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Fig. 3. Spatial expression patterns of aldolase A, B and C mRNAs examined in the wild-type Xenopus embryo at the tailbud stage (stage 35) by whole-mount in situ hybridization. Probes for aldolase A, B and C mRNA were, respectively, the HindIII fragment of XALDA (Hikasa et al., 1997), the AflII-XhoI fragment of XALDB2 (Kajita et al., 2000), and the SacI-KpnI fragment of XALDC (Atsuchi et al., 1994), which did not cross-hybridize to each other ( Kajita et al., 2000). All the signals were obtained with antisense probe, but not with the sense probe (data omitted). (a) An embryo hybridized with the XALDA antisense probe. Yellow lines indicate regions (b–d) where embryos were sectioned. h: heart anlage; p: pronephros, s: somites. In (b): b, brain; e, ear vasicle. In (c): s, somites; p, pronephros. In (d), somites are defferentially stained in dermatome (s (der)) and myotome (m (der)). (e) An embryo was fixed, cut into halves, and only the anterior half was hybridized with the XALDA antisense probe. Note the absence of difference in the staining between dermatome and myotome, indicating that the weaker staining in myotome in (d) is simply due to the lesser accessibility of the probe to it. (f) An embryo hybridized with the XALDB antisense probe. Yellow lines indicate regions (g–j), where embryos were sectioned. In (f–j): lr, liver rudiment; p, pronephros; ep, epidermis; pr, proctodeum. (k) An embryo hybridized with the XALDC antisense probe. Yellow lines indicate regions (l–n) where embryos were sectioned. In (k–n): br, brain; h, heart; p, pronephros; sc, spinal cord; rt, retina.
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Fig. 1. Calibration curves obtained from Northern blot analysis performed with in vitro transcribed aldolase RNAs and RNAs from embryos and adult tissues. In vitro transcribed aldolase A, B and C RNAs appeared as signals of ca. 1.5, 1.5 and 1.8 kb, respectively, and aldolase A, B and C mRNAs in embryos and adult tissues appeared as RNAs of similar sizes (insets). Strength of each signal was determined by denstometry in an image analyzer (BAS 2500). (a) A calibration curve for aldolase A mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase A RNA, total RNA of one fertilized egg, and total ovary RNA (10 μg) were analyzed. (b) A calibration curve for aldolase B mRNA. One-half, 5, 50, 200, and 500 pg of in vitro transcribed aldolase B RNA, total RNA from one-half embryo at stage 35, and total liver RNA (10 μg) were analyzed. A stage 35 tadpole contained ca. 150 pg of aldolase B mRNA. (c) A calibration curve for aldolase C mRNA. Five, 25, 50, 150 and 300 pg of in vitro transcribed aldolase C RNA, total RNA of one fertilized egg, and total ovary RNA (10 μg) were analyzed.
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Fig. 2. Absolute amounts of aldolase A, B and C mRNAs in Xenopus laevis embryos during development. (a) The graph was drawn based on the results of Northern blot analyses of RNAs from embryos at different stages (data not shown, but see Kajita et al., 2000) and calibration curves obtained in Fig. 1. Two or three independent determinations were performed. From cleavage to the late gastrula stage the amount of aldolase A mRNA per embryo decreased from 60 to 15 pg, but it increased sharply thereafter to the level of 600 pg (at the tadpole stage). The period of this sharp increase corresponded to the period of somitegenesis. The amount of aldolase B mRNA (1.5 pg/egg) did not change until the midgastrula stage, but increased slightly at the late gastrula stage, then increased sharply at the tailbud stage to reach the level of 140 pg/embryo at the tadpole stage. The amount of aldolase C mRNA per embryo decreased from ca. 45 pg (egg) to ca. 3 pg (gastrula), and did not increase greatly in post-gastrular stages (50 pg/embryo even at the tadpole stage). (b) Circle graphs were drawn based on the data in (a). The difference in the size of the circle graph represents the difference in the total amount of the three aldolase mRNAs (A plus B plus C) at each stage.
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Fig. 4. Amounts of aldolase A, B and C mRNAs expressed in different Xenopus adult tissues. (a) The bar graph was drawn based on the results of Northern blot analysis of RNAs from various tissues (not shown, but see Kajita et al., 2000) and the calibration curves shown in Fig. 1. Note that skeletal muscles expressed almost exclusively aldolase A mRNA, whereas cardiac muscles expressed much more aldolase C mRNA than aldolase A mRNA. Aldolase B mRNA constituted the major aldolase mRNA in liver and kidney, and also in stomach and intestine, which are necessary for dietary fructose metabolism ( Munnich et al., 1985). (b) Circle graphs showing the percent composition and relative amount of three aldolase mRNAs in different tissues, which were drawn based on the data in (a). The difference in the size of each circle graph represents the difference in the total amount of three aldolase mRNAs (A plus B plus C) per unit amount (1 μg) of the total RNA, which is nearly equal to rRNA.
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Fig. 5. Chromosomal localization of Xenopus aldolase genes. We examined at least 30 metaphase cells with clear twin-spot hybridization signals for each cDNA clone. There were 36 chromosomes in each nucleus. Top (a): a metaphase plate showing doublet signals (arrows) of aldolase A gene on the telomeric region of the long arms of a pair of acrocentric chromosomes. Middle (c): a metaphase plate showing doublet signals (arrows) of aldolase C gene on the middle portion of the short arms of a pair of large submetacentric chromosomes. Bottom (b): a metaphase plate showing doublet signals (arrow) of aldolase B gene on the short arm of a large submetacentric chromosome, which was confirmed by band pattern to be different from the chromosome which carries aldolase C gene. In the present data, we did not denote the number to each chromosome, since karyotype of Xenopus laevis has not yet been internationally standardized.
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