Cloning and characterization of a full-length cDNA coding for ovine aldolase B from fetal mesonephros

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Short Sequence-Paper

Cloning and characterization of a full-length cDNA coding for ovine aldolase B from fetal mesonephros Laurence Gianquinto a, Eric A. Pailhoux a Jacqueline Bezard b Nathalie Servel Marek Kirszenbaum c, Corinne Cotinot a,,

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a Laboratoire de Biologie Cellulaire et Mol~culaire, B~timent des Biotechnologies, INRA, 78352 Jouy-en-Josas, France b Station de Physiologie de la Reproduction, INRA Nouzilly, 37380 Monnaie, France c CEA, DSV, DPTE, Laboratoire d'Immunoradiologie, H3pital St. Louis, Centre Hayem, 75010 Paris, France Received 4 February 1994; revised 6 May 1994

Abstract

An ovine aldolase B cDNA was isolated from mesonephros (29 dpc). The sequence covers 1649 nucleotides. Comparison with human liver aldolase B cDNA shows a homology of about 86%. The deduced amino acid sequence is composed of 364 residues and exhibits 92% homology to the human protein. Northern blot analysis and in situ hybridization data show that during the first third of gestation in sheep, aldolase B expression is restricted to the mesonephros.

Key words: Aldolase B; cDNA sequence; Mesonephros; (Sheep)

Fructose-l,6-biphosphate aldolase (EC 4.1.2.13) is a glycolytic enzyme that catalyses the reversible conver•sion of fructose 1,6-biphosphate to glyceraldehyde 3phosphate and dihydroxyacetone phosphate [1]. In vertebrates, three isozyme forms exist: muscle type (A), liver type (B) and brain type (C). Aldolase B, has a high affinity for fructose 1-phosphate and it is mainly involved in the metabolism of dietary fructose [2,3]. Aldolase isoenzymes are encoded by separate genes and exhibit a tissue specific expression varying during development. In young rat foetuses, the liver mainly synthesizes both aldolases A and C and lacks aldolase B, which is produced only after birth [4]. In adult, aldolase B is selectively expressed in the liver, kidney and intestine; tissues that actively assimilate fructose. Until now, the structure of aldolase B genes of chicken [5], rat [6] and human [7, 8] have been determined. In human, the gene has been mapped to chromosome 9 [7] and consists of nine exons, the first of which is untranslated [9]. The coding region of the

The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession numbers Z23262 and Z29372. * Corresponding author. Fax: + 33 1 34652273. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 2 0 - R

corresponding mRNAs have a high degree of homology and amino acid sequences are 92% homologous between rat and human aldolase B [10]. Here we present the isolation and characterization of full length cDNA for sheep mesonephros aldolase B. The sequence covers 1649 nucleotides. The deduced amino acid sequence is composed of 364 residues and has 92% of homology with human liver aldolase B. The isolated cDNA clone hybridized to a 1800 + 50 nucleotides R N A from sheep adult kidney and liver. During the first third of gestation, aldolase B expression is mainly detected in mesonephros. Liver expression appears later and was clearly observed after the birth. In order to isolate transcripts preferentially expressed in urogenital ridges during gonadal differentiation, we used a subtractive screening strategy. A cDNA library was constructed by using poly(A) ÷ R N A isolated from male urogenital ridges (gonads plus mesonephroi) taken from sheep foetuses at 29 days of gestation. The cDNA clones were first selected according to their hybridization with a subtractive probe (male urogenital ridges minus female foetus without urogenital ridge). From 2.5.105 clones screened, 80 were first selected from the library and then submitted to a second differential screening. 30 differential clones

