, observations favored protein as the genetic material 15  For three reasons: 1- Protein are abundant in cells. 2- They are more diverse. 3- Much more was known about proteins than about nucleic acid chemistry. however, the chemical structure nature of nucleic acids was discovered during the early to mid- 1890 (nuclein) by Friedrick Miescher. Levene postulated incorrectly that identical groups of the 4 components were repeated over and over as 16 “Tetranucleotide hypothesis” 17 Three areas of most active research in genetics: 1.The study of Transmission genetics (Mendel) and 2. Mutation and variation for evolution (Darwin) 3. Experiment of Transformation 18 Evidence Favoring DNA as the Genetic Material Was First Obtained During the Study of Bacteria and Bacteriophages 19 ‫‪.1‬تجربة التحول الوراثى‬ ‫‪Transformation‬‬ ‫‪20‬‬ 1- Transformation ‫تجربة التحول الوراثى‬ Grifth 1927, Avery, Macleod, MaCarty 1944 21 22 The organism is now named Streptococcus pneumoniae 23 Transforming principle 24 25 2. The Hershey-Chase Experiment 26 ‫تجربة اإلستنقال بالفيرس ‪2. Transduction‬‬ ‫‪27‬‬ T-even bacteriophage 28 Lytic cycle 29 30 In 1952, Alfred Hershey and Martha Chase published the results of experiments designed to clarify the events leading to phage reproduction. Several of the experiments clearly established the independent functions of phage protein and nucleic acid in the reproduction process associated with the bacterial cell. 31 Hershey and Chase knew from existing data that: 1.T2 phages consist of approximately 50 percent protein and 50 percent DNA. 2. Infection is initiated by adsorption of the phage by its tail fibers to the bacterial cell. 3. The production of new viruses occurs within the bacterial cell. 32  ‫ النقل بالعدوى‬.3 Transfection (Protoplast or Spheroplasts) 33 In 1957, several reports demonstrated that if E. coli is treated with the enzyme lysozyme, the outer wall of the cell can be removed without destroying the bacterium. Enzymatically treated cells are naked, so to speak, and contain only the cell membrane as their outer boundary. Such structures are called protoplasts (or spheroplasts). 34 3- Transfection ‫النقل بالعدوى‬ (Protoplast or Spheroplasts) 35  John Spizizen and Dean Fraser independently reported that by using protoplasts, they were able to initiate phage reproduction with disrupted T2 particles. That is, provided protoplasts were used, a virus did not have to be intact for infection to occur. Thus, the outer protein coat structure may be essential to the movement of DNA through the intact cell wall, but it is not essential for infection when protoplasts are used. 36 4. Indirect and direct evidence that DNA is the genetic material in eukaryotes 37  a. Distribution of DNA 38 b. Mutagenesis The molecule serving as the genetic material is expected to absorb at the wavelength(s) found to be mutagenic. 39 40 Direct evidence Recombinant DNA Technology 41 42 RNA serves as the genetic material in some viruses 43  Some viruses contain an RNA core rather than a DNA core. In these viruses, it appears that RNA serves as the genetic material—an exception to the general rule that DNA performs this function. In 1956, it was demonstrated that when purified RNA from tobacco mosaic virus (TMV) was spread on tobacco leaves, the characteristic lesions caused by viral infection subsequently appeared. Thus, it was concluded that RNA is the genetic 44 material of this virus. 45  In 1965 and 1966, Norman Pace and Sol Spiegelman demonstrated that RNA from the phage Qβ can be isolated and replicated in vitro. Replication depends on an enzyme, RNA replicase, which is isolated from host E. coli cells following normal infection. When the RNA replicated in vitro is added to E. coli protoplasts, infection and viral multiplication (transfection) occur. Thus, RNA synthesized in a test tube serves as the genetic material in these phages by directing the production of all the components necessary for viral46 reproduction.  While many viruses, such the T2 virus used by Hershey and Chase, use DNA as their hereditary material, another group of RNA-containing viruses bears mention. These are the retroviruses, which replicate in an unusual way. Their RNA serves as a template for the synthesis of the complementary DNA molecule. The process, reverse transcription, occurs under the direction of an RNA-dependent DNA polymerase enzyme called reverse transcriptase. This DNA intermediate can be incorporated into the genome of the host cell, and when the host DNA is transcribed, copies of the original retroviral RNA chromosomes are produced. Retroviruses include the human immunodeficiency virus (HIV), which causes 47 AIDS, as well as several RNA tumor viruses 48 Knowledge of Nucleic Acid Chemistry is Essential to the Understanding of DNA Structure 49 50  Nucleotides: Building Blocks of Nucleic Acids 51 52  Purines: double ring bases Adenine (A) Guanine (G)  Pyrimidines:single ring bases Thymine (T) Cytosine (C ) change to Uracil (U) in RNA 53 54 55 Nucleotide= Nucleoside + Phosphate 56 Nucleotide 57 58 59 Nitrogen base + Sugar (Deoxyribose) +Phosphate = Nucleotides 60 Polynucleotides 61 The Structure of DNA Holds the Key to Understanding its Function 62 Base-Composition Studies 63 Base-Composition Studies 64 Hydrogen bonds 65 66 67 68 Direction of the two strands of DNA 69 70 X-Ray Diffraction Analysis 71 72 73 74 . 75 . 76 Space-filling diagram of DNA double helix 77 78 DNA structure 79 80 81  The Watson–Crick Model This model has the following major features: 1. Two long polynucleotide chains are coiled around a central axis, forming a right-handed double helix. 2. The two chains are antiparallel; that is, their C-5-to-C-3 orientations run in opposite directions. 82 3. The bases of both chains are flat structures lying perpendicular to the axis; they are “stacked” on one another, 3.4˚A (0.34 nm) apart, on the inside of the double helix. 4. The nitrogenous bases of opposite chains are paired as the result of the formation of hydrogen bonds; in DNA, occur. 83 5. Each complete turn of the helix is 34˚A (3.4 nm) long; thus, each turn of the helix is the length of a series of 10 base pairs. 6. A larger major groove alternating with a smaller minor groove winds along the length of the molecule. 7. The double helix has a diameter of 20˚A (2.0 nm). 84 85  The nature of the base pairing explaining its genetic functions. Antiparallel. Right direction. Complementarity. Base pair 10.4 rotation 34.6° instead of 34° Different bonds: 1- Hydrogen bonds between bases. 2- Covalent bonds; Phosphodiester bonds Between sugar and phosphate. 3- Covalent bonds; Glycocidic bonds between sugar and base. 86 4- Stacking force (Van dervals force). Thanks for your listening 87