Molecular Biology Lecture Notes PDF

University of Kerbala Title of the course: Molecular Biology College of Applied Medical Sciences Level: 2nd Class, 2nd Semeste Department of Medical Physics Lecturer : Dr. Noor Abdulameer Oudah Lecture 1: History and Introduction of M...

University of Kerbala Title of the course: Molecular Biology College of Applied Medical Sciences Level: 2nd Class, 2nd Semeste Department of Medical Physics Lecturer : Dr. Noor Abdulameer Oudah Lecture 1: History and Introduction of Molecular Biology : Molecular biology: is the field of biology that studies the composition, structure and interactions of cellular molecules – such as nucleic acids and proteins – that carry out the biological processes essential for the cell's functions and maintenance. Historical perspective DNA may be the most famous molecule in the world today, but it came to the attention of scientists rather late in the history of biology. Gregor Mendel found some of the underlying regularities of heredity almost a century before DNA was discovered. At the turn of the century scientists discovered similar principles then rediscovered Mendel’s work and rapidly realized that life was somehow encoded in genes. The discovery of the DNA structure was the start of a new era in biology, leading, over the next two decades, to the cracking of the genetic code and the realisation that DNA directs the synthesis of proteins. There were technical advances too, such as DNA sequencing , genetic engineering, and gene cloning. More recently, the complete sequences of many organisms have been solved - including the human genome in 2001. The next 50 years of the DNA story will be all about realising the practical benefits of Crick and Watson’s discovery for humanity - in industry, medicine, food and agriculture.( Table 1) 1 TABLE 1. The timeline of the principal discoveries in the field of molecular biology. Date Name of discoverer Event description (the “Father of Modern Genetics”) 1866 Gregor Mendel publishes his paper on inheritance of traits in peas isolates an acidic, phosphorus-rich Friedrich (Fritz) substance he called “ nuclein ” from the 1869 Miescher nuclei of white blood cells in pus from soiled bandages demonstrates a heritable “transforming 1928 Frederick Griffith principle” that transmits the ability of bacteria to cause pneumonia in mice characterizes and names the compounds ribonucleic acid and deoxyribonucleic acid, and a “ tetra nucleotide ” structure Phoebus Aaron of 1929 Levene DNA, in which the 4 bases of DNA are arranged one after another in a set of 4 heredity Alfred Hershey and 1952 DNA is genetic material. Martha Chase deduce DNA’s double helix 1953 Watson and Crick conformation 1970 Paul Berg Recombinant DNA Technology 1977 Frederick Sanger DNA sequencing 1983 Kary Mullis In Vitro Amplification of DNA (PCR) The leadership of American geneticist Francis Collins, with support from the 2003 The Human Genome Project (HGP) U.S. Department of Energy and the National Institutes of Health (NIH). 2  some of the classic experiments that led to the identification of DNA as the carrier of genetic information 1- Mendel’s Pea Plants: In one of his experiments on inheritance patterns, Mendel crossed plants that were true-breeding for violet flower color with plants true-breeding for white flower color (the P generation). The resulting hybrids in the F1 generation all had violet flowers. In the F2 generation, approximately three-quarters of the plants had violet flowers, and one-quarter had white flowers.( Figure 1) Mendel stated that each individual has two alleles for each trait, one from each parent. Thus, he formed the “first rule,” the Law of Segregation, which states individuals possess two alleles and a parent passes only one allele to his/her offspring. One allele is given by the female parent and the other is given by the male parent. The two factors may or may not contain the same information. If the two alleles are identical, the individual is called homozygous for the trait. If the two alleles are different, the individual is called heterozygous. The genotype of an individual is made up of the many alleles it possesses. An individual’s physical appearance, or phenotype, is determined by its alleles as well as by its environment. Figure 1: Mendel’s first experiment with pea plants. 3  2- Frederick Griffith: Bacterial transformation In 1928, British bacteriologist Frederick Griffith conducted a series of experiments using Streptococcus pneumoniae bacteria and mice (Figure 2). Griffith wasn't trying to identify the genetic material, but rather, trying to develop a vaccine against pneumonia. In his experiments, Griffith used two related strains of bacteria, known as R and S. When Griffith injected mice with live bacteria of an S strain, they invariably died of pneumonia. (The S- strain covers itself with a polysaccharide capsule that protects it from the host's immune system, resulting in the death of the host) When he injected mice with live R bacteria, the mice remained healthy. (R- strain doesn't have that protective capsule and is defeated by the host's immune system) Mice injected with heat-killed S- bacteria also remained healthy. However, when heat-killed S-bacteria and live R-bacteria were injected, the mice died. Griffith called this the “transforming principle.” He concluded there was transfer of some component of the pathogenic (S) bacteria which allowed the nonpathogenic (R) bacteria to make the polysaccharide coat and evade the mouse immune response. Figure 2: The transforming principle. Griffith’s experiment with Streptococcus pneumoniae. 4  3- The Hershey-Chase experiments An important event in the history of the characterization of DNA was the emerging availability and utility of radioisotopes in basic science research in the early post-World War II years. Radioisotopes allowed Alfred Hershey and Martha Chase to carry out a classic experiment in 1952 showing that the genetic material of a virus that infects bacteria, bacteriophage T2 (literally “bacterium eater”), is DNA.