Detection in situ of genomic regulatory elements in Drosophila

1
Detection in situ of genomic regulatory elements
in Drosophila

• Cahir J. OKane and Walter J. Gehring

2
Abstract

• Approach for in situ detection of genomic
  elements that regulate transcription in
  Drosophila.
• Analogous to random generation of operon fusions
  that enables the isolation and characterization
  of genes in prokaryotes.
• Expression of lacZ gene with a P-element promoter
  in germ-line transformant flies used to screen
  for chromosomal elements that can act at a
  distance to stimulate expression from the
  promoter.

3
Introduction

• Operon fusions in prokaryotes.
• The eukaryote system.
• Overview of the approach.

4
Operon fusions in prokaryotes.

• Promoterless reporter gene integrated into
  chromosomal DNA.
• The reporter gene would be controlled by a random
  selection of chromosomal promoters.
• Expression can be easily detected and assayed.
• Enables the isolation and characterization of
  genes.

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The eukaryotic system.

• For efficient expression of reporter gene
• 1. An active promoter
• 2. Reporter gene needs to be first in fusion
  transcript.
• Expression levels of reporter gene dependant on
  genomic position.
• Allow detection of genomic elements that regulate
  transcription at a distance.

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Overview of the approach.

• Reporter gene lac z gene of E. coli.
• P-element promoter-lac Z fusion.
• P-element trasformation into the Drosophila
  genome.
• Tissue specific expression of lac z visualized by
  X-gal staining for b-galactosidase activity.
• Expression of the reporter gene would reflect the
  activity of nearby enhancer elements in genome.

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Discussion

• Hypothesis
• Fusion construct Plac, ry
• Methods

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Hypothesis

• Promoter used to express lac z on the P element
  should have 3 properties
• Relatively weak
• Active constitutively in all cells throughout
  development
• Lie at one end of the P element
• Candidate P-element promoter

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Fusion construct Plac, ry

• 1. In frame translational fusion of lac z to the
  second exon of P element.
• 2. Both ends of P elements, including the P
  promoter at 5 end.
• 3. rosy gene as marker.
• 4. Trailer sequences and polyadenylation site of
  Drosophila hsp70 gene.

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Methods

• pLacA92 injected into ry506 Drosophila embryos
  along with helper P element.
• Helper P element produces transposase but itself
  incapable of transposing.

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Methods

• 171 injected survivors G0.
• 39 G0 gave rise to 348 ry G1.
• Transformant offspring (G1) pooled and
  intercrossed to obtain G2.
• G1 backcrossed to ry- if transformants were one
  sex only.
• Embryos collected overnight from 39 G2 pools, and
  x-gal stained.
• Individual G2 used to breed stocks homozygous for
  each Plac, ryA insertion.

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Results

• 20 of pools have no b-galactosidase activity.
• 80 give rise to mixture of lac and lac-
  activity.

13
Results

• 49 insertion strains with different patterns of
  lac Z expression bred from the G2 interbreeding.
• Of the 49 lines, 12 expressed lac Z in all or
  most of the embryo.
• 37 showed spatially regulated expression of lac Z.

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Results

• Examples of the pattern shown in Fig.2
• 23 strains showed expression in either the
  central or peripheral nervous system.
• 7 of these in all or most of CNS. (e,f)
• 13 shows distintive subset cells in CNS. (g,h)
• 8 in thoracic and abdominal peripheral nervous
  system.

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Results

• 13 in guts
• Others in epidermis (c,d,j), malpighian tubules,
  muscles and amnioserosa.
• 2 strains expressed in 2 segment periodicity.
• 10 homozygous lethal.

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Conclusion

• Expecting detection of enhancer-like elements.
• Could be transcriptional readthrough from
  Drosophila promoter.
• Expression of P promoter dependant upon
  integration site.
• The approach also allows detection of genes.
• Overall, this approach is found to be efficient
  for the generation of specific cell marker.