Chapter 1 Introduction to Molecular Biology Acknowledgement Centers

Chapter 1 Introduction to Molecular Biology

Acknowledgement • Centers for Disease Control and Prevention- Ethiopia (CDC -E) • American Society for Clinical Pathology (ASCP) • Addis Ababa Univesity • University of Gondar • University of Hawassa • Jimma University • Haromaya University

Learning Objectives: At the end of this chapter, students will be able to: • Describe eukaryotes & prokaryotic cells • Describe the phases of the cell cycle • Distinguish the difference between mitosis & meiosis • List macromolecules of cells • Discuss the composition, & chemistry of nucleic acids • Describe the central dogma of molecular biology

Outline 1. Historical overview of molecular biology 2. Overview of cells & macromolecules Cellular classifications Macromolecules: DNA, RNA DNA as Primary Genetic Material central dogma 3. Review questions 4. References

1. Introduction Molecular biology: • Molecular is the science that study all life processes within cells and at the molecular level. • It overlaps with genetics & biochemistry. Biochemistry: • It is the study of the chemical substances & vital processes occurring in living organisms

Genetics • It is study of heredity & the effect of genetic variation among generation of organisms. • Thus, Molecular biology use specific techniques native to molecular biology, but increasingly combine these techniques with techniques & ideas of genetics & biochemistry.

2. Molecular Biology History overview 1866, Gregor Mendel provide the fundamental principle of heredity. 1909, Johannse coined the term gene to denote the basic unit of heredity. 1910, Morgan describe the gene is contained in chromosome. • In 1944, Oswald Avery demonstrated that genes are made up of DNA

• In 1953, James Watson & Francis Crick discovered the double helical structure of the DNA molecule. • 1956, number of chromosomes in humans (46) understood. • In 1961, Francois Jacob & Jacques Monod hypothesized the existence of an intermediary between DNA & its protein products, which they called m. RNA • 1961 - 1965, the relationship between the information contained in DNA & the structure of proteins was determined:

• Correspondence between nucleotides in the sequence & a series of amino acids in proteins. • In 1966, gene transcription described • In 1975, Edward Southern discovered Southern blotting • In 1977, DNA sequencing methodology discovered

In 1981, Kan & Chang shown genetic diagnose of sickle cell anemia In 1985, PCR developed by Millis & co-workers In 2001 Draft of human genome sequence was revealed In 2003, Human Genome Project was launched

3. Overview of cells & Macromolecules Cells • All genetic diseases involve defects at the level of the cell • For this reasons, one must understand the basic cell biology to understand genetic diseases.

The three domains of life

The biological universe consists of two types of cells Prokaryotes • Archaea and Bacteria • Simplified internal organization • A distinct nucleus is absent/Nuclear material dispersed • DNA is in the form of a single circular chromosome. • Additional DNA is carried in plasmids. • Transcription and translation can be carried out simultaneously

Eukaryotes • Unicellular and Multi-cellular • DNA is carried on several chromosomes within a nucleus. • The nucleus is bounded by a nuclear membrane. • Transcription requires formation of messenger RNA (m. RNA) & movement of m. RNA out of the nucleus into the cytoplasm. • The cytoplasm is rich in membrane-bound organelles (mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes) which are absent in prokaryotes.

Cell cycle in eukaryotes • It alternates between interphase & cell division (mitosis and ctokinesis)

Cell division • Cell division in prokaryotes – Binary fission • Cell division in eukaryote – Mitosis – Meosis

Cell division in prokaryotes: binary fission

Mitosis and Meiosis Mitosis: - division of somatic (body) cells Meiosis - division of gametes (sex cells)

Mitosis • Interphase • Prophase • Metaphase • Anaphase • Telophase

Interphase • Interesting things happen! 1. Cell preparing to divide 2. Genetic material doubles

Prophase • Chromosome pair up! 1. Chromosomes thicken and shorten -become visible -2 chromatids joined by a centromere 2. Centrioles move to the opposite sides of the nucleus 3. Nucleolus disappears 4. Nuclear membrane disintegrate

Metaphase • Chromosomes meet in the middle! 1. Chromosomes arrange at equator of cell 2. Become attached to spindle fibres by centromeres 3. Homologous chromosomes do not associate

