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Ka-Lok Ng Dept. of Bioinformatics Asia University

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1 Ka-Lok Ng Dept. of Bioinformatics Asia University
Genome Science Ka-Lok Ng Dept. of Bioinformatics Asia University

2 The Core Aims of Genomics Science
(1) An integrated web-based database and research interface access to the enormous volume of data web interfaces Relational databases Generic Model Organism Database (GMOD) project  to develop reusable components suitable for creating new community databases of biology

3 The Core Aims of Genomics Science
(2) To assemble physical an genetic maps location of genes in a genome physical distance and relative position defined by recombination frequencies the map is crucial for comparing the genomes of related species related phenotypic and genetics data used in animal and plants breeding extend to more species with greater accuracy

4 The Core Aims of Genomics Science
To generate and order genomic and expressed gene sequences High-volume sequencing Basic technique is developed by Fred Sanger “Shotgun” approach  assemble into contigs, scaffolds (a set of contigs), then the whole chromosomes mRNA is unstable Coding parts  cDNA clones – cloned from mRNA transcripts Expressed sequence tags (ESTs) Obtain full length cDNA is not easy  because of mRNA structure

5 The Core Aims of Genomics Science
To generate and order genomic and expressed gene sequences mRNA  cDNA  EST Whole genome reconstruction Reverse transcription  cDNA EST - partial cDNA sequences sequenced either from 5' or 3‘ Alternative splicing  not a one-to-one correspondence between ESTs and genes

6 The Core Aims of Genomics Science
Identify and annotate the complete set of genes encoded within a genome From complete sequence of a genome  genes identification Alignment of cDNA, DNA and protein sequences – BLAST Gene finding software – ORFs, transcription start and termination sites, exon/intron boundaries Then gene annotation  linking sequence to genetic function, expression, locus information, comparative data from homologous proteins

7 The Core Aims of Genomics Science
(5) To characterize DNA sequence diversity Single-nucleotide polymorphisms (SNPs) About 90 percent of human genome variation comes in the form of single nucleotide polymorphisms (neither harmful nor beneficial) Theoretically, a SNP could have four possible forms, or alleles (different seq. alternative), since there are four types of bases in DNA. But in reality, most SNPs have only two alleles. For example, if some people have a T at a certain place in their genome while everyone else has a G, that place in the genome is a SNP with a T allele and a G allele. The human genome contains more than 10 million SNPs  once in every 100 to 300 bp ! Find associations between SNP variation and phenotypic variation,e.g. Sickle-cell anemia 鐮刀狀細胞貧血症 SNP mutation

8 Sickle-cell anemia and SNP

9 The Core Aims of Genomics Science
(5) To characterize DNA sequence diversity Characterize the level of haplotype structure due to linkage disequilibrium (LD) haplotype = a set of adjacent polymorphisms found on a single chromosome LD = groups of closely linked alleles that tend to be inherited together, can be used to map human disease genes very accurately Knowledge of LD are utilized to do disease locus mapping In the human genome, haplotypes tend to be approximately 60,000 bp in size and therefore contain up to 60 SNPs that travel as a group. Haplotype

10 The Core Aims of Genomics Science
Mendel's Laws enable the outcome of genetic crosses to be predicted. A and B on different chromosome

11 The Core Aims of Genomics Science
Genes on the same chromosome should display linkage. Genes A and B are on the same chromosome and so should be inherited together. Mendel's Second Law should therefore not apply to the inheritance of A and B, but holds for the inheritance of A and C, or B and C. Mendel did not discover linkage because the seven genes that he studied were each on a different pea chromosome. Partial linkage Partial linkage was discovered in the early 20th century. The cross shown here was carried out by Bateson, Saunders and Punnett in 1905 with sweet peas. The parental cross gives the typical dihybrid result (see Figure on the right ), with all the F1 plants displaying the same phenotype, indicating that the dominant alleles are purple flowers and long pollen grains. The F1 cross gives unexpected results as the progeny (後裔) show neither a 9 : 3 : 3 : 1 ratio (expected for genes on different chromosomes) nor a 3 : 1 ratio (expected if the genes are completely linked). An unusual ratio is typical of partial linkage

12 The Core Aims of Genomics Science
(5) To characterize DNA sequence diversity the farther apart two genes are, the more they tend to assort independently (randomly)  recombination frequency ↑ Higher freq.  farther apart Vermilion - 朱紅色

13 The Core Aims of Genomics Science
(6) To compile atlases of gene expression analyzing profiles of transcription and protein synthesis traditional method: Northern blots, hybridization modern technology – microarray relative level of expression (differential expression) patterns of covariation in gene expression  clues to unknown gene function (guilt by association)

14 The Core Aims of Genomics Science
(7) To accumulate functional data, including biochemical and phenotypic properties of genes Near-saturation mutagenesis (screening hundreds of thousands of mutants to identify genes that affect traits as diverse as embryogenesis, immunology, and behavior) high-throughput reverse genetics (methods to systematically and specifically inactivate individual genes). Yeast Genome Deletion Project Mouse Proteomics – detecting protein expression and protein-protein interactions Pharmacogenomicists – study the interactions between small molecules (i.e. potential drugs) and proteins Functional genomics – a crucial component is to study various model organisms Clone library – collections of DNA fragments that are cloned into a vector

15 The Core Aims of Genomics Science
With Smith's site-directed mutagenesis the researchers can study in detail how proteins function and how they interact with other biological molecules. Site-directed mutagenesis can be used, for example, to systematically change amino acids in enzymes, in order to better understand the function of these important biocatalysts. The researchers can also analyze how a protein is folded into its biologically active three-dimensional structure. The method can also be used to study the complex cellular regulation of the genes and to increase our understanding of the mechanism behind genetic and infectious diseases, including cancer. GTC  Valine GCC  Alanine Site-directed mutagenesis

