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We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.
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2210372
???displayArticle.link???Genes Dev
Figure 1. The nucleotide sequence combined
from the cDNAs PR22 and PR23
and the predicted amino acid sequence.
PR22 begins with the T residue in the initiation
codon and concludes at the end of
the sequence. The remainder of the 5'-end
sequence was derived from PR23. More
than 100 bp from the 5' end and 200 bp
from the 3' end of PR23 were sequenced.
PR23 is identical to PR22, with the exception
of a silent third position change of C
to T at the residue Gly-24 and the use of
an alternative polyadenylation site. The
identity of PR23 to PR22 was further supported
by the identical restriction patterns
of the cDNAs. The polyadenylation site of
PR23 starts at the T residue, following the
second polyadenylation signal, as underlined.
Presumably, PR23 uses the first polyadenylation
signal and PR22 uses the
second one.
Figure 2. Comparison of the amino acid sequence of X. laevis
and rat trypsinogen. The prepeptide (amino acids 1-15) is
shown in italics, and the activation peptide (amino acids
16-23) is underlined. The 12 conserved cysteine residues involved
in disulfide bond formation are indicated by carets.
Figure 3. Genomic Southern blotting analysis of the trypsin
gene. Two micrograms of homozygous diploid DNA per lane
was used. (Lanes 1-5) BamHI, BamHI + HindIII, HindIII, PstI,
and EcoRI digests, respectively. (A) A blot, washed twice in
0.05x SSC, 0.1% SDS, at 65~ for 30 min. (B) A blot, washed
twice in 1 x SSC, 0.1% SDS at 65~ for 30 min. The bars at
right of A and B indicate the positions of MHindIII DNA size
markers.
Figure 4. Developmental Northern blotting analysis of the
cDNA clone PR22. One microgram of total RNA was used in
each lane and hybridized with a2p-labeled cDNA insert. The
sizes of the mature mRNA (0.8 kb) and two higher-molecularweight
RNA bands that could be precursors (2.8 and 5.4 kb)
were determined by use of an RNA size ladder (BRL). (The sizes
of 18S and 28S RNAs are 1.8 and 4.1 kb, respectively.) (Lanes
1-7) Total RNA from ovary, tadpoles at stages 41, 44/45, 54, 58,
and 62, and 6-month-old frog (-4 grams).
Figure 5. Quantification of trypsin mRNA at different developmental stages plotted with the known endogenous thyroid hormone
level (Leloup and Buscaglia 1977). Each point is the average of four different measurements that were reproducible within a factor of
two. The mRNA was quantified by slot-blot hybridization of total RNA from ovary, 6-month-old frog, or different stage tadpoles with
known amounts of PR22 cDNA as a standard and PR22 eDNA as a probe. The hybridization signal was measured by Cerenkov
counting. (Insert) The trypsin mRNA increase plotted at stages 62, 64, and 66 and frog {ng mRNA/mg total RNA) on an expanded
scale. The amount of RNA used was determined by UV absorption and verified after blotting by methylene blue staining (Herrin and
Schmidt 1988}. Standardization was also monitored by hybridization with the eDNA PR28 whose mRNA does not change (within
threefold) in abundance during development from ovary to frog (data not shown).
Figure 6. Effect of thyroid hormone on the level of trypsin
mRNA. One microgram of total RNA was used in each lane
and probed with PR22 eDNA. (Lanes 1-7) RNA isolated from
control tadpoles and those treated for 1 week with hormone
starting at the developmental stage given. (Lanes 1 and 2) T 4-
treated (26 r~) and control tadpoles at stage 46/47, respectively;
(lanes 3-5) T 4 (26 nM), T a (5 nM), and control tadpoles at
stage 50, respectively; {lanes 6-8) T 3 (5 riM), T 3 (1 nJvl), and control
tadpoles at stage 54, respectively; (lane 9) stage 58tadpole;
(lane 10) 6-month-old frog. When the filter was reprobed with
the control clone PR28, no thyroxine-dependent regulation of
its mRNA was observed (data not shown).
Figure 7. Time course of the effect of thyroid hormone treatment
on the level of trypsin mRNA. Stage 51- 53 tadpoles were
treated with 5 n~ T3, and RNA was isolated from the tadpoles
at the time indicated and analyzed by slot-blot hybridization.
The radioactive slots were counted. The level of trypsin mRNA
in control tadpoles does not change appreciably during this period
(see Fig. 5, stages 51-55).
Figure 8. Developmental regulation of elastase and carboxypeptidase mRNA content. Ten micrograms of total RNA from ovary,
tadpoles at stages 41, 44/45, 54, 58, and 62, and 6-month-old small frog was used in lanes 1-7, respectively. The Northern filters were
hybridized with rat elastase I cDNA (A} and rat carboxypeptidase B cDNA (B) overnight in 5 x Denhardt's solution, 5 x SSPE, 1% SDS,
and 100 ~g/ml denatured salmon sperm DNA at 60~ The hybridized filters were washed twice with 0.5 x SSC, 0.1% SDS, at 50~
for 25 min.