Anaphase • Chromosomes get pulled apart 1. Spindle fibres contract pulling chromatids to the opposite poles of the cell

Telophase • Now there are two! 1. Chromosomes uncoil 2. Spindle fibres disintegrate 3. Centrioles replicate 4. Nucleur membrane forms 5. Cell divides

Meiosis • 4 daughter cells produced • Each daughter cell has half the chromosomes of the parent • 2 sets of cell division involved

Significance of meiosis • It Produce genetic variation, the raw material for evolution as follows: a) Independent Assortment • The orientation of homologous chromosomes on one side of the metaphase plate or the other in Meiosis I is random.

• The number of possible orientations is 2 n in daughter cells, where n is the haploid number. • For humans, the number is 223 = 8, 388, 608 ≈ 8. 4 million possible combinations. • Variation is added by crossing-over; if only one cross-over occurs within each bivalent, 423 or 70, 368, 744, 000 combinations are possible

b) Random fertilization • Any of a male’s 8. 4 million sperm can fertilize any of a woman’s 8. 4 million eggs resulting a total number of combinations over 70 trillion • Fertilization also contributes to genetic variation; (223)2 = 70, 368, 744, 000 possible combination without crossingover

c) Crossing over • When crossing over is considered with fertilization, the number of combinations is nearly infinite • (423)2 = 4, 951, 760, 200, 000, 000 combinations are possible.

Difference between mitosis & meosis Mitosis Meiosis Purpose Produces somatic cells (Body growth) produces reproductive cells Process cell duplication (Diploid -> diploid) reduction division (Diploid -> haploid) Number of Divisions One cell division Two cell division Product 1 -> 2 identical daughter cells To each other & mother cell 1 -> 4 cells (gametes) daughter cells different Occurrence More often At a certain time in the life cycle Crossing-over No Yes Chromosome separation Sister chromatids Meiosis I: Homologous chromosomes Meiosis II: sister chromatids

Biological Macromolecules • Cells are composed of biologically important macromolecules: – Polysaccharides – Phospholipids – Proteins – Nucleic acids: DNA and RNA

Nucleic Acid • Nucleic acids can be defined as any single or double stranded polynucleotides.

Constituents of Nucleic Acids • complete hydrolysis of nucleic acids yields pyrimidine and purine bases, a sugar component and phosphoric acid. The bases : pyrimidine and purine. – a flat, heterocyclic, nitrogen-containing organic bases – Pyrimidines have a six-member ring; – purines have fused five-member imidazole and sixmember pyrimidine rings. – Note that the numbering of the purine ring atoms differs from that used for the pyrimidine ring.

• Each nucleic acid is synthesized mainly from only four types of base. • The same two purines, adenine and guanine, are present in both DNA and RNA. • The two pyrimidines in DNA are cytosine and thymine; in RNA uracil is found instead of thymine. • The only difference between uracil and thymine is the presence of a methyl substituent at C 5.

• The bases are usually referred to by their initial letters; so DNA contains A, G, C, T while RNA contains A, G, C, U. • Certain modification of the bases may occur after their incorporation into nucleic acids

• The pyrimidine and purine bases can undergo keto-enol (C=O/COH) or amino-imino (NH 2/NH) tautomerism. • However, in the native nucleic acid at neutral or acidic p. H it will be the keto and the amino form which are definitely predominant.

2. Sugars • Two types of pentose, β-D-ribose and β -D-2 -deoxyribose, are found in nucleic acids. • These distinguish ribonucleic acid and deoxyribonucleic acid respectively, and give rise to the general names RNA or DNA for the two types of nucleic acid. • The difference between the two pentoses lies in the absence/presence of the hydroxyl group at position 2' of the sugar ring.

Sugars

• To avoid ambiguity between the numbering of the heterocyclic base and the sugar ring atoms, position on the pentose ring are given a prime ('). • In nucleic acids, the ribose and deoxyriboses occur in the furanose form (furan is a five-membered oxygen containing ring). • The orientation of the OH group at the C 1' in the ring is very important. Where the OH at the C 1' and C 4' are on opposite side of the ring are indicated as β. • The D indicates the configuration of the centre of asymmetry of the most remote end from the aldehydic end of the sugar molecule.

