Department of Biology/Faculty of Sciences/LU Dr. Fahd Nasr ... · III.3. Third base degeneracy and the wobble hypothesis 177 IV. Enzymology and mechanics of protein synthesis 178

Molecular Genetics and Genomics Department of Biology/Faculty of Sciences/LU

Dr. Fahd Nasr-All rights reserved

0



1



2

Table of Contents Preface 11 Boxes 15 Introduction 16

I. Preamble 16 II. DNA makes RNA makes proteins 16 III. Chromosomes 17 IV. A concise history of molecular genetics 17 V. Genomics and the human genome 23 VI. Post-genomics challenges 23 VII. Organization of the book 24

Chapter I- Genetic information: DNA and RNA 25

I. Opening remark 25 II. The one gene-one enzyme hypothesis 25 III. Properties of the genetic material 26 IV. The discovery that DNA is the genetic material 27

IV.1. Griffith discovers transformation 27 IV.2. The "Transforming principle" is DNA 28 IV.3. Experimental evidence that genes are made of DNA:

the Hershey and Chase experiment on bacteriophage T2 30 V. DNA quantity is constant 32 VI. The discovery of RNA as genetic material 33

Chapter II- The structure of DNA and RNA 34

I. Opening remark 34 II. Structure of nucleic acids 34

II.1. The structure of DNA 34 II.2. Nucleotides are called nucleoside phosphates 34 II.3. What about RNA 35 II.4. Nucleic acids are polynucleotides with directional sense 36 II.5. DNA is a double helix 38 II.6. Main features of the double helix 38

III. Classes of nucleic acids 40 IV. Significance of chemical differences between DNA and RNA 41 V. Physical features of DNA 42

V.1. Denaturation and renaturation of DNA 42 V.2. Thermal melting profile or hyperchromic shift 42 V.3. Buoyant density of DNA depends as well on G+C content 43

VI. Hydrolysis of nucleic acids 44 VI.1. Two major classes of nucleases 44 VI.2. Restriction enzymes as a specific class of DNases 46

VII. Conformation variation in the DNA double-helix 47 VIII. Tertiary structure of DNA and linking number 47



3

VIII.1. Supercoiling 48 VIII.2. Linking number 48 VIII.3. Intercalating agents 50 VIII.4. Cruciforms or palindromes 50

Chapter III- Anatomy of genomes in Eukaryotes and Prokaryotes 52

I. Opening remark 52 II. Anatomy of the eukaryotic genome 52

II.1. Chromatin structure 52 II.2. Organization of chromosomes 54 II.3. Special features of the metaphase chromosomes and Karyotype 54 II.4. Centromere and telomere: revisited 61

III. Anatomy of the prokaryotic genomes 62 III.1. The relatively small sizes of the prokaryotic genomes 62 III.2. Dynamism of bacterial genomes 62 III.3. The structure and organization of the prokaryotic genome 64

IV. Genomics as a new field in biology 65 IV.1. Definition 65 IV.2. Prokaryotic genomes 65

IV.2.1. The two kingdoms of prokaryotes: Bacteria and Archaea 66 IV.2.2. Bacterial genomes 66

IV.2.2.a. The complete genome of Escherichia coli K-12 67 IV.2.2.b. The Bacillus subtilis genome 68

IV.2.3. Archaeal genomes 68 IV.3. Eukaryotic genomes 72

IV.3.1. The genome project of the budding yeast S. cerevisiae 74 IV.3.2. The genome sequence of the Schizosaccharomyces pombe 75 IV.3.3. Drosophila melanogaster and Caenorhabditis elegans,

two models for molecular developmental genetics 75 IV.3.4. Arabidopsis thaliana, a model for plant molecular biology 76 IV.3.5. The human genome project 77 IV.3.6. Organelle genomes 78

IV.4. The post-sequencing era will be dominated by the new field of functional genomics 78

Chapter IV- DNA replication 80

I. Opening remark 80 II. General features of DNA replication 80

II.1. Three distinct issues are related to DNA replication 80 II.2. The plectonemic structure of DNA makes it difficult 81 II.3. DNA replication is semiconservative 81 II.4. Meselson and Stahl experiment 82 II.5. Harlequin chromosomes 84 II.6. The topological problem is solved by DNA topoisomerases 85

III. Enzymology and process of DNA replication 85 III.1. Initiation of DNA replication 85 III.2. DNA replication in outline 86 III.3. DNA replication in E. coli 87

III.3.a. Origin structure and initiation step 87



4

III.3.b. Elongation step 88 III.3.c. Termination step 93 III.3.d. Regulation of the E. coli DNA replication 93

III.4. DNA replication in eukaryotes 94 III.5. How to maintain the ends of a linear eukaryotic chromosome? 96

IV. Variations on the semi-conservative mode of replication 98 V. Regulation of eukaryotic DNA replication 99

V.1. Eukaryotic cell cycle and regulation of genome replication 99 V.2. Control of the Cell Cycle 100 V.3. Controlling DNA replication: getting molecular 100 V.4. Different checkpoints control the quality of the Cell Cycle 102 V.5. Cell cycle control involves oncogenes and tumor suppressor genes 103

VI. Variation of DNA replication: endoreplication 104 VI.1. Mitosis without cytokinesis 105 VI.2. Polyploidy 105 VI.3. Polyteny 105

Chapter V- Transcription: the first step in gene expression 107

I. Opening remark 107 II. How to access the genome 107 III. Chromatin structure and gene expression 109

III.1. Transcription initiation is a key control in gene expression 109 III.2. How chromatin condensation influences transcription 110 III.3. Subtle modifications with huge consequences 110

IV. Different types of RNAs 112 IV.1. Messenger RNA 112 IV.2. Ribosomal RNA 113 IV.3. Transfer RNA 114 IV.4. Specific types of RNA 114

IV.4.a. Small nuclear RNAs 114 IV.4.b. Small nucleolar RNAs 115 IV.4.c. Small cytoplasmic RNAs 115 IV.4.d. Transfer-messenger RNA 115 IV.4.e. Other non-coding small RNAs 116

V. Enzymology and mechanisms of transcription 119 V.1. RNA polymerases 119 V.2. Transcription in prokaryotes 120

V.2.1. Structure and function of the E. coli RNA polymerase 121 V.2.2. Binding of the RNA polymerase to promoter sequences 121 V.2.3. Properties of the prokaryotic promoters 121 V.2.4. The steps of transcription in prokaryotes 122

V.2.4.a. Initiation of polymerization 122 V.2.4.b. Transcription elongation 122 V.2.4.c. Chain termination 123

V.3. Transcription in eukaryotes 123 V.3.1. Properties of the eukaryotic promoters 124 V.3.2. Transcription initiation by the RNA polymerase I 125 V.3.3. Transcription initiation by the RNA polymerase II 125

V.3.3.a. RNA polymerase II promoter structure 125



5

V.3.3.b. The structure and function of the RNA polymerase I 125 V.3.3.c. Formation of the preinitiation complex 126

V.3.4. Transcription initiation by the RNA polymerase III 128 VI. Regulation of transcription in prokaryotes 128

VI.1 Operons are regulated by induction and repression 129 VI.2. The lac operon 129

VI.2.a. Structure and function of the lac operon 129 VI.2.b. Positive Control of Transcription: CAP 131

VI.3. Positive versus negative control systems 132 VI.4. The ara operon 133

VI.4.1. Structural and regulatory genes 133 VI.4.2. Regulation in details 135

VI.5. The trp operon 136 VI.5.1. Negative regulation by Trp repressor 138 VI.5.2. Regulation of trp operon by attenuation 138

VII. Regulation of transcription in eukaryotes 139 VII.1. Regulation of transcription initiation involves cis and trans components 139

VII.1.a. General features of the protein-encoding genes 140 VII.1.b. Core promoter versus upstream promoter or upstream control elements 140 VII.1.c. Enhancers, silencers and insulators 140

VII.2. The activity of transcription activators is controlled 140 Chapter VI- Synthesis and processing of RNA in prokaryotic and eukaryotic cells 142

I. Opening remark 142 II. Precursor RNA are modified in prokaryotes and eukaryotes 142 III. Synthesis and processing of bacterial mRNA 143 IV. Synthesis and processing of eukaryotic mRNA 145

