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Sequencing and characterization of the Xenopus laevis ribosomal protein L34 cDNA
Gene 318 (2003) 163 – 167 www.elsevier.com/locate/gene
Sequencing and characterization of the Xenopus laevis ribosomal $ protein L34 cDNA Maria Carmela Vaccaro * Department of Evolutionary and Comparative Biology, University of Naples Federico II, via Mezzocannone n.8, 80134, Naples, Italy Received 14 February 2003; received in revised form 30 May 2003; accepted 20 June 2003 Received by M. D’Urso
Abstract In this paper, a cDNA homologous to the mammalian ribosomal protein (r-protein) L34 was isolated from a Xenopus laevis oocytes library and named XL34. It encodes a protein of 116 residues with an Mr of 13.2 kDa and a highly basic sequence. The nucleotide (nt) and deduced amino acid (aa) sequence have been compared with the L34 sequence from other species. This analysis showed that the L34 is a protein greatly conserved from prokaryotes to eukaryotes. XL34 mRNA is abundantly present in the whole cytoplasm of oocytes at stages 1 and 2. D 2003 Elsevier B.V. All rights reserved. Keywords: Oocytes; Ribosome; Conserved sequence; Evolution
1. Introduction In eukaryotes, ribosomes contain about 80 structural proteins, besides rRNA. Their biosynthesis is a complex process implying the coregulated expression of the rRNA genes and ribosomal protein genes (rp-genes). The latter are coded for by housekeeping genes, since their activity is required for the growth and maintenance of all cell kinds (Wool, 1979). The study of the ribosomal proteins (r-proteins) belonging to a variety of organisms may improve our knowledge of ribosome structure and evolution, as well as elucidate the role of r-proteins in the basic mechanisms of protein synthesis. In this regard, the experimental model utilized in this paper, Xenopus laevis oocytes, is unique with respect to other tissues. In fact, since the ribosomes formed during X. laevis oogenesis are the order of 1012 (Rosbash, 1974), high levels Abbreviations: aa, amino acid(s); nt, nucleotide(s); bp, base pair(s); 3VUTR, 3Vuntranslated region; 5VUTR, 5Vuntranslated region; rp-genes, ribosomal protein genes; r-protein(s), ribosomal protein(s); rp-mRNA, ribosomal protein mRNA. $ The nucleotide sequence data reported in this paper have been submitted to the Gene Bank database under Accession number AY079177. * Tel.: +39-081-2535173; fax: +39-081-2535035. E-mail address: [email protected] (M.C. Vaccaro). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00771-6
of ribosomal protein mRNA (rp-mRNA) are expected for their construction. In this study, we have isolated and sequenced the fulllength cDNA of the X. laevis r-protein L34. The nucleotide (nt) and deduced amino acid (aa) sequences have been compared with the homologous sequences from several living organisms. Furthermore, we have analyzed the expression of the L34 r-protein mRNA in the X. laevis previtellogenic and vitellogenic oocytes. The Northern blot analysis indicates that the messenger is highly concentrated in the previtellogenic oocytes.
2. Materials and method 2.1. Animals and gametes Adult X. laevis females was obtained from Rettili (Varese, Italy). Groups of oocytes at various stages of oogenesis were excised from the ovaries of females anaesthetized with MS222 (Sigma, St. Louis, MO, USA). The stages of X. laevis oocyte growth stages (Dumont, 1972) are st. 1 (50 – 300 Am), st. 2 (300 –450 Am), st. 3 (450 – 600 Am), st. 4 (600 –1000 Am), st. 5 (1000 – 1200 Am), st. 6 (1200 –1300 Am).
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2.2. Cloning and sequencing of XL34 cDNA Unidirectional cDNA library in pBluescript SK( F ) phagemid vector (ZAP Express cDNA cloning kit, Stratagene) constructed using poly(A) mRNA isolated from X. laevis oocytes stages 1, 2, 3, gift from dott. W.J. Lennarz, were screened by standard hybridization methods under conditions of low stringency in a solution of 15% formamide, 10 mM EDTA, 200 mM NaHPO4, pH 7.0, 7% SDS, 1% bovine serum albumin (BSA) at 37 jC. The probe was the first 700 bp of the 5V region of D. melanogaster gurken cDNA and was labeled with the random primer DNA labeled kit (Roche) and 32P-labeled dCTP (NEN). Washes were performed with 0.5 SSPE and 0.5 SDS at 50 jC, and filters were exposed to Kodak YAR5 Film. Few clones were positive and one of these clones was similar to L34 cDNA of mammals, yeast, vegetables and mosquito. The clone was sequenced in both directions manually with the Thermo Sequenase Radiolabeled terminator cycle sequencing kit (USB, Ohio) using the 33P-dNTP (Amersham). All sequence information was processed using DNA Strider software and Clustal.
