GENOMICS & PROTEOMICS: GENOMICS & PROTEOMICS Presented by M.A. Zeeshan Ahmed, M.Pharmacy II Sem, Pharmaceutics. Contents : Genomics & Importance Proteomics & Importance Bioinformatics & Importance Human Genome Project Pharmacogenomics Conclusion References Contents GENOMICS: In 1920, H.Winkler coined the term genome, he described it as “the complete set of chromosomal and extra chromosomal genes of an organism”. Thomas Roderick in 1986 used the term genomics. “scientific discipline of mapping, sequencing and analyzing the genome”. “ The study of molecular organization of genome, their information contents and the gene products they encode”. GENOMICS TYPES OF GENOMICS: Structural Genomics: DNA sequencing, sequence assembly, sequence organization and management. Functional Genomics: Function of all gene sequences and their expressions in an organism. Comparative genomics: Amount of known and unknown function of genes in an organism E.g.: E.coli K12 TYPES OF GENOMICS Importance : It can provide insights into pathogenicity and suggest treatments for infectious diseases . By comparing a wide variety of genomes, the nature of horizontal gene transfer and microbial evolution can be studied. Used in industry to enhance the bioremediation of hazardous wastes and improve techniques for microbial production of methane and other fuels . Used to find out new biopesticides and improve agricultural practices through enhancement in N2-fixation , phosphate solubilization , and prod’n of IAA, HCN. New information about DNA and protein sequences , variation in mRNA, protein levels & protein interaction must be integrated. Used to study variation in drug metabolizing enzymes and individual response to medication. Importance Proteomics : The word " proteome " is a blend of " prote in " and " gen ome “. The term ‘proteome’ is used to describe the “ total set of proteins expressed from transcriptome of a cell”. ‘Transcriptome’ is the term used to describe the complement of mRNA transcribed from the genome of a cell. Proteomics is the study of identification , analysis and large scale characterization of proteome . Proteomics Slide 7: Objectives To characterize the post-transcriptional modifications in protein. To prepare the 3D map of cell – to indicate the exact location. Types of proteomics Structural proteomics: Study of structure and nature of protein complexes present in a cell. 2. Functional proteomics: Constitutes an emerging research area in the proteomic field that is focused to monitor and analyze the properties of the molecular networks formed in a living cell. 3. Expression proteomics: Comparative study of whole proteome between the samples. Slide 8:
Importance Proteomics provides information about genome function that mRNA studies cannot . Measurement of RNA levels show the changes that occur. This helps to identify proteins and generation of complete information about it because it governs phenotypic
of cells. “It can be applied to medicine”- Proteomic technologies will play an important role in drug discovery (target drugs), diagnostics and molecular medicine because is the link between genes, proteins and disease No other studies can provide the understanding of mechanism of disease development and various developmental changes occurring in organisms.
Bio informatics: The term bio informatics has been derived by combining bio logy and informatics Informatics includes computer science , mathematics and statistics in understanding the biology of life . It is the information technology applied to the management and analysis of biological data stored in database . Database – repository sequences Sequence databases: Involves sequence of both proteins and nucleic acids S tructural databases: Involves only protein databases Bio informatics How do we use Bioinformatics? : Store/retrieve biological information (databases) Retrieve/compare gene sequences Predict function of unknown genes/proteins Search for previously known functions of a gene Compare data with other researchers Compile/distribute data for other researchers How do we use Bioinformatics? Slide 11: It consists of three core areas Molecular biology database Sequence comparison and sequence analysis Emerging technology of microarrays Applications Processing raw information Prediction of Genes Complete information about the proteins from predicted genes Regulatory sequence can be identified and analyzed Prediction of function of unknown genes Human genome project: 1984 to 1986 - first proposed at US meetings. 