Genomics & Proteomics

1. Genomics & Proteomics • What is genomics? • GOALS of Genomics • How are genomes mapped? • Methods for sequencing genomes • The Human Genome Project • Finding answers w/advanced technology! • Bioinformatics • Comparative genomics & model organisms • DNA chips aka microarrays • Proteomics

2. Genome = the total genetic composition of an organism. Genomics = the molecular analysis of the entire genome of a species. Genome mapping = Segments of chromosomes are cloned and analyzed in progressively smaller pieces, the locations of which are known on the intact chromosomes Ultimately leads to the determination of the complete DNA sequence, and understanding of that sequence Genomes sequenced so far: 800+ organisms http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj 3181 sequenced or in progress… I. What is genomics?

3. Hemophilus influenzae – first bacterial genome sequenced in 1995. Composed of 1.83 bp circular chromosome, 1,743 genes.

4. A. GOALS of Genomics • Compile the genomic sequences of organisms • Establish the location of all genes and annotate the gene set in a genome • Find ORFs (start codon – stop codon) • Tells us the spatial relationships among genes • Establish the function of all genes • Generate gene expression profiles for cells under differing conditions • Compare genes and proteins between organisms to establish evolutionary relationships

5. Genomics has 3 subfields: • Structural genomics = genetic mapping, physical mapping, and sequencing of entire genomes • Functional genomics = comprehensive analysis of the functions of genes and nongene sequences in genomes • Comparative genomics = comparison of genomes of different species to determine the function of each genome and understand evolutionary relationships

6. B. How are genomes mapped? • Cytogenetic mapping FISH

7. 2)Linkage mapping • Testcross & Pedigree analysis • Molecular markers = segment of DNA found at a specific site along a chromosome, can be recognized using molecular tools, provides higher resolution. • RFLPs (restriction fragment length polymorphisms) • VNTR (variable number of tandem repeats) • STR (short tandom repeats) • SNP (single nucleotide polymorphism) • The distance between two linked markers can be determined by making crosses and analyzing the offspring (parentals v. nonparentals)

8. 3) Physical mapping • Physical map of a chromosome is constructed by creating a contiguous series of overlapping clones from a chromosome-specific library • Contig = collection of clones, found as overlapping regions within a group of vectors • The contigs are arranged relative to each other by comparing restriction maps & DNA markers • (YACs, BACs) can carry large genomic inserts • Genomic library generated, clone individually isolated and arranged to a grid pattern • Identify adjacent members that contain overlapping regions • Southern blotting • Molecular markers

9. YAC

11. A comparison of linkage, cytogenetic & physical maps

12. C. Methods for sequencing genomes • Clone by clone method (“top-down”) • Construction of genomic libraries of fragments covering the total DNA of an organism. Then using genetic markers, overlapping clones are assembled to establish maps that encompass the entire genome • Shotgun method • Genomic libraries are prepared and randomly selected clones are sequenced until all clones in library are analyzed. Software packages organize the nucleotide sequences.

13. overlapping clones are assembled to establish maps

14. libraries are prepared and randomly selected clones are sequenced Compiling the sequence = genome sequenced multiple times to ensure the sequence is accurate

15. D. The Human Genome Project International research effort to characterize the genome of the human, GOALS: • Complete mapping and sequencing of the DNA • Identify all the genes & store this information in a public database • Develop technologies for genome analysis & transfer these tools to the private sector • Examine ethical, legal and social implications of human genetics research • In U.S., Funded by NSF, global cooperative effort (GB, France, and Japan) – additionally, Celera (Craig Ventor) was involved: http://www.tigr.org/

16. Human Genome Project • Began in 1990… as of 2003, 99% of the euchromatic region was published: October issue of Nature • 2.85 billion nucleotides • ~45% of the genome consists of repetitive DNA • At least 50% is derived from transposable elements • < 5% protein coding genes • 20,500 known protein-coding genes and a further 4,000 additional sections of DNA predicted to be putative protein-coding genes • Chromosome 19 has the highest gene density, 55.8 million bases ~1500 genes; the Y chromosome the lowest (78 genes) http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606

17. II. Finding answers w/advanced technology! Annotation = identifying genes, their regulatory sequences, their function + identifying non-protein coding regions A. Bioinformatics = Annotating genomes Emerging field concerned with the development and application of computer software to the acquisition, storage, analysis and visualization of biological information • identifying protein coding genes (ORFs = open reading frames) • identifying non-protein coding regions • Characterizing Mobile elements • Analyzing homologous regions – comparative genomics • BLAST = basic local alignment search tool, computer program that allows you to start with a DNA or protein sequence and then locate homologous sequences in a huge database.

18. B. Comparative genomics & model organisms • So far, confirmed a common ancestor, similar gene sets for basic cellular functions • Can study inherited disorders, gene interactions and environment • To date: Yeast, Drosophila, C. elegans, mouse, dog • Orthologous = descended from a common anscestral gene, have same function • 240 between M. genitalium and H. influenzae • Paralogous – arise from a gene duplication event • w/ dog, humans share 400 single gene disorders, sex chromosome anueploidies, multifactorial diseases • Dogs share many genetic disorders w/humans

19. Pan troglodytes & Homo sapiens • Last common ancestor = 6mya, we’ve been diverging ever since! • Chromosome 22 – 24, only a 1.44% difference, however there are 68,000 indels • Tissue expression patterns differed – several genes found to be expressed in either one or the other, but not both • patterns of evolution in human and chimpanzee protein-coding genes are highly correlated and dominated by the fixation of neutral and slightly deleterious alleles

20. Functional genomics seeks to understand the function of genes and how they determine phenotypes. C. DNA chips aka microarrays • Analyzing genome wide expression patterns in different tissues • studying genome-wide patterns of gene expression • Can view cells as an array of expressed genes • DNA complementary to genes of interest are generated and laid out in microscopic quantities on a slide • Sample cDNA binds to complement, presence of bound DNA is detected by fluorescence • http://www.dnalc.org/ddnalc/resources/dnaarray.html

21. identify the genes that are active within a cell and help identify mutated genes

22. http://www.dnalc.org/ddnalc/resources/dnachip.html A hand-held DNA Chip device, (Nanogen, Inc). The circles at the top are sample ports. The wires guide electric fields over the DNA array, located on the light blue diamond.

23. III. Proteomics • Genomics 1st step… • Proteomics = the cataloging and analysis of a the proteome (a complete set of expressed proteins in a cell at a particular time) to determine when a protein is expressed, how much is made, and with what other proteins the protein can interact. • Goals: to identify every protein in the proteome, to determine the sequences of each protein, and to analyze globally protein levels in different cell types and at different stages in development.

24. Dot matrix can compare the degree of similarity between two primary sequences. Regions of homology are recognized, gaps can be inserted to align sequences. Too simple for very long sequences, dynamic programming methods used instead: Multiple sequence alignment.

25. Proteomics, big challenge! • Proteome – larger than the genome, • Changes in pre-mRNA structure may affect the primary sequence of a protein • Alternative splicing • RNA editing • Post-translational modifications • Methods: • Two-dimensional gel electrophoresis, used to separate cellular proteins • Mass spectrometry – used to identify proteins • Protein microarrays, protein expression & function