16 The Core Aims of Genomics Science
(8) To provide the resources for comparison with other genomes. Comparative maps  allow genetic data from one species to be used in the other species Comparative maps  local gene order along a chromosome tends to be conserved  Synteny (human and mouse genome) Even without synteny, the conservation of gene function is known (say from fly to primate靈長類動物) Gene order conservation (GOC)

17 Mapping Genomes – Genetic Maps
Genetic map – the relative order of genetic markers in linkage groups in which the distance between markers is expressed as units of recombination Genetic markers – sequences tags, repeats, restriction enzyme polymorphism (cutting sites) In diploid (具兩套染色體) organisms, genetic maps are assembled from data on the co-segregation (同時分離) of genetic markers either in pedigrees (家譜) or in the progeny (後代) of controlled crosses. Genetic distance unit  centriMorgan (cM) In human 1cM = 1% of recombination frequency Human, 1cM ~ 1Mbp 100 cM  1 crossover occurs per chromosome per generation Markers on different chromosomes have a chance of co-segregation 50cM (0.5 crossover occurs per generation) pgs2e-fig jpg

18 Mapping Genomes – Genetic Maps
A pair of different parental chromosomes (green and blue colors). (B) A table showing the frequency of recombinants between each marker. Larger number indicates that the genes are farther apart. (C) The most likely genetic map from the entire data. In this hypothetical example, two linkage groups are inferred, the top one is longer than 50 cM. pgs2e-fig jpg Genetic distance ~ 0.11  11cM 0.22  21cM, 0.25  24cM, 0.33  33cM Figure 1.1

19 Mapping Genomes – Genetic Maps
Software of the assembly of genetic maps Multiple factors lead to high variation in the correspondence between physical and genetic distances There is variability of recombination rate along a chromosome (centromeres and telomeres are less reconbinogenic than general euchromatin)  hot spots and cold spots of recombination

20 Exercise 1.1 (Part 1) Constructing a genetic map
Constructing a genetic map - four recessive loci – thickskin, reddish, sour, petite. After identifying two true-breeding trees that are either completely wild-type or mutant for all four loci, the breeder crosses them, and then plants an orchard of F2 (second generation) trees. Q. Based on the following frequencies of mutant classes, determine which loci are likely to be on the same chromosome and which are the most closely linked. pgs2e-exer jpg

21 Exercise 1.1 (Part 2) Constructing a genetic map
pgs2e-exer jpg Assume independent assortment for each recessive phenotype  ¼  242 petite ( ), 249 reddish, 247 sour and 236 thickskin Expect that unlinked loci would segregate independently ~ 60 trees (that is 1/4*1/4*968) produced each double mutants class

22 Exercise 1.1 (Part 2) Constructing a genetic map
Mapping Genomes – Genetic Maps Exercise 1.1 Constructing a genetic map four recessive loci – thickskin, reddish, sour, petite Q. Determine which loci are likely to be on the same chromosome and which are the most closely linked. Answer: Total number of 968 trees. Assume independent assortment for each recessive phenotype  ¼  242 petite, 249 reddish, 247 sour and 236 thickskin Expect that unlinked loci would segregate independently ~ 60 trees (that is 1/4*1/4*968) produced each double mutants class

23 Exercise 1.1 (Part 2) Constructing a genetic map
Mapping Genomes – Genetic Maps s r t p Approximate solution

24 Mapping Genomes – Physical Maps
is an assembly of contiguous stretches of chromosomal DNA – contigs – in which the distance between landmark sequences of DNA is expressed in kilobases the ultimate physical map is the complete sequence Applications (1) provide a scaffold upon which polymorphic markers can be placed (2) facilitating finer scale linkage mapping (3) confirm linkages inferred from recombination frequencies (4) resolve ambiguities about the order of closely linked genes (5) enable detailed comparisons of regions of synteny between genomes pgs2e-fig jpg

25 Mapping Genomes – Physical Maps
Two strategies used to assemble contigs Alignment of randomly isolated clones based on shared restriction fragment length profiles YAC – ~1Mbp long fragments BAC – ~100kbp long fragments Plasmid – ~ kbp long fragments Automatic restriction profiling (Ch. 2) assemble contigs (short for "contiguous sequences").

26 Genomic clone library Unlike the case of fX174, no large genome
could be completely sequenced without an extra round of fragmentation into manageable sized chunks. In other words it had to be transferred into one or more clone libraries from which individual clones were picked to be "subcloned" in M13 for sequencing. The general outline of the procedure is shown at right. You can see that fX174 bypassed the first stage, the construction of a clone library from the target genome. cDNA library – made from RNA that has been reverse transcribed into cDNA and are used for EST sequencing projects.

27 Cloning vectors

28 Mapping Genomes – Physical Maps
(2) Hybridization-based approaches – chromosome walking Chromosome walking is used as a means of finding adjacent genes (positional cloning), or parts of a gene which are missing in the original clone as well as to analyze long stretches of eukaryotic DNA. This task requires finding a set of overlapping fragments of DNA that spans the distance between the marker and the gene. Genomic DNA is shown in blue. Selected clones from a library of cloned genomic DNA fragments are shown in red. The initial probe, probe a, is specific to gene A or exon A and allows identification of clones 1 and 2. A new probe, probe b, is prepared from one end of clone 2 and used to isolate new clones 3 and 4 from the genomic library. Probe c, prepared from clone 4 is used to identify clone 5, etc. The orientation of the clones is determined by restriction mapping of the clones. Clone 6 contains the desired gene B or exon B.

