Determining Gene Sequence

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Chapter 7

• Part 2

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Determining Gene Sequence

• Single crossover leads to determining distance
  but not sequence
• require multiple crossovers to determine order
• Multiple crossovers between non-sister
  chromatids 2, 3 or more exchange may occur
• Double crossover (DCO) double exchange

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DCO

• 3 pairs of genes, heterozygous for 2 alleles
• DCO require 2 independent events to occur
  simultaneously
• depends on distance between the genes
• probability of 2 independent events is equal to
  the product of individual probabilities product
  law
• Single exchange recovered 20 - p0.20 between A
  and B
• Single exchange recovered 30 - p0.30 between B
  and C
• Together (0.2)(0.3)0.06
• to be less than either single exchange

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DCO Continued

• If A and B are 3 mu apart then p0.03
• If B and C are 2 mu apart then p0.02
• Double crossover (0.03)(0.02)0.0006 or 6
  events in 10,000 events
• Requires many offspring to find the DCO

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3 Point Mapping in Drosophila

• Successful mapping must meet 3 criteria
• 1) genotype of organism must be heterozygous at
  all loci being examined
• 2) Crosses must allow genotype to be determined
  by looking at phenotype
• 3) Sufficient offspring produced to recover
  representative sample of crossing over

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Linkages

• Yellow body color (y) white eye color (w) and
  echinus eye shape (ec)
• All 3 genes are on the X chromosome
• Assume a theoretical order not sure if it is
  correct
• for now we will order them ywec

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Crosses to Determine Map

• ? is hemizygous wild type y-w-ec/Y
• ? is homozygous recessive y-w-ec/y w ec
• This satisfies criterion 1
• F1 ? y-w-ec/Y and ? y-w-ec/ y-w-ec
• This satisfies criterion 2
• Determine crossover and non-crossover categories
• Determine F2 generation
• If crossover occurs, create a reciprocal class -
  1 chromosome will have 1 mutant allele and the
  other will have 2 mutant alleles

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P1 and F1 Generations
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Determining Gene Sequence

• Data supports our earlier arrangement y-w-ec
• Most of the time the sequence is not known and
  becomes another variable
• use simple methods to determine
• 3 genes so 3 possible orders
• I w-y-ec
• II y-ec-w
• III y-w-ec

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Method

• Determine arrangement of alleles on each homolog
  of heterozygous parents non-crossover /
  crossover (F1 female)
• Determine if DCO can occur within the arrangement
  to produce the DCO phenotypes
• Doesnt work try another arrangement

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Mapping Problem in Maize

• Gene sequence and interloci distance unknown
• 2 differences from fly problem
• fly is on the X-chromosome while this is
  autosomal genes
• change the symbols from flies use a to
  indicate wild type
• Still must meet 3 criteria
• Looking at brown midrib (bm) virescent seedling
  (v) and purple aleurone (pr)
• all linked to chromosome 5

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Estimates May Not be Accurate

• Assume crossover frequencies are directly related
  to distance between 2 alleles
• Cannot always detect all crossovers especially
  if the DCO didnt alter the phenotype of the
  organism
• Tend to underestimate the distance because of
  undetected crossovers further genes are apart
  the more likely there will be those
• Most accurate maps are between genes are close
  together

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Interference

• In maize example we saw 7.8 DCO
• Can calculate the expected percentage of DCO
• DCOexp (0.223)(0.434) 9.7
• Since the numbers are the same interference has
  occurred crossover events in one region of
  chromosome inhibits a second event in a nearby
  region

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Coefficient of Coincidence (C)

• Calculate C using the following formula
• Quantify interference I 1 C
• 1 0.804 0.196 interpretation 19.6 fewer
  DCO happened than expected
• I 1 if no interference
• If I is positive fewer observed than expected
  positive interference
• If I is negative more observed than expected
  negative interference

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Interference in Eukaryotes

• Positive interference in eukaryotes
• Closer genes are the more positive interference
• 10 mu cause complete interference in fruit fly,
  physical constraints present the formation of
  closely aligned chiasmata
• interference decrease when genes are further apart

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Drosophila Map

• Locus of mutant gene is 1st localized to 1 of 4
  or linkage group

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Lod Score Analysis/Somatic Cell Hybridization

• Techniques used to determine linkage in humans
• Lod scoring uses probabilities to determine if
  linkage occurs between 2 genes log of odds
• Somatic cell hybridization 2 cells are fused to
  form a single hybrid, can mix 2 species together,
  called a heterokaryon, 2 nuclei in common
  cytoplasm
• 2 changes occur 1) nuclei eventually fuse
  (synkaryon) and 2) chromosomes are gradually lost
  from parents
• in mouse/human heterokaryon, it is mainly the
  human chromosomes that are lost

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Human/Mouse Synkaryon

• Use cell culture to study which gene product is
  made and on what chromosome it may be on
• see protein, must come from one of the
  chromosomes present in the culture
• use data to determine the location of A, B, C and
  D on chromosomes 1 thru 8

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Map Human Chromosomes

• Use somatic cell hybridization to assign 100s of
  genes
• Use recombinant DNA and pedigree analysis to
  determine other gene locations
• Huntington disease 4
• cystic fibrosis 7
• neurofibromatosis 14

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Linkage Maps in Haploids

• Crosses made following fertilization and isolate
  meiotic structures that make spores called a
  tetrad (different from other tetrad talked about)
• Do tetrad analysis

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Gene-to-Centromere Mapping

• Single gene in Neurospora can be studied to
  determine the gene-to-centromere distance
• Determine the frequency of recombination using
  tetrad data
• 4 meiotic products are formed, mitotic division
  occurs and results in 8 products acrospores
  which appear as aaaa or aaaa
• 1st division segrgation 2 alleles separate
• If crossover occurs will alter pattern
  aaaa aaaa aaaa or aaaa
  depending on orientation during second meiotic
  division
• 2nd division segregation

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Distance Calculation

• a albino wild type
• 65 1st divisions 70 2nd divisions

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Other Aspects of Genetic Exchange

• Crossing over leads to genetic variation in
  gametes that allow for organisms to adapt
• In 1930s found some cytological markers to
  indicate that crossing over really occurs
• McClintock and Creighton studied maize 2 genes
  on chromosome 9
• colorless (c) and colored (C) endosperm
  coloration
• starchy (Wx) and waxy (wx) endosperm
  carbohydrate characteristic

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Knob and Tail Markers

• Saw a know at one end of chromosome and a
  translocated segment from chromosome 18 on parent
  chromosomes
• Only interested in the cytological markers to
  help prove that crossing over occurred

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Sister Chromatid Crossover

• Occurs during mitosis sister chromatids do
  sister chromatid exchanges (SCE) determined
  using bromosdeoxyuridine (BUdR), a fluorescent
  nucleotide analog
• brighter in single copy but less fluorescent if 2
  copies
• arrow points to SCE
• called harlequin chromosomes due to the
  patch-like appearance

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Bloom Syndrome

• See increase in SCE in Bloom Syndrome
• Mutation in BLM gene which is thought to be a DNA
  helicase
• Pre- and post-natal retardation of growth,
  sensitivity of skin to sunlight, immune
  deficiencies, predisposition to malignant and
  benign tumors and abnormal behavior patterns
• See increase in breaks (SCE) in the chromosomes