Hikasa H et al. (1997), Structure of aldolase A (muscle-type) cDNA and ...

XB-ART-15618

Biochim Biophys Acta 1997 Nov 20;13543:189-203.

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Structure of aldolase A (muscle-type) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis.

Hikasa H , Hori K , Shiokawa K .

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We obtained cDNA (XALDA; 1466 bp) for Xenopus laevis aldolase A gene (muscle-type), whose amino acid sequence had 88% similarity to those of mammalian aldolase A genes. XALDA mRNA occurred abundantly in skeletal muscle and at low levels also in other adult tissues, and such mRNA distribution was reflected in zymograms. In oocytes XALDA mRNA occurred at a relatively high level from stage I, and the mRNA level peaked at stage II, then decreased in later stages. XALDA mRNA in the full-grown oocyte was inherited as maternal mRNA throughout maturation and fertilization until midblastula stage, but its level became very low during gastrula and early neurula stages, and then increased greatly in later stages. While maternal XALDA mRNA was distributed uniformly in early embryos, mRNA zygotically expressed after late neurula stage occurred mainly in somites. In blastula animal caps XALDA mRNA occurred at a low level, but the expression was greatly enhanced by activin treatment. Thus, in Xenopus laevis aldolase A gene is actively transcribed in earlier phase of oogenesis, inherited as maternal mRNA in early embryos in a cell-type nonspecific way, then in later phases of embryogenesis, it is strongly expressed in somites with its concomitant ubiquitous expression at low levels in almost all the other cell types.

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Species referenced: Xenopus laevis
Genes referenced: aldoa

