Patterton D and Shi YB (1994), Thyroid hormone-dependent differential regulati...

XB-ART-20677

J Biol Chem 1994 Oct 14;26941:25328-34.

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Thyroid hormone-dependent differential regulation of multiple arginase genes during amphibian metamorphosis.

Patterton D .

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We have cloned three nonhepatic arginase genes in Xenopus laevis. The deduced amino acid sequences of the three arginases are almost identical and share about 60% identity with mammalian as well as Xenopus liver arginase. Both the liver and nonhepatic arginase genes are activated early during embryogenesis. The liver arginase gene is strongly expressed in tadpole liver, but weakly in other tissues. In contrast, the nonhepatic arginase genes have the strongest expression in the tadpole tail. During metamorphosis, the liver and nonhepatic arginase genes show distinct regulation patterns. In the intestine, both types of arginase genes are activated during the remodeling period. In the tail, the liver arginase gene is activated during tail resorption, whereas the nonhepatic ones are highly expressed throughout all stages examined. Finally, in the hindlimb, the liver arginase is up-regulated slightly during development, whereas the nonhepatic ones have low levels of expression until the end of metamorphosis. During 3,5,3'-L-triiodothyronine (T3)-induced metamorphosis, the nonhepatic arginase genes are activated very quickly, whereas the liver arginase gene is a late T3 response gene. These differential regulation patterns during normal and T3-induced metamorphosis suggest potential functions for the arginases during tissue remodeling.

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Species referenced: Xenopus laevis
Genes referenced: arg1 arg2 drg2 noct rpl8

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	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 3â-untranslated region of argl. The 5â- and 3â-end of arg3 are different from arg2 and in addition there are a few small deletionshnsertions in the overlapping 5â- and 3âuntranslated 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.