BIO302 Official Handouts

Chapter 1. Introduction

1. Molecular Biology

The term molecular biology was first used in 1945 by William Astbury who was referring to the study of the chemical and physical structure of biological macromolecules. By that time, biochemists had discovered many fundamental intracellular chemical reactions. The importance of specific reactions and of protein structure in defining the numerous properties of cells was also appreciated. However, the development of molecular biology had to await the understanding that the most advantageous approaches would be made by studying ―simple‖ systems such as bacteria and bacteriophages which yield information about the basic biological processes more readily than animal cells. In fact, the faith in the basic uniformity of life processes was an important factor in rapid growth of molecular biology. That is, it was believed that fundamental biological principles that govern the activity of simple organisms, such as bacteria and viruses, must apply to more complex cells; only the details should vary. This faith has been amply justified by experimental results. The roots of molecular biology were established in 1953 when an Englishman, Francis Crick and a young American, James Watson working at Medical Research Council Unit, Cavendish Laboratory, Cambridge, proposed a double helical model for the structure of DNA (deoxyribonucleic acid) molecule which was well known as the chemical bearer of genetic informations of certain microorganisms (bacteria, bacteriophages, etc.) due to pioneer discoveries made by Grifith (1928), Avery, Macleod and McCarthy (1944) and Hershey and Chase (1952). This discovery was followed by a thorough search of occurrence of DNA as the genetic material in other microorganisms, plants and animals and also by investigations of the molecular and atomic nature of different reactions of living cells. From all these studies has emerged the realization that the basic chemical organization and the metabolic processes of all living things are remarkably similar despite their morphological diversity and that the physical and chemical principles governing living systems are similar to those governing non-living systems. The present understanding of molecular biology is that in most organisms the phenotype or the body structure and function ultimately depend for their determination on the structural and functional (i.e., enzymatic) proteins or polypeptides. The synthesis of polypeptides is specified, directed and regulated by self-duplicating genes which are borne within molecules of DNA which is the universally accepted chemical bearer of genetic informations of most living organisms except certain viruses in which this function is carried by RNA, another nucleic acid. The genetic informations for polypeptide synthesis are initially dictated by the disposition of nitrogen bases in DNA molecule and are copied down by the process of transcription. During transcription stage copies (that is, transcripts) of an individual gene or genes are synthesized. These copies are molecules of RNA that include such familiar classes as ribosomal RNA, messenger RNA and transfer RNA. The biochemical interplay of these RNA copies which leads to the synthesis of a polypeptide chain, is called translation, meaning, literally, that the genetic message encoded in a messenger RNA molecule is translated into the linear sequence of amino acids in a polypeptide. The polypeptide in its turn determines the phenotype of the organism.