IV.1. Capping 146 IV.2. Polyadenylation 146

V. Intron splicing 148 V.1. Features of split or interrupted genes 149 V.2. Nuclear mRNA introns 150 V.3. Eukaryotic nuclear pre-rRNA introns belong to group I family 154 V.4. Group II introns 155 V.5. Eukaryotic nuclear tRNA introns 157

VI. Chemical modification of RNAs 163 VI.1. Processing of rRNAs and tRNAs by chemical modifications 163 VI.2. RNA editing 163

VII. Turnover of mRNAs 164 Chapter VII- Synthesis and processing of the proteome 168

I. Opening remark 168 II. The genetic code 168

II.1. Gene structure and protein structure are collinear 168 II.2. The genetic code is a triplet and non-overlapping 169 II.3. Elucidating the genetic code 169 II.4. General features of the genetic code 170 II.5. Natural variation in the standard genetic code 172



6

III. Aminoacylation and structure of aminoacyl-tRNAs 173 III.1. Structure of transfer RNA (tRNA) 173 III.2. Formation of aminoacyl-tRNAs by aminoacyl-tRNA synthetases 176 III.3. Third base degeneracy and the wobble hypothesis 177

IV. Enzymology and mechanics of protein synthesis 178 IV.1. Protein synthesis in prokaryotes 178

IV.1.a. Peptide chain initiation in bacteria requires an internal ribosome binding site 178

IV.1.b. Peptide chain elongation 179 IV.1.c. Peptide chain termination requires release factors 180

IV.2. Protein synthesis in eukaryotes 181 IV.2.a. Peptide chain initiation in eukaryotic cells 181 IV.2.b. Peptide chain elongation is similar in prokaryotes and eukaryotes 182 IV.2.c. Eukaryotic peptide chain termination requires one release factor 182

V. Post-translational processing of proteins 183 V.1. Protein folding 183 V.2. Proteolytic cleavage 183 V.3. Processing or proteins by chemical modification 184 V.4. Protein splicing 185

VI. Protein kinesis or movement of proteins to their destinations 187 VI.1. Prokaryotic cell has little to worry about 187 VI.2. Protein kinesis in eukaryotic cells 187

VI.2.a. Protein sorting 187 VI.2.b. Pathways through the Endoplasmic Reticulum (ER) 188 VI.2.c. Destinations of proteins synthesized within the ER 189 VI.2.d. Destinations of proteins synthesized by free ribosomes 189

VII. Protein turnover 190 VII.1. Structure of the Proteasome 191 VII.2. The ubiquitin-mediated pathway 191

Chapter VIII- Genetics of microorganisms 193

I. Opening remark 193 II. The SOS response 194

II.1. Control of the SOS system 194 II.2. Regulatory proteins: RecA and LexA 194 II.3. Prophage induction by RecA 196 II.4. Treatments inducing the SOS response 196 II.5. Functions of SOS gene products 196

III. Lysogeny versus lytic pathways of bacteriophages 197 III.1. Bateriophage Lambda (): structure and life cycle 197 III.2. Lambda lytic cycle relies on two antiterminators 199 III.3. How to make the right decision: lysogeny versus lysis 200 III.4. Lysogeny requires the integration of DNA into the E. coli genome 201 III.5. Prophage induction 202

IV. Transfer of genetic information between cells 203 IV.1. Lederberg and Tatum experiment 203 IV.2. Sexual conjugation 203 IV.3. Transduction 204 IV.4. Transformation 204



7

V. Genetic recombination 204 V.1. Definition and types 204 V.2. General recombination 205

V.2.1. The Holliday model for general recombination 207 V.2.2. Meselson and Radding model 208 V.2.3. Genetic recombination must be highly accurate 210 V.2.4. The double-strand break model of recombination

explains gene conversion in yeast 211 V.3. Site specific recombination 214 V.4. Transposition 214 V.5. Enzymology of homologous recombination in the bacterium E. coli 214

Chapter IX- Genetics of viruses 217

I. Opening remark 217 II. Viral genomes 218

II.1. DNA viruses 218 II.2. RNA viruses 219

II.2.a. Negative-sense single-stranded RNA viruses 219 II.2.b. Positive-sense single-stranded RNA viruses 220 II.2.c. Double-stranded RNA viruses 221

III. Retroviruses 221 III.1. Historical view 221 III.2.Taxonomy 222 III.3. Retrovirus Structure 222 III.4. Genome 223 III.5. Replication cycle 223 III.6. Integration 225 III.7. HIV: the causal agent of AIDS 225

III.7.1. Structure of HIV 227 III.7.2. Life cycle of HIV 227 III.7.3. Disease Progression 228 III.7.4. Genetic Variability of HIV 230

IV. Latent Viruses 230 V. Classification of viruses 231

V.1. Animal viruses 231 V.2. Plant viruses 232 V.3. Bacteriophages 232

Chapter X- Genetics of organelles 233

I. Preamble 233 II. Endosymbiotic theory and the origin of eukaryotes 234 III. Mitochondria 236

III.1. Location and structure 236 III.2. Growth and division 237 III.3. Mitochondrial genomes 239

III.3.1. The human mitochondrial genome 240 III.3.1.a. Mitochondrial genes 242 III.3.1.b. Mitochondrial DNA replication and transcription 243

III.3.2. The rice mitochondrial genome 243



8

III.3.3. Mitochondrial plasmids 244 III.4. Mitochondrial mutations 245 III.5. Mitochondrial protein import machinery 246

IV. Chloroplasts 247 IV.1. Structure 247 IV.2. How do chloroplasts divide? 248 IV.3. Function of the chloroplast 249 IV.4. Chloroplast genomes 250 IV.5. Chloroplast import machinery 251

V. End-notes 252 V.1. A deeper look of endosymbiosis 252 V.2. Why do mitochondria and chloroplasts have their own genetic systems? 253

Chapter XI- Epigenetics 256

I. Preamble 256 II. DNA methylation: a major epigenetic mark 256

II.1. Does DNA methylation affect gene expression? 256 II.2. Distribution of methylated cytosines and CpG islands 257 II.3. Enzymology of DNA methylation 258

II.3.1. De novo DNA methylation 260 II.3.2. Maintenance DNA methylation 261 II.3.3. DNA methylation: closing remark 261

II.4. Methyl CpG binding proteins and gene silencing 262 II.4.1. A family of MBD protein-encoding genes 263 II.4.2. Mechanisms of DNA methylation-mediated gene silencing 264

III. The interplay of histone modifications 265 III.1. Histone acetylation 266 III.2. Histone methylation 268 III.3. Histone phosphorylation 270 III.4. Histone ubiquitination 271

IV. Chromatin remodeling machines 273 IV.1. Remodeling complexes at work 274 IV.2. Activation and repression by SWI/SNF 275

V. Polycomb and trithorax proteins: maintenance of gene expression 278 V.1. Polycomb and trithorax group complexes 279 V.2. PcG proteins act in concert with trxG remodeling factors 280 V.3. PcG and trxG response elements 280 V.4. Remodeling and histone modifications links 281

VI. Small and non-coding RNAs in epigenetic regulation 283 VI.1. SiRNAs: the basic mechanism 283 VI.2. Micro RNAs: synthesis and biological functions 284 VI.3. RNAi and epigenetic modifications 286

VII. The epigenetic code: layers of non-genetic information 287 VII.1. Dynamic states of chromatin structure 287 VII.2. Decipher the versatile histone code… 288 VII.3. Remodeling and maintenance machines come into play 289 VII.4. Epigenomics is a challenge 291

VIII. End note 292



9

Chapter XII- Cloning: scientific and other considerations 293 I. Preamble 293 II. Cloning versus asexual reproduction 293

II.1. Asexual reproduction in plants 294 II.2. Asexual reproduction in animals 294 II.3. Sexual reproduction 294

III. Cloning in retrospect 296 III.1. Historical view of cloning 296 III.2. Gurdon's claims 299 III.3. Dolly the lamb 300

IV. Types of cloning 303 IV.1. Reproductive cloning 303 IV.2. Therapeutic cloning 305 IV.3. Embryo cloning 306 IV.4. Parthenogenesis 307

V. Gene therapy and gene targeting 311 V.1. Gene therapy techniques 312

V.1.1. Somatic gene therapy 312 V.1.2. Germ line gene therapy 313

V.2. Transgenic animals as models for diseases' research 313 V.2.1. DNA micro-injection versus gene transfer using embryonic stem cells 314 V.2.2. Selection of genetically modified embryonic stem cells 315 V.2.3. Production of transgenic animals by nuclear transplantation 317

