GENE MAPPINGGENE MAPPINGBy A.Arputha SelvarajBy A.Arputha Selvaraj

Comparative GenomicsComparative Genomics

Gene functions have been conserved Gene functions have been conserved across evolutionacross evolution

Nature solves a problem, it rarely solves it Nature solves a problem, it rarely solves it again.again.

Eyeless Mutation

http://www.ucm.es/info/genetica/AVG/practicas/Drosophila/Drosophila.htm

Drosophila Eyeless

Human Aniridia

Homologous Genes50% identical

Human gene expressed in correct location in fly carrying the mutant eyeless gene

http://www.geo.de/GEO/fotografie/portfolio_des_monats/2001_10_portfolio_meckes/page2.html?SDSID=

Ectopic Expression of Human Eyeless Gene in Drosophilahttp://as3.lib.byu.edu/~imaging/brad/set5/sl17.html

Flies with human eyeless homologdo not develop “human” eyes

Develop compound eyes, even with human homolog driving development

Normal Eyeless Gene

Developmental end product depends on

context

Human

Fruit fly

Normal Eyeless Gene

A

B

D

E

Evolution & GenesEvolution & Genes

The function of many genes is conserved across The function of many genes is conserved across immense evolutionary distances. immense evolutionary distances.

The function of a gene is affected by its environment.The function of a gene is affected by its environment.

Many gene products function as part of a cascade or Many gene products function as part of a cascade or pathway. pathway.

“Nothing in Biology Makes Sense Except in the Light of Evolution” (Theodosius Dobzhansky)

Map Genes in the Context of Map Genes in the Context of ChromosomesChromosomes

Uses of Gene MappingUses of Gene Mapping

Identify genes responsible for diseases.Identify genes responsible for diseases. Heritable diseasesHeritable diseases CancerCancer

Identify genes responsible for traits.Identify genes responsible for traits. Plants or AnimalsPlants or Animals Disease resistanceDisease resistance Meat or Milk ProductionMeat or Milk Production

Types of MapsTypes of Maps

Nucleotide Sequence MapsNucleotide Sequence Maps complete or partially sequenced organismscomplete or partially sequenced organisms

Cytogenetic MapsCytogenetic Maps Breakpoints in diseaseBreakpoints in disease Direct binding of probes to chromosomeDirect binding of probes to chromosome

Genetic Linkage MapsGenetic Linkage Maps MarkersMarkers

Physical MapsPhysical Maps

Mapping GenesMapping Genes

Essential element -- MarkersEssential element -- Markers Differences between two members of a Differences between two members of a

species.species. Typically between 1-400 nucleotides in Typically between 1-400 nucleotides in

length.length. Can also be gross chromosomal Can also be gross chromosomal

rearrangements.rearrangements.

DNA SequencingDNA Sequencing

Two methods originally developed:Two methods originally developed: Chemical or Maxim and Gilbert method.Chemical or Maxim and Gilbert method. Dideoxynucleotide or Sanger method.Dideoxynucleotide or Sanger method. Both Gilber and Sanger won Nobel prizes for Both Gilber and Sanger won Nobel prizes for

developing these techniques.developing these techniques. Dideoxynucleotide method is by far the Dideoxynucleotide method is by far the

most common employed today.most common employed today.

Automated SequencingAutomated Sequencing Each nucleotide is labeled with a Each nucleotide is labeled with a

different fluorescent chromophore.different fluorescent chromophore. Uses Taq polymerase to incorporate Uses Taq polymerase to incorporate

fluorescent dideoxy nucleotide.fluorescent dideoxy nucleotide. Analysis on a polyacrylamide gel or Analysis on a polyacrylamide gel or

capillary matrix that is can separate capillary matrix that is can separate DNA based upon a 1 nucleotide DNA based upon a 1 nucleotide difference.difference.

30-1000 nucleotides in length can 30-1000 nucleotides in length can theoretically be determined.theoretically be determined.

Deoxy

Dideoxy

OH

O

O

Base

CH2OP

O

O

O

Base

CH2OP

O

O

O

(3’2’)

Copyright Cold Spring Harbor Laboratories

G A T C G G A T C C

G A T C G G A T C C

T A G G

G A T C G G A T C C

T A G GCCdd

G A T C G G A T C C

T A G GCCdd

G A T C G G A T C C

T A G GCCGdd

Nucleotide SequencingNucleotide Sequencing

Complete Genome Sequencing Complete Genome Sequencing Bacterial Artificial Chromosomes (BAC)Bacterial Artificial Chromosomes (BAC)

• Can grow in bacteriaCan grow in bacteria• Have large inserts 100,000-300,000 Have large inserts 100,000-300,000

nucleotidesnucleotides Collect a large library of BACsCollect a large library of BACs Restriction Endonuclease (RE) MapRestriction Endonuclease (RE) Map

• RE - cuts DNA at specific sites RE - cuts DNA at specific sites • EcoRI cuts at GAATTC EcoRI cuts at GAATTC

meds.queensu.ca/~mbio318/ EXTRA_MATERIAL.html

BACs

Genomic SequencingGenomic Sequencing

Once BAC map completeOnce BAC map complete Sequence ends of BAC clones.Sequence ends of BAC clones.

