Copia Retrotransposable Element Host Control Mechanism in Drosophila melanogaster

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Copia Retrotransposable Element Host Control
Mechanism in Drosophila melanogaster

• Wendy Vu

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Eureka! Transposable Elements!

• Discovered in the mid-1940s by B. McClintock
• -Color Mosiacism in maize
• -Controlling elements

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TE Background info

• found in plants, animals and insects
• 10-90 of some genomes are TE derived sequences
• TE copy number can reach 100,000 per genome

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Transposable elements effects on the host

• Deleterious mutations
• Chromosomal breakage
• Host fitness decreases by 5-100
• Sterility

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Hybrid Dysgenesis
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Natural Variation in TE Activity

• In 1988, Eggleston observed copia transposition
  but none for I, gypsy and hobo in one genotype
• In 1990, Harada used a different genotype and
  observed transposition activity of hobo and I but
  none for copia

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Examples of TE Transposition Mechanisms

• Cut and Paste mechanism
• -P-element

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Examples of TE Transposition Mechanisms

• Transposition via an RNA intermediate
• -SINE-type elements Alu
• -LTR-retrovirus Copia, Gypsy

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Copia background info

• Abundant in plants, present in insects and
  uncommon in vertebras
• Transpositions occur only in male germ cells
• Forms virus like particles in the nucleus

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Copia is a long terminal repeat retrotransposon
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Copia Life Cycle
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Nature Reviews Genetics 8, 272-285 (April 2007)
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Piwi-interacting RNAs (piRNAs)
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Question to start with

• Is copia suppression mediated by
  post-transcriptional silencing?

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Isolating Copia plasmids

• We used isogenic D. melangaster flies
• -2b3 (active)
• -Ore (stable)
• Designed radioactive 33P primers to isolate the
  junction between the double-LTRs for PCR

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Results

• On the electrophoresis gel...
• -2b3 (active line) had an intense band of the
  expected size of 260 bp
• -Ore (stable line) had no bands corresponding to
  the expected size

260 bp
2b3
Ore
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98 Recombinant Inbred Lines (RILs)
Ore
2b
Backcross
F1
2b
Repeated full sib mating
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Copia plasmid level is refective of transposition
rate
-70 of RILs had no detectable plasmids -30 of
RILS had plasmid concentrations intermediate to
the parental lines -transposition was detected
in 5 RILs from 30
Freq. of Genotypes
Concentration of copia plasmids (log Scale)
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Plasmid concentration moderate correlation
between copy number and transcript level
r 0.314 P 0.002
Copia Plasmid Concen.
r 0.285 P 0.004
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QTL mapping

• Results
• -We detected a single QTL on the 2nd chromosome
  mapping to the pericentric region (33A-43E)

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PiRNA distribution along chromosome 2
Cell 128 (2007), pp. 10891103.
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Quantitative deficiency mapping analysis of copia
plasmid concentration on the 2nd Chromosome
2L
2R
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Deficiency Crosses
Def/CyO
Ore
Def/CyO
2b3
F1 Genotypes Males
Def/2b3 Cyo/2b3
Def/Ore Cyo/Ore
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Relative concentration of copia plasmids

• QPCR Results
• -2b3/Def, 2b3/CyO gt Ore/Def, Ore/CyO
• -Def 1054 showed elevated levels of copia
  plasmids in both 2b3 and Ore

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Nuage

• Vasa is a DEAD-box RNA Helicase
• -req. transports important mRNA required for
  Oogenesis and implicated in translational
  control.
• Aubergine aka Piwi protein
• -involved in RNAi transcriptional silencing
  pathway
• Tudor protein
• -implicated to have RNA-binding capacity

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Acknowledgements

• Sergey Nuzhdin (USC)
• Andrew Flavell (University of Dundee)
• Negar Kahen (USC)
• John Joseph (USC)

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Semi-Quantitative analysis of copia plasmid
concentration on RILs

• copia plasmid concentrations compared to a known
  concentration of pBB54 plasmids
• Relative copia plasmid concentrations were
  estimated as the ratio of signal intensity of the
  admix
• Assume that admix signal intensity represents
  zero

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Results

• Copia plasmid concentration
• -Intensity of 2b parental line 45-fold stronger
  than the admixed sample
• -Intensity of Ore parental line was close to the
  admixed sample
• -30 had intensities intermediate to 2b and Ore
• - we detected transposition in 5 RILs

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RNAi Transcriptional Silencing Pathway
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(No Transcript)
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Deficiency mapping Results

• Trends in copia plasmid concentration
• -F1 originating from 2b gt F1 from Ore
• -Cyo/2b Cyo/Ore gt Def/2b Def/Ore
• -1 deficency deviated from the general trend

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Future Directions

• Look at what specific role the vasa gene plays in
  suppressing copia transpositions.
• Look at whether the suppression mechanism is
  maternally inherited

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Recombinant Inbred Lines