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	Fig. 1. The nucleotide sequence of XALDA and the deduced amino acid sequence. This clone was 1466 nucleotides long with an open reading frame consisting of 1089 nucleotides. The boxes indicate an initiation codon ATG., termination codon TAA., and polyadenylation signal AATAAA. in this order. Arrows indicate the region used as aldolase A specific probe 507 bp. in Northern blot analysis and whole mount in situ hybridization.
	Fig. 2. Comparison of deduced amino acid sequences between XALDA and three types of aldolases from Xenopus and other animal species rat, human, rabbit, and chicken.. The strongly shadowed regions IâVII. are regions highly conserved among all types of aldolases, the weakly shadowed regions 1â4. being the isozyme group-specific regions. Dashes are amino acid residues identical to those of Xenopus muscle-type aldolase XALDA.. Asterisks ). in the rat aldolase C sequence indicates the position where an amino acid is deleted. Question marks ?. in chicken aldolase C are unknown amino acid residues. The literature sources are as follows: aldolase A, human w10x, rat w7x, rabbit w11x, and Xenopus this paper.; aldolase B, human w9x, rat w8x, and chick w43x; and aldolase C, human w12x, rat w13x, chick w6x, and Xenopus w21x.
	Fig. 3. Phylogenetic tree inference. On the basis of the alignment of 10 aldolases, a phylogenetic tree was inferred by the neighbor-joining method w25x, for the region where unambiguous alignment is possible. The inferred tree shows that Xenopus aldolase A reported in this paper shares a common ancestor with the aldolase A of rodent and primates. The length of the horizontal line is proportional to the number of amino acid substitutions per residue.
	Fig. 4. Distribution of XALDA mRNA in adult tissues. RNAs were extracted and subjected to Northern blot analysis using aldolase A-specific region of XALDA 3X-noncoding region. as a probe A,B.. The loading equivalency was clear from the occurrence of approximately the same amount of 18S and 28S rRNA on each lane A,B, bottom.. Autoradiographic film was exposed for 1 day A. or for 3 days B.. The same set of RNAs were also used for Northern blot analysis with the aldolase C-specific region PÃuIIâ EcoRI fragment. and the full-length XALDC w21x as probes C and D, respectively.. The loading equivalency was confirmed by the same amount of 18S rRNA C,D, bottom..
	Fig. 5. Temporal changes in the level of XALDA mRNA during oogenesis, oocyte maturation and embryogenesis. RNAs were extracted from oocytes at different stages, oocytes during maturation, and embryos at different stages and subjected to Northern blot analysis. A. Lanes 1â€“6, stages Iâ€“VI, respectively; lanes 7â€“10, stage VI oocyte treated with progesterone for 1.5, 3, 6, 9 h, respectively. Staging of oocytes was according to Dumont w27x. B. Lane 1, fertilized eggs; lane 2, stage 4 cleavage.; lane 3, stage 8 mid-blastula.; lane 4, stage 11 mid-gastrula.; lane 5, stage 13 early-neurula.; lane 6, stage 16 mid-neurula.; lane 7, stage 19 late-neurula.; lane 8, stage 22 tailbud.; lane 9, stage 26 late-tailbud.. Staging of embryos was according to Nieuwkoop and Faber w29x.
	Fig. 6. A summary of the changing level of XALDA mRNA during oogenesis, oocyte maturation and embryogenesis. Northern blot signals in Fig. 5A,B was analyzed in a densitometer, and the strength of signals was plotted against the ordinate on an arbitrary unit, taking the value for the sample in lane 10 A. and lane 1 B. as 1.0. The abscissa shows the changes in the developmental stages as indicated in Fig. 5.
	Fig. 7. Expression of XALDA mRNA as studied by the embryo dissection method at 8-cell and neurula stages. A. Diagram of the dissection of an 8-cell stage embryo. The embryo was separated into four different portions as illustrated. 1, dorsalâ€“animal part; 2, dorsalâ€“vegetal part; 3, ventralâ€“animal part; and 4, ventralâ€“vegetal part. B. Northern blot analysis of RNAs from four dissected portions of 8-cell stage embryo. The number of lanes corresponds to the region dissected in A.. C. Diagram of neurula dissection. The neural groove region of neurulae stage 19. was separated into four different parts: 1, notochord; 2, somite; 3, dorsal endoderm; and 4, dorsal ectoderm. D. Northern blot analysis of RNAs from dissected neurula tissues. The number of lanes corresponds to the region dissected in C..
	Fig. 8. Spatial expression pattern of XALDA at embryo as studied by whole mount in situ hybridization. In Aâ€“G., the aldolase A-specific region of XALDA was used as an antisense probe and in H as a sense probe. A. Stage 13 early-neurula.; B. stage 19 late-neurula.; C. stage 23 mid-tailbud.; D. stage 25 late-tailbud.; E. stage 26 late-tailbud.; F. stage 35 early-tadpole.; G. stage 40 tadpole.; and H. stage 26 late-tailbud.. Pronephros and heart anlage were indicated by small and large arrowhead, respectively.
	Fig. 9. Expression of XALDA and a-actin mRNAs in animal caps treated by activin A. Animal caps were treated with different concentrations of activin A for 30 min and, after culture for 20 or 36 h, RNAs were extracted and subjected to Northern blot analysis using either aldolase A-specific cDNA as in Fig. 4A and cDNA for a-actin as a probe B..
	Fig. 10. A zymogram of Xenopus aldolase in adult tissues. Extracts of different tissues were fractionated on a filter and the isozyme pattern was obtained. The tetramers of muscle-type aldolase A., brain-type aldolase C., and both types of aldolases were shown. Under the pH gradient conditions used, liver-type aldolase B. was not separated well on the filter.
	Fig. 11. Zymogram of Xenopus aldolase in embryos at various stages. Isozyme pattern was analyzed as in Fig. 10. Lane 1, fertilized eggs; lane 2, stage 4 cleavage.; lane 3, stage 8 mid- blastula.; lane 4, stage 11 mid-gastrula.; lane 5, stage 13 early- neurula.; lane 6, stage 13 early-neurula.; lane 7, stage 16 mid-neurula.; lane 8, stage 19 late-neurula.; lane 9, stage 22 early-tailbud.; lane 10, stage 26 late-tailbud.; and lane 11, stage 35 tadpole.. Data in left and right panels were from the same batch of embryos.