VI. Prospects and future challenges 317 Annex 1- Genetic engineering and Recombinant DNA 320

I. Opening remark 320 II. DNA cloning 320

II.1. Restriction enzymes 320 II.2. Plasmids 321

II.2.a. Definition and features 321 II.2.b. Cloning vectors 323

II.3. Recombinant DNA 324 II.4. Transcription and translation in vitro 324

III. Manipulation of nucleic acids 325 III.1. Isolation of DNA 325 III.2. Isolation of mRNA 325 III.3. Polymerase Chain Reaction (PCR) 325 III.4. Agarose Gel Electrophoresis 325 III.5. Probes 328 III.6. Southern Blotting 328 III.7. Northern Blotting 328 III.8. Western Blotting 329

IV. DNA libraries 329 IV.1 Genomic libraries 329 IV.2. Complementary DNA (cDNA) libraries 329 IV.3. How to screen a library 331

V. DNA sequencing methodology 331 V.1. Chain termination method 331



10

V.2. Chemical degradation method of Maxam and Gilbert 333 VI. Ultracentrifugation techniques 334

VI.1. Differential centrifugation and density gradient centrifugation 334 VI.2. Isopycnic centrifugation and buoyant density of DNA 335

VII. Fluorescence in situ hybridization or FISH 336 VIII. Differential display and SAGE 337

VIII.1. Differential display 338 VIII.2. Serial Analysis of Gene Expression (SAGE) 338

Annex 2- Nomenclature of genes in different organisms 339

I. Introductory remark 339 II. Microorganisms 339

II.1. Bacteria and Archaea (model: E. coli) 339 II.2. Protozoa (model Dictyostelium discoideum) 339 II.3. Fungi 340

II.3.a. The budding yeast S. cerevisiae 340 II.3.b. The fission yeast Schizosaccharomyces pombe 340 II.3.c. The filamentous fungus Aspergillus nidulans 340 II.3.d. The filamentous fungus Neurospora crassa 342

III. Plants 342 III.1. The corn plant Zea mays 342 III.2. Pea (Pisum sativum) 342 III.3. The mustard weed Arabidopsis thaliana 343

IV. Animals 343 IV.1. Invertebrates 343

IV.1.a. The nematode Caenorhabditis elegans 343 IV.1.b. The fruit fly Drosophila melanogaster 343

IV.2. Vertebrates 344 IV.2.a. The zebrafish Brachydanio rerio 344 IV.2.b. The chick Gallus domesticus 344 IV.2.c. The mouse Mus musculus 344 IV.2.b. The human Homo sapiens 345

Problems 346 Glossary 366



11

Preface to the fifth edition There is no area in science that moves as fast as biology and more particularly the field of Genetics. This field has known so many discoveries that we have yet to contemplate them. Further studies will be needed to realize the potential of the new discoveries' applications. Since 2001 I have written four versions of the Molecular Genetics and Genomics book with a constant commitment to update the information so that every new version sees the integration of recent and challenging data. I am writing a new preface for the fifth version for a very simple reason. I decided to add two new chapters, the first being concerned with Cloning and the second totally dedicated to Epigenetics. Both are old topics that gained recent and wide interest and much excitement among scientists for some of the related issues raised paradoxes and controversies. Cloning refers to the production of an identical copy of an existing organism. In this chapter I provide a historical view of cloning since Weismann's theory at the end of the eighteen century until the cloning of a dog a few weeks ago by a Korean team. Hallmarks in this history include the feat achieved by Spemann who ruled out the Weismann's hypothesis. Other crucial experiments were also cited and some depicted. In addition, I present a detailed view of the different types of cloning such as reproductive, therapeutic, etc. along with their applications and the ethical concerns they arouse. The chapter of cloning also describes the issue of stem cells research and cell-based therapies. The second chapter deals with epigenetic phenomenon. There has been a bulk of recent data to reconsider this sub-discipline in a separate chapter with the ultimate wish to fall in line with the progress in the understanding of complex biological processes. Epigenetics seeks to unravel a new code for gene expression, hence for life, lying outside the DNA sequence per se. DNA methylation, histone modifications, ATP-dependent remodeling complexes, and others are (heritable) changes that affect basic and more specific functions during morphogenesis and development. The new code for life deserves further attention. Scientists believe more and more that Genetics and Epigenetics contribute equally to the expression of the genetic information in normal conditions and disease. Henceforth, therapeutic strategies will be based on genetic and epigenetic approaches. I am confident that biology students still have real opportunities to make exciting breakthroughs and come up with new discoveries in the field of Genetics. However, only dedicated and smart people may expect to put potential opportunities into reach and keep the path open to push the limit of knowledge one step farther.

Dr. Fahd Nasr Baakleen, 15 October 2005



12

Preface This book aims at the molecular description of the different steps of gene expression with emphasis on the recent advances in the field of functional and structural genomics. The very definition of a gene, which corresponds to the so-called Mendelian factor, has been remodeled continuously during the last century to fit an ever-changing view of gene and genome anatomy. The simplest definition states that a gene is a fragment of nucleic acid molecule that specifies a polypeptide or a functional RNA. There is no doubt that this simple description encompasses the regulatory sequences needed for the correct expression of any gene i.e. promoter elements and termination sequences. A higher level of complexity was added with the discovery of split genes. These came into focus when it was found that the coding sequence of eukaryotic genes might be interrupted by non-coding regions. It was immediately proposed that highly specific machinery (spliceosome) must exist to remove precisely the intervening sequences, also called introns, and put together the coding regions, termed exons, in order for the genetic information of the split gene to be expressed correctly. In addition, the one gene-one enzyme hypothesis proposed by Beadle and Tatum in 1941 could not stand forever. The finding of genes that code for more than one enzyme was anticipated by Stark1 in 1977, thus suggesting new theories to account for gene and genome evolution. Moreover, a gene may lose its function and become dead gene or pseudogene. The gene story is still under consideration as some scientists proposed to extend the notion of gene to every regulatory segment (even though it is not expressed) that is required for genome-related functions such as stability (telomere), chromosome segregation (centromere), chromatin remodeling, etc. The main purpose of this book is to keep the biology student updated with every progress that occurs in the exciting field of molecular genetics and genomics. For instance, the RNA story may resume some related concerns: in the third quarter of the last century it was widely recognized that ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) are expected to fulfill all the RNA functions within the cell (prokaryotic and eukaryotic cells). However, in the last quarter the situation had completely changed with the discovery of many RNA species, having diverse functions ranging from structural through regulatory to catalytic. Among these non-coding RNAs we cite the transfer messenger RNA (tmRNA) that targets incomplete proteins for degradation in bacteria, the regulatory Xist RNA that is involved in dosage compensation mechanism in mammals, small nuclear RNAs (snRNAs) that mediate post-transcriptional processing event i.e. intron splicing, small nucleolar RNAs (snoRNAs), which act as guide RNAs to direct modification (pseudouridylation and 2'-O-ribose methylation) in rRNA, the small temporal RNAs (stRNA) and small interfering RNAs (siRNA) that are involved in RNA interference and gene silencing,

