Twice Nobel Prize Winner

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Twice Nobel Prize Winner

• FREDERICK SANGER
• HARD WORK IS PAID IN FORM OF AWARDS
• Prasanna Khandavilli

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Curiosity is the key for Scientific Discovery
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Frederick Sanger
The Nobel Prize in Chemistry 1958

• "for his work on the structure of proteins,
  especially that of insulin

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The Nobel Prize in Chemistry 1980
for their
contributions concerning the determination of
base sequences in nucleic acids
Walter Gilbert
Frederick Sanger
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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

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Basic Principles of Protein Chemistry

• Proteins - Amino Acid residues
• Physical and Biological Properties-
• Arrangement of the Amino Acid residues

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Bergmann and Niemann

• Periodic arrangement of Amino Acids
• Pure protein A random mixture of similar
• individuals

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Chibnall

• Studies on Insulin
• Simpler composition
• Tryptophan and Methionine absent
• Accurate analysis

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Van Slyke Procedure

• High content of free a-amino groups
• Short Polypeptide chains
• Jensen Evans
• Phenylalanine at the end of one of the chains

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Molecular weight of Insulin

• Physical methods 36,000 to 48,000
• Gutfreund 12,000
• Harfenist Craig 6,000

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Dinitrophenyl (DNP) method

• 124 flourodinitrobenzene (FDNB)

Alkaline conditions
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DNP method contd.

• Hydrolysis of DNP protein with Acid

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DNP method contd.

• Extraction with Ether
• Fractionation (Partition Chromatography)
• Comparison of Chromatographic rates (Silica-gel
  Chromatography or Paper Chromatography)
• Identification and Estimation Calorimetrically

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DNP labeling of Insulin

• Three yellow DNP-derivatives
• e-DNP-lysine (not extracted with Ether)
• DNP-phenylalanine
• DNP-glycine

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Edman phenyl isothiocyanate method

• Standard method for studying N-terminal
  residues

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Disulphide bridges

• Cystine residues
• Reduction to SH derivatives
• Polymerization gave insoluble products
• How to break these Disulfide bridges?

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Oxidation with Performic Acid
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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

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Acid hydrolysis of DNP-Phenylalanine
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Conclusions

• Position of residues
• Only two types of chains
• Molecular weight 12,000

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Fractionation

• Paper Chromatography for Fractionation of
• small peptides
• Consden, Gordon, Martin Synge worked on
• pentapeptide Gramicidin-S

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Fraction B studies

• Ionophoresis, Ion-exchange Chromatography,
• Adsorption on Charcoal
• 5-20 peptides
• Paper Chromatography
• Analysis of the constituent Amino Acids

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Results
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Conclusions

• Five sequences present in Phenylalanine Chain

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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

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Solution is

• Enzymatic Hydrolysis
• Use of Proteolytic enzymes
• More specific than acid hydrolysis

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Proteolytic Enzymes

• Pepsin Peptide Bp3 fragment
• Phe (CySO,H, Asp, Glu, Ser, Gly, Val, Leu,
  His)
• Trypsin, Chymotrypsin studies

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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

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Paper Ionophoresis

• pH 2.75
• -COOH groups uncharged
• -SO3H groups negative charge
• -NH2 groups positive charge
• pH 3.5
• -COOH groups charged

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Results of Paper Ionophoresis
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Sequence of Fraction A
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Acid Hydrolysis

• Ammonia produced from Amide groups on
• Aspartic and Glutamic acid residues
• Position of Amide groups
• Ionophoretic rates
• Amide contents of peptides

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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

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Disulphide interchange reaction
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Disulphide interchange reaction
Contd.