1. HISTORICAL BACKGROUND

The molecular biology is a very young biological discipline and has a very short history. Certain notable accomplishments of molecular biologists can be summarized as follows : 1928 F. Grifth discovered the phenomenon of transformation in bacteria. 1934 M. Schlesinger demonstrated that the bacteriophages are composed of DNA and protein. 1941 G.W. Beadle and E.L.Tatum published their classical study on the biochemical genetics of Neurospora. 1944 O.T. Avery, C.M. MacLeod and M. McCarthy recognized the DNA nature of transforming principle of pneumococcus bacteria. The fact suggested that it is DNA and not protein which is the hereditary chemical. 1948 A. Boivin, R.Vendrely and C. Vendrely showed that in the different cells of an organism the quantity of DNA for each haploid set of chromosome is constant. 1950 E. Chargaff demonstrated that in DNA the numbers of adenine and thymine groups are always equal and so are the numbers of guanine and cytosine groups. 1952 A.D. Hershey and M. Chase demonstrated that only the DNA of T2 bacteriophage enters the host, the bacterium Escherichia coli, whereas the protein remains behind. 1953 J. D. Watson and F.H.C. Crick proposed a model for DNA comprising of two helically intertwined chains tied together by hydrogen bonds between the purines and pyrimidines. 1956 A. Gierer and G. Schramm demonstrated that RNA is the genetic material of tobacco mosaic virus (TMV). 1957 H. Fraenkel-Contrat and B.Singer separated RNA from the protein of TMV viruses, produced hybrid RNA viruses and confirmed the view that RNA is the genetic material of some viruses. Mathew Meselson and Franklin W. Stahl performed a density-gradient experiment (using heavy isotope of nitrogen, 15N) in bacteria to confirm the Watson and Crick‘s semiconservative theory of DNA replication. 1958 G. Beadle and E. Tatum received Nobel Prize for their contribution in biochemical genetics of fungus. J. Laderberg got Nobel Prize for the discovery of bacterial recombination. 1959 R.L. Sinsheimer isolated singlestranded DNA from a small virus φ-X174 which attacks Escherichia coli. S. Ochoa ; A. Kornberg received Nobel Prize for artificial synthesis of nucleic acids. 1961 M.W. Nirenberg and J.H. Matthaei cracked the messenger RNA code. F.H.C.Crick and his colleagues showed that the genetic language is made up of three-letter words (i.e., triplet codons). F.Jacob and J. Monod put forward the operon concept. 1962 J. Watson and F. Crick ; M. Wilkens got Nobel Prize for the discovery of molecular nature of DNA. 1963 J.P. Waller reported that nearly one-half of all proteins in E.coli cells have the amino acid methionine in the N-terminal position. 1964 K.A. Marcker and F. Sanger discovered a peculiar aminoacyltRNA in E.coli, called N-formylmethionyl - tRNA and suggested that this molecule may play a role in the special mechanism of chain elongation. R.W. Holley and his colleagues gave detailed structure of alanyl tRNA (tRNA ala) from yeast. Holley died in 1993. 1965 F.H.C. Crick proposed the wobble hypothesis for anticodons of tRNA and explained how several codons meant for same amino acid are recognized by same tRNA. H. Wallace and M.L. Birnstiel isolated ribosomal RNA genes in Xenopus. F. Jacob., A. Lwoff, and J. Monod received Nobel Prize for the discovery of protein synthesis mechanism in virus. 1968 R.W. Holley ; H.G. Khorana and M.W. Nirenberg got Nobel Prize for deciphering the genetic code. 1969 A.D. Hershey, M. Delbruck and S.E. Luria shared Nobel Prize in medicine for their contribution to replication and recombination in viruses (bacteriophages). Britten and Davidson proposed the gene-battery model for regulation of protein synthesis in eukaryotes. 1970 Howard Temin and David Baltimore demonstrated the synthesis of DNA on RNA template tumour viruses. Both were awarded Nobel Prize in 1975 for the discovery of an enzyme called RNA directed DNA polymerase (or reverse transcriptase) which is present in the core of virus particle (rous sarcoma virus). Biotechnology emerged as a new discipline due to marriage of biological science with technology (see Dubey, 1995). Knippers ; Kornberg and Gefter ; Moses and Richardson isolated DNA-polymerase-II enzyme. 1972 Mertz and Davis in 1972 demonstrated that cohesive termini of cleaved DNA molecule could be covalently sealed with E.coli DNA ligase and were able to produce recombinant DNA molecules. Cohen et al., for the first time reported the cloning of DNA by using plasmid as vector. R. Porter; G.M. Edelman received Nobel Prize (physiology and medicine) for the discovery of chemical structure of antibodies. C.B. Anfinsen; S. Moore and W.H. Stein got Nobel Prize (chemistry) for the discovery of chemical structure and activity of the enzyme ribonuclease. 1973 S.H. Kim suggested three dimensional structure, i.e., L-shaped model, of tRNA. 1975 E.M. Southern developed a method, called Southern blotting technique for analysing the related genes in a DNA restriction fragment. D. Pribnow discovered Pribnow box or minus ten sequence in E. coli genome. 1977 P.A. Sharp and R.J. Roberts discovered split genes of adenovirus. D.S. Hogness, I.B. David and N. Davidson studied split genes for 28 S rRNA in Drosophila. P. Chambon, P. Leder and R.A. Flavell studied split genes of B'globin, ovalbumin and tRNA. Itakura et al., first of all produced human insulin (humulin) by means of recombinant technology. 1978-79 W. Gilbert first of all used the terms exon and intron (for split genes). 1978 Hinnen et al., first of all described the transformation of yeast (Saccharomyces cervisae) by the help of plasmid of E.coli. 1979 Khorana reported completion of the total synthesis of a biologically functional gene. Alwine et al., developed northern blotting technique in which mRNA bands are blot transferred from the gel onto chemically reactive paper. Towbin et al., developed the western blotting technique to find out the newly encoded protein by a transformed cell. 1980 Fredrick Sanger got the second Nobel Prize for discovering complete sequence of 5400 nucleotides of single stranded DNA of φ × 174 bacteriophage. 1982 A. Klug was awarded Nobel Prize in chemistry for providing three-dimensional structure of tRNAs. Rubin and Spradling for the first time introduced Drosophila gene of xanthine dehydrogenase into a P-element (= parental element) which then was microinjected into embryo deficient for this gene. R.D. Palmiter and R.L. Brinter produced transgenic mice by genetic engineering. 1983 Marilyn Kozak proposed the scanning hypothesis for initiation of translation by eukaryotic ribosomes. 1984 Robert Tijan identified a DNA-binding protein called SP1 which is involved in eukaryotic gene regulation.