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Twice Nobel Prize Winner FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli Curiosity is the key for Scientific Discovery Frederick Sanger The Nobel Prize in Chemistry 1958 "for his work on the structure of proteins, especially that of insulin” The Nobel Prize in Chemistry 1980 “for their contributions concerning the determination of base sequences in nucleic acids” Frederick Sanger Walter Gilbert Frederick Sanger Born: August 13, 1918 Place of Birth: Rendcombe, Gloucestershire, England Residence: U.S.A./Great Britain Affiliation: MRC Laboratory of Molecular Biology, Cambridge Basic Principles of Protein Chemistry Proteins - Amino Acid residues Physical and Biological PropertiesArrangement of the Amino Acid residues Bergmann and Niemann Periodic arrangement of Amino Acids Pure protein – A random mixture of similar individuals Chibnall Studies on Insulin: Simpler composition Tryptophan and Methionine absent Accurate analysis Van Slyke Procedure content of free α-amino groups Short Polypeptide chains High Jensen & Evans: Phenylalanine at the end of one of the chains Molecular weight of Insulin Physical methods 36,000 to 48,000 Gutfreund 12,000 Harfenist & Craig 6,000 Dinitrophenyl (DNP) method 1:2:4 flourodinitrobenzene (FDNB) *Alkaline conditions DNP method contd. Hydrolysis of DNP protein with Acid DNP method contd. Extraction with Ether Fractionation (Partition Chromatography) Comparison of Chromatographic rates (Silica-gel Chromatography or Paper Chromatography) Identification and Estimation Calorimetrically DNP labeling of Insulin Three yellow DNP-derivatives ε-DNP-lysine (not extracted with Ether) DNP-phenylalanine DNP-glycine Edman phenyl isothiocyanate method Standard method for studying N-terminal residues Disulphide bridges Cystine residues to –SH derivatives Polymerization gave insoluble products Reduction How to break these Disulfide bridges? Oxidation with Performic Acid Precipitation of Oxidized Insulin Fraction A : N-terminal residue Glycine Acidic Simpler composition (Lys, Arg, His, Phe, Thr, Pro were absent) Fraction B: N-terminal residue Phenylalanine Basic Amino acids Acid hydrolysis of DNPPhenylalanine Conclusions Position Only of residues two types of chains Molecular weight 12,000 Fractionation Paper Chromatography for Fractionation of small peptides Consden, Gordon, Martin & Synge worked on pentapeptide Gramicidin-S Fraction B studies Ionophoresis, Ion-exchange Chromatography, Adsorption on Charcoal 5-20 peptides Paper Chromatography Analysis of the constituent Amino Acids Results Conclusions Five sequences present in Phenylalanine Chain Problems How the 5 sequences are joined ? Hurdles in solving this mystery: Technical difficulty in fractionating peptides with non-polar residues (Tyr & Leu) Acid lability of the bonds involving Serine and Threonine Solution is……… Enzymatic Hydrolysis: Use of Proteolytic enzymes More specific than acid hydrolysis Proteolytic Enzymes Pepsin – Peptide Bp3 fragment Phe (CySO,H, Asp, Glu, Ser, Gly, Val, Leu, His) Trypsin, Chymotrypsin studies Fraction A studies Problems in applying fraction B studies to fraction A: Few residues that occur only once Less susceptible to enzymatic hydrolysis Water soluble peptides- difficult to fractionate on paper chromatography Paper Ionophoresis pH 2.75 -COOH groups uncharged -SO3H groups negative charge -NH2 groups positive charge pH 3.5 -COOH groups charged Results of Paper Ionophoresis Sequence of Fraction A Acid Hydrolysis Ammonia produced from Amide groups on Aspartic and Glutamic acid residues Position of Amide groups: Ionophoretic rates Amide contents of peptides Arrangement of Disulphide bridges Assumptions and hypothesis: Harfenist & Craig Mol Wt 6000 Two chains with three disulphide bridges: Two bridges connecting the two chains One intrachain bridge in fraction A Disulphide interchange reaction Disulphide interchange reaction Contd. Two types of disulphide interchange reactions In neutral & alkaline solution catalyzed by –SH compounds Enzymic Hydrolysis Chymotrypsin action -CySO3H.AspNH -Leu.Val. CySO3H.Gly.Glu.Arg.Gly.Phe.Phe Cystine peptide structure The Structure of Insulin Sequenced Insulin supports Protein chemistry theories Hofmeister & Fischer – Classical peptide hypothesis No evidence of periodicity Random order Unique & most significant order Insulin from different species Determination of Nucleotide Sequences Smallest DNA molecule - Bacteriophage φX174 – 5,000 nucleotides tRNA - 75 nucleotides 32P-labelled Fractionation of oligonucleotides G.G.Brownlee and B.G.Barrell method: Partial degradation by enzymes Separation of smaller products Determination of sequence Applied to RNA sequences Disadvantages Slow and tedious Requires successive digestions and fractionations Not easy to apply to larger DNA molecules Copying Procedures C.Weissmann: Bacteriophage Qβ -Qβ Replicase – Complementary copy -Pulse-labeling with radio actively labeled nucleotides DNA Polymerase substitutes Replicase -Primer, Triphosphates containing 32P in α position - Sanger Copying Procedure Primer Source Synthetic Oligonucleotides Restriction enzymes Copying procedure Results Short specific regions of labeled DNA were obtained Unable to obtain individual residues for sequencing How to obtain individual nucleotide residues? Solution is ……… Incorporation of ribonucleotides in DNA Sequence by DNA Polymerase Splitting of ribonucleotide residues later by action of alkali Technique put forth by Berg, Fancher & Chamberlin The ‘Plus and Minus’ method