Genetics PCB 3063

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Genetics - PCB 3063

• Todays focus
• Polyploidy, Aneuploidy, Transposons and
  Population Genetics
• We will focus on 4 major questions today
• What are the consequences of changes in
  chromosome number and structure?
• Which types of genetic elements are capable of
  movement in the genome?
• How is genetic variation assessed?
• What frequencies of alleles do we expect in
  populations?

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Polyploidy and Aneuploidy - Terms

• Although diploid organisms are common, not all
  organisms are diploids

• Often, monoploid organisms (like our old friend
  Neurospora crassa) are called haploid.
• Organisms that contain only complete sets of
  chromosomes are called euploid.
• If an organism does not have one or more complete
  sets of chromosomes, it is called aneuploid.
• In many cases, aneuploidy is associated with
  pathological phenotypes.

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Some Polyploids are Infertile

• Triploids and other polyploids with odd numbers
  of chromosomes are often infertile.
• This reflects the fact that the unpaired
  chromosomes will segregate randomly.
• For example, a triploid organism with 4
  chromosomes will give rise to different classes
  of gametes, e.g.
• The probability of 2N gamete is 0.0625 based upon
  the multiplication rule.
• In some cases, this infertility can reflect
  abnormalities of gametes with incomplete sets of
  chromosomes.
• This infertility may be limited to male or female
  gametes.

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Tetraploids from Triploids

• Triploids can give rise from tetraploids.
• This is typically called passage through the
  triploid block.

2N
2N
2N
1N
Pollen
3N
Pollen Various Chromosome Numbers
4N
Most common viable offspring
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Types of Polyploids

• Chromosome sets in polyploids can have a number
  of distinct origins.
• If the chromosome sets are sufficiently similar
  that they can pair, usually reflecting fusion of
  unreduced (2N) gametes within a species, the
  polyploid is called an autopolyploid.
• If the chromosome sets are dissimilar and unable
  to pair, usually reflecting the fusion of gametes
  from different species, the polyploid is called
  an allopolyploid.
• In some cases, usually when parental species are
  known, allotetraploids are called amphidiploids.
• Allopolyploids can be of two major types
• If the chromosomes cannot pair at all, they are
  often called genomic allopolyploids.
• Result from the fusion of gametes from fairly
  distinct species.
• If the chromosomes pair partially, so there are
  regions that cross-over, they are often called
  segmental allopolyploids.
• Reflect fusion of gametes from relatively closely
  related species.

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Types of Polyploids - Allopolyploids

• Allopolyploidy can allow crosses of relatively
  distinct species...

• But sometimes the crosses arent valuable.
• E.g., the Rabbage was an attempt to cross the
  radish (2N18) and cabbage (2N18). The cross was
  infertile but allowing the chromosome number to
  double generated fertile plants. However, rabbage
  plants had small roots and small leaves...

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Plants, Animals and Polyploids

• Polyploidy has played different roles in the
  evolution of plants, animals and fungi.
• Polyploidy is very common in plants, but rare in
  the animals and fungi.
• This may reflect the ability of plants to
  reproduce vegetatively, at least in part.
• The paucity of polyploid fungi is unclear.
• More than 70 of angiosperms and 95 of ferns
  have polyploid ancestors.
• Both maize and Arabidopsis are diploids but they
  show clear evidence of being polyploids that
  reverted to disomic inheritance.
• In both of these organisms, many genes are
  duplicated and the duplications occur in blocks.
• For maize, more than 85 of loci appear to have
  been duplicated.
• See http//www.agron.missouri.edu/mnl/73/47braun.h
  tml for examples of a few duplicated maize loci,
  if you are interested.

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Plants, Animals and Polyploids

• Despite the paucity of polyploid animals and
  fungi, these processes have played a role in the
  evolution of these groups.
• S. cerevisiae appears to have been a tetraploid
  that reverted to disomic inheritance.
• Like Arabidopsis, there are a number of
  duplicated blocks in the S. cerevisiae genome.
• So the ancestor of S. cerevisiae was probably a
  tetraploid that reverted to disomic inhertiance
  and lost many duplicate loci.
• Vertebrates also have a large number of duplicate
  genes.
• These have been proposed to reflect two rounds of
  duplication due to the reversion of tetraploids
  to disomic inheritance.
• This idea is still controversial -- the
  duplicated genes in vertebrates could simply
  reflect a period of active gene duplication.