29 Mapping Genomes – Cytogenetic Maps
Historically – aid in the alignment of physical and genetic maps Cytogenetic maps are the banding patterns observed through a microscope on stained chromosome spreads Traditional preparation – salivary gland polytene chromosomes 唾液腺多線染色體 (greatly enlarged relative to their usual condition) of insects and Giemsa-banded mammalian metaphase karyotypes Chromosomes  the genetic material  phenotypes or medical conditions correlate with the deletion or rearrangement of chromosome sections Cytogenetic map are aligned with the physical map through in situ (在原位置) hybridization – a clone fragment is annealed to a single location on the cytogenetic map NCBI Genomic Biology Keyword: HOX AND homo[ORGN] Karyotypes pgs2e-fig jpg

30 Mapping Genomes – Cytogenetic Maps
Alignment of cytological, physical, and genetic maps. Cytological map – a representation of a chromosome based on the pattern of staining of bands Physical map – the location of transcripts and sites of insertions and deletions Genetic map – recombination rates vary along a chromosome, typically reduced near the telomere and centromere Distances between genetic, physical and cytological markers are not uniform How to search for genes on a genome map ? See my lecture notes on Bioinformatics class.

31 Comparative Genomics Synteny – conservation of gene order between chromosome segments of two or more organisms. Homologes – highly conserved loci derived form a common ancestral locus Orthologs – similar genes that arose as result of duplication subsequent to an evolutionary split Paralogs – similar genes that arose as result of duplication speciation pgs2e-fig jpg Conservation of gene order is an inverse function of the times since divergence from the ancestral locus. Note – rates of divergence vary considerably at all taxonomic levels. Japanese pufferfish – 7.5 times smaller than the human genome, show extensive gene order similarity with humans, around 50% - 80% is in the same order as is found in the human genome

32 Comparative Genomics Chromosome painting – used to define regions of Synteny cover regions (~0.1 of a chromosome arm) Each chromosome of one species is labeled with a set of fluorescent dyes, and hybridized to chromosome spreads of the other genome. Uses the fluorescent in situ hybridization (FISH) technique to detect DNA sequences in metaphase spreads of animal cells. The fluorescently labeled hybrid karyotype is shown in bottom.

33 Comparative Genomics Synteny between cat and human genomes. Ideograms (染色體模式圖) for each of the 24 chromosomes shown on the right in each pair are aligned against color-coded representations of corresponding cat chromosomes. CAT – six groups (A – F) of 2 – 4 chromosomes each. Top row – 12 autosomes that are essentially syntenic along, except for some rearrangements Bottom row – 10 autosomes that have at least one major rearrangement The two sex chromosomes are essentially syntenic between cat and human pgs2e-fig jpg

34 Comparative Genomics Sequence conservation = functional importance
High-resolution comparative physical mapping – found ~1Mbp synteny region between human and mouse May contain hundreds of genes, local inversions and insertions/deletions involving one or a few genes Families of genes organized in tandem clusters Considerable size variation in intergenic “junk” DNA

35 Comparative Genomics Identifying genes and regulatory regions in seq. genomes is challenging ORF are usually good

36 Comparative Genomics Identifying genes and regulatory regions in sequenced genomes is challenging Open reading frames (ORFs) are usually good indication of genes However, it is difficult to determine which ORFs belong to a gene Many mammalian genes have small exons and large introns Regulatory sequences even more difficult One of the major problems facing genomic researchers is to annotate sequenced genomes. A first step in this process is the identification of genes and the regulatory sequences that govern their expression. In general, the presence of an open reading frame (ORF) is a good indicator that a peptide is encoded in the DNA. However, it is frequently not straightforward to identify which ORFs belong to a gene. This problem is particularly acute in mammalian genomes, in which genes tend to have a lot of small exons (an average of 9-10) and large introns. Regulatory sequences such as transcription-factor binding sites are particularly difficult to identify when only a single organism’s genomic sequence is available.

37 Comparative Genomics Computer programs analyze genomic sequence GRAIL
GeneFinder Look for ORFs, splice sites, poly A addition sites, etc. Predict gene structure Frequently wrong Usually miss exons at beginning or end of gene Sometimes predict exon when one doesn’t really exist It is not easy to know whether an ORF is really part of a gene or is there fortuitously. Computer programs like GRAIL and GeneFinder will search DNA sequences for ORFs. In addition, these programs search for regulatory sequences that can help in the identification of genes. In particular, the sequences found around splice junctions are very useful in distinguishing real exons from ORFs that happen to be present in an intron. Nevertheless, most computer programs designed to identify genes by using only a single genomic sequence are wrong at least 10% of the time. They frequently miss exons at the beginning or end of a gene, or they predict an exon where one doesn’t really exist.

38 Comparative Genomics When comparing genomes of different species, the genes normally have the same exon–intron structure Look for conserved ORFs in both genomes Frequently permit accurate identification of genes Fugu–human comparison found >1,000 genes Mouse–human comparison indicates only 25,000 genes in genome When genomes of more than one species are compared, coding regions and exon–intron structures tend to be conserved. Thus, it becomes fairly straightforward to look for ORFs that are conserved in both genomes and found in similar positions relative to surrounding ORFs. Cross-genome comparisons have been shown to be an effective means of accurately annotating genes as well as identifying new genes. For example, in the comparison of the Fugu genome with the human genome, over 1,000 putative genes were discovered that had been missed by the annotation programs. Comparison of the mouse genome with the human genome indicates that there are only about 25,000 genes in both genomes. This finding is consistent with the predictions made from the draft human sequence, but runs contrary to earlier pregenome predictions that set the number of genes as closer to 100,000.