• Two types of disulphide interchange reactions
• In neutral alkaline solution catalyzed by
• SH compounds

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Enzymic Hydrolysis

• Chymotrypsin action
• -CySO3H.AspNH
• -Leu.Val. CySO3H.Gly.Glu.Arg.Gly.Phe.Phe

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Cystine peptide structure
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The Structure of Insulin
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Sequenced Insulin supports Protein chemistry
theories

• Hofmeister Fischer Classical peptide
  hypothesis
• No evidence of periodicity
• Random order
• Unique most significant order

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Insulin from different species
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Determination of Nucleotide Sequences

• Smallest DNA molecule - Bacteriophage fX174
  5,000 nucleotides
• tRNA - 75 nucleotides

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Fractionation of 32P-labelled oligonucleotides

• G.G.Brownlee and B.G.Barrell method
• Partial degradation by enzymes
• Separation of smaller products
• Determination of sequence
• Applied to RNA sequences

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Disadvantages

• Slow and tedious
• Requires successive digestions and fractionations
• Not easy to apply to larger DNA molecules

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Copying Procedures

• C.Weissmann Bacteriophage Qß
• -Qß Replicase Complementary copy
• -Pulse-labeling with radio actively labeled
• nucleotides
• DNA Polymerase substitutes Replicase
• -Primer, Triphosphates containing 32P in a
  position - Sanger

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Copying Procedure
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Primer Source

• Synthetic Oligonucleotides
• Restriction enzymes

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Copying procedure

• Results
• Short specific regions of labeled DNA were
  obtained
• Unable to obtain individual residues for
  sequencing
• How to obtain individual
• nucleotide residues?

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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

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The Plus and Minus method

• a32P-dNTP labeling and sequence specific
• termination
• J.E.Donelson - Ionophoresis of products on
• acrylamide gels

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The Dideoxy method

• Quicker and more accurate
• fX174
• Bacteriophage G4
• Mammalian mitochondrial DNA

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Dideoxynucleoside triphosphates

• Lack 3 hydroxyl group
• Incorporated into growing DNA chain by DNA
  polymerase
• Chain terminating analogues

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Dideoxy nucleotide triphosphate
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Chain Termination with ddNTP
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Chain-Terminating Method
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Autoradiograph DNA sequencing gel
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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

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Cloning in single-stranded Bacteriophage

• Method to prepare template DNA
• Based on studies of bacteriophage M 13 and
  restriction fragments provided by others

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Cloning

• Gronenborn Messing M13 Bacteriophage
• Insert of ß-galactosidase gene with an EcoRI
  restriction enzyme site in it
• Heidccker 96-nucleotide long restriction fragment
  from M13 vector flanking EcoRI site

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Cloning
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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

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Bacteriophage fX174 DNA

• First DNA sequenced by Copying procedure
• Single-stranded circular DNA
• 5,386 nucleotides
• Ten genes
• Genes are overlapping

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Gene Map
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Reading Frames
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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

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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

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Findings

• TGA - Tryptophan (not termination codon)
• ATA Methionine (not isoleucine)
• Is it Species variation (?)
• Young Anderson-isolated bovine mtDNA
• - Confirmed Uniqueness of mtDNA

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mtDNA Genetic Code
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(No Transcript)
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Transfer RNAs

• Cytoplasmic tRNAs
• Clover-leaf model
• Invariable features
• Mammalian mt-tRNA
• Invariable features missing
• Serine tRNA lacks loop of cloverleaf structure

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Cytoplasmic Transfer RNAs

• Wobble effect forming Family boxes

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Mitochondrial Transfer RNAs

• 22 tRNA genes in Mammalian mtDNA
• For all family boxes-
• Only one which had a T in the position
• corresponding to the third position of the
  codon
• One tRNA-Recognizes all codons in a family
• box

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Distribution of Protein genes

• Cytochrome oxidase
• ATPase complex
• Cytochrome b

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Gene Map of Human mtDNA
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Mitochondrial DNA Conclusions

• Very compact structure
• Reading frames coding for proteins and rRNA genes
  are flanked by tRNA genes
• Simple model for transcription

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• TRENDS AND PROGRESS
• IN
• SEQUENCING FIELD

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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.

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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
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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

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Automated Sequencing (Contd)
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Automated Sequencing (Contd)
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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/

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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)

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Other Genome Databases

• A lot of Organism specific databases at NCBI
• Allows for Comparative Genomics studies
• Phylogenetic Analysis studies
• Gene Annotation and Identification issues
• Drug therapy and Gene Therapy- Cystic Fibrosis
  etc.
• DNA Vaccines

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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

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Insulin Production in E.coli
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3D STRUCTURE OF INSULIN
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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

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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

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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/FrederickSange
  r.html
• The wellcome Trust Sanger Institute

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Questions and Suggestions
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