1988 - endorsed by US National Research Council. U.S. govt. project coordinated by the Department of Energy and the National Institutes of Health , launched in 1990 by Charles DeLisi. This is a genome sequencing project . About 90% of the human genome has been sequenced and is composed of 3.2 billion base pairs. Human genome project Objectives: To identify the approximate 1,00,000 genes in the human DNA. Store this information in databases . Develop tools for data analysis . Provides opportunities to exploit the sequence for desired experimentation. Identify the entire set of genes & map them all to their chromosomes. Objectives Genome sequencing techniques: Maxam And Gilbert Technique Sanger Technique Whole genome shotgun sequencing Random Shotgun sequencing DNA chips (MICROARRAYS) Direct sequencing technique Genome sequencing techniques 1 Maxam And Gilbert Technique : The first DNA sequencing technique using chemical reagents , was developed by Maxam and Gilbert in 1977. In this method end labeled DNA is subjected to base specific cleavage reaction before gel separation and then followed by auto radiography . In this method the DNA molecule can be radio labeled at either 5’ end by using poly nucleotide kinase , or 3’ end by terminal transferase. 1 Maxam And Gilbert Technique Cleavages of purines and pyrimidines: Cleavage of purines The mixture is separated at four sets, each treated wit different reagents which degrade only G, C or A+G, T+C. In one set DNA is treated with acid followed by dimethyl sulphate , this causes methylation of A at 3’ and G at 7’ . Subsequently addition of alkali (-OH) and piperidine results in cleavage of DNA and removal of purines . Cleavage of pyrimidines Similar to cleavage of purine, pyrimidine(C or T) is also cleaved in the presence of 1-2 M NaCl solution and Hydrazine results in the removal of pyrimidines . Cleavages of purines and pyrimidines Procedure: A strand of DNA is labeled at one end with radiolabel led 32 PO4. The one end of a DNA is separated by using endonuclease then labeled DNA is distributed into four samples . Each sample is subjected to the chemical treatment that specifically destroys one (G,C) or two bases (A+G, T+C) in the DNA. Thus the DNA strands are partially digested in four samples at G, A+G, T+C and C, this results in the formation of series of labeled fragments of varying lengths. The labeled DNA fragments obtained in four tubes are subjected to electrophoresis side by side and they are detected by autoradiograph. Procedure 2.Sanger Technique : Frederick Sanger et al. developed this enzymatic method of DNA sequencing. He utilized a single stranded DNA as template. It is also called as “dideoxynucleotide chain termination method”. There are 4 types of deoxynucleotide triphosphates (dNTPs) such as dATP, dCTP, dGTP, dTTP. Similarly there are 4 types of dideoxynucleotide triphosphates (ddNTPs) such as ddATP, ddCTP, ddGTP, ddTTP. 2.Sanger Technique Deoxy(dNTP) and dideoxy(ddNTP): Deoxy(dNTP) and dideoxy(ddNTP) Procedure : Four reaction tubes are labeled with A , G , T , C each containing single stranded DNA template (obtained by NaOH hydrolysis), 5’ radiolabelled DNA primer, and all 4 radiolabel led dNTPs ( dATP , dGTP , dTTP , dCTP ). A small amount of ddATP is added to tube 1 , ddGTP to tube 2, ddTTP to tube 3, ddCTP to tube 4. DNA polymerase is added to each tube , DNA synthesis starts and chain elongates Then ddNTPs are added randomly in each tube whose conc’n should be about 1% of conc’n of dNTPs, this leads to termination of DNA synthesis. The length of each fragment depends on the position of incorporation of ddNTPs. Procedure Slide 24: Then the fragments of each tube are separated by electrophoresis in 4 different lanes of high resolution polyacramide gel . Then the gel is dried and autoradiography is done so that position of different bands (having radiolabelled 5’ end) in each lane is observed. DNA sequence is obtained by reading ( from bottom to top gel ) the bands on autoradiogram of four lanes. Slide 25: Limitations: Two limitations in this technique 1) Need of single stranded DNA template 2) Use of a primer to a unknown sequence Automatic DNA sequencers : Improvement of Sanger method Using this technique a DNA sequence containing thousands of nucleotides can be determined in a few hours 3 Whole genome shotgun sequencing : Before 1995, whole genome sequencing was not possible because of lack of computer software to assemble a genome from thousands of DNA fragments Craig and Smith developed whole genome shotgun sequencing and sequenced the genome of two bacteria, H. influenzae and M. genitalium. This approach was divided into four stages 1) Library construction 2) Random sequencing 3) Fragment-alignment and Gap closure 4) Editing 3 Whole genome shotgun sequencing Slide 28: 1.Library construction : The chromosome is isolate d from the desired cells and randomly broken into small fragments using ultrasonic waves , then they are purified and attached to plasmid vectors . A library of plasmid clones are prepared by transforming E.coli strains with plasmid that lack restriction enzymes. 