genome science ka-lok ng dept. of bioinformatics asia university
TRANSCRIPT
Genome Science
Ka-Lok Ng
Dept. of Bioinformatics
Asia University
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 http://www.gmod.org/ to develop reusable components suitable for creating new community databases of biology
(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
The Core Aims of Genomics Science
(3) To generate and order genomic and expressed gene sequencesHigh-volume sequencingBasic technique is developed by Fred Sanger“Shotgun” approach assemble into contigs, scaffolds (a set of contigs), then the whole chromosomesmRNA is unstableCoding parts cDNA clones – cloned from mRNA transcriptsExpressed sequence tags (ESTs)Obtain full length cDNA is not easy because of mRNA structure
The Core Aims of Genomics Science
The Core Aims of Genomics Science
(3)To generate and order genomic and expressed gene sequences
mRNA cDNA EST
Reverse transcription cDNA
EST - partial cDNA sequences sequenced either from 5' or 3‘
Alternative splicing not a one-to-onecorrespondence between ESTs and genes
Whole genome reconstruction
(4) Identify and annotate the complete set of genes encoded within a genomeFrom complete sequence of a genome genes identificationAlignment of cDNA, DNA and protein sequences – BLASTGene finding software – ORFs, transcription start and termination sites, exon/intron boundariesThen gene annotation linking sequence to genetic function, expression, locus information, comparative data from homologous proteins
The Core Aims of Genomics Science
(5) To characterize DNA sequence diversitySingle-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
The Core Aims of Genomics Science
mutation
SNP
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
Sickle-cell anemia and SNP
(5) To characterize DNA sequence diversityCharacterize the level of haplotype structure due to linkage disequilibrium (LD)haplotype = a set of adjacent polymorphisms found on a single chromosomeLD = groups of closely linked alleles that tend to be inherited together, can be used to map human disease genes very accuratelyKnowledge of LD are utilized to do disease locus mappingIn 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.
The Core Aims of Genomics Science
Haplotype
Mendel's Laws enable the outcome of genetic crosses to be predicted.
The Core Aims of Genomics Science
A and B on different chromosome
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.
The Core Aims of Genomics Science
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
(5) To characterize DNA sequence diversitythe farther apart two genes are, the more they tend to assort independently (randomly) recombination frequency ↑
The Core Aims of Genomics Science
Vermilion - 朱紅色
Higher freq. farther apart
(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)
The Core Aims of Genomics Science
(7) To accumulate functional data, including biochemical and phenotypic properties of genesNear-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 http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.htmlMouse http://www.bioscience.org/knockout/knochome.htmProteomics – detecting protein expression and protein-protein interactionsPharmacogenomicists – study the interactions between small molecules (i.e. potential drugs) and proteinsFunctional genomics – a crucial component is to study various model organismsClone library – collections of DNA fragments that are cloned into a vector
The Core Aims of Genomics Science
Site-directed mutagenesis
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.
The Core Aims of Genomics Science
GTC Valine GCC Alanine
(8) To provide the resources for comparison with other genomes.Comparative maps allow genetic data from one species to be used in the other speciesComparative 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)
The Core Aims of Genomics Science
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 50-50 chance of co-segregation 50cM (0.5 crossover occurs per generation)
Mapping Genomes – Genetic Maps
(A) A pair of different parental chromosomes(green and blue colors).
(B)A table showing the frequency of recombinantsbetween 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.
Genetic distance ~ 0.11 11cM0.22 21cM, 0.25 24cM,0.33 33cM Figure 1.1
• Software of the assembly of genetic maps
http://linkage.rockefeller.edu/soft/list.html
• 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
Mapping Genomes – Genetic Maps
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.
Exercise 1.1 (Part 2) Constructing a genetic map
Assume independent assortment for each recessive phenotype ¼ 242 petite (127+42+38+12+10+8+3+2), 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
Exercise 1.1 (Part 2) Constructing a genetic map
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
Mapping Genomes – Genetic Maps
Exercise 1.1 (Part 2) Constructing a genetic map
s r t p
Mapping Genomes – Genetic Maps
Approximate solution
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 sequenceApplications(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
Mapping Genomes – Physical Maps
Two strategies used to assemble contigs(1) 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").
Mapping Genomes – Physical Maps
Unlike the case of X174, 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 X174 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.
Genomic clone library
Cloning vectors
(2) Hybridization-based approaches – chromosome walkingChromosome 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.
Mapping Genomes – Physical 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
http://book.tngs.tn.edu.tw/database/scientieic/content/1970/00100010/images/0053b.jpg
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 http://www.ncbi.nlm.nih.gov/Genomes/
Keyword: HOX AND homo[ORGN]
Mapping Genomes – Cytogenetic Maps
Karyotypes
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 bandsPhysical 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 centromereDistances 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.
