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Twice Nobel Prize Winner Twice Nobel Prize Winner FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli.

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Presentation on theme: "Twice Nobel Prize Winner Twice Nobel Prize Winner FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli."— Presentation transcript:

1 Twice Nobel Prize Winner Twice Nobel Prize Winner FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli

2 Curiosity is the key for Scientific Discovery

3 Frederick Sanger "for his work on the structure of proteins, especially that of insulin” "for his work on the structure of proteins, especially that of insulin” The Nobel Prize in Chemistry 1958

4 The Nobel Prize in Chemistry 1980 “for their contributions concerning the determination of base sequences in nucleic acids” Walter GilbertFrederick Sanger

5  Born: August 13, 1918  Place of Birth: Rendcombe, Gloucestershire, England  Residence: U.S.A./Great Britain  Affiliation: MRC Laboratory of Molecular Biology, Cambridge

6 Basic Principles of Protein Chemistry Proteins - Amino Acid residues Physical and Biological Properties- Arrangement of the Amino Acid residues

7 Bergmann and Niemann Periodic arrangement of Amino Acids Pure protein – A random mixture of similar individuals

8 Chibnall Studies on Insulin:  Simpler composition  Tryptophan and Methionine absent  Accurate analysis

9 Van Slyke Procedure  High content of free α-amino groups  Short Polypeptide chains Jensen & Evans: Phenylalanine at the end of one of the chains

10 Molecular weight of Insulin  Physical methods 36,000 to 48,000  Gutfreund 12,000  Harfenist & Craig 6,000

11 Dinitrophenyl (DNP) method 1:2:4 flourodinitrobenzene (FDNB) *Alkaline conditions

12 DNP method contd. Hydrolysis of DNP protein with Acid

13 DNP method contd. Extraction with Ether Extraction with Ether Fractionation (Partition Chromatography) Fractionation (Partition Chromatography) Comparison of Chromatographic rates (Silica-gel Chromatography or Paper Chromatography) Identification and Estimation Calorimetrically Identification and Estimation Calorimetrically

14 DNP labeling of Insulin Three yellow DNP-derivatives  ε-DNP-lysine (not extracted with Ether)  DNP-phenylalanine  DNP-glycine

15 Edman phenyl isothiocyanate method Standard method for studying N-terminal residues Standard method for studying N-terminal residues

16 Disulphide bridges  Cystine residues  Reduction to –SH derivatives Polymerization gave insoluble products Polymerization gave insoluble products How to break these Disulfide bridges? How to break these Disulfide bridges?

17 Oxidation with Performic Acid

18 Precipitation of Oxidized Insulin  Fraction A : N-terminal residue Glycine N-terminal residue Glycine Acidic Acidic Simpler composition (Lys, Arg, His, Phe, Simpler composition (Lys, Arg, His, Phe, Thr, Pro were absent) Thr, Pro were absent)  Fraction B: N-terminal residue Phenylalanine N-terminal residue Phenylalanine Basic Amino acids Basic Amino acids

19 Acid hydrolysis of DNP- Phenylalanine

20 Conclusions  Position of residues  Only two types of chains  Molecular weight 12,000

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

22 Fraction B studies  Ionophoresis, Ion-exchange Chromatography, Adsorption on Charcoal Adsorption on Charcoal  5-20 peptides  Paper Chromatography  Analysis of the constituent Amino Acids

23 Results

24 Conclusions Five sequences present in Phenylalanine Chain

25 Problems How the 5 sequences are joined ? Hurdles in solving this mystery: TTTTechnical difficulty in fractionating peptides with non-polar residues (Tyr & Leu) AAAAcid lability of the bonds involving Serine and Threonine

26 Solution is……… Enzymatic Hydrolysis: Use of Proteolytic enzymes More specific than acid hydrolysis

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

28 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

29 Paper Ionophoresis  pH COOH groups uncharged -SO 3 H groups negative charge -NH 2 groups positive charge  pH 3.5 -COOH groups charged

30 Results of Paper Ionophoresis

31 Sequence of Fraction A

32 Acid Hydrolysis Ammonia produced from Amide groups on Aspartic and Glutamic acid residues  Position of Amide groups: Ionophoretic rates Ionophoretic rates Amide contents of peptides Amide contents of peptides

33 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

34 Disulphide interchange reaction

35 Disulphide interchange reaction Contd.  Two types of disulphide interchange reactions  In neutral & alkaline solution catalyzed by –SH compounds –SH compounds

36 Enzymic Hydrolysis  Chymotrypsin action -CySO 3 H.AspNH -Leu.Val. CySO 3 H.Gly.Glu.Arg.Gly.Phe.Phe

37 Cystine peptide structure

38 The Structure of Insulin

39 Sequenced Insulin supports Protein chemistry theories  Hofmeister & Fischer – Classical peptide hypothesis No evidence of periodicity No evidence of periodicity Random order Random order Unique & most significant order Unique & most significant order

40 Insulin from different species

41 Determination of Nucleotide Sequences  Smallest DNA molecule - Bacteriophage φX174 – 5,000 nucleotides  tRNA - 75 nucleotides