39 Example of sequence comparison
Comparison of the human and mouse spermidine synthase genes revealed an additional intron in the human gene that is not found in the mouse homologue Human Mouse When the human spermidine synthase gene involved in the synthesis of polyamines was compared with the homologous gene in the mouse genome, it was found that there is an additional intron in the human gene that interrupts the fifth exon in the mouse gene. This is an example of a comparison that highlights how gene structure can change as species evolve. 5,500 bp

40 The Human Genome Project (HGP)
Objectives Generation of high-resolution genetic and physical maps that will help in the localization of disease-associated genes. The attainment of sequence benchmarks, leading to generation of a complete genome sequence by the year (A draft version was achieved in May 2000, but finished sequence required an error rate of less than 1 in 10,000 bp) Identification of each and every gene in the genome by a combination bioinformatics identification of open reading frame (ORFs), generation of voluminous EST databases, and collation(對照)of functional data including comparative data from other animal genome projects. Compilation of exhaustive polymorphism databases, in particular of SNPs, to facilitate integration of genomic and clinical data, as well as studies of human diversity and evolution. pgs2e-fig jpg

41 The Human Genome Project (HGP)
Table 1.1 Initial Goals of the HGP From the First 5-Year Plan: Table 1.2 A Blueprint for the Future of the HGP 15 Grand Challenges in the Third 5-Year Plan: 2003 – 2005 HGP budget – set aside for research on the ethical , legal, and social implication of genetic reserach (the ELSI project)

42 pgs2e-table jpg

43 pgs2e-table jpg

44 pgs2e-table jpg

45 pgs2e-table jpg

46 pgs2e-table jpg

47 The Human Genome Project
The architecture of the Human Genome Project in the twenty-first century. Three major themes for future genome research are founded on six pillars of genome resources.

48 ELSI Box 1.1 The Ethical, Legal, and Social Implications of the HGP
Funding – The National Human Genome Research Institute (NHGRI)  5% of its annual budget to ELSI Funding three types of activities: regular research grants, education grants, and intramural programs at the NIH campus Web sites: 4 major objectives 4 main subject areas

49 ELSI Great concern is the privacy and confidentiality of genetic information. Especially – Iceland (介於格陵蘭與挪威間 and Estonia (愛沙尼亞共和國  government-sponsored databases of medical records have been supplied to medical research companies. Psychological impact and potential for stigmatization (給帶來恥辱,使貼上標籤) inherent in the generation of genetic data  racial mistrust and socioeconomic differences in gathering of and access to genetic information Reproductive issues Potential moral (possible legal) obligations once data has been obtained. Philosophical discussions – human responsibility, human right to “play God” with genetic material, meaning of free will in relation to genetically influenced behaviors Genetically Modified Organisms (GMOs) 1998 – Five new major aims

50 pgs2e-table jpg

51 1.7 (Part 1) Whose genome was sequenced?
The content of the Human Genome Completion of the first draft of the HGP was announced at press conference in May 2000, but publication of the result was delayed until Feb. of 2001. Need refinment of the seq. assembly, including gap closure, gene annotation, and prediction It is estimated that the total number of genes is somewhere around 25,000 (~ two times greater than gene contents of the fruit-fly and C.elegans, and five times greater than yeast, see Table 1.3 for more details) Table 1.3 Comparison of Gene Content in some Representative Genomes No dramatic differences in gene content between humans and other mammals. Sep – the first high-resolution genetic map of the complete genome – 23 linkage groups (one per chromosome) with 1200 markers at an average of 1cM intervals Around 1995 – physical map – sequence tag sites (STS) at ~60 kbp intervals 1998 – 3000 SNPs Middle of 2004 – 1.8 million mapped SNP, see The SNP Consortium (TSC) Providing polymorphic markers at 2kb intervals and placing 85% of all exons within 5kp of a SNP. 2000 – the first draft of the smallest human chromosome, chromosome 21 was published pgs2e-fig jpg

52 pgs2e-table jpg

53 The content of the Human Genome
Two questions for the HGP Whose genome was sequenced ? The sequence is derived from a collection of several libraries obtained from a set of anonymous donors. Both the IHGSC and the private firm Celera Genomics assembled their seq. from multiple libraries of ethnicaly diverse individuals One particular indiveidual’s DNA contributed 3/4 and 2/3 of the raw seq. respectively. Size of shaded sector ~ amount of seq. contributed by a single individual

54 The content of the Human Genome
The Celera sample included at least one individuals from each of four ethnic groups, as well as both males and females. Craig Venter admitted that his own DNA contributed substantially to the Celera sequence Their own poodle (獅子狗) contributed to the first-draft canine (犬科動物) genome seq.

55 The Human Genome Project
(2) When can we regard it as finished ? The complete seq. of 99% of human euchromatin has been published to an estimated error rate of ~ 1 event in 100,000 bases. Human polymorphism is an order of magnitude greater than this  at least 10 SNPs for each seq. error Extensive tracts of heterochromatin (there are few or no genes, such as centromeres and telomeres), mostly associated with centromeres that may account for as much as 20% of the total genome, will probably never be sequenced. Since the completion of the first draft  HGP focus on characteristing human diversity. International HapMap project – map all of the major haplotypes in the human genome and characterize their distribution among populations, as a step toward identification of human disease susceptibility factors, see pgs2e-fig jpg

56 Internet Resources – NCBI and Ensembl NCBI
Ensemble – a collaboration between EMBL-EBI and the Sanger Center in the UK. Both sites provide high-resolution physical maps of any segment of the genome. Several genome views UCSC Genome Browser Commercial web sites - Incyte Genomics, Celera, Rosetta Inpharmatics, Informax, and LION Biosciences Figure 1.8 The National Center for Biotechnology Information (NCBI) Web site. pgs2e-fig jpg

57 Internet Resources – NCBI and Ensembl
Ex. 1.2 Use the NCBI and Ensemble genome browser to examine a human disease gene. Use OMIM to identify a gene that is implicated in the etiology (病因學) of the disease. Ans. Go to  Asthma (氣喘)  find one of the interest  for example, Interleukin 13 (IL13). This page gives a lot of textual information + link to other sites, including Human Gene Mutation Database (HGDB) or Entrez Gene What are the various identifiers of the gene ? *147683 (b) Where is the gene located on the chromosome (cytologically and physically) ? The cytological location is 5q31 (chromosome 5, long arm, Click on Gene map locus  5q13  click location 5q13  click NCBI MapViewer  position Mb, Gene ID for IL13 is 3596  Gene aliases: ALRH; P600; IL-13; MGC116786; MGC116788; MGC116789 (c) What is the RefSeq for the gene ? The RefSeq is NM_002188, an mRNA seq.

