CO 10

The entire collection of genes encoded by a Genome: The entire collection of genes encoded by a particular organism. Determination of a entire genome sequence is a prerequisite to understanding the complete biology of an organism.

Genomics: Structural: construction of sequence data and gene map. Functional: functions of genes, and their regulation and products. Comparative: compare genes from different genomes to elucidate functional and evolutional relationship.

History 1990: International Human Genome project begins. To generate physical, genetic, and sequence map of the human genome. To sequence the genome of a variety of model organisms. To develop improved technologies for mapping and sequencing. To develop computational tools for capturing, storing, analyzing, displaying, and distributing map and sequence information.

History 1990: International Human Genome project begins. 5. To sequence EST (expressed-sequence tag) fragments of cDNA, and eventually full-length cDNA in different cell types of human and mice. 6. To consider the ethical, social, and legal challenges posted by genomic information.

Fig. 10.1

What in this chapter? Challenges and strategies of genome analysis Major insights emerging from complete genome sequences High throughput tools for analyzing genome and their products.

The genomes of living Organisms vary enormously in size Table 10.1 The genomes of living Organisms vary enormously in size

Sequences and polymorphisms Challenges and strategies of genome analysis Sequences and polymorphisms Sequence error rate: 1% per sequence read Good genomic sequence errors: 1/10,000 Polymorphisms: 1/500 bp. Repeated sequences may be hard to place Unclonable DNA cannot be sequenced

A divide and conquer strategy Fig. 10.2 A divide and conquer strategy

10-fold sequence coverage Sequencing of every chromosomal region from 10 independent inserts can generate an error rate of less than 1/10000. Random sequence error:1/10 sequence fragments Polymorphisms: 5/10 sequence fragments

Major techniques in genome characterization Cloning hybridization PCR amplification sequencing Computational tool

Three types of maps used in the analysis of human genome Linkage map (DNA markers) Physical map (divide and conquer) Sequence map Human genome: 3X109

The making of large-scale linkage maps Fig. 10.3 The making of large-scale linkage maps Two common types of polymorphisms used or mapping (expand or contract during replication) DNA markers

Genomewide identification of genetic markers Identification of SSR by specific pairs of PCR primers

Human Linkage Map 20,000 SSRs, 4 million SNPs.

Physical Maps Overlapping DNA fragments that are ordered and oriented Fig. 10.4 Physical Maps Overlapping DNA fragments that are ordered and oriented and span each of the chromosomes in a genome The molecular counterparts of linkage maps In human: 1 cM= 1 Mb In mice: 1 cM= 2 Mb

How to build the long-range physical maps: Bottom-up and Top-Down approaches

A Hypothetical physical map generated by the analysis of sequence tagged sites STS: sequence tagged sites

metaphase Dark band: gene poor, AT rich Light band: gene rich, CG rich Fig. 10.5 metaphase Dark band: gene poor, AT rich Light band: gene rich, CG rich

Chromosome 7 at three levels of banding resolution

FISH (fluorescent in situ hybridization) Fig. 10.6 FISH (fluorescent in situ hybridization)

Advantages of FISH compared to linkage mapping All clones can be mapped by FISH, but those that detect polymorphisms can be mapped by linkage analysis. FISH can be done on any clone locus in isolation, but linkage requires the analysis of one locus in relation to another. 3. FISH requires only a single sample, linkage requires genotype information from a large cohort of individuals. Disadvantages: low resolution, 4-8 Mb

A sequencing map is the highest-resolution genomic map Hierarchical shotgun approach Whole-genome shotgun approach

Hierarchical shotgun approach Fig. 10.12 Hierarchical shotgun approach 200kbX10/2Kb=1000 10X coverage across The BAC insert minimal overlapping BACs

Whole genome shotgun approach Fig. 10.13 Whole genome shotgun approach 10-fold sequence coverage 3X109X6/2000

Whole genome shotgun approach Advantages: no construction of physical map. Disadvantage: some genomic sequences can not be cloned.