Comparative Genomics
Synteny – conservation of gene order between chromosome segments of two or more organisms.Homologes – highly conserved loci derived form a common ancestral locusOrthologs – similar genes that arose as result of duplication subsequent to an evolutionary splitParalogs – similar genes that arose as result of duplication
speciation
• 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
Comparative Genomics1. Chromosome painting – used to define regions of Synteny cover regions (~0.1
of a chromosome arm)2. Each chromosome of one species is labeled with a set of fluorescent dyes, and
hybridized to chromosome spreads of the other genome.3. 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.
Comparative GenomicsSynteny between cat and human genomes. Ideograms ( 染色體模式圖 ) for each ofthe 24 chromosomes shown on the right in each pair are aligned against color-codedrepresentations 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 rearrangementsBottom row – 10 autosomes that have at least one major rearrangementThe two sex chromosomes are essentially syntenic between cat and human
• 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
Comparative Genomics
• Identifying genes and regulatory regions in seq. genomes is challenging
• ORF are usually good
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
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
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
Comparative Genomics
• 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
5,500 bp
Example of sequence comparison
The Human Genome Project (HGP)
Objectives
1. Generation of high-resolution genetic and physical maps that will help in the localization of disease-associated genes.
2. The attainment of sequence benchmarks, leading to generation of a complete genome sequence by the year 2005. (A draft version was achieved in May 2000, but finished sequence required an error rate of less than 1 in 10,000 bp)
3. 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.
4. 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.
Table 1.1 Initial Goals of the HGPFrom the First 5-Year Plan: 1993-1998
Table 1.2 A Blueprint for the Future of the HGP15 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)
The Human Genome Project (HGP)
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.
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: http://www.genome.gov/10001618http://www.ornl.gov/sci/techresources/Human_Genome/research/elsi.html
4 major objectives4 main subject areas
ELSI
Great concern is the privacy and confidentiality of genetic information.Especially – Iceland ( 介於格陵蘭與挪威間
http://www.tita.org.tw/view/iceland.html) and Estonia ( 愛沙尼亞共和國 http://www.suntravel.com.tw/zone/Europe/Estonia-136.htm)
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 issuesPotential 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
ELSI
1.7 (Part 1) Whose genome was sequenced?
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 GenomesNo dramatic differences in gene content between humans and other mammals.Sep. 1994 – 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 – 52000 sequence tag sites (STS) at ~60 kbp intervals
1998 – 3000 SNPsMiddle of 2004 – 1.8 million mapped SNP, see The SNP Consortium (TSC)
http://snp.cshl.orgProviding 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
The content of the Human Genome
The content of the Human GenomeTwo questions for the HGP(1) 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
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.
The content of the Human Genome
(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 http://www.hapmap.org
The Human Genome Project
Figure 1.8 The National Center for Biotechnology Information (NCBI) Web site.
Internet Resources
– NCBI and Ensembl
NCBI http://www.ncbi.nlm.nih.govEnsemble http://www.ensembl.org – 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 viewsUCSC Genome Browser http://genome.cse.ucsc.eduCommercial web sites - Incyte Genomics, Celera, Rosetta Inpharmatics, Informax, and LION Bioscienceshttp://consert-lpg.obs.ujf-grenoble.fr/html/en/rosetta_section2_wrapper.shtml
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 http://www.ncbi.nlm.nih.gov 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
(a) 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
position132.02 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.
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_002188 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 http://www.ensembl.org, select gene type IL-13 Ensembl gene ID ENSG00000169194
GeneView show that the Exons: 4 Transcript length: 1,282 bps Protein length: 146 residues
Internet Resources – NCBI and Ensembl
Internet Resources - OMIM
Figure 1.9 The Mendelian Inheritance in Man (OMIM) Web site
• 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 geneticistsdisease 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 geneexpression database that have similar seq, and then compare levels of expression of the genes across species and experimental conditions.
Internet Resources - OMIMOMIM http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
Use OMIM help
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 ) dominant2 = autosomal recessive 3 = X-linked locus or phenotype4 = Y-linked locus or phenotype5 = mitochondrial6 = autosomal locus or phenotype
• 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
Internet Resources - OMIM
Example: 604896 (MKKS)
Internet Resources - OMIM
allelic variants – description is given after each allelic variant of the clinical or biochemical outcome of that particular mutation
allelic variant for MKKS
Displayallele variant
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.
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.