2.Random sequencing : After the clones are prepared and the DNA purified, thousands of DNA fragments are sequenced with automatic sequencers , employing special dye labeled primers . These recognize the plasmid DNA sequences adjacent to DNA insert. The whole genome is sequenced several times and this increases the accuracy of the final results. Slide 29: 3. Fragment alignment and gap closure : By using special computer programme , the sequence DNA fragments are clustered and assembled into longer stretches of sequence by comparing nucleotide sequence overlaps between fragments. Two fragments are joined to form a large stretch of DNA, if the sequences at their ends overlap and match. This overlap comparison process results in a set of larger contiguous nucleotide sequence called contigs . Finally, the contigs are aligned in proper order to form the complete genome sequence. If there exists gaps between the two contigs , these are analyzed and gap filled in with their sequences. Slide 30: 4. Editing / Proof reading The sequence is then carefully proofread in order to resolve any ambiguities in the sequence. Using this approach, it took less than 4 months to sequence M.genitalium genome (about 500,000 base pairs in size) 4.Random Shotgun sequencing : In genetics, shotgun sequencing , also known as shotgun cloning , is a method used for sequencing long DNA strands. Since the chain termination method of DNA sequencing can only be used for fairly short strands (100 to 1000 base pairs) This sequencing entails randomly cutting the DNA segment of interest into more appropriate sized fragments , sequencing each fragment , and arranging the pieces based on overlapping sequences. The overlapping sequences are identified through a computer programme which joins all the sequences into one contiguous stretch. Thus the complete information of the sequence is generated 4.Random Shotgun sequencing Technique : Technique 5.DNA chips (MICROARRAYS) : These are recent developments for DNA sequencing .A DNA chip carrying an array of different oligonucleotides can be used for DNA sequencing. First the cells are chosen and RNA is isolated from two samples. For this purpose, a fluorescently labeled DNA test molecule whose sequence has to be determined is applied to the chip . Hybridization takes place between complementary sequences of test molecule and oligonucleotides of the chip . The positions can be determined by scanning and interpreting the scanned image. The sequence of the test DNA molecule can be deduced from the overlaps between the sequences of the hybridizing oligonucleotides . 5.DNA chips (MICROARRAYS) Technique : 1) Choosing Cell Populations 2) mRNA Extraction and Reverse Transcription 3) Fluorescent Labeling of cDNA's 4) Hybridization to a DNA Microarray 5) Scanning the Hybridized Array 6) Interpreting the Scanned Image Technique 6.Direct sequencing technique : BAC (Bacterial Artificial Chromosome) vectors are stable and introduce a foreign DNA of 80-100 kb in E.coli cells. Therefore BAC is used in construction of genomic library . Screening of genomic library is done through searching of common restriction fragments. Then BAC clone mapping is done just to determine the arrays of contigs which overlap. The large DNA fragments are broken into small pieces and the mapped contigs are sequenced . 6.Direct sequencing technique Pharmacogenomics : It is the study of different parameters (disposition, targets ) arised due to genetic variations in an invidual. It is the study of— The inherited variations in genes that control the body’s response to drugs. These variations can be used to predict whether a patient will have a good or bad response to a drug, or no response at all. Pharmacogenetics The use of genomic methods to determine what causes variations in individual response to drug treatments Pharmacogenomics Importance : Adverse drug reactions account for over 2 million hospitalizations and 100,000 deaths annually in the U.S. Some promising medicines never come to market because they probably only work well on people with certain genotypes. Patients with different genetic variations may require different amounts of a drug to achieve the same effect. Drugs may be toxic in patients of one genotype and helpful to patients of another. Importance Genetic variations ?: Mutations A permanent , structural change in the DNA. Polymorphisms A common (>1%) variation in the sequence of DNA among individuals. Single Nucleotide Polymorphism (SNP) A common (>1%) single base variation in the DNA. or base substitution. b Deletions: A certain part is lost. ( abc-----ac ) c Insertions: A certain part is added . ( ac-----abc ) Genetic variations ? Single Nucleotide Polymorphism (SNP): Single base variation in the DNA Single Nucleotide Polymorphism (SNP) Consequences Such a single nucleotide difference, which may