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were again isolated. The inserted fragments from these 30 clones were amplified by PCR for further analysis. One of them was 32p-labelled and hybridized on the 30 PCR-products. 14 presented a strong hybridization signal indicating their common origin. Their size ranged from 0.4 kb to 1.7 kb. To establish the specificity of the cDNA inserts, two of the 14 clones (1.8 and 6.3) were 32P-labelled and hybridized on Southern blots containing sheep genomic DNA. After high stringency washings, the autoradiograms have revealed one single restriction fragment with both probes using EcoRI restriction enzyme (Fig. 1A). This band at 6.5 kb is common with male and female DNA. The two cDNA clones 1.8 and 6.3 were sequenced. The 454 bases pairs (bp) of the 1.8 sequence were included within the 6.3 sequence. Comparison to the Genbank database revealed a high degree of homology with aldolase B mRNA sequences. The 1.8 clone corresponds to the 3' untranslated region with an overall homology of 77% between sheep and human. No alignment was obtained with 1.8 clone and human aldolase A and C, this region appears specific to the isoenzyme of type B. It was deposited with the E M B L accession No. Z23262. The nucleotide sequence of the 6.3 cDNA clone with the corresponding amino acid sequence are shown in Fig. 2. This sequence is available from E M B L / G e n Bank Data Libraries under the accession number Z29372. The sheep aldolase B cDNA contains 1649 nucleotides including the whole coding region (1095 nucleotides); starting at a methionine residue in position 67 and ending with a T A G nonsence codon in position 1162; a 5' non coding region (67 nucleotides) and a 3' untranslated region (454 nucleotides from the termination codon to the poly(A) tail). A canonical polyadenylation signal (AATAAA) is present 23 bases before the poly(A) tail. In contrast to human and mouse no additionnal signals were contained in the 3' non coding region. Comparison with human liver aldolase B cDNA shows a homology of about 86%. The deduced amino acid sequence is composed of 364 residues as the human and rat aldolase B proteins. There is an overall homology of 92% between human and sheep aldolase B (Fig. 3). In the region around the active site (amino acids 215-242), the homology increases to 100%. The aldolase B isoenzymes have residues in common that distinguish them from aldolase A and C. At position 150, aldolase B from various species have an alanine, at position 218 an asparagine, and at position 242 there is a lysine. The derived sequence of our sheep cDNA clone possesses

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Fig. 1. (A) Southern blot analysis of EcoRI-digested sheep DNA hybridized with 32P-labelled 1.8 insert. A single 6.5 kb band was obtained with male (M) and female (F) DNA. A similar hybridization pattern was observed with 4.2 clone (data not shown). (B) Total RNA (10 /xg) from male (m) and female (f) gonads (G), foetuses without gonads (F) and liver (L) at 29 dpc was used and also testis (T), ovary (O) and liver (L) at 12 dpp. After transfer, membranes probed with 3zp-labelled 1.8 insert show a hybridized band ( ---, ) of 1.8 kb. The presence of equal amounts of RNA in each lane was confirmed by ethidium bromide staining of agarose gel before and after transfer to the membrane. (C) Northern blot analysis of sheep adult organs for aldolase B expression. Total RNA (10 ~g) from male liver (L), testis (T) and ovary (O) was electrophoresed, transferred and hybridized with 1.8 probe. A hybridization signal was observed only in liver.

these residues, confirming that it corresponds to the B isoenzyme. In order to determine the pattern of tissue expression of this clone, Northern blots of RNA from a range of organs were hybridized. Under stringent conditions 1.8 insert (3' untranslated region) detected a single m R N A species, whose length approximated 1.8 kb (Fig. 1B, C). As expected by the screening strategy used, the transcripts are detected in the fetal urogenital ridges of both sexes in an intense manner and

Fig. 2. Nucleotide sequence of sheep aldolase B cDNA and its corresponding amino acid sequence. Numbering of amino acid begins with the first ATG codon. The star indicates the stop codon and the polyadenylation site is underlined. The oligonucleotides used for in situ hybridization are boxed.

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absent in the remaining part of the 29 days old foetus indicating a regionalized expression (Fig. 1B). Nevertheless when the liver was tested separately, a very weak signal was observed. In the neonate (12 d p p ) , a dramatic reduction in expression of gonadal transcripts was observed, nevertheless in ovary the hybridization signal was remain detectable. In contrast, the liver showed a high level of expression after the birth (Fig. 1B). In the adult, expression cannot be detected in the gonads however a high level of expression was observed in liver (Fig. 1C). The tissular distribution of aldolase B transcripts during the fetal period was determined by in situ hybridization. At day 27 of gestation, the urogenital ridges contained the sexually indifferent gonads plus the mesonephroi. At this stage and until day 35, gonads and mesonephroi cannot be physically separated. After hybridization with 1.8 B oligonucleotide (antisense) the gonad was negative and only the

