|
FIG.1 . Organization of three nonhepaticxenopus arginasec DNAs ( zArgl4) .T he numbers are relative to the first nucleotiodfe z Arg2.
The lengthso f cDNAclones are 2.7,3.1, an1d. 6 kilobase pairsf or argl, arg2, and arg3, respectively. Each clone has ano pen reading framee ncoding
360 amino acids( shaded area) with the initiation( ATG) and termination (TAG) codons as indicated. argl and arg2 are highly homologous except
for a 400-nucleotide deletion (between nucleotides 2551 and 2975 of rArg2) and a few small deletions/insertions in the 3‘-untranslated region of
argl. The 5’- and 3’-end of arg3 are different from arg2 and in addition there are a few small deletionshnsertions in the overlapping 5’- and
3“untranslated regions compared witha rg2. All numbers are relative to thfei rst nucleotide of drg2. The arrows indicate the boundarieso f the
coding regions or the positions where the sequences of the cDNAs diverge from each other.
|
|
FIG. 2. The nonhepatic arginases in
share homology with yeast arginase
(yArg, Sumrada and Cooper, 1984)
and vertebrate liver arginases, including
those from human (hArgL,
Haraguchi et al., 1987), rat (rArgL,
Kawamoto et al., 1987), and Xenopus
(xArgL, Xu et al., 1993). Gaps (indicated
by dots) were introduced into the sequences
for a better fit. The hatched burs
indicate several domains that are well
conserved across species
|
|
FIG. 3. The Xenopus liver (ArgL) and nonhepatic arginase
(Argl-3) have different tissue specific expression patterns. A,
the expression o f a r g l 3w as stronger in the intestinel i vthera onf stage
52-54 premetamorphic tadpoles, opposite ttoh at for argL. In addition,
argl-3 could be up-regulated by 18 h T, treatment in both tissues,
whereas ArgL could not. Five pgof total RNA from each tissue was used
per lane. The same or duplicated blots were probed with an argl-3
probe, argL or rpL8, which serveda s a loading control.B , expression of
Xenopus arginase genes in different tissues/regions of stage 60 metamorphosing
tadpoles. Ten pg of total RNA was used per lane. The
positions of the 28 and 18 S rRNA are indicated.
|
|
FIG. 4. Northern blots showing that Xenopus argl-3 and argL
are activated early during embryogeneEsaisc.h lane contained1 0
pg of total RNA from whole embryos or tadpoles at different developmental
stages. argL mRNA was detected by stage 10/11 (gastrula) and
argl-2 by stage 16/17 (neurula). Under the hybridization conditions,
arg3 was not detectable until stage 33/34 (tailbud). Equal loading was
confirmed by staining the membranfeo r total RNAwith methylene blue
(not shown, Henin and Schmidt, 1988).
|
|
FIG5. . Northern blots showing differential regulation of the
liver and nonhepatic arginase genes in the intestine, tail, and
hindlimb during metamorphosis. The most dramatic period of intestinal
remodeling is around stage6s0 -62 and thato f tail resorption is
stage 62 and later (Nieuwkoop and Faber, 1956; Dodd and Dodd, 1976;
McAvoy and Dixon, 1977). The tail is completely resorbed by stage 66,
the end of metamorphosis. Hindlimb morphogenesis is completed
around stages 56 and undergoes rapid growth in the remaining metamorphic
period. Ten pg of total RNA was loaded per lane, except for
stage 64 tail and stage 56 hindlimb, which contained only 5 pg. The
same or duplicated blots were probed with an argl-3 probe, argL, or
rpL8. The upper band detected with the rpL8 probe is likely to be a
precursor of the mRNA, as it was not observed in oocytes where no
transcription takes place (not shown). Slight vardiaoteiso noc cur in the
levels of rpL8 mRNA during development, especially for the precursor
(Shi and Liang, 1994).
|
|
FIG6. . Northern blots showing that a r g l 3 and argL responded
to Ts differently. Ten pg of total RNA from intestine or tail
of stage 56 tadpoles treated with 5 nM T, for indicated number of days
was probed with argl-3, argL, or rpL8. argl-2 were activated after
1-day treatment and reached a peak expression after 2-3 days in the
intestine, and in the taila, r g l 3 were all up-regulated by about 2-fold
after 1-7 days. The activationo f arg3 in the intestine is not apparent in
this figure due to its low levels of expression. But it could also be
up-regulated in the intestine at least after 2-3 days of treatment (not
shown). In contrast, argL was up-regulated in both tissues only after
5-7 days. The signal for rpL8 varied slightly in some samples. It was
likely due to unequal loading. However, it clearly does not affect the
conclusions above.
|
|
FIG. 7. A Northern blot showing that a r g l 3 responded to T,
within several hours. Each lane had 10 pg of total intestinal RNA
from stage 52-54 tadpoles treated with 5 nM T, for indicated number of
hours.
|
|
FIG8. . The activation of argl3 appear to be a dirercets ponse
stage 52-54 tadpoles treated with or withou5t 0 nM T3i n thep resence
or absence of protein synthesis inhibitors (CHX). Cycloheximide (CHX)
stabilized the mRNA, but a short exposure (top panel) showed that a
slight up-regulation by T3 was produced even in the presence of CHX.
|