not cause a change in amino acid sequence of a protein, may cause changes in the levels of its transcription or translation which could affect response to a pharmaceutical. Proportion of Drugs Metabolized by CYP Enzymes : Proportion of Drugs Metabolized by CYP Enzymes Genetic polymorphisms influencing drug disposition : Pharmacogenetics of phase I drug metabolism Eg : 1) CYP2D6 “CYP - cytochrome P450 “2” - family “D” - subfamily “6” - enzyme/gene Genetic polymorphisms influencing drug disposition Debrisoquin : It is an anti hypertensive agent. Inherited differences in CYP2D6 leads to marked hypotension following a dose of antihypertensive agent. Subsequent studies determined that a significant proportion of general population had an impaired ability to hydroxylate , and therefore no deactivation of Debrisoquin. Approx 5 - 10% of white subjects were found to have a relative deficiency in their ability to have oxidize debrisoquin. It is due to decreased CYP2D6 activity or one with no activity at all. Debrisoquin 2.Dihydropyrimidine dehydrogenase: It is an enzyme that metabolizes 5 - flurouracil and endogenous pyrimidines. 5 - fluorouracil is a antineoplastic agent. Severe fluorouracil toxicity occurs in individuals with reduced DPD activity (below 100 pmol/min/mg). The accumulation of 5 - fluorouracil leads to fatal CNS toxicity. 2.Dihydropyrimidine dehydrogenase Pharmacogenetics of phase II drug metabolism : N - Acetyl transferase Isoniazid is a anti TB drug. It is metabollized to acetyl isoniazid via NAT2. Acetyl isoniazid undergoes further chemical hydrolysis to acetyl hydrazine, which is then metabolized by CYP2E1 to a hepatotoxic intermediate. The hydrolysis product is also acetylated by NAT2 to form non - toxic metabolite. Acute or chronic hepatitis is a commonly encountered toxicity in patient receiving Isoniazid. Pharmacogenetics of phase II drug metabolism 2. Uridine diphosphate glucuronyl transferases: These are microsomal phase II enzyme that catalyse numerous exogenous and endogenous substrates. UGT1A1 plays several roles in metabolic inactivation of anticancer drug Irinotecan . Irinotecan is a camptothecin derivative used in metastatic colorectal cancer. It is a prodrug, since it requires activation by carboxyl esterases to SN - 38 (7 - ethyl - 10 - hydroxy camptothecin ) to exert antitumor activity mediated by topoisomerase I. SN - 38 is inturn glucuronidated to form inactive SN - 38 glucuronide . SN - 38 is associated with severe episodes of diarrhea occuring shortly. Glucuronidation of SN - 38 to inactive SN - 38G via UTG1A1 protects against Irinotecan induced intestinal toxicities. 2. Uridine diphosphate glucuronyl transferases Genetic polymorphism in drug targets: Associated with effects on drug response and toxicity ß2 - adrenergic receptor It is a G - protein coupled receptor which interacts with endogenous catecholamines and various medications. They are widely distributed in regulating cardiac, vascular, pulmonary and metabolic functions. In heart, activation of it results in ↑ HR and force of cardiac muscle. In lungs, its stimulation acts to relax airway smooth muscle. Alteration at aminoacid 16 (Arg > Gly) appears to have relevance in pulmonary disease, with patients homozygous for Arg exhibiting a greater response to ß2 - agonist medications. Genetic polymorphism in drug targets Slide 50: 2) Thymidilate synthase It is a key enzyme in synthesis of pyrimidine nucleotides , by catalyzing the methylation of dUMP to dTMP . The TS reaction is the sole source of de novo thymidilate in the cell and is essential for DNA replication. The critical role of TS is nucleotide metabolism has made it an important target for a variety of anticancer drugs like 5 - fluorouracil, methotrexate . Inhibition of TS by these agents causes tumour cell death . Conclusion :
Genomics provides new information about DNA and protein sequences, variation in mRNA, protein levels & protein interaction. The success of proteomics (structural, functional, expressional) hinges almost entirely on successful sample preparation and separation. Bioinformatics is needed for better, faster research
. HGP address the ethical, legal, and social issues that arise from genome research Pharmacogenomics offers the potential to target medicines more effectively, improving patient outcomes, reducing adverse effects and could produce significant saving. Conclusion
References : A textbook of biotechnology by R.C Dubey . Department of pharmaceutical technology hand book Remington vol I. http://www.google.co.in/images?hl=en&q=dna+nucleotide&um=1&ie=UTF&source=univ&ei=ky9YTNa4PMivrAfhqbyOBQ&sa=X&oi=image_result_group&ct=title&resnum=1&ved=0CCgQsAQwAA&biw=1024&bih=604 http://en.wikipedia.org/wiki/Polymorphism_%28biology%29 http://en.wikipedia.org/wiki/Bioinformatics References Slide 53: THANK YOU