1 Stark, G. (1977) Multifunctional proteins: one gene more than one enzyme.Trends Biochem. Sci., 2:64-66.



13

etc. The list has not been closed yet! Hence, it is of general importance to integrate the new knowledge into the old one to provide a comprehensive and updated view of genetics. Finally, The advent of genome projects in the last decade of the last century has produced a huge amount of information that will impact the different areas in biology. The accumulated knowledge is expected to double every two years (maybe less), so that it would be theoretically impossible to keep pace with the new discoveries and their consequences. By definition, a genome is the complete set of all genes in a given organism. It contains the biological information required to specify all the functions that maintain the life of that organism. In the late 80’s the scientific community has arrived to a kind of conviction that the characterization of the molecular mechanisms of life, as well as their regulatory networks, needs a comprehensive set of all the proteins involved. To achieve this goal, they decided to launch out on an enterprise that we call now "Genomics". The first step in this enterprise is a genome project that aims at the determination of the complete DNA sequence for the model organism being studied. However, the genome sequence is not an end; it should be further analyzed and submitted to functional analysis in an attempt to answer the question: how genome functions? This requires that the results of mapping and sequencing must be combined with the activities of the genomes in living cells. The latter need first to express the biological information contained within their genomes. This step of genome expression determines the make-up of the cellular RNA, termed transcriptome, which is restructured according to the prevailing conditions. Finally, the end result of genome expression is the proteome, the complete set of functioning proteins in living cells. The accomplishment of many genome projects for dozens of model organisms, including human, the fruit fly Drosophila melanogaster, the worm Caenorhabditis elegans, the plant Arabidopsis thaliana, the yeast Saccharomyces cerevisiae, The fission yeast Schizosaccharomyces pombe and many others, will enable us to understand the language of genes. Now, the genomes of 694 viruses, 37 viroids, 90 phages, 248 naturally-occurring plasmids, 202 organelles, 15 Archaea, 60 Bacteria prokaryotes, 8 eukaryotes, including human, have been sequenced. For instance, the complete sequence of the yeast S. cerevisiae genome was achieved in 1996 and published in Nature2. The analysis of the 12008kb sequence has revealed 5885 protein-encoding genes, 140 ribosomal RNA genes, 40 small RNA genes (snRNA, snoRNA, etc.), and 275 transfer RNA genes. Among the 5885 genes more than 50% are encoding proteins of unknown function. In order to unravel the function of the so-called orphan genes, many approaches and strategies have been devised. Some are concerned with the analysis of individual genes and others, termed large-scale approaches are devoted to the analysis and comparison of entire genomes from a large number of species. Furthermore, when genes of different and distant species show significant similarities, there is every reason to believe that they are related. This means that by comparing the sequence of many genomes with each other it should be possible to establish the evolutionary relationships between them. Therefore, biology students have now a unique opportunity to pursue a scientific career in this field that will have a number of applications in every area of biology mainly in medicine where scientists are working hard to characterize

2 Goffeau, A., Nasr, F., et al. (1997) The yeast genome directory. Nature, 387 (Suppl.) 1-105.



14

genes that contribute to many diseases such as cancer, diabetes, mental illness etc. It seems obvious that molecular genetics and functional genomics will revolutionize the life sciences during and 21st century, with the promise of new and fascinating discoveries to come.

Dr. Fahd Nasr Baakleen, 01 October 2002



15

Boxes Deeper look on some topics

Chapter-Box Title Page Chapter II-Box 1 Every biological molecule is informational 38

Chapter III-Box 1 Although histones are the major constituents of chromatin in most eukaryotes, there is an exception to the rule

53

Chapter III-Box 2 Human cytogenetics out of ages 60

Chapter III-Box 3 A deeper look of Archaea 69

Chapter V-Box 1 Small RNAs and DNA metylation in gene silencing

116

Chapter VI-Box 1 Splicing and cancer 157

Chapter VI-Box 2 Nonsense-mediated mRNA decay in mammals 165

Chapter X-Box 1 Functions of mitochrondria 238

Chapter X-Box 2 Gene transfer 244

Chapter X-Box 3 Rickettsia prowazekii as potential ancestor of mitochondria

246

Chapter X-Box 4 Photosynthesis consists of both "light" and "Dark" reactions

249

Chapter X-Box 5 What are the possible fates of genes in endosymbiosis

254

Chapter XI-Box 1 Methylation and demethylation 269

Chapter XI-Box 2 Phosphorylation at stake 270

Chapter XI-Box 3 Closing remark with RNAi and polycomb group 272

Chapter XI-Box 4 Imprinting 276

Chapter XI-Box 5 RNAi technology 287

Chapter XI-Box 6 Epigenetic reprogramming: struggle of the clones 290

Chapter XII-Box 1 In vitro fertilization (IVF) 295

Chapter XII-Box 2 The technique for nuclear transfer 301

Chapter XII-Box 3 Cell based therapies 309



16

Introduction I. Preamble A genome is the complete set of instructions that specify all the functions needed to maintain the life of an organism. Most genomes are made up of DNA but some viruses have RNA genomes. Nuclein, which corresponds to DNA, was first isolated in 1869 by Frederich Miescher however, it had to wait 83 years to show unambiguously that it is the genetic material of life. Both RNA and DNA are polymeric molecules, called polynucleotides, made up of monomeric subunits termed nucleotides. Each nucleotide comprises a sugar, a nitrogenous base, and a phosphate group (or more). The sugar is the ribose in RNA and deoxyribose in DNA. Four nitrogenous bases are present in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T). In RNA we find the bases A, G, and C but T is replaced with uracil (U). Nucleotides are linked together via phosphodiester bonds to form polynucleotides. In 1953, Francis Crick and James Watson unraveled the three dimensional structure of DNA3. Their model indicated that DNA is a double helix in which the two polynucleotides are wound around each other in a right-handed and antiparallel fashion. The two strands are maintained together by hydrogen bonds between A and T on the one hand, and G and C one the other hand. These base-pairing rules indicate that the two strands in DNA have complementary sequences i.e. the sequence information in one strand is conserved in the other. In DNA the sugar-phosphate-sugar backbone provides stability, whereas the succession of bases generates identity and diversity. II. DNA makes RNA makes protein According to the "central dogma of molecular biology", formulated by Crick in 1958, there is a directional flow of information from DNA to proteins. The genetic information contained in the genome is specified by the sequence of nucleotides. DNA, that specifies all the instructions for life, should contain information for its own maintenance and replication to ensure that two correct copies of the genome are available when the cell divides. This represents the first flow of information from DNA to DNA via the replication process. Further, DNA must specify the biochemical signature of the cell in which it resides. Each DNA molecule entails many genes that are accessible to proteins, which initiate a two-step process called gene expression. The latter comprises transcription of DNA and generation of RNA molecules (directional flow from DNA to RNA), then translation of RNA resulting in the synthesis of proteins whose amino acid sequence is dictated by the sequence of nucleotides via the genetic code (directional flow from RNA to proteins). In addition, a directional flow from RNA to DNA exists through the process of reverse transcription generating a complementary DNA molecule (e.g. telomerase and retroviral reverse

3 Watson, J.D. and Crick, F.H.C. (1953) Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature, 171: 737-738.



17

transcriptase). Eventually, a new directional flow from protein to protein has recently been reported via the phenomenon of prion. Prion is a new term designating an infectious particle made up of protein only. III. Chromosomes The 3 billion bp in the human genome are organized into 24 distinct, physically separate microscopic units called chromosomes. All genes are arranged linearly along the chromosomes. The nucleus of most human cells contains 2 sets of chromosomes, 1 set given by each parent. Each set has 23 single chromosomes—22 autosomes and an X or Y sex chromosome. A normal female will have a pair of X chromosomes; a male will have an X and Y pair. Chromosomes contain roughly equal parts of protein and DNA; chromosomal DNA contains an average of 135 million base pairs (bp). During cell division, Chromosomes become highly condensed so that they can be seen under a light microscope and, when stained with certain dyes, reveal a pattern of light and dark bands reflecting regional variations in the amounts of A and T versus G and C. Differences in size and banding pattern allow the 24 chromosomes to be distinguished from each other, an analysis called a karyotype. A few types of major chromosomal abnormalities, including missing or extra copies of a chromosome or gross breaks and rejoinings (translocations), can be detected by microscopic examination; Down’s syndrome, in which an individual's cells contain a third copy of chromosome 21, is diagnosed by karyotype analysis. Most changes in DNA, however, are too subtle to be detected by this technique and require molecular analysis. These subtle DNA abnormalities (mutations) are responsible for many inherited diseases such as cystic fibrosis and sickle cell anemia or may predispose an individual to cancer, major mental illnesses, and other complex diseases. IV. A concise history of Molecular Biology and Genetics The discovery of double-helix structure of DNA is considered as the major contribution to biology in the past century. Since then, a number of discoveries in the biological sciences, more particularly in the area of molecular genetics, have shaped our knowledge and helped usher in the new area of genomics, which is the discipline of the 21st century. Molecular genetics is a discipline that seeks to fully understand the molecular basis of heredity, genetic variation, and the expression patterns of individual units of heredity called genes. Genes contain the instructions for making cells and for the work that goes on inside them. Here I will present a timeline of key discoveries that have culminated in the human genome project, which holds an extraordinary trove of information about human development, physiology, medicine and evolution. This was the result of an international effort to decipher the sequence of the 3 billion base pair subunits of DNA, which reside in the chromosomes of human beings (see below). 1865- Gregor Mendel published the results of breeding experiments on the garden

pea Pisum sativum. By noting the appearance or disappearance of traits, such as pod and flower color, over several generations Mendel postulated a generalized set of rules