glycine buffer. The sections were hybridized overnight at 60 jC in 40% formamide, 1 Denhardt’s solution, 5 SSC, 200 Ag/ml tRNA (Sigma) and 100 ng of sense or antisense digoxigenin-labeled RNA on each slide. The slides were exposed to 0.5 SSC, 20% formamide for 1 h at 60 jC and to the following treatments: (1) NTE (10 mM Tris –HCl, pH 7.0, 0.5 M NaCl, 5 mM EDTA) at 37 jC for 15 min; (2) NTE containing RNAse A (10 Ag/ml) at 37 jC for 30 min; (3) NTE at 37 jC for 15 min; (4) 0.5% SSC, 20% formamide at 60 jC for 30 min; (6) 2% Roche blocking solution in 100 mM maleic acid, 150 mM NaCl, pH 7.5 for 30 min. Sections were incubated overnight in anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche), diluted 1:2000 in 2% Roche blocking solution in 100 mM maleic acid, 150 mM NaCl, pH 7.5. Following washing in 0.1% Tween-20, 0.5 mg/ml levamisole (Sigma), sections were incubated at room temperature in the developing solution, BM purple color substrate (Roche) in 2% Tween-20 and 1 mM levamisole. After stopping the reaction in 1 mM EDTA in PBS, sections were mounted in PBS-glycerol and observed on a Leitz Photomicroscope. Photographs were taken using a Kodacolor ASA 100 film.
2.3. Northern blot analysis 3. Results and discussion For analysis of transcripts, total RNA from X. laevis previtellogenic and vitellogenic oocytes was isolated using a Trireagent solution (Sigma). Twenty micrograms of total RNA from each one was separated on 1% formaldehyde agarose gel and blotted on a nylon membrane (Roche). The XL34 cDNA was labeled with the random primer DNA labeled kit (Roche) and 32P-labeled dCTP (NEN), and the RNA blots were hybridized in a mixture of 0.5 M NaHPO4, 7% SDS, 1% BSA, 1 mM EDTA at 60 jC. The filter was washed at 60 jC in 2 SSC, 2% SDS, 0.2 SSC, 0.2% SDS before exposure to X-ray film (Kodak). 2.4. In situ hybridization on sections The XL34 cDNA was digested with EcoRI and XhoI and used as template to synthesize digoxigenin-labeled antisense and sense, with digoxigenin-labeled UTP and, respectively, T3 and T7 RNA polymerase according to the manufacturer recommendations (RNA T3-SP6 transcription and labeling kit, Roche). X. laevis oocytes were fixed in 2% flavin adenine dinucleotide (FAD), 250 mM NaCl, 5% acetic acid for 1 h at 4 jC and processed for embedding in paraffin according to standard techniques. Following attachment onto Superfrost Plus slides (BDH), the 7-Am-thick sections were deparaffinized in hystolemon and rehydrated in graded ethanol concentration. Sections were then fixed in 4% paraformaldehyde in PBS, washed in PBS and digested with proteinase K (Roche) (10 Ag/ml in 20 mM Tris/HCl, pH 7.0, containing 1 mM EDTA, pH 7.2) at 37 jC. After refixing with 4% paraformaldehyde, the sections were rinsed in PBS and incubated in 0.1 M Tris/
3.1. Structural and evolutive analysis A clone of 441 bp was isolated from a cDNA library of X. laevis oocytes at stages 1, 2, 3 and the DNA sequenced (Fig. 1). Through a Gene Bank search, the clone was identified as an rp-cDNA homologous to L34 rp-cDNA of Homo sapiens (84% nucleotide sequence identity) and Rattus rattus (82% nucleotide sequence identity) (Aoyama et al., 1989). In consideration of this, the clone was named XL34. The size is similar to that of the corresponding mRNA as estimated by Northern blot analysis (see Fig. 4). XL34 cDNA has an open reading frame of 354 bp. The start codon ATG is enclosed in the AAAATGG sequence, where purine (A) is three nt upstream from the start codon, a position that is highly conserved in the mRNA of several eukaryotes, and guanine (G) is four nt downstream from AUG. This sequence pattern is considered to be optimal for efficient recognition and initiation of translation by eukaryotic ribosomes (Kozak, 1986). The 3Vuntranslated region (3VUTR) is made of 60 nt and has the polyadenylation signal (AATAAA) 22 nt downstream from the stop codon TAA (Fig. 1). The 5Vuntranslated region (5VUTR) consists of 27 nt with a brief repeat of four pyrimidines (TTTC). As the cDNA 5V start site was not determined, the repeat of pyrimidines might be of longer extension. A stretch of 8 –20 pyrimidines is typical of the mRNA 5VUTR of several eukaryotic rproteins and of the corresponding genes. Studies conducted both in X. laevis (Amaldi et al., 1995) and in other organisms, such as mouse (Levy et al., 1991) and Dictyostelium (Steell and Jacobson, 1991), indicate that the 5VUTR of the r-
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Fig. 1. Nucleotide and deduced amino acid sequence of cDNA XL34. The sequence AAAATGG that encloses the start codon ATG is underlined. The pyrimidine repeat in the 5Vuntranslated region, the stop codon TAA and the polyadenylation signal are in boldface. The basic residues are marked by the symbol z. The three acidic residues are marked by the symbol ..