3. Phosphoric acid • a negatively charged phosphate group, which gives the polymer its acidic property • the sugars are connected to each other in the polymerised RNA and DNA molecules. Two pentose rings are joined by a phosphoric acid molecule which is forming an ester bond with the 5' and 3' C. O HO P O OH

4. Nucleosides. • When a purine or pyrimidine base is linked to ribose or deoxyribose the resulting compound is known as a nucleoside. • Depending on whether A, G, C or U (T) are condensed with a ribose (deoxyribose) we talk about adenosine (deoxyadenosine), guanosine (deoxyguanosine), cytidine (deoxycytidine) or uridine (deoxythymidine). The ribonucleoside from hypoxanthine is named inosine.

• In the pyrimidine nucleosides, the sugar and base are joined by a β-glycosidic link from the C 1' of the pentose to the N 1 of the pyrimidine base • In the purine nucleosides, the C 1' of the sugar is connected through the β-glycosydic link to the N 9 of the purine base.

5. Nucleotides • the polymeric forms of RNA and DNA contain phosphoric acid esters of the nucleosides. These esters are called nucleotides. • When DNA is broken into its constituent nucleotides, the cleavage may take place on either side of the phosphodiester bonds. So, two types of nucleotides can be released : the nucleoside-3'-monophosphate and the nucleoside-5'-monophosphate.

• The ribonucleoside 5'-phosphates may be further phosphorylated at position 5' to yield 5'-di- and -triphosphates. • The bonds between the first (α) and the second (b) and between the second (β) and the third (γ) phosphate groups are energy rich and are used to provide an energy source for various cellular activities. • The triphosphates are the forms from which the nucleic acids are synthesized.

DNA as Primary Genetic Material • Nucleic acids are genetic material of different organisms. • DNA is the genetic material of prokaryotes(virus, bacteria) and eukaryotes(plants, animals, etc). • RNA is also genetic material of some viruses, for instance HIV. • There are experimental basis that proved DNA or RNA to be the genetic material of different organisms.

• Bacteria transformation provided the first poof that DNA is the genetic material of the bacteria. • genetic properties can be transferred from one bacterial strain to another by extracting DNA from the first strain and adding it to the second strain.

• Inactive heat killed S bacteria and the ineffectual variant R bacteria together have quite different effect from either bacterium by itself. • virulent S bacteria can be recovered from the mouse postmortem.

• Phage infection proved that DNA is the genetic material of viruses • When the and protein bacteriophages (bacterial viruses) different transmitted DNA radioactive isotopes, to infected bacteria. the progeny components are only labeled the of with DNA is phages produced within

DNA • Transfection of eukaryotic cells is the acquisition of new genetic markers by incorporation of added DNA. • When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid enters the cells, and in some of them results in the production of new protein.

The Central dogma of molecular biology • It is the general pathway for the flow of genetic information from the nucleotide sequence of genes to the structure of proteins. DNA Transcription RNA Translation Protein

Summary • All genetic diseases involve defects at the level of the cell such as errors of cell division • DNA is a polymer of nucleotides, storing genetic information in the order of the nucleotide sequence • Nucleotides consist of a nitrogenous base, five carbon sugar, and a phosphate group • Genetic information flows from generation to generation through DNA replication • Genetic information flows with in a cell through transcription & translation

Review questions 1. Define molecular biology 2. Why do you study cells? 3. List the three domain of life 4. List the types of cells. 5. What is cell cycle and its components. 6. Compare and contrast mitosis and meiosis 7. Describe the components of nucleotides 8. Indicate central dogma of molcular biology

References • Robert F. weaver, Philip W. Hedrick. Genetics. • Darnel, Lodish, Baltimore. Molecular Cell Biology • James D. Watson: Recombinant DNA • Robert F. Weaver. Molecular biology • Richard J. Epistein: Human Molecular Biology • P. K. Gupta: Cell and Molecular Biology • Tarek H. EL-Metwally. Basic Medical Molecular Biology: A Comprehensive update.