Yatsuki H et al. (1998), Cloning of the Xenopus laevis aldolase C gene a...

XB-ART-14020

Biochim Biophys Acta 1998 Nov 08;14422-3:199-217.

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Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells.

Yatsuki H , Outida M , Atsuchi Y , Mukai T , Shiokawa K , Hori K .

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A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

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Species referenced: Xenopus laevis
Genes referenced: aldoc camp ins mt-tr trna

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	Fig. 1. Nucleotide and deduced amino acid sequences of the Xenopus laevis aldolase C gene. Nucleotide sequence of the 12.2 kb EcoRI fragment is shown. Exons are underlined and numbered consecutively from 1 to 9 (EX1–EX9) (to the right), whereas 8 introns are named from a to h (INT a–h), the length of each intron being indicated in parentheses. The sequence for each intron is not shown but is deposited in the DDBJ database. Transcriptional start site is shown by +1. All the nucleotides are numbered according to the +1 site. The deduced amino acids are numbered by bold letters. The poly(A) signal is indicated by double underlines. For explanations of the sequence elements, ACSE (AGTCACGTAG-CTCTGC), PRE (AACCGCAAGTGTCCAGT), ERE (GGTCA), IPE (GGGCCC), GAGA (GAGA) and CRE (TGACGTCA), all indicated here by bold underline, see the text and the legend to Fig. 4.
	Fig. 2. Comparison of exon/intron structure among Xenopus, rat and human aldolase C genes. Top: a restriction map of isolated genomic DNA fragment, containing the Xenopus aldolase C gene. Exons (1–9) are indicated by boxes. Coding and non-coding parts are indicated by closed and open boxes, respectively. Introns are denoted alphabetically (a–h). Open triangle on exon-2 and closed triangle on exon-9 indicate the translational initiation (ATG) and termination (TGA) site, respectively. The lower two: rat and human aldolase C genes. Scales for all the three genes are the same. Inset: partial sequence of exon-2 of Xenopus, rat and human aldolase C genes. In the 5′-side, a short untranslated sequence is included, which is followed by the coding sequence in all three genes. Nucleotides ATG for translational initiation is underlined.
	Fig. 3. Primer extension analysis of aldolase C mRNA. 32P-labeled DdeI–HinfI fragment (+29 to +103) of aldolase cDNA was hybridized with 10 μg of Xenopus oocyte poly(A)+ RNA and extension reaction carried out. (A) The products extended along Xenopus oocyte poly(A)+ RNA and yeast tRNA were electrophoresed on a 5% denaturing gel. pBR322 DNA digested with HinfI or HpaII is given as size markers. (B) The extension product was electrophoresed on a 6% sequencing gel with a sequence ladder of SacI–HinfI fragment (SacI site located in the vector sequence which is upstream of the 5′-end of the Xenopus aldolase C cDNA[7]). Lane 1, Maxam–Gilbert sequencing products (G+A); lane 2, the same reaction products (T+C); lane 3, reaction products with oocyte poly(A)+ RNA; lane 4, reaction products with yeast tRNA.
	Fig. 4. Comparison of the promoter regions of Xenopus, human, and rat aldolase C genes. The 5′-flanking region is divided conventionally into six portions (regions A–F). Potentially negative and positive elements found by computer search are schematically indicated by ovals and rectangular boxes, respectively. PRE, a proximal repressor element of rat growth hormone; ERE, a half palindromic estrogen-responsive element motif; IPE, an insulin gene promoter element; GAGA, a GAGA box; CRE, cAMP-responsive element; CAT, the CCAAT box; GC, the GC box; TATA, the TATA-like element; and ACSE, the aldolase C-specific element. The transcriptional initiation site is shown by +1. For comparison, proximal promoter regions (−120 to +1) of human and rat aldolase C genes are shown.
	Fig. 5. Sequences of 5′-proximal promoter regions of Xenopus, rat and human aldolase C genes. CCAT and GC boxes, the TATA-like element and ACSE sequences are indicated by bold letters. The GC box and the ACSE are enclosed in boxes. The transcriptional start site is indicated by +1. Nucleotides identical to those of the Xenopus gene are indicated by vertical lines. Hyphens given in the sequences of human and rat genes indicate nucleotides which are tentatively thought to be missing in these genes in comparison with the Xenopus sequence.
	Fig. 6. Transcriptional activation of pXAC-CAT in Xenopus embryos during development. About 300 pg of circular pSV2CAT or pXAC-CAT was injected together with 200 pg of pRSV/L into the animal side of both of the blastomeres of two-cell stage embryos. Embryos were collected at indicated stages [24] and whole embryos were used for CAT assay. The amount of each sample applied to thin layer chromatography was subjected to minor adjustment depending on the activity of luciferase determined as an internal control. C, AC1 and AC2 are for chloramphenicol, 1-acetylated chloramphenicol, and 3′-acetylated chloramphenicol, respectively.
