Download Differential Gene Expression in the Gastrula of Xenopus Laevis

Differential Gene Expression in
the Gastrula of Xenopus Laevis
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Differential Gastrula
mRna – DG mRNA
Transcribed during
Gastrula stage;
Selectively used
Maternal mRNA –
Passed from female
parent to the egg.
Background: Early Development
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Egg > Embryo >
Blastula > Gastrula >
Neurula > Tadpole
Gastrula - First
appearance of three
germ layers:
Endoderm,
Mesoderm, &
Ectoderm
Experimental Goal:
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Determine and Isolate the genes
responsible for early differentiation
Separate, study DG mRNA
Method & Obstacle:
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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:
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Use of modified
cDNA cloning
procedure
Use highly enriched
DG cDNA Library
Purify sequences by
hybridizing to ovary
mRna
Methodology Cont…
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Enriched DG cDNA
inserted to ClaI site
of pBR322 plasmid
vector.
Results in 150,000
clones in pBR322
vector.
Dot Blot Hybridization:
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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:
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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
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DG Clones and r5
(probes) hybridized
to Gastrula RNA
Lane 42 proof of
possible nuclear
precursor molecules
(in kilobases)
Nuclease Protection Assays
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DG mRNA is
hybridized to labeled
ss DG 42 DNA
excess probes
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Unhybridized mRNA
degraded by
nucleases
Hybridized mRNA
visualized via
Autoradiogram
NPA continued…
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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:
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DG Gastrula Calculated to be 48
picograms per Gastrula
Supports figure 2 data that DG mRNA is
synthesized de novo.
Conclusions:
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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.