Internet Resources - OMIM
Figure 1.10 (Part 1) A gallery of animal genome sequencing projects
The International Sequencing Consortium (ISC) http://www.intlgenome.org
- A database of animal and plant genome sequencing projects
- Some of these organisms are shown in Figure 1.10
Animal Genome 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
Figure 1.10 (Part 2) A gallery of animal genome sequencing projects
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 polymorphismThe same gene may be represented by multiple
different seq. or annotations in the genome database
Refseq – hand curation by expertsExample – human HoxA1, 11421562Go to http://www.ncbi.nlm.nih.gov/1. LOCUS: XM_004915, GI:147512462. Followed by the reference, ….3. Features section (CDS, misc_feature, .. etc),
links to GeneID, MIM, CDD4. Next comes the seq. in FASTA format,
‘Display’ in XML or ASN.1 file format
GenBank Files – Box 1.2
Use Entrez Gene – HOXA1 Two isoforms
GenBank format Graph display – HOXA1
GenBank Files – Box 1.2
Ensembl - http://www.ensembl.org/index.html
Gene – HOXA1
UCSC Genome Browser http://genome.cse.ucsc.eduGene – HOXA1
GenBank Files – Box 1.2
Figure 1.11 The Mouse Genome Informatics (MGI) Web site
Rodent Genome ProjectsMouse Genome Informatics (MGI)
http://www.informatics.jax.org/
Three major advantages of rodent research are1. 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
2. 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 genome2004 – draft of the rat genome
Functional genomic analysis of rat has been stimulated by three major advances achieved in the 1990s
1. The technology for targeted (Site-directed) mutagenesis by homologous recombination of the wide-type locus with a disrupted copy
2. Saturation random (unbiased) mutagenesis programs - Gathers information about entire “sequence space” – i.e., relationship between aa sequence, 3D protein structure and function
3. 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
Rodent Genome Projects
Figure 1.12 Mouse-human synteny and sequence conservation
Conservation of gene order and DNA seq. between the human and mouse genomes
http://www.ncbi.nlm.nih.gov/Homology/(A)Blocks of synteny between mouse (chr.
11) and parts of five different human chromosomes
(B)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.
(C)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 http://nog.cse.psu.edu/pipmaker
Rodent Genome Projects
Exercise 1.3 Compare the structure of a gene in a mouse and a human
Use NCBI http://www.ncbi.nlm.nih.gov choose Genome biology mouse chr.11 use Maps and options
add human gene map
Rodent Genome Projects
Mouse Genome Informatics (MGI)
http://www.informatics.jax.org
- Integrate physical and genetic maps
- Search for ortholog genes
- Online comparison of the mouse and human genome
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 http://www.ensembl.org
– type in human IL13 (ENSG00000169194) ‘Orthologue Prediction’ view all genes in ‘MultiContigView’ IL13 is on mouse chr.11, human chr. 5, and rat chr.10
Rodent Genome Projects
Box 1.2 (Part 2) GenBank Files
Other Vertebrate Biomedical Models2004 – chicken (G. gallus) and dog (C. familiaris) genomes are fully sequencedMotivation – biomedicalChickens – model for oncogenesis and virologyDog – model for complex diseases such as asthma, parasite infection, cancerarthritis ( 關節炎 ), diabetes, and behavioral disordersApplications • Artificial selection on breed diversity• Research into avian ( 鳥類的 ) evolutionVertebrate development Zebrafish • transparent embryogenesis, ease of culture, existence of dense genetic map• Found ~ thousands of genes are required for proper development of organs• http://zfin.org• a variety of ecologically and commercially fish species, such as sticklebacks 刺魚 , cichlids 慈鯛 , salmonids
狗基因圖譜 定序完成華盛頓郵報 2005/12/8 電 http://www.udn.com/2005/12/9/NEWS/WORLD/WOR4/3052845.shtml
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。
Other Vertebrate Biomedical Models
克隆羊「桃莉」http://scc.bookzone.com.tw/sccc/sccc.asp?ser=302
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
Other Vertebrate Biomedical Models
OMIA (Australia) – genome maps for over a dozen species of agricultural importance
http://www.angis.org.au/Databases/BIRX/omia• 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)
http://www.thearkdb.org• genomes resources for ~10 species
Animal Breeding Projects
Invertebrate Model Organisms
Figure 1.13 The GMOD project
Generic Model Organism Database (GMOD)http://www.gmod.org
- 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
Invertebrate Model Organisms
Figure 1.14 Drosophila gene annotation
A 40 kb region of cytological band 43E of fruit fly, centered on the saxophone gene.