	Fig. 7. Regionally restricted expression of pXAC-lacZ in embryos. About 500 pg of pXAC-lacZ was injected into the animal side of both blastomeres of two-cell stage embryos as in Fig. 6. Injected embryos were allowed to develop until the early tail-bud stage (stage 22) (A), late tail-bud stage (stage 26) (B), and early tadpole stage (stage 37) (C), and X-gal staining was carried out. Arrowheads indicate the regions where injected gene was expressed strongly.
	Fig. 8. Autoradiogram showing the activities of variously truncated promoter in embryos. CAT genes with variously deleted promoters of the Xenopus aldolase C gene were injected together with pRSV/L into the animal side of both blastomeres of two-cell stage embryos as in Fig. 6. pO/ACAT was the promoter-less control. Embryos were collected at 22 h after injection (at neural tube stage; stage 22) and then assayed for CAT activity as in Fig. 6.
	Fig. 9. Summary of the activities of CAT fusion genes with 5′-side-deleted promoters assayed in Xenopus embryos. CAT genes with variously deleted promoters of the Xenopus aldolase C gene are schematically indicated (left). These genes were injected together with pRSV/L into the animal side of both blastomeres of two-cell stage embryos as in Fig. 6. pO/ACAT was the promoter-less control. Embryos were collected at 22 h after injection (at the neural tube stage; stage 22) and assayed for CAT activity as in Fig. 6. The promoter was conventionally divided into six regions (regions A–F) as in Fig. 4. Activities are shown by taking the value for pXAC-CAT as 100%. All data are mean values of four independent experiments, and error bars are indicated.
	Fig. 10. Northern blot analysis of RNAs from A6 cells and other adult tissues. Ten μg each of total RNA from Xenopus A6 cells and adult muscle, liver, kidney, testis and oocytes, and 1 μg of total RNA of brain were electrophoresed on a 1% formaldehyde agarose gel. The use of 1/10 amount of brain RNA is to reduce its strong signal to the level comparable to those of other RNAs. The 3′-non-coding region of the Xenopus aldolase C cDNA which does not crosshybridize with aldolase A and B mRNAs was used as a probe.
	Fig. 11. Summary of the expression studies of CAT fusion genes with 5′-side-deleted promoters in A6 cells. A6 cells were co-transfected with a series of deletion constructs used in the experiment in Fig. 9 and pRSV/L as an internal reference plasmid, and CAT activities were measured and summarized as in [9]. Results indicate mean values of three independent experiments, and error bars are indicated.
	Fig. 12. Construction of fusion genes with the normal and deleted intron a and their expression in A6 cells. (A) −68CAT, −92CAT, and −92aCAT were assayed in A6 cells as in the experiments in Fig. 11. (B) −92aCAT, −92aΔBCAT, and −92aΔSK were assayed in A6 cells as in (A). All the bar graphs are the mean values of three independent experiments and error bars are shown. Activities are shown by taking the value for −92CAT and −92aCAT as 100% in the experiments in (A) and (B), respectively.
	Fig. 13. Summary of the promoter activities of −68CAT constructs with mutated basal elements. The CCAT and GC boxes, the TATA-like element and ACSE were mutated site-directedly in −68CAT. The mutated plasmids are indicated as −68CAT(CCAT)m, −68CAT(GC)m, −68CAT(TATA)m, and −68CAT(ACSE)m, respectively as in Table 1. These were cotransfected with pRSV/L into A6 cells as in Fig. 11. CAT assays were carried out, and results summarized as in Fig. 11, by taking the value for −68CAT as 100%. Results indicate mean values of three independent experiments with error bars.
	Fig. 14. Gel sift assays with a DNA fragment from the proximal promoter region. A6 cell nuclear extract (11 μg) was incubated with 32P-labeled PE probe (the DNA fragment corresponding to −93 to +10) in the presence of increasing amounts of the unlabeled PE. The numbers above the lanes indicate the fold molar excess of unlabeled wild-type competitor (PE) over the labeled probe (PE). Lane 1, probe alone; lane 2, incubated with the labeled PE in the absence of competitor; lanes 3–6, same as in lane 2, but in presence of the competitor, 5-fold, 50-fold, 125-fold, 250-fold, respectively. Four retarded bands (arrowheads 1–4) were obtained.
	Fig. 15. DNase I foot-printing analysis with the proximal promoter region. A fragment of the Xenopus aldolase C promoter (from −68 to +10) was used as a probe. The probe was digested with DNase I after being incubated either in the presence (lane 1) or absence (lane 2) of the A6 cell nuclear extract. The sequence ladder of the probe DNA was obtained by Maxam–Gilbert A+G reaction (lane 3). The sequence of the region of the DNA used as the probe is schematically depicted to the left. The thick line represents the protected region. The sequence in the square is GC box. The four nucleotides in the box constitute a part of CCAT box.