TOPICS IN (NANO) BIOTECHNOLOGY Human Genome Project Lecture 9

1. PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Human Genome Project Lecture 9 15th April, 2004

3. Nuclear Human Genome Mitochondrial Remember what the genome is? • Human Genome organisation • Human genome contains ~ 40,000 genes • Nuclear genome 3000 Mb • 30,000 to 40,000 structural genes • 24 different types of DNA duplex • 22 autosomes, 2 sex chromosomes

4. Let’s define it. • DEFINITION: The entire genetic makeup of the human cell nucleus. Includes non-coding sequences located between genes, which makes up the vast majority of the DNA in the genome (~95%)

5. What is the Human Genome Project? • DEFINITION: The Human Genome Project is a multi-year effort to find all of the genes on every chromosome in the human body and to determine their biochemical nature. • SPECIFIC GOALS: • Identify all the genes in human DNA • Determine the sequences of the 3 billion bps • Save the information in databases • Improve tools for data analysis • Transfer related technologies to the private sector • Address the ethical, legal and social issues that may arise from the project

6. Sequencing the Human Genome

7. Importance and Impact Why are genome projects important? • The key to continued development of molecular biology, genetics and molecular life sciences • a catalogue containing a description of the sequence of every gene in a genome is seen as immensely valuable, even if the function is not known • aid in isolation and utilisation of new genes • stretch technology to its limits What is the potential impact? • Improved diagnosis/therapy of disease • prokaryotic genomes: vaccine design, exploration of new microbial energy sources • plant and animal genomes: enhance agriculture

8. The primary HGP sequencing sites • The Whitehead Institute for Biomedical Research (Eric Lander, Massachusetts, USA) • The Sanger Centre (Cambridge, GB) • Baylor College of Medicine (Richard Gibbs, Houston, USA) • Washington University (Robert Wayerston, St. Louis, USA) • DoEs Joint Genome Institute, JGI (Trevor Hawkins, Walnut Creek, California, USA) • …and other genome centres worldwide...

9. The Human Genome Project- Timelines - President announcesgenome working draft completed Celera Genomics Formed High Resolution Maps ofSpecific Chromosomes Announced Conferenceon HGPFeasibility HGPOfficially Begins 1st HumanChromosome Sequenced E.coliGenome Completed 1985 1989 1991 1993 1995 1997 1999 2001 1987 1986 1988 1990 1992 1994 1996 1998 2000 FlyGenome Completed LowResolution LinkageMap of HG Published Congress Recommends 15 year HGP Project S. cerevisiae Genome Completed C. elegans Genome Completed Human Genome Published Science (Feb. 16, 2001) - Celera Nature (Feb. 15, 2001) - HGP

10. History of Human Genome Project • 1983 Los Alamos Labs and Lawrence Livermore National Labs, both under the DOE, begin production of DNA cosmid libraries for single chromosomes • 1986 DOE announces HUMAN GENOME PROJECT • 1987 DOE advisory committee recommends a 15-year multi-disciplinary undertaking to map and sequence the human genome. NHS begins funding of genome projects • 1988 Recognition of need for concerted effort. HUGO founded (Human Genome Organisation) to coordinate international efforts DOE and NIH sign the Memorandum of Understanding outlining plans for co-operation

11. History of Human Genome Project • 1990 DOE and NIH present joint 5-year Human Genome Project to Congress. The 15 year project formally begins • 1991 Genome Database (GDB) established • 1992 Low resolution genetic linkage map of entire human genome published, High resolution map of Y and chromosome 21 published • 1993 DOE and NIH revise 5-year goals • IMAGE consortium established to co-ordinate efficient mapping and sequencing of gene-representing cDNAs (Integrated Molecular Analysis of Genomes and their Expression)

12. History of Human Genome Project • 1994 Genetic-mapping 5-year goal achieved 1 year ahead of schedule • Genetic Privacy Act proposed to regulate collection, analysis, sorage and use of DNA samples (endorsed by ELSI) • LLNL chromosome paints commercialised • 1994-98 Tons of stuff happens that continues to advance the project • 1998 Celera Genomics formed • New 5-year plan by DOE and NIH • 1999 First chromosome completely sequenced (Chromosome 22) • 2000 June 6, HGP and Celera announce they had completed ~ 97% of the human genome.