18

governing inheritance. He proposed the presence of discrete units of heredity (which today we call genes) that are transmitted stably from generation to generation. These traits are not necessary observed in each generation (dominance and recessiveness notions). 1869- Frederick Miescher isolated DNA for the first time. 1885- Flemming showed that nuclei contained chromosomes. 1900- De Vries, Correns, and Von Tschermak-Seysenegg independently

performed breeding experiments confirming Mendel's principles of heredity. 1902- Sutton and Boveri proposed the chromosome theory of heredity stating that

the segregation of chromosomes is the basis of the gene segregation. 1911- Morgan proposed that genetic linkage was the result of the genes involved

being located on the same chromosome. 1927- Muller reported that genetic damage is inheritable and proved that X-rays

cause mutations that pass from one generation to the next. 1928- Griffith unwittingly discovered transformation, a process involving the

uptake of genetic material by a living organism. 1931- Creighton and McClintock showed that genetic recombination in maize

results from physical exchange between homologous chromosomes. 1941- Beadle and Tatum proposed the one gene-one enzyme hypothesis. They

jointly published the results of their experiments with the fungus Neurospora crassa. They concluded that ultraviolet light treatment somehow caused a mutation in a gene that controlled the synthesis of an enzyme involved in the synthesis of an essential nutrient. They also showed that the defect was inherited in a typical Mendelian fashion. These results ultimately led to the "one gene-one enzyme" concept of biology. 1943- Luria and Delbruck demonstrated that mutational alterations occur randomly

and before exposure to exogenous mutagenic influences and that inheritance in bacteria is subject to natural selection. 1944- Building upon the earlier work of Griffith, Avery, MacLeod, and McCarty

showed that transfer of DNA is responsible for the transformation of Streptococcus pneumoniae from an avirulent phenotype to a virulent phenotype. 1945- Luria and Hershey demonstrated that bacterial viruses (bacteriophages)

mutate, suggesting the possibility that other virus types (such as the causative agents of influenza and colds) mutate spontaneously as well. 1946- Lederberg and Tatum demonstrated sexual exchange of genetic material in

bacteria (sexual conjugation). 1950- McKlintock publishes proof of mobile genetic elements (transposons) in

corn. 1952- Lederberg and Zinder described transduction, which is the transfer of

genetic information by viruses. Dulbecco showed that single particles of an animal virus can produce areas of cellular lysis called plaques. Hershey and Chase published data suggesting that only DNA is required for T2 bacteriophage replication. Luria and



19

Human, and independently Weigle, described a non-genetic host-controlled modification system in bacteriophage which ultimately led to the study of bacterial systems of restriction and modification; these studies in turn eventually led to the discovery of restriction endonucleases, essential tools in the development of genetic engineering methodologies. 1953- Crick and Wilkins, together with Watson, proposed the double-helix

structure of DNA. The proposed structure was based on X-ray crystallography studies on DNA performed by Franklin. 1957- Benzer, in an analysis of mutations of the rII gene in bacteriophage T4,

showed that recombination can occur between mutations in the same gene and that genes consist of linear arrays of subunits that can be altered. Jacob and Wollman presented evidence of the circular nature of the Escherichia coli chromosome by analyzing the results of interrupted mating experiments between conjugating bacteria. Kornberg discovered DNA polymerase. 1958- Meselson and Stahl used density gradient centrifugation to demonstrate that

the two parental strands of DNA unwind during replication with each single strand becoming a template for synthesis of a new, complementary strand. Each daughter molecule, consisting of one old and one new DNA strand, is an exact copy of the parent molecule. This indicated that replication proceeds via a semi-conservative model, exactly as predicted by Watson and Crick's DNA replication model. 1960- Jacob, Perrin, Sanchez, and Monod proposed the operon concept of gene

regulation in bacteria. Jacob and Monod later proposed that a protein repressor blocks RNA transcription of a specific set of genes, termed the lac operon, unless an inducer, lactose, binds to the repressor, thus altering its conformation and preventing the repressor from binding to the operon site. 1961 Crick, Brenner, and colleagues proposed the existence of a transfer RNA that

utilizes a three base code and participates directly in the synthesis of proteins. Nirenberg and Matthaei demonstrated that the polynucleotide poly (U) directs the synthesis of a polypeptide containing only phenylalanine residues. They concluded from these experiments that the triplet UUU must code for the amino acid phenylalanine. This was the beginning of the ultimately successful effort to decipher the genetic code. Brenner, Jacob, and Meselson demonstrated that ribosomes are the site of protein synthesis and confirmed the existence of messenger RNA. 1966- Nirenberg, Ochoa and Khorana elucidated the genetic code. 1967- Gilbert isolated the lac repressor regulatory protein postulated by Jacob and

Monod. Ptashne isolated the lambda repressor protein from bacteriophage. 1970- Smith and Wilcox reported on the characteristics of restriction enzymes,

which are enzymes that protect bacteria from the potentially harmful effects of foreign DNA. They showed that a restriction enzyme isolated from Haemophilus influenzae had the ability to recognize and cleave specific DNA sequences. Temin and Baltimore independently reported the discovery of reverse transcriptase in RNA viruses.



20

1972- Mertz and Davis confirmed that the EcoR1 restriction endonuclease from Escherichia coli cuts DNA at a specific site six nucleotides long. The DNA sequence that is cut by the restriction enzyme is complementary to other DNAs cut by the same enzyme. This observation paved the way for splicing together of dissimilar sequences and other forms of genetic engineering. Berg and coworkers reported the construction of a recombinant DNA molecule comprised of viral and bacterial DNA sequences. 1973- Cohen, Chang, Helling, and Boyer demonstrated that if DNA is fragmented

with restriction endonucleases and combined with similarly restricted plasmid DNA, then the resulting recombinant DNA molecules are biologically active and can replicate in host bacterial cells. Plasmids can thus act as vectors for the propagation of foreign cloned genes. This discovery was a major breakthrough in the development of recombinant DNA technologies and genetic engineering. 1975- Southern described a new analytical tool involving the capillary transfer of

restricted DNA fragments from a sizing gel to a nitrocellulose membrane, resulting in an exact replica of the DNA fragments in the gel on the membrane. A specific radiolabeled probe is then applied to the membrane under hybridizing conditions. Subsequent exposure of the membrane to photographic film reveals which DNA fragments are homologous to the specific probe used in the experiment. Southern blotting, as this technique is now referred to, allows researchers to determine a physical map of restriction sites within a gene in its normal chromosomal location and provides an estimate of the copy number of a gene in the genome along with information on the degree of similarity of the gene in question to other known homologous sequences. 1977- Chow and Roberts, and independently Sharp showed that in eucaryotic

organisms (and not procaryotes) genes are not continuous but instead are interspersed with stretches of non-coding sequence which do not code for protein structure (these interspersed regions of sequence are called introns). Maxam and Gilbert at Harvard University and Sanger at the MRC developed independent methods for determining the exact nucleotide sequence of DNA. Sanger and his colleagues used their own sequencing method to determine the complete nucleotide sequence of the bacteriophage X174, the first genome ever completely sequenced. Nester, Gordon, and Dell-Chilton demonstrated the transfer of genes on the Agrobacterium tumefaciens plasmid into infected plant cells, paving the way for genetic engineering of plant species. 1980- Botstein, Davis, Skolnick and White proposed a method to map the entire

human genome based on RFLPs. First commercialization of Molecular biology kits. 1981- Eli Lilly receives FDA approval to market the first recombinant protein,

human insulin, for the treatment of diabetes. 1983- Cech and Altman discovered self-splicing of an intron RNA. 1985- Mullis introduced the polymerase chain reaction (PCR), a novel method of

amplifying large amounts of a specific DNA fragment starting with very small amounts of source DNA. Mullis used a DNA polymerase enzyme from the heat-stable organism Thermus aquaticus to replicate specific DNAs of interest using oligonucleotide primers