58 Internet Resources – NCBI and Ensembl
(d) How many exons are there in the major transcript, and how long is it? From Entrez Gene  Display ‘Gene table’  4 exons, 1282 bp long and encodes a 146 amino acid protein, or use NCBI MapViewer  Consensus CDS (ccds) From RefSeq ID is NM_ link to GeneBank signal peptide (interleukin 13 precursor), 34 aa (seq. 15 – 116), mat_peptide (interleukin 13 precursor) 98 aa (e) What is known about the function of the gene? See NCBI description - This gene encodes an immunoregulatory cytokine produced primarily by activated Th2 cells. This cytokine is involved in several stages of B-cell maturation and differentiation. (f) Do the two annotations agree? Which browser do you prefer, and why? Ensemble select gene  type IL-13  Ensembl gene ID ENSG GeneView show that the Exons: 4 Transcript length: 1,282 bps Protein length: 146 residues

59 Internet Resources - OMIM
Online Mendelian Inheritance in Man A database that provides text summarizing recent genetic research in response to a query about a particular disease, as well as links to MedLine and GenBank and other information. Intended for physicians and human geneticists disease types such as muscle, metabolism, cardiovascular, and physiological disorders. OMIM lists in excess of 15,000 known disease-causing Mendelian disorders. GEO BLAST tool – search for all genes in the gene expression database that have similar seq, and then compare levels of expression of the genes across species and experimental conditions. pgs2e-fig jpg Figure 1.9 The Mendelian Inheritance in Man (OMIM) Web site

60 Internet Resources - OMIM
Use OMIM help

61 Internet Resources - OMIM
OMIM has a defined numbering system – certain positions within that number indicate information about the genetic disorder itself. The first digit – the mode of inheritance of the disorder 1 = autosomal (常染色體) dominant 2 = autosomal recessive 3 = X-linked locus or phenotype 4 = Y-linked locus or phenotype 5 = mitochondrial 6 = autosomal locus or phenotype

62 Internet Resources - OMIM
The distinct between 1 or 2 and 6 is that entries cataloged before May 1994 were assigned either a 1 or 2, whereas entries after that date were assigned a 6 regardless of whether the mode of inheritance was dominate or recessive. * = the phenotype caused by the gene at this locus is not influenced by genes at other loci; however, the disorder itself may be caused by mutations at multiple loci # = the phenotype is caused by two or more genetic mutations

63 Internet Resources - OMIM
Example: (MKKS) Display allele variant allelic variants – description is given after each allelic variant of the clinical or biochemical outcome of that particular mutation allelic variant for MKKS

64 Internet Resources - OMIM
The OMIM indicates that the gene SRY encodes a transcription factor that is a member of the high-mobility group-box family of DNA binding proteins. Mutations in this gene give rise to XY females with gonadal dysgenesis(女性生殖腺發育不全症), as well as translation of part of the Y chromosome containing this gene to the X chromosome in XX males. Q 1a. An allelic variant of SRY causing sex reversal with partial ovarian function has been cataloged in OMIM. What was the mutation at the amino acid level and what is observed in XY mice carrying this mutation? Ans. Use “SRY AND human” for the OMIM search  then view list of allelic variants. Variant 0020 is the correct entry. Mutation is Gln2Ter; XY mice are fertile females, although fertility is reduced and ovaries fail early.

65 Internet Resources - OMIM
Q1b. Follow the Gene Map link in the left sidebar to access the MIM gene map, one other gene is found at the same cytogenetic map location. What is the name of this gene, and what methods were used to map the gene to this location? Ans. Click GeneMap in the left sidebar. Correct gene is ZFY. Under the Methods columns, REn and A are listed. Clicking on the Methods hyperlink at the top of the column shows the key to the abbreviations. REn stands for neighbor analysis in restriction fragments; A stands for in situ hybridization.

66 Figure 1.10 (Part 1) A gallery of animal genome sequencing projects
Animal Genome Projects The International Sequencing Consortium (ISC) A database of animal and plant genome sequencing projects Some of these organisms are shown in Figure 1.10 pgs2e-fig jpg Figure 1.10 (Part 1) A gallery of animal genome sequencing projects

67 Figure 1.10 (Part 2) A gallery of animal genome sequencing projects
Animal Genome Projects At the National Human Genome Research Institute (NHGRI), the decision to commit the tens of millions of dollars required for any new genome is made by a council of senior genome scientists – a 10 page “white paper” Weigh the expected impact of the sequence on enabling biomedical research and the annotation of sequence function A draft genome can be produced for most animals within 3-6 months pgs2e-fig jpg Figure 1.10 (Part 2) A gallery of animal genome sequencing projects

68 1.10 (Part 3) A gallery of animal genome sequencing projects
GenBank Files – Box 1.2 There are may ways to present the structure and annotation of a gene or seq. due to alternative splicing and TSS, the small errors occur during cDNA cloning all genomes are full of polymorphism The same gene may be represented by multiple different seq. or annotations in the genome database Refseq – hand curation by experts Example – human HoxA1, Go to LOCUS: XM_004915, GI: Followed by the reference, …. Features section (CDS, misc_feature, .. etc), links to GeneID, MIM, CDD Next comes the seq. in FASTA format, ‘Display’ in XML or ASN.1 file format pgs2e-fig jpg

69 GenBank Files – Box 1.2 Use Entrez Gene – HOXA1 Two isoforms
GenBank format Graph display – HOXA1

70 GenBank Files – Box 1.2 Ensembl -
Gene – HOXA1

71 GenBank Files – Box 1.2 UCSC Genome Browser
Gene – HOXA1

72 Figure 1.11 The Mouse Genome Informatics (MGI) Web site
Rodent Genome Projects Mouse Genome Informatics (MGI) Three major advantages of rodent research are Existence of a large number of mutant strains that, combined with whole genome mutagensis  lead to genetic analysis of every identified locus in the genome Existence of a panel of approximately 100 commonly used lab. mouse strains with well-characterized genealogy – a resource for the study of genetic variation 3. The existence of conserved seq. blocks is generally an indicator of functional constraint 2002 – draft of the Mouse genome 2004 – draft of the rat genome pgs2e-fig jpg Figure The Mouse Genome Informatics (MGI) Web site