13. People of Human Genome Project • James Watson Original Head of HGP • Francis Collins • Craig Venter

14. DNA sequencing • The Sanger dideoxy termination method (remember?) • Nucleotide analogs (ddNTP) are incorporated into DNA during its synthesis together with normal nucleotides (dNTP) - when a ddNTP is inserted, the reaction stops = chain termination • Radioactively labeled ddNTPs • four different reactions are performed, each reaction contains ddA, ddG, ddC, ddT • Autoradiography enable analysis of different fragment lengths which correspond to different termination points • Fluorescently labeled ddNTPS • one reaction carried out, all four ddNTPs are incorporated but each ddNTP is labelled with a different fluourescent dye • automated DNA sequencers interfaced with computers determine the order of the dyes and hence the DNA sequence

15. Mapping the Human Genome: Low Resolution Mapping • The Gene Linkage Map • Identifies position of genes by locating marker base sequences associated with RFLPs • Based on how close together two genes are • the closer together two genes are, the less likely they are to separate during meiotic recombination in germ cells • the frequency of recombination between two genes can help to decipher the distance between them on a gene linkage map • genes separated by more than 50cM (50 million bps) are not considered linked • Studies of families affected by genetic disease have proven useful for genetic linkage analysis

16. Mapping the Human Genome: High Resolution Mapping • The Physical Map • Provides the actual distances in bps between genes on a given chromosome • Prepared by aligning the sequences of adjacent DNA fragments from small overlapping clones to form a contiguous map (a contig map) • Sequence tag sites (STGs) mark sites on chromosomes and help to locate adjacent segments of DNA • if two DNA fragments share an STS they overlap and are contiguous

17. Determining genome sequences • The aim, obviously, is to determine the entire genome sequence • A sequence has to be constructed from a series of shorter fragments • Shotgun technique • break molecule into smaller fragments • determine sequence of each one • use a computer to search for overlaps and build a master sequence

18. Chromosome walking • Analysis of DNA sequences of chromosomes by extending the sequenced region a little bit further each time until the tips of the chromosome are reached • The next round of sequencing is based on the results of the previous round by synthesising appropriate DNA primers to extend further

19. Results of Human Genome Project • The International Human Genome Sequencing Consortium published their results in Nature, 409(6822):860-921, 2001 • Initial Sequencing and Analysis of the Human Genome • Celera Genomics published their results in Science, 291(5507), 1304-1351, 2001 • The Sequence of the Human Genome

20. Results of Human Genome Project • The Human genome contains 3146.7 million bases • The average gene size is 3,000 bases • Total number of genes is between 30-40,000 • The order of 99.9% of the nucleotides is the same in all people • Of the discovered genes, the function for more than half is unknown • > 30 genes have already been associated with human disease (e.g. Cancer, blindness)

21. Results of Human Genome Project • About 2% of the genome encodes instructions for the synthesis of proteins • Repeated sequenes make up 50% of the genome • There are urban centres that are gene rich: stretches of C and G bases repeats (CpG islands) occur adjacent to gene rich areas • Chromosome 1 has 2,968 genes; the Y has 231 • Humans: • only twice number of genes of the fly • 3 times as many proteins as fly or worm • share the same gene families as fly or worm

22. Completed genomes • Microbial genomes • Haemophilus influenzae • Escherichia coli • Bacillus subtilus • Helicobacter pylori • Streptococcus pneumonaie • Saacharomyces cerevisiae • Archaeglobus fulgidus • Methanbacterium thermoautotropicum • Methanococcus jannaschil • Mycobacterium tubercolosis • Staphylococcus aureus • and more….. • Insect genomes • Arabidopsis thaliana • Drosophilia melanogaster • Mus musculus

23. Results of Human Genome Project

24. Ethical, legal and societal issues • The DOE and the NIH spend between 3-5% of their annual HGP budgets toward studying the ELSI associated with availability of genetic information • This budget is the world’s largest bioethics program, and has become a worldwide model • Examples of ELSI are: • privacy legislation • gene testing • patenting • forensics • behavioural genetics • genetics in the courtroom