Invertebrate Model Organisms
Flybasehttp://www.flybase.org/- Search for the gene symbol : sax- click the ‘gene region map’http://www.flybase.org/cgi-bin/gbrowse_fb/dmel?ref=2R;id=FBgn0003317
- 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)
• The first multicellular eukaryotes to be sequenced completely is C. elegans at 1998 http://www.wormbase.org
• 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
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
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
Invertebrate Model Organisms
Figure 1.15 Human disease genes in model organisms
>60% of a sample of 289 human disease genes have an orthologous genes in the fly<60% in nematode~20% in yeastFig. 1.15 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 significanceConservation of genetic interactions across the animal kingdom uncover genes that are interact with known disease-promoting lociPharmaceutical companies – interested in invertebrate genomics for its potential to identify drugs that affect neural functionExample: fluoxetine resistance in nematodes, alcohol tolerance in filesMolecular interactions between gene products can be conserved allows the functional comparison of genes across species
Invertebrate Model Organisms
http://www.udn.com/2006/10/31/NEWS/WORLD/WOR4/3581547.shtml
蜜蜂 (Honey Bee) 基因定序
海膽 (Sea urchin) 基因定序http://tw.news.yahoo.com/article/url/d/a/061110/2/6cqy.html
Internet technology is essential for genomic scientistsNCBI, 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 formOODB – 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 languagehttp://www.geocities.com/SiliconValley/Vista/2207/sg17.htmlScripting language (no need to compile) = PERL = good for
extracting and processing text fileshttp://bio.perl.org
Box 1.3 Managing and Distributing Genome Data
Box 1.3 Managing and Distributing Genome Data
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
Plant Genome Projects
Figure 1.16 Chromosome duplications in the Arabidopsis thaliana genome
>30 Segmental duplications(A) 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
(B) Anther two dozen inter-chromosomal segmental duplications. A twist in the band inversion accompanied the duplication event
Plant Genome Projects
Plant genomes – plant-specific genesEnzymes required for cell wall biosynthesisTransport 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
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 http://www.arabidopsis.org
• UK CropNet http://ukcrop.net/
Plant Genome Projects
>50 different plant species are under wayThe 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 http://www.maizegdb.orgwaxy rice 糯米
Grasses and Legumes 豆莢
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.
Rice-Arabidopsis synteny
rice
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
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.
Grasses and Legumes
Figure 1.18 Teosinte branched 1 and the evolution of maize
• >90 angiosperm genome projects are listed on the US department of Agriculture web site http://www.nal.usda.gov/pgdic/Map_proj
• 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
Other Flowering Plants
Figure 1.19 Forest genomics
Other Flowering PlantsForest trees – potential for economic impactHigh-density genetic maps of spruce, loblolly and several pines, a few species of EucalyptusTrait – wood quality, growth and flowering parametersDendrome web site http://dendrome.ucdavis.edu
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, cottonAnalyzing the genome diversity affect productivity, yield and quality improvements
No plant equivalent of the HGP’s ELSI initiative has been established.
t
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 http://www.tigr.org
Microbial Genome Projects
Microbial Gene contentsM. genitalium 0.6 Mb, 471 genesH. influenzae 1.8 Mb, 1750 genesE. coli K12 4.6 Mb, 4288 genes average gene length ~ 1.1 kbGene duplication and divergence in large genomes, gene loss in small genomes
Exercise 1.4 Compare two microbial genomes using the CMR
– the minimum complement of genes that are necessary and sufficient to maintain a living organism
To define genetically ‘What is life’?Two general strategiesBioinformatics 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
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 )
The minimal genome
Figure 1.20A Describing the minimal genome
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.
Figure 1.20B Describing the minimal genome
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.
1.21 TIGR representation of a typical microbial genome
TIGR – Comprehensive Microbial Resource (CMR) http://www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl
New site http://pathema.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi
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 – blueBLAST – yellow, Omniome – pink
Click ‘align genome’ – MUMMERClick ‘Analyses’ – for more tools, such as
COG/TIGRFAM/PFAM
Sequenced Microbial Genomes
Box 1.2 (Part 1) GenBank Files
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
Environmental Sequencing
Completed at 1997
MIPS http://mips.gsf.de/genre/proj/yeast/index.jsp
SGD http://www.yeastgenome.org
Yeast
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
Parasite Genomics
Aims
1. Identify species-specific genes
2. Understanding the developmental genetics
3. Polymorphism surveys that address the population biology of the parasites
4. Mapping the genomics of the mosquito
Parasite Genomics
100 genomes, 10 days and 10 million dollars Awards2006 News, http://www.biotechnews.com.au/index.php/id;1321634104
全球首見 實驗室做出人類精子 2009/07/09http://udn.com/NEWS/WORLD/WOR4/5008159.shtml
The End