With dense BAC map, large regions may be With dense BAC map, large regions may be covered by overlapping the sequences.covered by overlapping the sequences.

Individual BACs can be completely Individual BACs can be completely sequenced.sequenced.

Individual BACs can be hybridized to Individual BACs can be hybridized to chromosomes to identify chromosome of chromosomes to identify chromosome of origin.origin.

RecombinationRecombination Permits Mapping by providing:Permits Mapping by providing:

Linkage groupsLinkage groups DistancesDistances

During MeiosisDuring Meiosis

RecombinationRecombination

Linkage Groups - markers that tend to Linkage Groups - markers that tend to remain together.remain together.

Distance - the further apart two markers Distance - the further apart two markers lie, the more often recombination will occur lie, the more often recombination will occur between those markers.between those markers.

Markers on the same chromosome can be Markers on the same chromosome can be so far apart that they appear in different so far apart that they appear in different linkage groups.linkage groups.

Cytogenetic MappingCytogenetic MappingPhiladelphia ChromosomePhiladelphia Chromosome

Chronic myelogenous leukemia (CML) is Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation characterized by a reciprocal translocation between chromosomes 9 and 22 that between chromosomes 9 and 22 that produces the Philadelphia chromosome. produces the Philadelphia chromosome. Invariably there is disease progression, Invariably there is disease progression, with loss of the capacity for terminal with loss of the capacity for terminal differentiation by the hematopoietic stem differentiation by the hematopoietic stem cell, resulting in an acute leukemia.cell, resulting in an acute leukemia.

www.clubstewart.co.uk/cml.htmwww.clubstewart.co.uk/cml.htm

Genetic Linkage Maps Genetic Linkage Maps PolymorphismPolymorphism

PolymorphismsPolymorphisms Polymorphism - an allele present in a Polymorphism - an allele present in a

population that exhibits multiple forms.population that exhibits multiple forms. Monomorph - Single form in a population.Monomorph - Single form in a population. Common.Common. Typically result in no effect on survival of Typically result in no effect on survival of

individual.individual.

Types of MarkersTypes of Markers Single Nucleotide Polymorphisms (SNPs)Single Nucleotide Polymorphisms (SNPs)

Occur approximately every 100-300 bp in Occur approximately every 100-300 bp in humans.humans.

Names: rs8111765

MarkersMarkers

Microsatellites or Tandem RepeatsMicrosatellites or Tandem Repeats acgCACACAtgcacgCACACAtgc acgCACACACAtgcacgCACACACAtgc

Physical MapsPhysical Maps Sequence Tagged Sites (STSs)Sequence Tagged Sites (STSs)

Use Polymerase Chain Reaction (PCR).Use Polymerase Chain Reaction (PCR). Primers based upon:Primers based upon:

• random sequencerandom sequence• expressed sequence tagexpressed sequence tag

Amplify from library of clones containing Amplify from library of clones containing large inserts (BAC). large inserts (BAC).

Relate to BAC map.Relate to BAC map. If more than one on same clone, then close If more than one on same clone, then close

togethertogether

Types of questionsTypes of questions

Where does gene X exist within the genome of organism Y? Where does gene X exist within the genome of organism Y? What are some flanking markers? What are some flanking markers?

Which genes exist on a chromosome, and in what order do they Which genes exist on a chromosome, and in what order do they appear? appear?

Show the genes that exist in region R of the chromosome. Show Show the genes that exist in region R of the chromosome. Show me the corresponding sequence data for that region. me the corresponding sequence data for that region.

Display the region of a chromosome between points A and B. Display the region of a chromosome between points A and B. Show both the cytogenetic and sequence map for that region, Show both the cytogenetic and sequence map for that region,

aligned to each other based on markers that have been placed aligned to each other based on markers that have been placed on both maps. on both maps.

What is the distance between two genes? (Note: scale depends What is the distance between two genes? (Note: scale depends on the type of map on which those genes have been placed.)on the type of map on which those genes have been placed.)

I know the cytogenetic location of gene X. What is the I know the cytogenetic location of gene X. What is the corresponding physical location? corresponding physical location?

http://www.ncbi.nlm.nih.gov/mapview/static/MapViewerHelp.html

Genes and MapsGenes and Maps

Bcl2Bcl2 Map LinkMap Link OMIM LinkOMIM Link Syntenic Relationships - tendency of Syntenic Relationships - tendency of

closely linked genes to remain linked closely linked genes to remain linked during evolution.during evolution.

GO evidence codesGO evidence codes

PathwaysPathways

KEGG pathwaysKEGG pathwaysExamine Cell Adhesion MoleculesExamine Cell Adhesion Molecules-What does JAM3 appear to interact with? -What does JAM3 appear to interact with? -What information is available if you click on -What information is available if you click on

JAM2?JAM2?What motifs are found in JAM2?What motifs are found in JAM2?Click on Tight JunctionClick on Tight Junction-What molecules appear to interact with the JAM--What molecules appear to interact with the JAM-

JAM dimer? What is ZO-1JAM dimer? What is ZO-1

Thank YouThank You