42 Fractionation of 32 P-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

43 Disadvantages  Slow and tedious  Requires successive digestions and fractionations  Not easy to apply to larger DNA molecules

44 Copying Procedures  C.Weissmann: Bacteriophage Qβ -Qβ Replicase – Complementary copy -Qβ Replicase – Complementary copy -Pulse-labeling with radio actively labeled -Pulse-labeling with radio actively labeled nucleotides nucleotides  DNA Polymerase substitutes Replicase -Primer, Triphosphates containing 32 P in α position - Sanger -Primer, Triphosphates containing 32 P in α position - Sanger

45 Copying Procedure

46 Primer Source  Synthetic Oligonucleotides  Restriction enzymes

47 Copying procedure Results Results  Short specific regions of labeled DNA were obtained  Unable to obtain individual residues for sequencing How to obtain individual nucleotide residues?

48 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

49 The ‘Plus and Minus’ method α[ 32 P]-dNTP labeling and sequence specific termination J.E.Donelson - Ionophoresis of products on acrylamide gels

50 The Dideoxy method Quicker and more accurate  φX174  Bacteriophage G4  Mammalian mitochondrial DNA

51 Dideoxynucleoside triphosphates  Lack 3’ hydroxyl group  Incorporated into growing DNA chain by DNA polymerase  Chain terminating analogues

52 Dideoxy nucleotide triphosphate

53 Chain Termination with ddNTP

54 Chain-Terminating Method

55 Autoradiograph DNA sequencing gel

56 Chain terminating method  Problem: Requires single stranded DNA as template stranded DNA as template  Solution A.J.H.Smith Exonuclease III Fragments cloned in plasmid vectors and Human mitochondrial DNA

57 Cloning in single-stranded Bacteriophage  Method to prepare template DNA  Based on studies of bacteriophage M 13 and restriction fragments provided by others

58 Cloning  Gronenborn & Messing – M13 Bacteriophage Insert of β-galactosidase gene with an EcoRI restriction enzyme site in it 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

59 Cloning

60 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

61 Bacteriophage φX174 DNA  First DNA sequenced by Copying procedure  Single-stranded circular DNA  5,386 nucleotides  Ten genes  Genes are overlapping

62 Gene Map

63 Reading Frames

64 Mammalian mitochondrial DNA  Two ribosomal RNAs (rRNAs)  transfer RNAs (tRNAs)  inner mitochondrial membrane proteins Transcription and translation machinery of mitochondria is different from other biological systems

65 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

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

67 mtDNA Genetic Code

68

69 Transfer RNAs  Cytoplasmic tRNAs: Clover-leaf model Clover-leaf model Invariable features  Mammalian mt-tRNA: Invariable features missing Invariable features missing Serine tRNA lacks loop of cloverleaf structure Serine tRNA lacks loop of cloverleaf structure

70 Cytoplasmic Transfer RNAs Wobble effect forming Family boxes

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

72 Distribution of Protein genes  Cytochrome oxidase  ATPase complex  Cytochrome b

73 Gene Map of Human mtDNA

74 Mitochondrial DNA Conclusions VVVVery compact structure RRRReading frames coding for proteins and rRNA genes are flanked by tRNA genes SSSSimple model for transcription

75 TRENDS AND PROGRESS TRENDS AND PROGRESS IN IN SEQUENCING FIELD SEQUENCING FIELD

76 Trends  1974 Conventional Sequencing Method Sanger, Conventional Sequencing Method Sanger, Maxam & Gilbert Maxam & Gilbert  1986 A regiment of scientists and technicians – A regiment of scientists and technicians – Caltech and Applied Biosystems Inc.,invented Caltech and Applied Biosystems Inc.,invented the Automated DNA Fluorescence Sequencer. the Automated DNA Fluorescence Sequencer.

77 Trends  Craig Venter's Sequencing Method  In 1991, working with Nobel laureate Hamilton Smith, Venter's genomic Nobel laureate Hamilton Smith, Venter's genomic research project (TIGR) created a new sequencing process coined ‘shotgun technique’. research project (TIGR) created a new sequencing process coined ‘shotgun technique’. “Trend Setter” & “Gene Hunter” Dr. Craig Venter Dr. Craig Venter

78 Automated DNA Sequencing  Smith et al  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

79 Automated Sequencing (Contd)

80

81 Genome Projects   1999 “Celera genomics”– Rockville, Maryland Drosophila genome   2000 Completed Human Genome Project   2002 Mouse Genome Project

82 Human Genome Project  The Human Genome Project Started in 1988, Public Domain Collaborative work between Celera Genomics Collaborative work between Celera Genomics and NIH and NIHAccomplishments:  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)

83 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

84 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

85 Insulin Production in E.coli

86 3D STRUCTURE OF INSULIN 3D STRUCTURE OF INSULIN

87 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

88 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

89 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  nger.html nger.html nger.html  The wellcome Trust Sanger Institute

90 Questions and Suggestions Our View changes our World


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