73 Rodent Genome Projects
Functional genomic analysis of rat has been stimulated by three major advances achieved in the 1990s The technology for targeted (Site-directed) mutagenesis by homologous recombination of the wide-type locus with a disrupted copy Saturation random (unbiased) mutagenesis programs - Gathers information about entire “sequence space” – i.e., relationship between aa sequence, 3D protein structure and function Emergence of ‘phenomic’(表現性狀) analysis, in which mutagenized lines are subject to biochemical, physiological, immunological, morphological, and behavioral tests in parallel  large-scale identification of genes required for non-lethal (非致命的) phenotypes

74 Figure 1.12 Mouse-human synteny and sequence conservation
Rodent Genome Projects Conservation of gene order and DNA seq. between the human and mouse genomes Blocks of synteny between mouse (chr. 11) and parts of five different human chromosomes Enlarged view of a small region – human 5q31. In this approximately 1 Mb region there is almost perfect correspondence in the order, orientation, and spacing of 23 putative genes, including four interleukins. Enlargement of the alignment of 50kb that includes the genes KIF-3A, IL-4 and IL-13. Blue dots show the distribution of conserved seq. (with 50%-100% identity). Two of the conserved blocks (red bars) fall between genes, whereas most of the others (blue bars) are in the introns and exons of the genes. Use PipMaker pgs2e-fig jpg Figure Mouse-human synteny and sequence conservation

75 Exercise 1.3 Compare the structure of a gene in a mouse and a human
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76 Rodent Genome Projects
Use NCBI choose Genome biology mouse chr.11 use Maps and options add human gene map

77 Rodent Genome Projects
Mouse Genome Informatics (MGI) Integrate physical and genetic maps Search for ortholog genes Online comparison of the mouse and human genome

78 Rodent Genome Projects
Ex. 1.3 Use either NCBI or Ensembl browser, explore the structure of the gene used in Fig. 1.2 in a mouse and a human (and other vertebrates) Ans. Ensembl – type in human IL13 (ENSG ) ‘Orthologue Prediction’  view all genes in ‘MultiContigView’  IL13 is on mouse chr.11, human chr. 5, and rat chr.10

79 Box 1.2 (Part 2) GenBank Files
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80 Other Vertebrate Biomedical Models
2004 – chicken (G. gallus) and dog (C. familiaris) genomes are fully sequenced Motivation – biomedical Chickens – model for oncogenesis and virology Dog – model for complex diseases such as asthma, parasite infection, cancer arthritis (關節炎), diabetes, and behavioral disorders Applications Artificial selection on breed diversity Research into avian (鳥類的) evolution Vertebrate development  Zebrafish transparent embryogenesis, ease of culture, existence of dense genetic map Found ~ thousands of genes are required for proper development of organs a variety of ecologically and commercially fish species, such as sticklebacks刺魚, cichlids慈鯛, salmonids

81 Other Vertebrate Biomedical Models
狗基因圖譜 定序完成 華盛頓郵報2005/12/8電 可以用狗當作探討人類基因疾病的主要工具。因為某些狗罹患某些疾病的機率遠高於其他的狗,如 薩摩耶犬易得糖尿病, 羅威納犬易得骨癌, 西班牙獵犬是癲癇症的高危險群, 杜賓犬罹患嗜睡症的比率遠高於其他的狗,這些疾病人類也很常見。

82 克隆羊「桃莉」

83 Other Vertebrate Biomedical Models
Sequencing nonhuman primates, such as rehsus macaque (獮猴), chimpanzee(黑猩猩) – intend to understand the origins of diversity in the immune system as well as mechanisms of pathogen resistance Comparison of human and chimp seq. Many genes seems to have been positively selected Huamn are differentiated from chimps by small deletions up to 10kb in length, which occur on average every 500kb along chromosome 21 pgs2e-fig jpg

84 Animal Breeding Projects
OMIA (Australia) – genome maps for over a dozen species of agricultural importance Access data on inheritance patterns for species other than human and mouse Benefits of breeding programs lie in improvements in yield, infectious disease resistance adaptation to climatic conditions, improved food quality, maximizing the benefits of transgenic technology These goals will be met both through enhanced genetic map development and association studies using SNP technology ArkDBs (UK, Roslin Institute in Edinburgh) genomes resources for ~10 species pgs2e-fig jpg

85 Invertebrate Model Organisms
Generic Model Organism Database (GMOD) A coordinated effort of the mammalian, invertebrate, and plant genome communities to standardize web tool construction and implementation and to provide open source software for database management Figure The GMOD project

86 Invertebrate Model Organisms
A 40 kb region of cytological band 43E of fruit fly, centered on the saxophone gene. Figure Drosophila gene annotation

87 Invertebrate Model Organisms
Flybase Search for the gene symbol : sax click the ‘gene region map’ each gene either has a number beginning with CG or is identified by its standard name (e.g. sax) show gene and mRNA transposable element insertions (Burdock, one is shown in pink)

88 Invertebrate Model Organisms
The first multicellular eukaryotes to be sequenced completely is C. elegans at 1998 Fruit fly –sequences completed at 2000 Decades of genetic analysis have led to the molecular characterization of up to 20% of the complement of genes in these two organisms Over 90% of the true genes seem to have been identified Assigned a tentative function based on seq. similarity 1/3 ~ 1/4 of the predicted genes remain ‘orphans’ with no known seq. similarity to genes in any other organism  without functional data