25. Societal Concerns • Who should have access to this information? • Employers • Insurers • Schools • Courts • Adoption agencies • Military • Philosophical Implications • Human responsibility • Free will versus genetic determinism • Who owns and controls genetic information? • How is privacy and confidentiality managed? • Psychological impact and stigmatisation • Effects on the individual • Effects on society’s perceptions and expectations of the individual

26. Clinical Issues • Clinical Issues • Growing demand to educate health care workers • Public needs to gain scientific literary and understand the capabilities, limitations and risks • Standards need to be established including quality controls to ensure accuracy and reliability • Regulations? • Genetic Counselling • Informed consent for complex procedures • Counseling about risks, limitations and reliability of genetic screening techniques • Reproductive decision making based on genetic information • Reproductive rights • Multifactorial diseases and environmental factors • Genetic predispositions do not mandate disease development • Caution must be exercised when correlating genetic tests with predictions

27. Commercialisation and patents • Who owns genes and DNA sequences? • The person (or company) who discovered it, or the person whose body it came from • Should genetic information be the property of humanity? • Is it ethical to charge someone for access to a database of genetic information? • Is it time to raise the bar concerning patents? • Will patent protection slow the advance of research and be detrimental to society as a whole in the long run

28. Diagnostic & therapeutic applications Gene therapy applications Agriculture & Bioremediation Industries Medicine & pharmaceutical industries Preventative measures Microarray Technology Proteomics DNA chip technology Developmental Biology Evolutionary & Comparative Biologists Pharmacogenomics Benefits of Human Genome Project Biotechnology Medicine Bioinformatics

29. Single nucleotide polymorphisms • These occur when a single nucleotide in the genome sequence is altered (1 bp difference) • 66% of SNPs involve a C to T change and they occur every 100-300 bases in either coding or non-coding regions • Evolutionary stable, there are between 2 and 3 million SNPs in the human genome • Many SNPs have no effect on cell function, but: • some SNPs could be responsible for variations in how many humans respond to disease, environmental factors, drugs and other therapies • SNPs may help identify multiple genes involved in complex diseases

30. Single nucleotide polymorphisms • SNPs are NOT the same things as alleles (or so we believe so far) • Researchers have found that most SNPs are not responsible for a disease state • They serve as markers for pinpointing a disease on the human genome map, being located near a gene found to be associated with a certain disease • Occasionally, SNPs may actually cause a disease and can to be used to search for and isolate the disease-causing gene • SNPs travel together - i.e. Variations in DNA are linked • To date, Celera & Orchid Biosciences have largest databases

31. Single nucleotide polymorphisms • Goals: • Develop large scale technologies • Identify common variants in the coding regions • Create a SNP of at least 100,000 markers • Develop the intellectual foundation for studies of sequence variation • Create public resources of DNA samples and cell lines • SNP Consortium: • Ten large pharmaceutical companies and the UK Wellcome Trust • Headed by Arthur Holden • Find and map 300,000 common SNPs • Generate a widely accepted, high-quality, publically available map

32. What next? • High quality genome sequencing and annotation (2003) • Complete sequencing the genomes of other model organisms (e.g. Mouse) • The next step: Functional Genomics • Determine what our genes do through systematic studies of function on a large scale • Transcriptomics - Comparative analysis of mRNA expression /splicing • Proteomics - Comparative analysis of protein expression and post-translational modifications • Structural genomics - Determine 3-D structures of key family members • Intervention studies - Effects of inhibiting gene expression • Comparative genomics - Analysis of DNA sequence patterns of humans and well studies model organisms

33. Is it ethical for the government to invest such a large fraction of its research budget in the Human Genome Project when the result is denial of funding for other worthy projects? • Do such possibilities as finding the cause of many genetic diseases and identifying criminals outweigh such concerns as the possibility of using the genetic information to renew the types of eugenics programs practiced before and during World War II or to deny health insurance coverage? • Given the huge investment of public funds in the Human Genome project, is the government responsible to assure that the benefits will be equally available to people of all socioeconomic levels and ethnic or racial backgrounds? • Should genetic testing be made available to people who have not received the genetics counseling they need in order to fully understand and respond to the results?