21

on either side of the gene to be amplified. The primers are allowed to anneal to their homologous targets and the reaction is repeated in order to amplify the target DNA exponentially. PCR has revolutionized modern biology and has widespread applications in the areas of forensics, diagnostics, and gene expression analysis. 1986- Hood and Smith at Caltech announce the first automated DNA sequencing

machine. 1987- Burke, Olson and Carle of Washington University develop YACs (yeast

artificial chromosome) for cloning, thus increasing insert size by a least 10-fold. DuPont scientists develop a system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides; at the same time Applied Biosystems commercialize the first automated sequencing machine, based on Hood and Smith's technology. 1989- Collins finds gene for cystic fibrosis. 1990- Lipman and Myers at the NCBI publish the BLAST algorithm designed to

align sequences. Human Genome Project The 15-year Human Genome Project formally began, and represents an effort to "find all the genes on every chromosome in the body and to determine their biochemical nature". The first approved gene therapy is performed with some success. Immunoglobulin genes are inserted into harvested white blood cells that are then returned to the patient and confer some immunity. 1992- Gene Sequence Published, the entire 315,000 bp sequence of chromosome

III, one of the sixteen chromosomes of the yeast Saccharomyces cerevisiae was published. 1995- Venter, Smith, and Fraser and colleagues reported the first complete genome

sequence of a nonviral microorganism, Haemophilus influenza. Amersham Company develops improved sequencing dyes and thermostable polymerase. Brown and colleagues publish first paper using a recent technology termed DNA microarray. 1996- The genome sequence of the yeast S. cerevisiae was completed by an

international consortium. The sequence of 12,068 kb specify about 6000 potential protein-encoding genes, approximately 140 ribosomal RNA genes, 40 small nuclear RNA genes, and 275 transfer RNA genes4. 1997- The Roslin Institute in Edinburgh reported the birth of Dolly the lamb5, the

first mammal to be cloned from an adult using the modern techniques of transgenic and reproductive cloning. The successful cloning of Dolly suggested the possibility that similar techniques could be utilized to clone humans. Blattner, Plunkett, and colleagues complete the genomic sequence of the Gram-negative bacterium6 Escherichia coli, 4.6 Mb.

4 Goffeau, A. et al. (1996) Life with 6000 genes. Science, 274: 546-567. 5 Campbell, K.H.S. et al. (1997). Sheep cloned by nuclear transfer from a cultured cell line. Nature, 285: 810-813. 6 Blattner, F.R. et al. (1997) The complete genome sequence of Escherichia coli. Science, 277: 1453-1462.



22

1998- Green and Ewing of Washigton University publish a program called PHRED7 for automatically interpreting sequencer data. Both PHRED and its sister's program PHRAP8 (used to assemble sequences) had been in wide use since 1995. Sulston of the Sanger center, Waterston of Washington University and colleagues complete the genomic sequence of the nematode9 Caenorhabditis elegans. 1999- Researchers in the Human Genome Project reported the complete sequencing

of the DNA making up human chromosome 22. 2000- Celera and academic collaborators sequence the 180-Mb genome of the fruit

fly10 Drosophila melanogaster, the largest genome yet sequenced. The human genome project (HGP) publishes the complete sequence of chromosome 21. An international consortium completes the genome sequencing of the first plant11, Arabidopsis thaliana, 125 Mb. Human Genome Project leaders announced the completion of a "working draft" DNA sequence of the entire human genome. The post-genomic era begins. 2001- The Human Genome Project consortium publishes the human working

sequence draft in Nature and the private company Celera publishes its draft in Science12. 2003- Dolly the first cloned sheep was put to death in 14 February 2003 after

developing progressive lung disease and arthritis, diseases that are most common of older animals. The death of Dolly at the age of six-and-a-half years refuel the debate over the health and life-expectancy of cloned animals. It also exacerbated the ethical controversy concerning the human cloning issue. Zeng, Marra and colleagues13, publish The complete genomic nucleotide sequence (29.7kb) of a Hong Kong severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) strain HK-39. The completion of the genomic sequence of the virus will help understand the pathogenecity of the virus and assist in tracing its origin.

7 PHRED is A widely used computer program designed to analyze raw sequences (individual unassembled sequence readings) and produce a consensus or a "base call" with an associated score for each position in the sequence. 8 PHRAP is a computer program devised to assemble raw sequences into sequence contigs and assign a score for each position in the sequence. 9 The C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: platform for investigating biology. Science, 282:2012-2018. 10 Adams, M.D., et al. (2000) The genome sequence of Drosophila melanogaster. Science, 287: 2185-95. 11 The Arabidopsis genome initiative. (2000) Analysis of the genome of the flowering plant Arabidopsis thaliana. Nature, 408: 796-815. 12 Venter, G. et al. (2001) The sequence of the human genome. Science, 291: 1304-1351. International Human Genome Sequencing Consortium. (2001) Initial sequencing and analysis of the human genome. Nature, 409: 860-921. 13 Zeng, F.Y. et al. (2003). The Complete Genome Sequence of Severe Acute Respiratory Syndrome Coronavirus Strain HKU-39849 (HK-39). Exp. Biol. Med., 228: 866-873. Marra, M.A. et al. (2003). The Genome sequence of the SARS-associated coronavirus. Science, 300:1399-404.



23

V. Genomics and the human genome project Genomics is a new field in biological sciences that aims at the analysis of the entire genetic information of a given genome. The rediscovery of Mendel's laws of heredity at the very beginning of the 20th century initiated a series of discoveries in the new biological discipline, termed genetics, that aimed at the understanding of the nature and content of genetic information. The last decade of the 20th century has been marked by a general tendency to decipher entire genomes, spawning the field of genomics. A primary goal of the Human Genome Project is to make a map of each human chromosome at increasingly finer resolutions. Mapping involves (1) dividing the chromosomes into smaller fragments that can be amplified and characterized and (2) ordering (mapping) them to locate their positions on the chromosomes. A perfect collection of fragments should cover the whole genome. After mapping is completed, the next step is to determine the base sequence of each of the ordered DNA fragments. The ultimate goal of genome research is to generate the complete sequence of the 24 different chromosomes and to find all the genes in the DNA sequence and to develop tools for using this information in the study of human biology and medicine. The human genome project was the result of a fruitful collaboration involving 20 groups from different countries to produce a draft sequence that covers more than 94% of the human genome. Much work remains to be done to produce a complete finished sequence, but most of the information has become available, thus allowing a global perspective on the human genome. The estimated number of genes is 30,000, but there are also 740 identified genes that makes non-protein-coding RNAs. This number should be compared with 4,300 genes for the bacterium E. coli, 6,000 for a yeast cell, 13,000 for a fly, 18,000 for a worm and 26,000 for a plant14. VI. Post-genomes challenges The working draft DNA sequence represents a huge achievement. The human genome has been joined by genome projects concerning other eukaryotes such as the fruit fly Drosophila melanogaster, the worm Caenorhabditis elegans, the plant Arabidopsis thaliana, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe15. These genome projects have generated a huge amount of information that requires genomic-scale technologies to study and compare entire genome sequences, identify gene families from a large number of species, and analyze variation among individuals. The ever-growing field of biotechnology, which deals with the analysis of the crude information, should move in parallel with functional analysis in order to face the future challenges in genetics. Among these challenges we cite: Determination of gene number, exact locations, functions and regulation. Structure and organization of chromosomes, and chromatin remodeling.

14 Note that none of the gene numbers for the multicellular organisms is highly accurate because of the limitations of gene finding programs. 15 Wood, V. et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature, 415: 871-880.



24

Coordination of gene expression, protein synthesis and processing, and protein conservation among organisms. Disease-susceptibility prediction based on gene sequence variation and identification

of genes involved in complex traits and multigene diseases. Developmental genetics. Identify the complexes of multiproteins that carry out the functions of living systems. Characterize the gene-regulatory networks.

VII. Organization of the book The main goal of the present book is to provide an overview of the molecular mechanisms of gene expression in prokaryotic and eukaryotic organisms. We will focus on four principal issues: replication and maintenance of DNA, synthesis and processing of RNA, regulation of gene expression, and synthesis and maturation of the proteome16. We will also concentrate on the endosymbiotic theory and the origin of eukaryotes. We devote one chapter to the cloning topic focusing on the different ethical and scientific aspects. Also, with the reevaluation of the epigenetic modifications we decided to present a comprehensive synthesis of a new discipline, simply termed Epigenetics, to try to describe the ways whereby heritable marks are written outside the DNA sequence information per se. Furthermore, we will describe some of the procedures that are widely used in molecular genetics as well as some approaches and strategies that are usually employed to solve biological problems in both fundamental and applied research.