89 Invertebrate Model Organisms
Ongoing EST sequencing, gene structure and mutational analysis Unexpected – there may be 50% more genes in C.elegans genome (19,000) than there are in the fly genome (13,500), despite the fact that the fly is much more complex at several levels, including (1) the number of cells, (2) number of cell types, and (3) organization of the nervous system Nematode – a surprising surplus of steroid類固醇-hormone receptors Fruit fly – olfactory嗅覺的 receptor family There is no simple relationship between gene number and tissue complexity The high degree of conservation of all the major regulatory and biochemical pathways, most of all are identifiable not only in both nematode and flies but also in the unicellular eukaryote yeast and in vertebrate genomes

90 Invertebrate Model Organisms
Functional genomics  a major impact of the invertebrate genome projects is the prospect of obtaining mutations in every single gene of the genomes In fly – by a combination of saturation mutagenesis + a library of overlapping deficiencies (deletion) that remove every segment of each chromosome In nematode - saturation mutagenesis + RNAi (a double-strand RNA fed to the worms

91 Figure 1.15 Human disease genes in model organisms
Invertebrate Model Organisms >60% of a sample of 289 human disease genes have an orthologous genes in the fly <60% in nematode ~20% in yeast Fig shows the fraction of human disease genes in each of six categories that have orthologs in the fly, nematode and yeast genome, as detected by seq. similarity at three level of significance Conservation of genetic interactions across the animal kingdom  uncover genes that are interact with known disease-promoting loci Pharmaceutical companies – interested in invertebrate genomics for its potential to identify drugs that affect neural function Example: fluoxetine resistance in nematodes, alcohol tolerance in files Molecular interactions between gene products can be conserved allows the functional comparison of genes across species pgs2e-fig jpg Figure Human disease genes in model organisms

92 蜜蜂(Honey Bee)基因定序

93 海膽(Sea urchin)基因定序

94 Box 1.3 Managing and Distributing Genome Data
Internet technology is essential for genomic scientists NCBI, EBI, LIMS (laboratory information management systems) DB – RDB (relational DB) and OODB (object-oriented DB) RDB – very effective for sorting, searching, and distributing data that fits into table form OODB – good at handling complex data structures and are useful for performing analyses on sequence ‘objects’ (data + with functions for operating on the data)  a very efficient programming approach DB query language = SQL = structured query language Scripting language (no need to compile) = PERL = good for extracting and processing text files

95 Box 1.3 Managing and Distributing Genome Data
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96 Plant Genome Projects Arabidopsis Thaliana – the first plant genome to be sequenced between 1999 and 2000 ~115 Mb, ~25,000 genes, ~2 times (no. fly genes) Evolved via two rounds of whole genome duplication  shuffling隨意混和 of chromosome regions and considerable gene loss >1500 tandem arrays (generally 2 or 3 copies) of repeated genes have been identified, ~11,000 gene families Some geneticists regard this number as representative of the minimal complexity required to support multicellularity It is believed that all plant and animal genomes represent modifications of a ‘toolkit’ of gene families that evolved >109 years ago

97 Figure 1.16 Chromosome duplications in the Arabidopsis thaliana genome
Plant Genome Projects >30 Segmental duplications 7 intra-chromosomal duplication are shown as duplicated blocks of color within three of the five chromosomes; five duplications occur in the first chromosome and the fourth and fifth chromosomes display one duplication piece Anther two dozen inter-chromosomal segmental duplications. A twist in the band  inversion accompanied the duplication event pgs2e-fig jpg Figure Chromosome duplications in the Arabidopsis thaliana genome

98 Plant Genome Projects Plant genomes – plant-specific genes
Enzymes required for cell wall biosynthesis Transport proteins that move organic nutrients, inorganic ions, toxic compounds, metabolites, and even proteins and nucleic acids between cells Enzymes required for photosynthesis, such as Rubisco and electron transport proteins Products involved in plant turgor 細胞之正常膨脹, phototrophic趨光性 and gravitrophic趨地性 Enzymes and cytochromes involved in the production of second metabolites found in flowering plants A large number of pathogen resistance R genes, as are mammalian immune system. R genes are dispersed throughout the genome rather than localized in a single complex

99 Plant Genome Projects Plants share with animals many of the gene families - Intercellular communication, transcriptional regulation, signal transduction A. Thaliana lacks homologs of the Ras G-protein family and tyrosine kinase receptors, Rel, forkhaed, nuclear steroids receptor transcription factors TAIR – The Arabidopsis Information Resource UK CropNet

100 Grasses and Legumes豆莢 >50 different plant species are under way
The most important – major feed crops – the grasses maize, rice, wheat, sorghum高粱, barley大麥, the forage飼料的 legumes soybean, alfalfa紫花苜蓿, forage rye黑麥 grasses, fescues(羊茅,酥油草)  several genomes are very large  whole genome sequencing is impractical Both rice (Oryza sativa) and maize (Zea mays) have relatively small genomes Two major rice genome cultivars培育品種, japonica rice禾更米 and indica rice秈米 MaizeGDB waxy rice糯米

101 Figure 1.17 Rice-Arabidopsis synteny
Comparison of genome sequences of rice and arabidopsis extensive complex patterns of synteny 20 of 54 genes in a 340 kb long of the rice genome (top) retain the same order in five different 80- to 200-kb regions of the Arabidopsis genome (below). Conserved genes (red and green boxes) are found on both rice and Arabidopsis strands, but are interspersed by a variable number of different genes (yellow boxes) in Arabidopsis. Shaded boxes above the rice chromosome indicate that the conserved genes is in the opposite relative orientation on the Arabidopsis chromosomes. pgs2e-fig jpg rice Figure Rice-Arabidopsis synteny

102 Grasses and Legumes Economically important traits include resistance to a broad range of pathogens; flowering time, seed set, grain morphology, and related yield traits; tolerance to drought, salt, heavy metals and other extreme environmental circumstances; and measures of feed quality such as protein and sugar content. Improved through genetic engineering + specialized plant breeding techniques Genome projects  reveal much information regarding the evolution of domesticated species

