Identifying%20Genes

1
Identifying Genes
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Genes for Complex Traits

• Polygenic
• Quantitative Trait Locus (QTL)
• Need molecular genetics to identify
• Multiple genes involved for trait
• Additive contribution from individual genes
• E.g., height, skin colour, autism, cancer
• QTLs probabilistic risk factor for disorder

3
QTLs

• Not necessarily genes themselves
• Stretches of DNA closely linked to genes
  underlying the trait in question
• QTLs often on different chromosomes
• Could be
• many genes, each of small effect
• a few genes of large effect

4
Inheritance

• Mendelian inheritance at many loci
• Normally-distributed trait
• If n (loci involved) sufficiently high with
  binomial expansion (ab)2n
• Multifactorial inheritance
• When 2 genes involved in trait, with or without
  environmental activators

5
Genetic Mapping

• Based on principle of genetic linkage
• Particular loci or alleles are jointly inherited
• Loci on same chromosome
• Physically connected
• Tend to segregate together in meiosis, so linked

6
Crossing Over

• Where parts of chromosomes swap (recombination)
• Separates genes on same chromosome
• The further apart the gene loci, the greater the
  chance of separation through crossover
• Relative distance between two genes used to
  calculate degree of linkage

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Linkage Mapping

• Alfred Sturtevant
• By calculating number of recombinants get measure
  of distance between genes
• Genetic map unit (m.u.) or centrimorgan (cM)
• Distance between genes for which there is a 1
  chance of recombination due to crossover in a
  generation
• 1 cM is about 1 million base pairs in humans

8
Linkage Map

• Shows positions of known genes and/or markers
  relative to each other in terms of recombination
  frequency
• RF of 1 is equal to 1 m.u.
• Greater the frequency of recombination, the
  further apart the markers are

9
Single-Gene Linkage

• Use family pedigrees
• Cotransmission of marker allele and trait (e.g.,
  disorder) can be traced

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Hypothetical Linkage Study
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Actual Pedigree Data

• Portion of Venezuelan HD pedigree
• Illustrates inheritance of HD gene on a
  chromosome bearing the C haplotype at the G8
  locus
• A single individual (arrow) representing a
  recombination event was found in this portion of
  the pedigree

12
Affected Sib-Pair Linkage Design

• Use families with two affected siblings
• Share 0, 1, or 2 alleles for DNA marker
• If marker not linked to gene, probabilities are
  25, 50, and 25 for sharing 0, 1, or 2 alleles,
  respectively
• If marker is linked, more than 25 of affected
  sib pairs will share two alleles for marker

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Candidate Genes

• Allelic association studies
• Gene that may influence the development of a
  disease or trait of interest
• Positional candidate genes selected for
  association studies based on their location in a
  genomic region linked to the trait of interest

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Whole Genome Scan
Genetic Map
Reference Markers
Physical Map
Distribution of SNPs
Chromosome 1

• Start with genome scan of 400 to 1000 markers
  spread across all chromosomes
• Markers are a dragnet
• Pinpoint location of disease genes
• Human chr. 1 with different types of genetic
  markers overlaid on the map

15
Dense Mapping

• Initial genomic scan carried out at low marker
  density
• Next, identify chromosome regions which may be
  linked to disease of interest
• Investigate by typing more markers (dense
  mapping)
• This is where SNPs are becoming popular and
  useful
• Etc.

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Limitations

• May not be possible to select potential candidate
  genes for some disorders
• Functional candidate genes selected for
  association studies if it seems plausible that
  they influence the disorder
• But
• Large number of possible candidate genes
• Functional candidates might not really be
  involved
• Expensive to carry out dense mapping

17
QTL Mapping

• Determine degree of association of specific
  region on genome to inheritance of trait of
  interest
• If high, region probably has something to do with
  trait expression
• Remember, QTLs for trait can be on multiple
  chromosomes

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LOD Score

• Logarithm (base 10) of Odds
• Statistical test for linkage analysis
• Steps
• Establish pedigree
• Estimate recombination frequency
• Calculate LOD for each estimate
• Estimate with highest LOD considered best
  estimate
• LOD score greater than 3.0 considered evidence
  for linkage
• 3.0 means likelihood of observing pedigrees if
  two loci not linked is less than 1 in 1000