*****

16 The proteome is a term that was designed to represent the whole set of functioning proteins in a given cell under specific conditions.



25

Genetic information: DNA and RNA

I. Opening remark Although it is now known that the DNA is the genetic material (the most common) that carries the genetic information, this fact was neither accepted nor defended as recently as 50 years ago. At that time Genetics or the science of heredity was an abstract science. The molecular basis of heredity, which was defined as the tendency of the offspring to inherit the characteristics of the parents, was not obvious. Despite the lack of information, the genes, the elusive but real units carrying and transmitting the genetic information from one generation to another, were found by the geneticists to be contained within the nuclei of the cells in association with the chromosomes. Nevertheless, the molecular nature of the genetic material (the nature of the genes) was not obvious despite the realization that chromosomes are made up of proteins and DNA. In this chapter we will deal with those experiments which were devised to try to unravel the nature of the genetic material. These attempts were fruitful in demonstrating what the genes are, how they function and later how they are organized. II. The one-gene, one-enzyme hypothesis In 1941 George Beadle and Edward Tatum performed some experiments on the metabolism of the orange bread mold Neurosopora crassa. This fungus can grow very well on a minimal growth medium, which contains the basic set of nutrients (sucrose, inorganic salts and the vitamin biotin). Firstly, they irradiated spores of the fungus with X-rays to cause mutations. Secondly, they tested the progeny derived from the irradiated spores for their growth requirements. Cells were first grown on complete medium (theoretically, this consists of minimal medium supplemented with all the 20 amino acids and other nutrients) and then tested for growth on minimal medium. Those that failed to grow on minimal medium were thought to be defective in enzymes involved in the synthesis of essential metabolites. Thirdly, by testing different minimal media supplemented with various amino acids they were able to identify mutants that grow normally (as wild type) if provided with a specific nutrient (Fig. 1). They concluded that these mutants were unable to synthesize certain essential substance. Presumably, the mutation caused by X-rays has hit the gene, which is responsible for an enzyme in the biosynthesis pathway for the needed supplement. The analysis of these mutants and the identification of the defective biosynthetic steps have led to the hypothesis that one gene codes for one enzyme. This was deduced from the simple correspondence between a mutation and the absence of a specific enzyme (Fig. 2). Despite these studies the molecular nature of the gene remained unknown.



26

Figure 1. One gene-one enzyme concept. Mutant strains unable to grow on minimal medium unless supplemented with an essential metabolite, such as an amino acid, are identified. Such mutations are called auxotrophic. If a mutant is auxotroph for leucine, for instance, this means that this mutant requires leucine for growth (cells that have no special requirements and are able to grow on minimal medium are said to be prototrophic).

Mutants Growth on different media: Wild type Class I Class II Class III Minimal medium Yes No No No Minimal medium + product 1 Yes Yes No No Minimal medium + product 2 Yes Yes Yes No Minimal medium + product 3 Yes Yes Yes Yes

Figure 2. As stated in the previous figure, mutants in classes I, II and III are unable to grow on minimal medium unless provided with product 3. However, each of these mutant classes was defective in a single gene encoding a specific enzyme of the metabolic pathway of product 3 synthesis. According the growth requirements the class I mutants lack enzyme 1, the class II lack enzyme 2 and class III are defective in enzyme 3.

III. Properties of the genetic material Although the nature of the genetic material was still controversial at the very beginning of 1950s, geneticists accepted unanimously the proposal that the material of heredity should have the following properties:

Precursor Product 1 Product 2 Product 3 E 1 E 2 E 3

Irradiation with X-rays

Culture of Spores

Progeny derived from sporesTest progeny from irradiated culture for growth on complete medium

Growth requirements

Test progeny for growth on minimal medium and different amino acids

Test progeny for growth on minimal medium

Analysis of auxotrophs



27

1. It must be very stable and contains all the genetic information for an organism's structure. The genetic information included in the postulated material is transmitted indefinitely to the next generations.

2. It must be capable of accurate replication so that the progeny cells have the same genetic information as those from which they derive.

3. Although stable, it must also be subject to variation in order to account for change, adaptation and evolution.

IV. The discovery that DNA is the genetic material IV.1. Griffith discovers transformation The first evidence that DNA might be the genetic material came from investigations involving the bacterium Streptococcus pneumoniae (also called pneumococcus), that causes pneumonia. The English microbiologist, Frederick Griffith, who compared the properties of two strains of pneumococcus, performed these experiments in 1928. The smooth strain (or type S for smooth colonial morphology) is virulent (infectious) because it is surrounded by a polysaccharide coat or capsule; this protects the bacteria from the immune system of its host17. The rough strain (or type R for rough-looking colonies) lacks the enzyme needed for the biosynthesis of the polysaccharide coat and, as a consequence, is not virulent; the R strain cannot resist attack by the host's immune system.

In 1928 Griffith injected mice with type R bacteria, which lack the polysaccharide coat and found that the injected mice were not affected instead, the bacteria disappeared from the animals bloodstream. In the second experiment he injected type S bacteria into mice and found that the blood had become filled with S bacteria and the mice died. Heat-killed type S bacteria had no effect on the mice however, if mice were injected with a mixture of heat killed S bacteria and type R bacteria, the mice died and virulent type S bacteria could be recovered from their blood. Somehow, the type R bacteria were transformed by extracts of heat-killed type S bacteria into infectious type S bacteria, which were genetically stable (Fig. 3). The S strains can be subdivided further into IIS and IIIS, which have differences in terms of the chemical composition of the polysaccharide coat. These differences are controlled genetically and, as a consequence, they are stable. Virulent type IIS can change to non-virulent type R by mutation. Although the reversion of type R to type S is a rare event, the S colony that develops from the type R mutant has the same capsule type as the S strain from which the R mutant was originally derived; this means that the reversion of the R strain that derives from type IIS will be IIS and not IIIS. In his last experiment, Griffith injected mice with a mixture of living R bacteria (derived from IIS) and heat killed type IIIS bacteria. The injected mice died and living type IIIS bacteria were found in the blood; these bacteria could not have arisen from type R by mutation, since mutation would have produced type IIS bacteria (Fig. 4). The unknown agent

17 The capsule that surrounds the type S cells prevents the pneumococci from being engulfed (by phagocytosis) and destroyed by the scavenging cells - neutrophils and macrophages - of the body. In contrast, the R forms are completely at the mercy of phagocytes.



28

responsible for the change in the genetic material was referred to as the transforming principle. Griffith believed that this agent was a protein.

Figure 3. Griffith’s experiment with Streptococcus pneumoniae. a) Injection of type S bacteria killed the mice and virulent type S bacteria were found in the animals’ bloodstream. b) Mice injected with type R bacteria survived and no living bacteria could be recovered from the blood. c) Injection of heat-killed type S bacteria did not affect mice. d) When heat-killed type S bacteria and avirulent type R bacteria were injected into the mice in the same time, the mixture has the capacity to kill mice and virulent type S bacteria were recovered from the blood.

IV.2. The "Transforming principle" is DNA In 1944, Oswald T. Avery and his colleagues Colin M. Macleod and Maclyn McCarty working at the Rockefeller institute, made a major contribution when they demonstrated that the agent responsible in transforming type R into type S was DNA. This finding was quite surprising since most scientists believed that proteins, which are chemically more complex and diverse than nucleic acids, were the genetic material. Avery, Macleod and McCarty performed their experiments using a system in which the transformation of bacteria was done in a test tube. After they have lysed type IIIS bacteria and separated the extracts into the various macromolecular components, they showed that highly purified preparations containing no detectable protein were able to transform type R (derived from IIS bacteria) into virulent type IIIS bacteria. The other macromolecular components such as lipids, polysaccharides and proteins could not transform type R into type IIIS and, subsequently, do not contain the transforming principle. Instead, only the nucleic acids (RNA and DNA) component was shown to have the transforming principle and was able to transform the R cells into IIIS. Avery, Macleod and McCarty treated the nucleic acids components with ribonuclease (which degrades RNA but has no effect on DNA) and showed that the transforming capacity was still present. However, treatment with deoxyribonuclease (which degrades DNA but not RNA) resulted in the disappearance of the transforming principle and no transforming activity could be detected (Fig. 5). These results strongly suggested that DNA

(a) (b) (c) (d)



29

was the genetic material. Despite the fact that the transformation of type R into type S was stably inherited, this did not lead to general acceptance of DNA as the genetic material because it was possible to criticize some aspects of Avery's work. Indeed, doubts were raised about the purity of the deoxyribonuclease enzyme.

