Twice Nobel Prize Winner frederick sanger

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

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

The Nobel Prize in Chemistry 1980

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 Properties-

• Arrangement 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

• High content of free α-amino groups

• Short Polypeptide chains

• 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)

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

• Reduction to –SH derivatives

• Polymerization gave insoluble products

• 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 DNP-Phenylalanine

Conclusions

• Position of residues

• Only 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

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

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 to prepare template DNA

• Based on studies of bacteriophage M 13 and restriction fragments provided by others

Cloning

• Gronenborn & Messing – M13 Bacteriophage

• Insert of β-galactosidase gene with an EcoRI restriction enzyme site in it

• 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 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

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’.

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 studies

• Gene Annotation and Identification issues

• Drug 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

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/FrederickSanger.html

• The wellcome Trust Sanger Institute

Questions and Suggestions