( Figure 3) Experiments:  They depend on the differences between protein &DNA chemical structure (DNA contains : C, H,O,N and P while protein :C,H.O.N,S)  In their first set of experiments, Hershey and Chase labeled the DNA of phages with radioactive Phosphorus- 32P (the element phosphorus is present in DNA but not present in any of the 20 amino acids from which proteins are made). They allowed the phages to infect E. coli, and through several elegant experiments were able to observe the transfer of 32P labeled phage DNA into the cytoplasm of the bacterium  In their second set of experiments, they labeled the phages with radioactive Sulfur-35 (Sulfur is present in the amino acids cysteine and methionine, but not in DNA). Following infection of E. coli they then sheared the viral protein shells off of infected cells using a high-speed blender and separated the cells and viral coats by using a centrifuge.  After separation, the radioactive 35S tracer was observed in the protein shells, but not in the infected bacteria, supporting the hypothesis that the genetic material which infects the bacteria was DNA and not protein.  After synthesis of phage components from the phage genetic material and their assembly, lysis of the bacteria occurred. Isolated progeny phage particles only contained 32P, showing that all the information required to make new phage was contained within the injected DNA. 5 Figure 3. DNA is the genetic material of bacteriophage T2. (A) Bacteriophage T2 on E. coli. Colorenhanced transmission electron micrograph (TEM) of bacteriophages (green-blue) attacking a bacterium (brown). The bacteriophage uses its tail as a syringe to inject its own DNA (blue) into the bacterium. The bacteriophage then replicates within the bacterium. (B) The Hershey–Chase experiment using 35S- and 32P-labeled bacteriophage T2. The DNA label ( 32P) enters the E. coli bacterium during infection. The protein label (35S) does not. 6  Experimental Modeling in Molecular Biology Bacteria :Prokaryotes unicellular free living cells. only one single chromosome not enclosed inside nucleus but it is free within the cytoplasm called nucleoid. the Escherichia coli(E.coli) represent the best model to be used for many reason like easily to be cultured , relatively simple in their needs , short generation time (20 min for E.coli),best growth temperature 37cº so it complete DNA replication ,RNA transcription and Protein synthesis within few minutes Bacteriophage : they represent the simplest form of life These infect the bacteria (there are animal , plant and human viruses) unlike the bacteria, they are not free living (completely inert )once they enter the host they start replication depending on the machines of the host cell. it now used as cloning vector Yeast: another experimental model but for Eukaryotic cell thus it contains chromosomes within a true nucleus surrounded with nuclear membrane.great deal of early biochemical research was carried out specially fermentation process now for molecular biologist, mutant strains of yeast often used to discover genes that control growth , division ,and cell behavior. Animal and plant cell : also could be used as a model in genetic experiments. 7  Human Genome Project Human genome project (HGP) was an international scientific research project which got successfully completed in the year 2003 by sequencing the entire human genome of 3.3 billion base pairs. The HGP led to the growth of bioinformatics which is a vast field of research. The successful sequencing of the human genome could solve the mystery of many disorders in humans and gave us a way to cope up with them. Goals of the human genome project Goals of the human genome project include:  Optimization of the data analysis.  Sequencing the entire genome.  Identification of the complete human genome.  Creating genome sequence databases to store the data.  Taking care of the legal, ethical and social issues that the project may pose. Methods of the human genome project In this project, two different and significant methods are typically used. 1. Expressed sequence tags wherein the genes were differentiated into the ones forming a part of the genome and the others which expressed RNAs. 2. Sequence Annotation wherein the entire genome was first sequenced and the functional tags were assigned later. Features Features of the Human genome project include:  Our entire genome is made up of 3164.7 million base pairs.  On average, a gene is made up of 3000 nucleotides.  The function of more than 50 percent of the genes is yet to be discovered.  Proteins are coded by less than 2 percent of the genome.  Most of the genome is made up of repetitive sequences which have no coding purposes specifically but such redundant codes can help us better understand of genetic development of humanity through the ages 8  Definition Of Some Terms Genetics is a branch of biology concerned with the study of genes, genetic variation, and heredity in organisms. The process of transmission of characters from one generation to next, either by gametes–sperms and ova–in sexual reproduction or by the asexual reproductive bodies in asexual reproduction, is called inheritance or heredity. It is the cause of similarities between individuals. This is the reason that brothers and sisters with the same parents resemble each other and with their parents. The term genome refers to all the DNA present in a cell. A cell’s genome includes its chromosomes and non-chromosomal sites, for example, bacteria and some fungi contain extra tiny pieces of DNA called plasmids and the mitochondria of eukaryotes are equipped with their own functional DNA. The gene is the fundamental unit of heredity. It is a Segment of DNA that has the information (the code) for a protein or RNA. these unit of information that encodes a genetic characteristic come in multiple forms called alleles. A single molecule of DNA has thousands of genes on the molecule. Alleles : Alleles are a pair of genes that occupy a specific location on a particular chromosome and control the same trait. OR / variant of a gene controlling the same trait and occupying a specific region on a chromosome (called the locus). Genotype : is the genetic makeup of an individual cell or organism that determines or contributes to its phenotype Phenotype :The term refers to an individual’s observable traits, such as height, eye color and blood type. A person’s phenotype is determined by both their genomic makeup (genotype) and environmental factors. 9 Figure 4. Explains the concept of genotype, phenotype, gene and allele 10