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Aneuploidy

• In normal diploids, individuals are said to be
  disomic for all chromosomes.
• If three copies of a specific chromosome are
  present, the individual is said to be trisomic
  for that chromosome.
• Likewise, if one copy of a specific chromosome
  are present, the individual is monosomic for that
  chromosome.
• Cases in which two chromosomes are present in
  unusual numbers are referred to as cases where
  the organism is doubly trisomic or monosomic.
• With the exception of sex chromosomes, humans
  tolerate trisomy and monosomy poorly.
• Down syndrome is trisomy 21, and it is associated
  with a number of phenotypes.
• Trisomics for the larger chromosomes in humans
  are lethal during development, and embryos that
  have these abnormalities undergo spontaneous
  abortion.

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Aneuploidy

• Thus, most aneuploidies in humans involve the sex
  chromosomes
• Down syndrome - trisomy 21.
• Turner syndrome - single X chromosome.
• Short stature, limited sexual development, mental
  retardation
• Klinefelter syndrome - multiple X chromosomes
  along with a Y
• Males, but some female characteristics.
• XYY - male, no clear phenotype.
• The basis for the viability of these individuals
  is likely to be the fact that dosage compensation
  mechanisms exist for the sex chromosomes.
• Also, the Y has very few genes, so little
  potential to disrupt development.
• The fact that these syndromes do have phenotypes
  indicates that dosage compensation is imperfect.

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Aneuploidy of Sex Chromosomes

• The potential for viable sex chromosome
  aneuploids is also seen in other organisms.

• This shows a bilateral gynandromorph -- an
  organism in which a mitotic nondisjunction has
  occurred.
• Thus, this organism is a somatic aneuploid.
• Since this is a moth, what would you call the sex
  chromosomes that were involved?

• At a cellular level, aneuploidy can play a major
  role in the development of cancer.
• E.g., loss of chromosomes or chromosome regions
  with tumor suppressors.

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Chromosomal Rearrangements

• A number of chromosomal rearrangements are
  described in your text.
• Rearrangements can lead to pathological
  conditions.
• Deletions of regions lead to specific phenotypes.
• This has been used to map genes in some organisms
  (e.g., Drosophila).
• Alternatively, fusion of a strong promoter from
  one gene to another gene can cause unusual
  phenotypes.
• The yellow allele of the mouse agouti gene
  described in chapter 4 (page 105) is a deletion
  that results in the overexpression of the agouti
  gene. The lethality of the allele actually
  reflects deletion of a gene adjacent to the
  agouti gene that is essential for viability.
• Burkitt's lymphoma (BL) is characterized by
  translocations between chromosome 8 and 14 (90
  of cases). The c-myc protooncogene maps to the
  chromosome 8 breakpoint and Ig (immunoglobulin)
  genes map to the chromosome 14 breakpoint.
  Placing the c-myc gene near the Ig promotor
  (active in B lymphocytes) results in increased
  expression of c-myc - which activates cellular
  proliferation.

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Chromosomal Rearrangements

• However, it is important to stress that these
  rearrangements occur during evolution and
  represent on mechanism isolating species.
• For example, two species of dik-diks were
  recognized until people examined karyotypes,
  revealing three species.
• The cryptic species of dik-dik cannot hybridize,
  indicating that the karyotypic differences are
  meaningful.

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Transposons

• Transposons - jumping genes
• The phenomenon of transposition was originally
  noted by Marcus Rhoades and Barbara McClintock in
  maize.
• Rhoades noted an unusual pattern of reversion at
  the A1 locus.
• A1 (anthocyaninless1) is necessary for formation
  of the purple anthocyanin pigments in maize.
• The dominant allele (A1) is colored while the
  recessive (a1) is clear. NOTE THAT THE TEXT IS
  INCORRECT HERE.
• Rhoades noted that a specific a1 mutant actually
  reverted multiple times within a kernel of corn -
  giving a dotted appearance

Note the spotted kernels - the spots are due to
the transposon jumping out of the A1 gene.
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McClintock and the Ac-Ds System

• Barbara McClintock noted that recessive markers
  on maize chromosome 9 could be revealed at a high
  rate in a specific mutant.
• This phenotypic change was accompanied by a
  cytological change - loss of part of the short
  arm of chromosome 9!

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McClintock and the Ac-Ds System

• McClintock called the breakpoint in these lines
  Ds, for dissociation.
• The break would not occur unless an Ac element
  (activator) was crossed in.

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What are Ac and Ds?