mesonephros was labelled (Fig. 4A). An intense signal was observed in mesonephric tubules and absent in giant nephron (Fig. 4B). These data revealed that in sheep at a developmental stage corresponding to the gonadal differentiation, aldolase B expression is restricted to the mesonephros. At this stage, the metanephros (kidney) is not yet functional and the mesonephros is the main site of fructose 1-phosphate degradation. In early human foetus, the liver mainly synthetizes both aldolase A and B, development being associated with a progressive disappearance of these isoenzymes correlated with a parallel increase of aldolase B [10]. This is in agreement with our findings from fetal and post-natal liver samples where aldolase B expression was clearly observed only after the birth. We thank B. Bloch (Laboratoire d'Endocrinologie Exp6rimentale, Universit6 de Bordeaux II, France) for expert assistance in applying the In situ hybridization

I0 20 30 40 50 60 70 MAHQFPALTSEQKKALSETARRIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFRELLFTVDSSV MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSI i0 20 30 40 50 60 70 80 90 i00 ii0 120 130 140 SQSIGGVILFHETLYQKDGQGKLFRDILKEKGIVVGIKLDQGVAPLAGTNKETTVQGLDGLSERCAQYKK NQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKK 80 90 i00 ii0 120 130 140 150 160 170 180 190 200 210 DGADFGKWRAVLKIDNQCPSHLAIQENANTLARYASIYQQNGLVPIVEPEVIPDGSHDMEHCQYVTEKVL DGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVL 150 160 170 180 190 200 210 220 230 240 250 260 270 280 AAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDA AAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDA 220 230 240 250 260 270 280 290 300 310 320 330 340 350 TLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAENKKATQEAFMKRALANSQAAKGQYVHMGSSD TLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSG 290 300 310 320 330 340 350 360 SASTQSLFTASYTY AASTQSLFTACYTY 360

Fig.3.C•mparis•n••sheepandhumana•d••aseBdeducedpr•teins.Thehumansequen•eusedf•r••mparis•nis•educe•fr•m •6••bpss-mRNA(GenBank).Thesequenceswerea•ignedusingapr•ceduredescribedbyKanehisa[••].

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technique. We gratefully acknowledge A. Locatelli and R. Lorentz for laparotomies and S. Ruffini for technical assistance. Also, I. Blondeau for typing manuscript. This work was supported in parts by a I.N.R.A. grant No. 4862.

References

D Fig. 4. In situ hybridization to sections of 27 dpc sheep gonad (G) and mesonephros using an anti-sense oligonucleotide probe specific for the aldolase B transcripts. Bright-field illuminations (A, C) and dark-field illuminations (B, D) respectively. GN, giant nephron; T, mesonephric tubules ( × 10 and 40). Isolated urogenital ridges were fixed with a 1% solution of paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) overnight at 4°C. Samples were then embedded in a tissue freezing medium and frozen by holding them at the surface of liquid nitrogen. The sections (5-7 /xm thick) were treated for 5 min with 10/xgml i proteinase K and for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Oligodeoxynucleotides 1.8 A (sense) and 1.8 B (anti-sense) corresponding to the 3' non-coding region of aldolase B cDNA were labeled with [3sS]dATP (1200 Ci/mmol) using terminal deoxynucleotidyl transferase (Boehringer) and then purified on G50 Sephadex column (Pharmacia). The hybrization step and buffers used were identical to those described by Guitteny et al. [12]. In brief, 20/zl of hybridization buffer (4 x SSC, 1 x Denhart, 10% dextran sulfate, DTT 200 /~gml - l , 1% sarcosyl, 0.02 M NaH2Po 4, salmon sperm DNA 100/xg m l - 1, tRNA 200/~g ml-1 and formamide 50% (v/v)) containing 0.5 ng of 3ss-labeled probe were deposited on each slide. Hybridization was performed for 16 h at 40°C in moistened Petri dishes. Coverslips were removed in 4 x SSC and the sections were subjected to serial washes with 1 × SSC and high-stringency washes in 0.1 x SSC at 40°C for 45 min. Sections were left 3-5 days in contact with X-ray film (Kodak X-Omat) and then coated with Ilford K-5 emulsion, exposed for 2-4 weeks, developed and stained with toluidine blue.

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