1. 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.

2. Background: Early Development Egg > Embryo > Blastula > Gastrula > Neurula > Tadpole Gastrula - First appearance of three germ layers: Endoderm, Mesoderm, & Ectoderm

3. Experimental Goal: Determine and Isolate the genes responsible for early differentiation Separate, study DG mRNA

4. 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

5. Solution & Methodology: Use of modified cDNA cloning procedure Use highly enriched DG cDNA Library Purify sequences by hybridizing to ovary mRna

6. Methodology Cont… Enriched DG cDNA inserted to ClaI site of pBR322 plasmid vector. Results in 150,000 clones in pBR322 vector.

7. 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

8. 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)

9. Northern Blot DG Clones and r5 (probes) hybridized to Gastrula RNA Lane 42 proof of possible nuclear precursor molecules (in kilobases)

10. 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

11. NPA continued… Measurements were compared to a control and DG clone concentration was calculated. Calculated concentration adjusted to dot blot data in Fig. 5

12. DG Abundance Table: DG Gastrula Calculated to be 48 picograms per Gastrula Supports figure 2 data that DG mRNA is synthesized de novo.

13. 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.