• Ds elements can also move in the genome.
• Thus, they did not behave in the way genes have
  been shown to behave previously - they did not
  have defined loci in the genome!
• In addition, they relied upon the Ac element for
  activity. Why?
• When Ac and Ds elements were cloned, it was shown
  that they were related.
• Ac has two open reading frames (ORFs), while Ds
  elements have deletions covering at least part of
  the first ORF.

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What are Ac and Ds?

• When Ac and Ds elements were cloned, it was shown
  that they were related.
• Ac has two open reading frames (ORFs), while Ds
  elements have deletions covering at least part of
  the first ORF.
• The first ORF is a transposase...
• It is necessary for insertion at novel loci,
  excision, and chromosome breakage.
• Ac will supply the transposase in trans -
  activating the Ds elements.

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Examining Transposons in Maize
From Feschotte, Jiang and Wessler (2002) Nature
Reviews Genet. 3 329-41.
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Types of Transposons

• Ac and Ds are DNA transposons.
• The transposition does not involve an RNA
  intermediate.
• Ac is an example of an autonomous element (can
  transpose by itself).
• Ds is a non-autonomous element (requires
  trans-acting factors from another element).
• In this case, Ac supplies the transposase - the
  trans-acting factor.
• Bacterial insertion sequences and Drosophila P
  elements are DNA transposons.
• A particularly interesting group of DNA
  transposons are the MITEs --
• MITEs predominate in the non-coding regions of
  grass genes.
• MITEs elements are structurally reminiscent of
  non-autonomous DNA transposons, are small (lt 600
  bp) and have terminal inverted repeats. However,
  their high copy number, target-site preference
  (TA or TAA) and the uniformity of related
  elements distinguished them from the previously
  described transposons.

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Types of Transposons

• Other transposons are retrotransposons.
• These elements have an RNA intermediate.
• In many cases, they resemble retroviruses but
  lack the env (envelope) gene.
• Some elements transpose through an RNA
  intermediate but are distinct from retroviruses.
• These include the AluI elements and L1 elements
  of humans.
• The human genome has more than 500,000 AluI
  elements and 100,000 L1 elements.

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Assessing Genetic Diversity

• A variety of methods have been used to examine
  genetic variation in populations
• Observation of phenotypes
• This is inexpensive and straightforward
• Problematic for several reasons
• Some genes contribute to continuous variation.
• Some genes have unknown phenotypes.
• Examination of proteins
• Typically done using electrophoresis.
• Direct examination of DNA variation
• Can be accomplished by sampling a subset of sites
  in DNA, either at a specific locus or across the
  genome.
• Can be accomplished by sequencing loci.
• Sequencing is still relatively expensive.

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Protein Electrophoresis

• Protein variation is often examined using gel
  electrophoresis
• Usually, either starch or cellulose acetate gels
  are used.
• The gels are run under non-denaturing conditions,
  so enzymes can be detected by stained with
  substrates that change color.
• Migration is determined by charge and size of the
  proteins - most variation is charge.
• Many proteins will migrate to the anode () but
  the same principle works for the cathode as well

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How do Variants Migrate?

• Imagine a population that has two different
  alleles of a gene encoding a protein we are
  examining
• If the alleles encode proteins with different
  charges, the are likely to migrate at different
  rates.
• Typically, these variants are called fast and
  slow. If so, we will see
• If the enzyme is not a monomer, more complex
  patterns will result.
• For example, a dimer would yield
• What are the bands composed in this gel?

A - slow homozygote. B - heterozygote. C - fast
homzygote.
A
B
C
A
B
C
A - slow homozygote. B - heterozygote. C - fast
homzygote.
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RFLP - Examining DNA Variation

• RFLPs are Restriction Fragment Length
  Polymorphisms.
• Restriction enzymes cut at defined sequences in
  DNA. For example, EcoRI cuts at
• If an EcoRI site is present in some members of a
  population but not others, it is an RFLP.
• Alternatively, the polymorphism my reflect a
  large indel between two EcoRI sites.
• For a single locus, one can detect RFLPs using
  either PCR or Southern blotting.
• For a Southern, DNA is sized (using agarose gel
  electrophoresis)
• Then the DNA is transferred to a membrane.
• Labeled probe DNA (corresponding to the locus of
  interest) is hybridized to the membrane.
• Alternatively, one PCR amplifies the locus and
  cuts with the enzyme of interest.