34. Functional Genomics • Whole genome • Once the whole genome is truly known and the whole genome sequences become available for an organism, the challenge turns from identifying parts to understanding function • Functional genomics • The post-genomic era is defined as functional genomics • Assignation of function to identified genes • Organisation and control of genetic pathways that come together to make up the physiology of an organism

35. Functional Genomics • 42% of human genes of unknown function have been found in the human genome • assigning function to these genes using systematic high throughput methods is required

36. The Periodic Table: Functional grouping of Chemical Elements

37. System for classifying genes Organism’s Gene Biologist’s Periodic Table Genomics • Will not be two-dimensional • Will reflect similarities at diverse levels • Primary DNA sequence in coding and regulatory regions • Polymorphic variation within a species or subgroup • Time and place of expression of RNAs during development, physiological response and disease • Subcellular localisation and intermolecular interaction of protein products

38. Gene Expression analysis • Array of hope? Arrays offer hope for global views of biological processes • Systematic way to study DNA and RNA variation • Standard tool for molecular biology research & clinical diagnostics • Labelled nucleic acid molecules can be used to interrogate nucleic acid molecules attached to solid support (remember Southern Blotting?) (Refer to January 1999, Nature Genetics Supplement, Volume 21)

39. Gene Expression analysis • DNA chipsAlso known as gene chips, biochips, microarrays…basically DNA-covered pieces of glass (or plastic) capable of simultaneously analysing thousands of genes at a time – they can be high density arrays of oligonucleotides or cDNA • Chips allow the monitoring of mRNA expression on a big scale (i.e many many genes at the same time) Pre-1995, Northern Blots used to look at gene expression

40. Gene Expression analysis Incyte

41. Gene Expression analysis Affymetrix

42. Determining gene function sequence homology sequence motif tissue distribution chromsme localisation function . expression in disease proteomics . biochemical assays expression in models

43. Protein synthesis

44. RNA synthesis and processing

45. Alternatively spliced mRNA

46. The transcriptome • DEFINITION: The mRNA collection content, present at any given moment in a cell or a tissue, and its behaviour over time and cell states (Adam Sartel, COMPUGEN). The complete collection of mRNAs and their alternative splice forms is sometimes referred to as the trancriptome. The transcriptome is teh set of instructions for creating all of the different proteins found in an organism. (From Genome to Transcriptome, Incyte)

47. The Genome Genome, proteome and transcriptome The Transcriptome Golden path: Proteome information in DNA technology The Proteome - Index to a range of possible proteins - Useful as a map and for inter-organisms analysis - Describes what actually happens in the cell - Complex tools, partial results

48. Use of transcriptome analysis • Discovery of new proteins: • that are present in specific tissues • that have specific cell locations • that respond to specific cell states • Discovery of new variants: • of important genes • that work to increase/decrease the activity of the ‘native’ protein • The transcriptome reflects tissue source (cell type, organ) and also tissue activity and state such as the stage of development, growth and death, cell cycle, diseased or healthy, response to therapy or stress..

49. Beyond genomics…proteomics • Proteomics…where the genome hits the road • Proteomics refers to the simultaneous, large scale analysis of all (or many) of the proteins made in a cell at one time to get a global picture of what proteins are made in cells and when • Hopefully then we can determine the ‘whys’ and what we can thus do about it – very important for drug development • The proteome is the protein complement encoded by a genome and the term was first proposed by an Australian post-doc, Marc Wilkins in 1994

50. Beyond the genome: Proteomics • Genomics involves study of mRNA expression-the full set of genetic information in an organism contains the recipes for making proteins • Proteins constitute the “bricks and mortar” of cells and do most of the work • Proteins distinguish various types of cells, since all cells have essentially the same “Genome” their differences are dictated by which genes are active and the corresponding proteins that are made • Similarly, diseased cells may produce dissimilar proteins to healthy cells • However task of studying proteins is often more difficult than genes (e.g. post-translational modifications can dramatically alter protein function)