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QTL-Scan for Genome
1,100 microsatellite markers for patients with
osteoporosis used for genome-wide scan.
ltbiology.plosjournals.org/perlserv/?requestslides
howtypefiguredoi10.1371/journal.pbio.0000069i
d3630gt
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QTL-Scan on Single Chromosome
A. chromosome 20 scan. B. Detail of linkage
peak showing microsatellite markers (STRs), SNP,
and gene locations.
ltbiology.plosjournals.org/perlserv/?requestget-do
cumentdoi10.1371/journal.pbio.0000069gt
21
Markers

• Originally, detectable phenotypes from coding DNA
  sequences used eye colour, protein production,
  etc.
• More recently, noncoding DNA sequences,
  microsatellites, fragment length polymorphisms
  (e.g., AFLPs, RFLPs), etc. used

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Genetic Marker

• Really, any known DNA sequence
• An observable variation in genomic loci
• Mutation, alteration
• Short sequence
• E.g., single nucleotide polymorphism
• Long sequence
• E.g., microsatellites

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Requirements

• Easily identifiable
• Associated with specific locus
• Highly polymorphic

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Detection

• Co-dominant markers
• Analyze one locus at a time
• Dominant markers
• Can analyze many loci at once
• Direct detection by RNA sequencing
• Indirect detection using allozymes
• Variant forms of an enzyme coded by different
  alleles at the same locus

25
Amplified Fragment Length Polymorphisms (AFLPs)

• Restriction enzymes cut genomic DNA
• Subset of fragments then amplified
• Highly sensitive method for detecting DNA
  polymorphisms
• High resolution and sensitivity at whole genome
  level
• Can amplify 50-100 fragments at once
• Dont need to know prior sequencing information

Electropherogram trace from AFLP analysis
lten.wikipedia.org/wiki/ImageElectropherogram_trac
e.jpggt
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Microsatellites

• Also called simple sequence repeats (SSRs)
• Polymorphic loci consisting of 1-4 base pairs
  repeating 10-100 times
• Serve as excellent gene markers
• Common throughout genome and specific sequences
  closely linked to particular loci
• Can be amplified through PCR

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Polymerase Chain Reaction (PCR)

• DNA polymerase used to amplify piece of DNA by in
  vitro enzymatic replication
• DNA polymerase, an enzyme, adds free nucleotides
  to 3 end of newly-forming strand
• Get millions of copies of DNA piece
• Gene, part of a gene, non-coding sequence
• PCR can be modified to perform specific genetic
  manipulations

28
Transgenic Organism

• Genetically modified organism
• DNA from different sources is combined in vitro,
  then transferred to live organisms
• Causes expression of modified or novel trait
• Can study effects of genes with unknown function
  on phenotypic traits
• Also used to make money

GloFish first GMO sold as pets
ltwww.glofish.com/images/glofish_005.jpggt
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Terms

• Chimera
• Organism having two or more different populations
  of genotypes in cells originating from different
  zygotes
• Genetic mosaic
• Two populations of cells with different genotypes
  in single organism developed from single
  fertilized egg
• Usually mutation during development only affects
  subset of adult cells
• Intersex condition some cells XX, others XY
• Trisomies from nondisjunction in early mitosis

Calico cats gene for fur colour on X chromosome
express both dominant (orange) and recessive
(black) alleles lten.wikipedia.org/wiki/ImageCurly
cat02.jpggt
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Knockout

• Useful for learning effects of genes that have
  been sequenced but have unknown or incompletely
  understood function
• Gene is made inoperative
• Compare knockout organism with normal

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Technique

• Isolate/identify gene
• New DNA sequence of gene is engineered (it will
  be inoperable)
• Typically also include marker gene (e.g.,
  flurorescence)
• Isolate stem cells from blastocyst and introduce
  new DNA sequence
• Insert modified stem cells back into a blastocyst
  and allow embryo to develop
• Organisms will be a chimera parts of their
  bodies will be from their original stem cells,
  other parts from engineered stem cells