----GAATTC---- ----CTTAAC----
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Multilocus (RFLP) Fingerprinting

• However, a much more powerful approach was
  proposed by Alec Jeffries in the 80s.
• This is multilocus RFLP analysis, often called
  DNA fingerprinting.
• One uses a probe to VNTR (variable number tandem
  repeat) sequences and then examines a Southern
  blot of DNA cut with various enzymes.
• Usually the repeat number changes sufficiently
  rapidly that most populations will show
  differences among individuals.
• This method is commonly used in forensics and for
  paternity analysis.

A DNA fingerprint blot
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PCR

• In contrast, PCR methods are limited to known
  loci.
• This reflects the need for primer sequences.
• PCR is popular because little template DNA is
  necessary.
• Most of the template in later rounds was
  generated in early rounds.

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RAPD PCRExamining Multiple Loci

• Because so little DNA is necessary, PCR is
  desirable for population surveys.
• Likewise, PCR is very useful for forensics.
• Can it be used to survey multiple loci?
• One method is to use short primers with arbitrary
  sequences, but anneal at a lower temperature.
• Typically, one uses specific primers that are 18
  to 30 mers and anneals at temperatures over 50C.
• Under these conditions, typically only one locus
  will anneal to both primers and amplify.
• However, RAPD PCR uses shorter primers (12 mers
  in some cases) and lower temperatures.
• Many loci in the genome will anneal to these
  primers and amplify.

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RAPD PCR Related Methods

• RAPDs still requires regions that match the
  primers.
• But the low annealing temperature and short
  sequence length allow the primers to anneal at
  many more places than in a standard (locus
  specific) PCR reaction.
• Since the specificity is so low, one actually
  uses a single primers.
• This will amplify only loci that have two
  flanking sites that can anneal to the primer.
• Do you think the loci amplified by RAPD PCR will
  correspond to known loci? or to protein coding
  loci?
• Can you distinguish homozygotes from
  heterozygotes using RAPDs?
• A number of similar techniques have been
  developed (e.g., ISSRs) as have methods that use
  PCR to scan polymorphic restriction sites (AFLPs).

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SNPs - Sequence Variation

• One can also amplify specific loci by PCR and
  examine the sequences.
• Nucleotide sites that are polymorphic are called
  single nucleotide polymorphisms or SNPs.
• This method is a much more powerful method to
  examine all variation at a specific locus.
• However, it is expensive.
• It can only examine a single locus at a time.

• Sequencing can detect heterozygotes.
• Heterozygotes will show both peaks in a
  sequencing reaction.

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Microsatellites - Fragment Analysis

• Like VNTRs (minisatellites) used in DNA
  fingerprinting, other types of repeat loci are
  popular targets in population studies.
• Microsatellites are repeats of 2 or 3 nucleotides
  (like ...GTGTGTGTGTGTGTGT)
• Microsatellites often vary in length due to
  slippage of DNA polymerase during replication.

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Microsatellites - Fragment Analysis

• Microsatellite repeat loci are popular targets in
  population studies.
• If one designs primers to a specific
  microsatellite locus (in the flanking
  non-repetitive region) one can amplify the
  microsatellite by PCR.
• Then, one can separate the fragments by size
  using a DNA sequencer to look for size
  polymorphisms. (dont actually sequence)

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What do we do with this data?

• Information about genetic variation in not very
  useful in the absence of methods to analyze the
  data.
• Typically one examines specific patterns that one
  expects for the variation.
• The simplest expectation is given by
  Hardy-Weinberg equilibrium.
• If we have a population in which individuals are
  randomly mating and there are two alleles at a
  locus, the expected proportions of homozygotes
  and heterozygotes is
• We expect p2 homozygotes for allele 1, where p is
  the frequency of allele 1.
• Likewise, we expect q2 homozygotes for allele 2,
  where q is the allele 2 frequency.
• Finally, we expect 2pq heterozygotes, where p and
  q are defined above.

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What do we do with this data?

• The simplest expectation is given by
  Hardy-Weinberg equilibrium.
• If we have a population in which individuals are
  randomly mating and there are two alleles at a
  locus, the expected proportions of homozygotes
  and heterozygotes is
• We expect p2 homozygotes for allele 1, where p is
  the frequency of allele 1.
• Likewise, we expect q2 homozygotes for allele 2,
  where q is the allele 2 frequency.
• Finally, we expect 2pq heterozygotes, where p and
  q are defined above.

It is easy to think of Hardy-Weinberg equilibrium
graphically, like this...
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TestingHardy-Weinberg Equilibrium

• How do we test whether the data we have collected
  fit Hardy-Weinberg equilibrium?
• One way is to use our old friend, the c2 test!