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Unit VI CHEMICAL BASIS OF INHERITANCE MLT, FAMS, TU Dr. Nabil MTIRAOUI, M.Sc, Ph.D Molecular Genetics The branch of genetics that deals with hereditary transmission and variation on the molecular level. Deals with the expression of genes by studying the DNA sequences of chromosomes The study of the molecular structure of genes, involving DNA and RNA. Lecture 11 DNA Structure & Properties I. DNA’s Discovery & Structure What is a Genetic “Factor”? From Mendel: we now accepted that there was genetic transmission of traits. Traits are transmitted by “factors” Organisms carry 2 copies of each “factor” The question now was: what is the factor that carries the genetic information? Requirements of Genetic Material Must be able to replicate, so it is reproduced in each cell of a growing organism. Must be able to control expression of traits Traits are determined by the proteins that act within us Proteins are determined by their sequences Therefore, the genetic material must be able to encode the sequence of proteins It must be able to change in a controlled way, to allow variation, adaptation, thus survival in a changing environment. Chromosomes – The First Clue First ability to visualize chromosomes in the nucleus came at the turn of the century construction of increasingly powerful microscopes the discovery of dyes that selectively colored various components of the cell Scientists examined cellular nuclei and observed nuclear structures, which they called chromosomes Observation of these structures suggested their role in genetic transmission Implications Chromosomes behaved like Mendel’s “factors” Mendel's hereditary factors were either located on the chrs or were the chromosomes themselves. Proof chromosomes were hereditary factors – 1905: The first physical trait was linked to the presence of specific chromosomal material conversely, the absence of that chromosome meant the absence of the particular physical trait. Discovery of the sex chromosomes "X" and "Y." distinguished from other chromosomes and from each other What Carries the Genetic Information? Chromosomes are about 40% DNA & 60% protein. Protein molecules are composed of 20 different subunits DNA molecules are composed of only four Therefore protein molecules could encode more information, and a greater variety of information Protein is the larger component protein had the possibility for much more diversity than in DNA Therefore, scientists believed that the protein in chromosomes must carry the genetic information 1. A History of DNA F. Griffiths (1928) Tried to determine what genetic material was made of. The Transforming Principle Fredrick Griffith - 1928 Discovered that different strains of the bacterium Strepotococcus pneumonae had different effects on mice One strain could kill an injected mouse (virulent) Another strain had no effect (avirulent) When the virulent strain was heat-killed and injected into mice, there was no effect. But when a heat-killed virulent strain was co-injected with the avirulent strain, the mice died. Concluded that some factor in the heat killed bacteria was transforming the living avirulent to virulent? What was the transforming principle and was this the genetic material? Griffiths’ Experiment Griffiths’ Experiment Pneumococcus bacteria on mice 2 STRAINS S-type Smooth colonies Virulent R-type Rough colonies Avirulent Innoculate into mice Innoculate into mice Dead from pneumonia Not killed Avery, MacCleod & McCarthy (1944) Tried purifying the transforming principle to change R-type Pneumococcus to S-type The Transforming Principle is DNA Avery, Macleod, & McCarty – 1943 Attempted to identify Griffith’s “transforming principle” Separated the dead virulent cells into fractions The protein fraction DNA fraction Co-injected them with the avirulent strain. When co-injected with protein fraction, the mice lived with the DNA fraction, the mice died Result was IGNORED Most scientists believed protein was the genetic material. They discounted this result and said that there must have been some protein in the fraction that conferred virulence. The Hershey-Chase Experiment Hershey & Chase – 1952 Performed the definitive experiment that showed that DNA was the genetic material. Bacteriphages = viruses that infect bacteria Bacteriphage is composed only of protein & DNA Inject their genetic material into the host The Hershey-Chase Experiment The Experiment Prepared 2 cultures of bacteriophages Radiolabeled sulphur in one culture there is sulphur in proteins, in the amino acids methionine and cysteine there is no sulphur in DNA Radiolabeled phosphorous in the second culture there is phosphorous in the phosphate backbone of DNA none in any of the amino acids. So this one culture in which only the phage protein was labeled, and one culture in which only the phage DNA was labeled. Experiment Summary Performed side by side experiments with separate phage cultures in which either the protein capsule was labeled with radioactive sulfur or the DNA core was labeled with radioactive phosphorus. The radioactively labeled phages were allowed to infect bacteria. Agitation in a blender dislodged phage particles from bacterial cells. Centrifugation pelleted cells, separating them from the phage particles left in the supernatant. Results Summary Radioactive sulfur was found predominantly in the supernatant Radioactive phosphorus was found predominantly in