Figure 4. The cells of pneumococcus are usually surrounded by a capsule made of a polysaccharide. When grown on the surface of a solid culture medium, the capsule causes the colonies to have a glistening, smooth appearance. These cells are called "S" cells. Prolonged cultivation of the S strain on artificial medium causes some cells to lose the ability to form the capsule, and the surface of their colonies become wrinkled and rough (R). With the loss of their capsule, the bacteria also lose their virulence. Injection of a single S pneumococcus into a mouse will kill the mouse in 24 hours or so. But an injection of R cells is entirely harmless. Pneumococci also occur in about 80 different types: I, II, III and so on. The types differ in the chemistry of their polysaccharide capsule. Unlike the occasional shift of S R, the type of the organism is constant. Mice injected with a few type II S cells will soon have their bodies teeming with descendant cells of the same type i.e. SII cells. However, Griffith found that when living R cells (deriving from SII strain) and dead S cells (which also should have been harmless) were injected together, the mouse became ill and living S cells could be recovered from its bloodstream. Furthermore, the type of the cells recovered from the mouse's body was determined by the type of the dead S cells. In the experiment shown, injection of living RII cells and dead SIII cells produced a dying mouse with its body filled with living SIII pneumococci. Something in the dead SIII cells had made a permanent change in the phenotype of the RI-I cells. The process was named transformation. Later, Oswald Avery and his colleagues at The Rockefeller Institute in New York City eventually showed that the "something" or the transforming principle was DNA.

Capsules SIII SII

Select RII strain Heat

Mix live RII and dead SIII

Dead SIII Live R

Live SIII



30

Figure 5. In pursuing Griffith's discovery, Avery, Macleod and McCarty found that they could bring about the same kind of transformation in vitro using an extract of the bacterial cells. Treating this extract with enzymes to destroy all polysaccharides (including the polysaccharide of the capsule), a lipase to destroy any lipids, proteases to destroy all proteins, and RNase to destroy RNA did not destroy the ability of the extract to transform the bacteria. On the other hand treating the extracts with DNase to destroy the DNA in the extract did abolish its transforming activity. So DNA was the only material in the dead cells capable of transforming cells from one type to another. DNA was the substance of genes.

IV.3. Experimental evidence that genes are made of DNA: the Hershey and Chase experiment on bacteriophage T2 In 1952, Alfred Hershey and Martha Chase devised an elegant experiment to trace the fates of the two major components of bacteriophage18 T2 (coat protein and DNA) to see which one is required to complete the life cycle of the phage (Fig. 6). The life cycle of T2 involves attachment of the bacteriophage to the bacterial cell wall and then, injection of its genes into the cytoplasm leaving the protein coat behind on the surface of the bacterium. The life cycle culminates with the lysis of the bacterium and the release of the phage progeny. Hershey and Chase took advantage of the fact that nucleic acids lack sulfur and proteins lack phosphorus to label bacteriophage DNA with radioactive isotope 32P and bacteriophage proteins with 35S. They grew cells of Escherichia coli in media containing either radioactive isotope 32P (through the radioactive 32P-labeled inorganic phosphate) or radioactive isotope 35S (through radioactive 35S-labeled methionine). This resulted in two batches of T2: one had the proteins labeled with 35S and one had the DNA labeled with 32P.

18 Bacteriophage or phage is a virus that infects a bacterium.

S strain

Prepare extractsTreat to destroy polysaccharides, lipids, proteins, and RNA

Treat todestroy DNA

Transform live R cells

S colonies R colonies



31

The authors adopted a smart strategy in which they infected E. coli bacteria with radiolabeled T2 phages, left the culture for a few minutes to allow the phages to inject their genes into the bacteria and then agitated the culture in a blender. The blending shears the phage coats from the bacterial surface, enabling the bacteria plus phage genes to be collected by centrifugation. First, they infected E. coli cells with 35S -labeled batch of T2 and, after centrifugation of the culture, they collected the infected bacteria in the pellet, whereas the phage coats (called also ghosts) containing most of the 35S isotope remained in the suspension. In contrast, when they infected E. coli cells with 32P -labeled batch of T2 phages, the bacterial pellet contained most of the 32P. Moreover, after completion of the life cycle and lysis of the infected bacteria, 30% of the original 32P but only traces (less than 1%) of the 35S was recovered in the bacteriophage progeny (Fig. 7). As the DNA seems to be sufficient for bacteriophage reproduction, they concluded that DNA must be the genetic material. Although the bacteriophage is very different from a cell and it might be wrong to extrapolate from one system to the other, this experiment had a big impact on many biologists, Watson and Crick in particular, who were alerted that DNA might be the genetic material that hold the secret of life.

Figure 6. Bacteriophage T4. T2, T4 and T6, also called T-even phages, form a group of virulent dsDNA-containing bacteriophages. These phages resemble one another morphologically and their genomes can undergo recombination. Each consists of an elongated head attached to a long complex tail. The phage genome located in the head consists of a linear dsDNA molecule that is approximately 170kb in length.



32

Figure 7. Hershey and Chase experiments on bacteriophage T2. They demonstrated that the DNA component of T2 carried the genetic information needed to complete the life cycle of the bacteriophage.

V. DNA quantity is constant Other lines of evidence are consistent with the role of DNA as the material of heredity.

First, the amount of DNA in the cell of a given organism does not vary from cell to cell. The other biomolecules such as proteins or RNA vary in terms of amounts depending on the metabolic conditions of the cell, whereas the DNA content is constant. Second, the amount of DNA in the cells of different species is somehow proportional to the complexity of the organism (Table 1).

Table 1. DNA content per cell is constant. Species Base pairs pg of DNA per cell Flowering plants 0.3-100 x 109 0.6-24 Mammals 2-4 x 109 4-9 Fungi 1-3 x 107 1.1-3.3 x 10-2 Bacteria 2-9 x 106 2.2-10 x 10-3



33

VI. The discovery of RNA as genetic material Although DNA is the genetic material of most of the organisms, some animal viruses, most of the plant viruses and several bacteriophages have RNA as their genetic material. Tobacco mosaic virus (TMV), an RNA plant virus (Fig. 8), was used in experiments which provided a proof that nucleic acids are the substance of heredity. TMV, as well as T2 phages, contains two major components: RNA and protein coat. The RNA genome is packaged in a coat made up of 2130 identical proteins organized in a helical configuration (the RNA chain is located half way from the central axis). TMV virus infects the tobacco plant (Nicotiana tabacum) causing lesions on the leaves. In 1956, A. Gierer and G. Schramm demonstrated that the RNA itself was able to produce typical viral lesions on the surface of the leaves. This indicated strongly that RNA is the genetic material and was supported by the observation that no lesions could be detected when RNA were previously treated with ribonuclease.

Figure 8. Tobacco mosaic virus (TMV). It is the type member of the tobamoviruses. TMV virion is a rod-shaped tubular filament 20nm in diameter and 300nm long consisting a ssRNA genome of 6390 nucleotides coated with 2130 identical protein subunits each of which is about 17.5kD.

In 1957, Fraenkel-Conrat and Singer used two different strains of tobacco mosaic virus, termed HR and TMV, and reconstructed virions particles in vitro by mixing the isolated two components, RNA and proteins, in the four possible combinations:

HR RNA + HR proteins HR RNA + TMV proteins TMV RNA + HR proteins TMV RNA + TMV proteins

These reconstructed virus particles were found to be infective and used in infection of host plants. The resulting progeny virus particles were analyzed and it was found that the protein coat of progeny viruses was determined by the source of RNA in the virus infecting the plant. For instance, TMV RNA + HR (or TMV) proteins always yielded progeny particles with TMV protein coats and HR RNA + TMV (or HR) proteins gave HR protein coats in the progeny. These experiments provided the proof that nucleic acids, not proteins, are carrying the genetic information.

*****

Documents

Department of Biology/Faculty of Sciences/LU Dr. Fahd Nasr ... · III.3. Third base degeneracy and the wobble hypothesis 177 IV. Enzymology and mechanics of protein synthesis 178