α[32P]-dNTP labeling and sequence specific termination J.E.Donelson - Ionophoresis of products on acrylamide gels The Dideoxy method Quicker and more accurate φX174 Bacteriophage G4 Mammalian mitochondrial DNA Dideoxynucleoside triphosphates Lack 3’ hydroxyl group Incorporated into growing DNA chain by DNA polymerase Chain terminating analogues Dideoxy nucleotide triphosphate Chain Termination with ddNTP Chain-Terminating Method Autoradiograph DNA sequencing gel Chain terminating method Problem: Requires single stranded DNA as template Solution A.J.H.Smith Exonuclease III Fragments cloned in plasmid vectors and Human mitochondrial DNA Cloning in single-stranded Bacteriophage Method Based to prepare template DNA on studies of bacteriophage M 13 and restriction fragments provided by others Cloning Messing – M13 Bacteriophage Insert of β-galactosidase gene with an EcoRI restriction enzyme site in it Gronenborn & Heidccker 96-nucleotide long restriction fragment from M13 vector flanking EcoRI site Cloning Advantages Same primer on all clones Very efficient and rapid method of fractionating Each clone represents progeny of a single molecule and is therefore pure No theoretical limit to the size of DNA that could be sequenced Bacteriophage φX174 DNA First DNA sequenced by Copying procedure Single-stranded circular DNA 5,386 nucleotides Ten genes Genes are overlapping Gene Map Reading Frames Mammalian mitochondrial DNA Two ribosomal RNAs (rRNAs) 22-23 transfer RNAs (tRNAs) 10-13 inner mitochondrial membrane proteins Transcription and translation machinery of mitochondria is different from other biological systems The genetic code in mitochondria Steffans & Buse - Sequence of Subunit II of Cytochrome Oxidase (COII) from bovine mitochondria Barrel, Bankier & Drouin – DNA sequence for protein homologous to the above amino acid sequence in human beings Findings TGA - Tryptophan (not termination codon) ATA – Methionine (not isoleucine) Is it Species variation (?) Young & Anderson-isolated bovine mtDNA - Confirmed Uniqueness of mtDNA mtDNA Genetic Code Transfer RNAs Cytoplasmic tRNAs: Clover-leaf model Invariable features Mammalian mt-tRNA: Invariable features missing Serine tRNA lacks loop of cloverleaf structure Cytoplasmic Transfer RNAs Wobble effect forming Family boxes Mitochondrial Transfer RNAs 22 tRNA genes in Mammalian mtDNA For all family boxesOnly one which had a T in the position corresponding to the third position of the codon One box tRNA-Recognizes all codons in a family Distribution of Protein genes Cytochrome oxidase ATPase complex Cytochrome b Gene Map of Human mtDNA Mitochondrial DNA Conclusions Very compact structure Reading frames coding for proteins and rRNA genes are flanked by tRNA genes Simple model for transcription TRENDS AND PROGRESS IN SEQUENCING FIELD Trends 1974 Conventional Sequencing Method Sanger, Maxam & Gilbert 1986 A regiment of scientists and technicians – Caltech and Applied Biosystems Inc.,invented the Automated DNA Fluorescence Sequencer. Trends Craig Venter's Sequencing Method In 1991, working with Nobel laureate Hamilton Smith, Venter's genomic research project (TIGR) created a new sequencing process coined ‘shotgun technique’. “Trend Setter” & “Gene Hunter” Dr. Craig Venter Automated DNA Sequencing Smith et al. 1986 DNA molecules labeled with fluorescent dyes Products of dideoxy-sequencing reactions separated by gel electrophoresis Dye molecules are excited by laser beam Fluorescent signals are amplified and detected by Photomultiplier tubes (CCD Camera) Computer software identifies each nucleotide based on the distinctive color of each dye Automated Sequencing (Contd) Automated Sequencing (Contd) Genome Projects 1999 “Celera genomics”– Rockville, Maryland Drosophila genome 2000 Completed Human Genome Project http:// www.genome.gov/ 2002 Mouse Genome Project www.informatics.jax.org/ Human Genome Project The Human Genome Project Started in 1988, Public Domain Collaborative work between Celera Genomics and NIH Accomplishments: Identify all the approximately 35,000 genes in human DNA Determine the sequences of the 3 billion chemical bases that make up human DNA (completed July 2000) Other Genome Databases A lot of Organism specific databases at NCBI Allows for Comparative Genomics studies Phylogenetic Analysis Gene Annotation Drug studies and Identification issues therapy and Gene Therapy- Cystic Fibrosis etc. DNA Vaccines Insulin and Biotechnology 1978: Genentech, Inc. - Genetic engineering techniques used to produce human insulin in E. coli 1983: Genetech, Inc. licensed Eli Lily to make insulin Insulin Production in E.coli 3D STRUCTURE OF INSULIN Insulin Trends Insulin was first isolated from the pancreas of cows and pigs in the early 1920s In 1978, a synthetic version of the human insulin gene was constructed and inserted into the bacterium Eschericia coli, in the laboratory of Herbert Boyer at the University of California at San Francisco Insulin Trends in Medicine Recombinant human insulin was developed by Boyer's fledgling company, Genentech, in October of 1982, the first product of modern biotechnology Humulin Various modes of delivering Insulin to the Tissue Less Adverse reactions, More strict glucose control in diabetics References Nobel e-Museum The Nobel Prize Internet Archive Britannica Nobel Prizes, Guide to the Nobel Prizes Michigan State University, Department of Chemistry Science Daily http://www.geocities.com/jdelaney25/FrederickSa nger.html The wellcome Trust Sanger Institute Questions and Suggestions Our View changes our World