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Effects

• The knockout gene is permanently inoperable
• Can examine for phenotypic changes
• Assess functionality of gene

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Limitations

• Certain percentage of gene knockouts are
  developmentally lethal
• Embryo cant develop to adulthood
• Some genes have different effects at different
  developmental points
• May not produce observable change

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Your Friend, the Transgenic Mouse

• Science of Transgenics, Part 1
• Science of Transgenics, Part 2

35
Knockdown

• GMO with reduced expression of one or more genes
• Typically refers to temporary effect from using
  siRNA or RNAi gene silencing
• Degrades mRNA transcript or blocks mRNA
  translation
• RNA Interference video

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Knock-In

• Specific allele, locus, or base sequence is
  manipulated to observe specific functions within
  the gene
• Typically leads to over expression of some trait
• Allows study of effect upon morphology and
  physiology of organism

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Non-human Animal Models

• Have the advantage of being able to induce
  polymorphisms
• Many non-human animal genes are homologous to
  those of humans
• Can serve as effective controlled models for
  human comparison at a variety of levels
• Cellular to organ to organisms

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Dictyostelium discoideum

• Social amoebae
• Grow as separate independent cells
• Interact to form multicellular structures under
  adverse environmental conditions
• Useful for studying genetic regulation of
  fundamental cellular processes
• Cytokinesis, motility, phagocytosis, chemotaxis,
  cell sorting, pattern formation, cell-type
  determination

ltdictybase.org/Multimedia/chemotaxis/ColorcAR.avigt
ltdictybase.org/Multimedia/LarryBlanton/dev.htmlgt
ltdictybase.org/Multimedia/LarryBlanton/culmBW.html
gt
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Applications

• Many issues of human health and disease depend
  upon individual cell behaviour
• E.g., cytokinesis is critical in cell
  proliferation integral part of immune response,
  tissue maintenance, and cancer
• E.g., chemotaxis important in inflammation,
  arthritis, asthma

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Molecular Genetics

• Genomic sequencing underway
• 6 chromosomes, 10,000 genes
• Several thousand genes have homologues to higher
  eukaryotes
• Over 400 genes identified so far for cell
  motility, signal transduction, cell
  differentiation

D. discoideum genomic information from chromosome
1
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Caenorhabditis elegans

• Nematode (round) worm
• 1 mm long
• 959 cells (302 nerve cells)
• About 19,000 genes, half of which have been
  identified by purpose

ltwww.nih.gov/science/models/c_elegans/worm.jpggt
42
C. elegans Neurons

• Cells visible during development
• Neuronal pathways of many behaviours known
• Genetic regulation determinable through knockout,
  etc.

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Observation in the Living System

• Can visualize gene expression in living worms
• Links gene expression to identified neurons
• Expression of fusion between mec-4 and GFP
  (highlights touch cells)

ltFrom Rankin (2002)gt
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Mice

• Main mammalian species for mutational screening
• Hundreds of mutant mice strains
• NIH Knockout Mouse Project (KOMP)
• Mouse embryonic stem cells containing a null
  mutation in every gene in mouse genome

Homozygous for piebald spotting allele of
endothelin receptor type B. ltwww.informatics.jax.o
rg/gt
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Flint et al. (1995)

• Utilized allelic associations to study QTLs in
  mice
• Open field activity (OFA)levels
• F2 mice derived from cross between high and low
  lines selected for OFA subsequently bred for 30
  generations
• Each F2 mouse has unique allele set from original
  parental strain
• One recombination, on average, per chromosome
  inherited from F1 strain

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Shuffling Markers
F2 intercross and recombinant inbred strains. Two
inbred strains (shown as two chromosome pairs,
black and white) are crossed to form an F1, and
then intercrossed to form an F2 to one of the
parental strains. Because the parental strains
are inbred there will be just two alleles
segregating at each locus in the F2, one from
each parental strain, and consequently the
genotype at any locus in the F2 must be one of
AA, BB or AB. Recombinant inbred strains are
generated by brother-sister mating a pair of F2
animals and inbreeding for at least 20
generations until the animals are homozygous at
all autosomal loci.
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QTL Mapping
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Other QTL Depictions