differential gene expression in the gastrula of xenopus laevis
DESCRIPTION
Differential Gastrula mRna – DG mRNA Transcribed during Gastrula stage; Selectively used Maternal mRNA – Passed from female parent to the egg. Differential Gene Expression in the Gastrula of Xenopus Laevis. Egg > Embryo > Blastula > Gastrula > Neurula > Tadpole - PowerPoint PPT PresentationTRANSCRIPT
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Differential Gene Expression in the Gastrula of Xenopus Laevis
Differential Gastrula mRna – DG mRNA
Transcribed during Gastrula stage; Selectively used
Maternal mRNA – Passed from female parent to the egg.
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Background: Early Development
Egg > Embryo > Blastula > Gastrula > Neurula > Tadpole
Gastrula - First appearance of three germ layers: Endoderm, Mesoderm, & Ectoderm
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Experimental Goal:
Determine and Isolate the genes responsible for early differentiation
Separate, study DG mRNA
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Method & Obstacle:
Plan Of Action: Hybridize mRNA to cDNA library (Maternal & DG mRNA) via Colony Hybridization.
Problem: 0.05% of 10000 mRNA too rare for detection
Examples: If Blastula is tested,
Maternal RNA has strong signal
If Gastrula is tested DG RNA has strong signal
Rare mRNA not detected
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Solution & Methodology:
Use of modified cDNA cloning procedure
Use highly enriched DG cDNA Library
Purify sequences by hybridizing to ovary mRna
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Methodology Cont…
Enriched DG cDNA inserted to ClaI site of pBR322 plasmid vector.
Results in 150,000 clones in pBR322 vector.
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Dot Blot Hybridization:
Six clones were picked from “reference cDNA library” (+) control)
pBR322 fragment (-) Hybridized to labeled
probes from Egg, Blastula, Gastrula & Tadpole stage RNA Fig. 2
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Southern Blot:
DNA from 9 nonhomologous clones labeled by nick translation
Hybridized by Southern Blot using Eco-RI digest of Xenopus genomic DNA (Fig. 3)(in kb)
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Northern Blot
DG Clones and r5 (probes) hybridized to Gastrula RNA
Lane 42 proof of possible nuclear precursor molecules
(in kilobases)
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Nuclease Protection Assays
DG mRNA is hybridized to labeled ss DG 42 DNA excess probes
Unhybridized mRNA degraded by nucleases
Hybridized mRNA visualized via Autoradiogram
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NPA continued…
Measurements were compared to a control and DG clone concentration was calculated.
Calculated concentration adjusted to dot blot data in Fig. 5
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DG Abundance Table:
DG Gastrula Calculated to be 48 picograms per Gastrula
Supports figure 2 data that DG mRNA is synthesized de novo.
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Conclusions:
Enrichment Cloning technique was a success
Confirmed the presence of Differential Gastrula mRNA separate from Maternal mRNA
Gradually disappear after Gastrula; Implication that it has little preceding stages. Some increase in concentration.