the cell fraction, from which a new generation of infective phage was generated. Thus, it was shown that the genetic material that encoded the growth of a new generation of phage was in the phosphorouscontaining DNA. Chargaff’s Rule Chargaff’s rule is a rule about DNA, Chargaff’s Rule Once DNA was recognized as the genetic material, scientists began investigating its mechanism and structure. Erwin Chargaff – 1950 discovered the % content of the 4 nucleotides was the same in all tissues of the same species percentages could vary from species to species. He also found that in all animals (Chargaff’s rule): %G = %C %A = %T This suggested that the structure of the DNA was specific and conserved in each organism. The significance of these results was initially overlooked The Double Helix: Watson & Crick Watson and Crick shared the 1962 Nobel Prize for Physiology and Medicine with Maurice Wilkins. Rosalind Franklin died before this date. The Double Helix: Watson & Crick James Watson and Francis Crick – 1953 Presented a model of the structure of DNA. It was already known from chemical studies that DNA was a polymer of nucleotide (sugar, base and phosphate) units. X-ray crystallographic data obtained by Rosalind Franklin, combined with the previous results from Chargaff and others, were fitted together by Watson and Crick into the double helix model. 2. Chemical Bases in DNA Two types of nucleic acid can be recognized: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is mostly found in the nucleus where it forms the principal substance of the chromosomal material, the chromatin. In addition to DNA, chromatin contains proteins, mainly histones, and little RNA. 2. Chemical Bases in DNA In prokaryotes, DNA is present in a single chromosome in the nucleoid. Little DNA is also found in mitochondria and in chloroplasts. Many viruses are made up of DNA, mostly double stranded, but some are single stranded. 3. Primary Structure: Nucleotide & Nucleoside The addition of a pentose sugar to a base produces a nucleoside . If the sugar is ribose, a ribonucleoside is produced; if the sugar is 2deoxyribose, a deoxyribonucleoside is produced Addition of phosphate group to nucleoside produces nucleoside mono-phosphate (NMP) like AMP or CMP or a nucleotide 3. Primary Structure: Mononucleotide 3. Primary Structure: Nitrogenous Bases PURINES 1. Adenine (A) 2. Guanine (G) PYRIMIDINES 3. Thymine (T) 4. Cytosine (C) T or C 3. Primary Structure: Dinucleotide 3. Primary Structure: Polynucleotide 3. Primary Structure: Polynucleotide O A 5’-End H H3C N Thymine O H 5’ I O=P-O-CH2 O I O4’ H 1’ H N 3’ O N 2’ N Adenine N H O 5’ I O=P-O-CH2 O I O4’ N N 3’ H 1’ 2’ NH2 H N H O 5’ I O=P-O-CH2 O I O4’ O N O 1’ N 2’ HN 3’ Cytosine H N O 5’ I O=P-O-CH2 O I O4’ 3’ N 3’ 5’ Phosphodiester bond NH2 1’ 2’ OH 3’-End Guanine 4. Secondary structure: double helical structure The 2 strands are twisted about each other, coiled around a common axis, forming a righthanded double helix. The hydrophilic sugar- phosphate backbone of each chain lies on the outside of the molecule. The hydrophobic nitrogenous bases project inwards from the outer sugar-phosphate framework, perpendicular to the long axis of the helix and are stacked one above the other. The stacking of bases is held by hydrophobic bonds .This helps in holding the helical structure. 4. Secondary structure: double helical structure The nitrogenous bases of the 2 strands meet each other near the central axis of the helix where they become connected by hydrogen bonds between the amino, or imino, hydrogen and the ketonic oxygen atoms. The hydrogen bonding between the bases helps to hold the 2 strands of the DNA together. Thymine Adenine Cytosine Guanine 4. Secondary Structure: Chargaff’s Rule A nitrogen-containing ring structure called a base. The base is attached to the 1' carbon atom of the pentose. In DNA, four different bases are found: two purines, called adenine (A) and guanine (G) two pyrimidines, called thymine (T) and cytosine (C) *A always pairs with T : two hydrogen bonds *C always pairs with G : three hydrogen bonds 4. Secondary Structure: Direction of Strands The 2 strands of the double helical molecule are antiparallel, i.e., they run in opposite direction; one runs in the 5’ to 3’ direction, while the other runs in the 3’ to 5’ direction. 5. DNA Conformations B conformation (B-DNA): A-DNA: The most common form of DNA. The minor groove and major groove, are of different widths on the outside of DNA. Forms under conditions of low salt and low humidity. There can be transient shifts from B to A form. Z-DNA: Consists of alternating purines and pyrimidines Found infrequently. Z-DNA is: long and thin Left-handed, Phosphate backbone has a zig-zag appearance. 6. Key Features of a DNA molecule 7. Key Features of a RNA molecule 8. Biochemistry of DNA DNA is the carrier of genetic information, which is stored in the Replication DNA form of a nucleotide Transcription sequence. DNA has 2 important functions: “replication” and RNA Translation “transcription”. Protein 9. What is Gene ? The gene, the basic units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus. 9. What is Gene ? A gene in relation to the double helix structure of DNA and to a chromosome (right). Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA): only the exons encode the protein. This diagram labels a region of only 40 or so bases as a gene. In reality most genes are hundreds of times larger.