103 Grasses and Legumes Teosinte墨西哥類蜀黍 versus Maize玉蜀黍
Modern maize is a derivative of the wild progenitor teosinte, which had multiple tillers. Throughout the coding region of tb1, the level of polymorphism is substantially the same in a sample of maize and teosinte. However, in the 5’ UTR, there is a dramatic reduction in the level of polymorphism in maize relative to that seen in teosinte. Figure Teosinte branched 1 and the evolution of maize

104 Other Flowering Plants
>90 angiosperm genome projects are listed on the US department of Agriculture web site African, Australian, European, US projects Genetic maps and search for a common set of plant genes For some species, large EST seq. projects are also in place  enable comparative genomic analysis Arabidopsis + grasses + several model organisms  shed light on plant evolution

105 Figure 1.19 Forest genomics
Other Flowering Plants Forest trees – potential for economic impact High-density genetic maps of spruce, loblolly and several pines, a few species of Eucalyptus Trait – wood quality, growth and flowering parameters Dendrome web site Comparative analyses and transcription profiling of genes involved in wood properties including lignins木質素 and enzymes that regulate cell wall biosynthesis Crops plants – potato, tomato, tobacco, beans, cotton Analyzing the genome diversity affect productivity, yield and quality improvements No plant equivalent of the HGP’s ELSI initiative has been established. pgs2e-fig jpg Figure Forest genomics

106 Microbial Genome Projects
The minimal genome 1995 – the 1st complete genome, H. influenzae  M. genitalium  3 other bacteria 1997 – E. coli Seq. information – genome structures (GC content, transposable elements, recombination), genome content (total number of genes, conserved gene families) Gene annotation for prokaryotes are more straightforward – ORF tend to be uninterrupted and genes tend to be closely spaced; however the assignment of genes to operons is not trivial ~3/4 microbial genome can be assigned a function based on their similarity to genes on other organisms or by identifying protein domains TIGR

107 Microbial Gene contents
M. genitalium 0.6 Mb, 471 genes H. influenzae 1.8 Mb, 1750 genes E. coli K Mb, 4288 genes  average gene length ~ 1.1 kb Gene duplication and divergence in large genomes, gene loss in small genomes pgs2e-table jpg

108 Exercise 1.4 Compare two microbial genomes using the CMR
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109 The minimal genome – the minimum complement of genes that are necessary and sufficient to maintain a living organism To define genetically ‘What is life’? Two general strategies Bioinformatics strategy – identify which genes are present in each and every sequenced genome Some functions can be performed by non-orthologous genes Conserved orthologs + a small number of alternatives ~256 genes

110 The minimal genome Experimental strategy – systematically knock out the function of individual genes: mutations that cannot be recovered define genes that are likely to be components of the minimal genome M. genitalium – recovered 120 of the 470 genes B. subtilis (~4100 genes) – 271 genes are indispensable (必要的) under favorable growth conditions, metabolism, cell division and shape, synthesis of cellular envelope Synthetic lethal (綜合的致命) – the nonviability (無存活能力) in combination of two or more individually viable mutations Infer that life can be supported by a genome of between 250 and 350 genes Build a viable organism from scratch by stitching (組在一起) together artificially synthesized genes – build a poliovirus (脊髓灰質炎病毒)

111 Figure 1.20A Describing the minimal genome
Deeper color  presence of a gene Pale color  the genes is absent in that species Gene a, d, f are present in all species, so are inferred to be necessary for life. pgs2e-fig jpg Figure 1.20A Describing the minimal genome

112 Figure 1.20B Describing the minimal genome
Mutagenesis experiments Establish which genes are essential by systematically knockout each functional genes and seeing whether the organism can survive without it. The overlap of these two approaches may define the minimal genome. pgs2e-fig jpg Figure 1.20B Describing the minimal genome

113 1.21 TIGR representation of a typical microbial genome
Sequenced Microbial Genomes TIGR – Comprehensive Microbial Resource (CMR) New site 39 genomes were generated by TIGR, and the rest by Brazil, Japan … Omniome DB Streptococcus pneumoniae TIGR4 The outer and inner circles represent genes encoded on the two strands of the chromosomes Genes from HMM – blue BLAST – yellow, Omniome – pink Click ‘align genome’ – MUMMER Click ‘Analyses’ – for more tools, such as COG/TIGRFAM/PFAM pgs2e-fig jpg

114 Box 1.2 (Part 1) GenBank Files
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115 pgs2e-table jpg

116 pgs2e-table jpg

117 Environmental Sequencing
Sequencing DNA extracted form an environment such as ocean, soil, or intestinal flora (腸道微生物) The main reason is that the vast majority of bacteria cannot be cultured in vitro  our knowledge of microflora is both limited by and biased by sampling Pilot projects – identify novel genes has the potential to change oceanographers’ understanding of the mechanisms of photosynthesis and global carbon and nitrogen cycling Proteorhodopsin genes – suggesting that light harvesting need not be coupled to chlorophyll in cyanobacteria C. Venter – identified >1M new genes !!, almost 150 new types of bacteria Fecal material – human gut contains > 500 different species of bacteria, < 30% can be cultured outside the body

118 Yeast Completed at 1997 MIPS SGD

119 Parasite Genomics World Health Organization (WHO) 10 tropical diseases that affect billions of people worldwide Eradicating (根除) the pathogenic agents Crop damage caused by parasitic plant nematodes costs billions of dollars

120 Parasite Genomics Aims Identify species-specific genes Understanding the developmental genetics Polymorphism surveys that address the population biology of the parasites Mapping the genomics of the mosquito

121 pgs2e-table jpg

122 100 genomes, 10 days and 10 million dollars Awards
2006 News,

123 全球首見 實驗室做出人類精子 2009/07/09

124 The End

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