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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 Presentation
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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.
Background: Early Development
Egg > Embryo > Blastula > Gastrula > Neurula > Tadpole
Gastrula - First appearance of three germ layers: Endoderm, Mesoderm, & Ectoderm
Experimental Goal:
Determine and Isolate the genes responsible for early differentiation
Separate, study DG mRNA
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
Solution & Methodology:
Use of modified cDNA cloning procedure
Use highly enriched DG cDNA Library
Purify sequences by hybridizing to ovary mRna
Methodology Cont…
Enriched DG cDNA inserted to ClaI site of pBR322 plasmid vector.
Results in 150,000 clones in pBR322 vector.
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
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)
Northern Blot
DG Clones and r5 (probes) hybridized to Gastrula RNA
Lane 42 proof of possible nuclear precursor molecules
(in kilobases)
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
NPA continued…
Measurements were compared to a control and DG clone concentration was calculated.
Calculated concentration adjusted to dot blot data in Fig. 5
DG Abundance Table:
DG Gastrula Calculated to be 48 picograms per Gastrula
Supports figure 2 data that DG mRNA is synthesized de novo.
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.