The human genome project has changed the practice of Biology, genetics, and genomics Gene finding and gene-function analyses: Through comparative genomics, Identification of genes and gene functions in second genome is facilitated by sequence homology. Genes often encodes one or more protein domains. These information provide insights into the functions of a protein.

Fig. 10.14

Fig. 10.15 Syntetic blocks

Major insights from the Human and model organism genome sequence There are approximately 30,000 human genes. Genes encodes either noncoding RNAs or proteins Non-coding RNAs: tRNA, snoRNA (small nucleolar RNAs) snRNA (small nuclear RNAs)

3. Higher complexity of proteome in human: more genes, more paralogous, alternative splicing. Homologous genes: genes with enough sequence similarity to be evolutionarily related. Orthologous genes: defined by their sequence similarities, are genes in two different species that arose from the same gene in the two species’ common ancestor. Paralogous genes: arise by duplication within the same species.

Major insights from the Human and model organism genome sequence 4. More Domain architecture:

5. Chemical modification of proteins 400 different chemical modification 1 million different proteins

Major insights from the Human and model organism genome sequence 6. Repeated sequences constitute more than 50% of the human genome. Transposon-derived repeats, pseudogenes, or simple sequence repeats

Major insights from the Human and model organism genome sequence 6. The genome contains distinct types of gene organization A). gene family: multiple related genes olfactory gene family (1000 genes), histones, hemoglobins,

Olfactory receptor gene family Fig. 10.19 Olfactory receptor gene family One gene undergoes duplication to generate 20 paralogs. Massive duplication created 30 sites of the original 20-paralog family.

B). Gene rich region 60 genes/700 kb C). Gene deserts Fig. 10.20 B). Gene rich region 70% DNA is transcribed 60 genes/700 kb C). Gene deserts 82 gene deserts: no identifiable gene within a megabase

Combinational strategies may amplify genetic Fig. 10.21 Combinational strategies may amplify genetic Information and generate diversity at DNA level Antibody or T-cell receptor genes: VDJ recombination

Combinational strategies may amplify genetic Fig. 10.22 Combinational strategies may amplify genetic Information and generate diversity At the RNA level

High throughput genomic and proteomic platforms permit the global analysis of gene product

Sanger sequencing scheme Fig. 10.23 Sanger sequencing scheme

DNA arrays Macroarray: cDNA on nylon membrane Microarray: PCR amplified product on glass-slide Oligonuclotide array: chemically synthesized 20- 60 nt of DNA or RNA

Two-color DNA microarray Fig. 10.25 Two-color DNA microarray Normal tumor Normal tumor

Fig. 10.27 Protein analyses Mass/charge ratios

MPSS: methods to identify transcriptome Fig. 10.28 MPSS: methods to identify transcriptome (multiple parallel signature sequencing)

Protein-protein interaction: affinity purification and Fig. 10.31 Protein-protein interaction: affinity purification and mass spectrometry

Fig. 10.32 The yeast two-hybrid

System Biology Global study of multiple components of biological systems and their simultaneous interaction

System Biology approaches Formulate a computer-based model based on current understanding. To define as many of the system’s element as possible by discovery science. Perturb the system either genetically or environmentally and measure changes.

Perturb the system and measure changes Fig. 10.33 Perturb the system and measure changes

Fig. 10.34

4. Integrate the biological information, and compare Fig. 10.35 4. Integrate the biological information, and compare these data against prediction of the model

5. Formulate hypothesis to explain disparities between experimental data and the model, and use these hypothesis as the basis for a second round of perturbation 6. Refine the model until model and experiment are in accord with one another.

TABLES

Table 10.2a

Table 10.2b

Table 10.3

Fig. 10.9b

Fig. 10.11

Fig. 10.16

Fig. 10.26

Fig. 10.36

Fig. 10.29

Fig. 10.30

Basic procedures in building a whole chromosome physical map Fig. 10.7 Basic procedures in building a whole chromosome physical map