Differential Gene Expression in the Gastrula of Xenopus Laevis Differential Gastrula mRna – DG...

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.