Kousteni S et al. (1997), Sequence and expression analysis of a Xenopus l...

XB-ART-16515

Gene 1997 May 06;1902:279-85. doi: 10.1016/s0378-1119(97)00013-9.

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Sequence and expression analysis of a Xenopus laevis cDNA which encodes a homologue of mammalian 14-3-3 zeta protein.

Kousteni S , Tura F , Sweeney GE , Ramji DP .

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We report the cloning and characterisation of a cDNA that encodes a novel member of the Xenopus laevis 14-3-3 protein family. Sequence analysis reveals that the cDNA-encoded protein shares 84% identity with the rat, human or sheep 14-3-3zeta isoform, and between 66% and 77% identity with bovine, human or rat beta, bovine gamma, human tau, Drosophila 14-3-3 and a previously isolated Xenopus member. The corresponding mRNA is present in all adult tissues examined with the highest levels in the brain. Although the gene is expressed throughout embryogenesis, higher levels of mRNA accumulate after gastrulation. Whole-mount in situ hybridisation on tailbud stage embryo reveals strong expression of the gene in the head, optic vesicles, spinal cord and branchial arches with weaker expression in the somites. In addition, expression along the notochord is observed at stage 45 (tadpole). This spatial and temporal expression profile along with recent studies implicating the importance of 14-3-3 proteins in the regulation of signal transduction pathways argues for a key role of this isoform in embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: camp gtf2a1l kit lipa mapt mt-tr nr2f5 odc1 tf trna ywhaz

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	Fig. 1. The nt sequence of the cDNA insert and the deduced aa sequence. The DNA was sequenced by the dideoxy chain-termination method using either a Sequenase Version 2.0 sequencing kit (US Biochemical, Cleveland, OH, USA) or the ALF automated sequencer (Pharmacia). Multiple primers were used to completely sequence the DNA on both strands. (*) denotes the termination codon and the arrows indicate the positions of primers used for RT-PCR (Fig. 3) and to produce 3' UTR probes for in situ hybridisation (Fig. 4). The nt sequence reported here has been deposited in the GenBank data base (accession number X95519).
	Fig. 2. Alignment of the aa sequence of the six putative domains (A-F) in x14-3-3~ with other characterised members. The sequences compared with x14-3-3~ (1) are: (2), rat ~ (Watanabe et al., 1994); (3), sheep ~ (Toker et al., 1992); (4), human ~ (Zupan et al., 1992) and (5), the previously isolated Xenopus homologue (Martens et al., 1992). The homology comparison was generated using the CLUSTAL V algorithm (SERC Daresbury Seqnet Series). (.) represent gaps introduced by the computer program to improve alignment. Putative phosphorylation sites are indicated by asterisks (Ser 57 and 58, PKC; Ser 58, cAMP-dependent protein kinase; Ser 63, Ca2+/calmodulin kinase; Thr 185, corresponds to the Ser residue that is phosphorylated in mammalian and avian brain 14-3-3 ~ and 8, respectively).
	Fig. 3. RT-PCR analysis of RNA from several adult tissues and different embryonic stages. Total RNA samples (1 tag) were treated with RNase-free DNase (Promega) and used as templates to generate random-primed cDNA according to the manufacturer's instructions (Clonetech). 50 lal PCR reactions were carried out using Taq DNA polymerase (2.5 U/reaction) and PCR buffer, with 0.2 mM dNTPs, 100 pmol of each primer, 1.5 or 2 mM MgC12 (ODC or x14-3-3~, respectively), and 1/50th (ODC) or 1/10th (x14-3-3~) original cDNA template. The initial denaturation was carried out at 94°C for 2 min and was followed by 30, three-temperature cycles: denaturation at 94°C for 45 s (x14-3-3~) or 1 min (ODC), annealing for 45 s at 60°C (x14- 3-34) or 1 min at 50°C (ODC), and extension at 72°C for 1 min. The final extension was 5 min. These conditions were in the exponential phase of amplification as judged by the use of three different amounts of cDNA template. The sequences of the x14-3-3~ primers are shown in Fig. 1 and those for ODC were 5'-AATGGATTTCAGAGACCA-3' and 5'-CCAAGGCTAAAGTTGCAG-3' (Bassez et al., 1990). The PCR products were size-fractionated on a 2% agarose gel, transferred onto nylon membranes and probed, at high stringency, with radiolabelled probes corresponding to the region of PCR amplification. The x14-3-3~ and ODC signals for each cDNA sample were determined by densitometric scanning of the autoradiographs, and the relative x14- 3-3~:ODC ratios are shown. The specificity of amplification was confirmed by sequencing of products following subcloning into the pGEM-T vector (Gacesa and Ramji, 1994). In addition, parallel control samples in which reverse transcriptase had been omitted were negative in each case. (A) RT-PCR with tissue RNA isolated from female Xenopus laevis: kidney (KI), muscle (MU), lung (LU), liver (LI), brain (BR), ovary (OV), adipose tissue (AD) and heart (HE). (B) RT-PCR with RNA from different embryonic stages: 2 (cleavage), 8 (blastula), 11 (mid gastrula), 18 (mid neurula), 23 (early tailbud), 33 (late tailbud) and 45 (tadpole). The data shown are representative of two independent experiments.
	Fig. 4. The spatial pattern of x14-3-3~ expression. Whole-mount in situ hybridisation was carried out according to the protocol of Harland (1990). The probe was synthesised using T7 (antisense) or T3 (sense) RNA polymerase in the presence of digoxigenin-UTP using recombinant pGEM-T template containing the PCR ampification product from the 3'UTR of x14-3-3~ cDNA insert (see Fig. 1). Hybridisation was carried out for 16 h at 52°C in 50% formamide, 2 x SSC, 1 mg/ml bacterial tRNA, 2% Boehringer-block (Boehringer), 0.1% Tween-20, 0.1% CHAPS and 0.1 mg/ml heparin. Washes were carried out at 60°C in 2 x SSC, 0.3% CHAPS (3 x 20 rain each) and then in 0.2% SSC, 0.3% CHAPS (2 x 10 min each). These represent high stringency conditions as demonstrated by our recent studies on the COUP-TF-A and COUP-TF-B genes, which show 94% identity at the aa level (van der Wees et al., 1996). (A, B) Tailbud embryo (stage 36) probed with antisense and sense transcripts, respectively; (C, D) early tadpole (stage 45) probed with antisense and sense transcripts, respectively, ba, branchial arches; n, notochord; ov, optic vesicles; s, somites; sp, spinal cord. The result is representative of at least six independent embryos analysed.