proteins mRNA sequence has elements responsible for controlling the translation of these proteins. The pyrimidine stretch may play a key role in regulating the expression of rpgenes. However, Chen et al. (1992) have shown that the presence of the pyrimidine stretch in the 5V region of rpgenes is not necessary for their transcription and translation in Xenopus oocytes. Yet, this finding does not eliminate a possible regulatory control confined to specific stages of embryonic development. XL34 cDNA encodes a protein of 117 residues with an Mr of 13.29 kDa, quite similar to the size of other L34 proteins, as isolated from a variety of living organisms, with two exceptions, i.e. a bacterial L34-like protein of only 88 aa, and the insect Aedes albopictus (130 aa), where an additional
sequence, absent from other L34-like proteins, is present in the C-terminus of the protein (Lan et al., 1994) (Fig. 2). The analysis of the deduced aa sequence indicates that XL34 is rather hydrophobic (42 hydrophobic aa over 117 aa) and has a high percentage of basic aa (16 arginines, 16 lysines, 1 histidine) scattered in the sequence. Acidic aa are poorly represented (one aspartic acid and two glutamic acids) and are located only in the C-terminus half of the deduced protein sequence (Fig. 1). The strong basic character of L34 is a characteristic shared with other mouse r-proteins (Dudov and Perry, 1984; Wiedemann and Perry, 1984), with L29, a yeast r-protein (Kaufer et al., 1983), and with several histones, where basic aa are generally organized in clusters. It has been hypothesized that the high basicity of this protein
Fig. 2. Sequence comparison of X. laevis ribosomal protein L34 with homologous protein of the six organism. The position in the alignment perfectly conserved is indicated by an asterisk (*) and in boldface.
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N. tabacum) and at the C-terminus region from position 89 to 101. A marked difference is present in position 30 –88 at the centre of the sequence (46%, 40% and 30%, respectively). The dendogram of the alignment (Fig. 3) confirms the clustal analysis and indicates that the sequence of L34 protein
Fig. 3. Sequence dendogram of X. laevis ribosomal protein L34 with homologous protein of the six organisms.
may be instrumental for its binding to rRNA in the 60S subunit of eukaryotic ribosomes (Dudov and Perry, 1984; Wiedemann and Perry, 1984; Ulbrich et al., 1979). Further search of the Gene Bank showed that the deduced aa sequence of XL34 has a high identity percentage to the L34-like proteins in the following species: H. sapiens (95% identity) and R. rattus (94%), R. norvegicus (93%), Sus scrofa (88%), Schizosaccaromyces pombe and D. melanogaster (59%), Aedes albopticus (54%); Arabidopsis thaliana, Nicotiana tabacum and Pisum sativum (47 – 49%). Furthermore, XL34 shows a 35% identity to the L34 protein of the bacterial 50S subunits. The high conservativeness of the L34 aa sequence from prokaryotes to eukaryotes suggests that this protein might have been subjected to a strong selective pressure during evolution, and that its role might have a substantial biological meaning from prokaryotes to eukaryotes. XL34 was aligned with six protein sequences, namely H. sapiens, R. rattus, A. albopticus, S. pombe and Methanothermobacter thermautotrophicus. The results indicate high homology of XL34 with the sequences of H. sapiens and R. rattus. By contrast, for the rest of the species compared, the homology is high in the first 30 residues of the N-terminus region (74% identity to S. pombe and A. albopictus, 50% to
Fig. 4. The Northern blot analysis on total RNA forms stages 1 and 2 previtellogenic oocytes (lane A) and vitellogenic oocytes (lane B) with the cDNA XL34 as probe (upper panel). The same filter was reprobed with histone H2 cDNA as control (lower panel). The size was determined with 0.24 – 9.5 Kb RNA ladder (Invitrogen).
Fig. 5. In situ hybridization on sections of paraffin-embedded oocytes with XL34 antisense and sense riboprobe. The signal is evident in stages 1 and 2 oocytes (a). In oocytes at stage 3, the signal decreases (b). Section hybridized with sense riboprobe as negative control (c). The bar indicates the unit length: 100 Am.
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from X. laevis and mammals is very similar. The similarity between the mosquito A. albopictus and the vegetable N. tabacum, two organisms from different phyla, probably results from event of convergence. 3.2. Gene expression in the amphibian oogenesis In X. laevis, as in mammals, gene expression regulation has been reported to occur at transcriptional, posttranscriptional and translational levels (Amaldi et al., 1995). In particular, during X. laevis oogenesis, there is a typical modulation of transcription that is common to all the rpgenes. At the beginning of oogenesis, there is a relatively rapid accumulation of transcripts that peaks at stage 2 followed by a similarly rapid decrease of the transcription activity (Bagni et al., 1990). In this paper, the Northern blot analysis showed that the messenger is abundantly present in the previtellogenic oocytes (stages 1 and 2), in contrast, the signal is lower in the vitellogenic oocytes (stages 3, 4, 5, 6) (Fig. 4). The ‘‘in situ’’ hybridization on paraffin sections, utilizing XL34 antisense and sense riboprobe, indicates that messenger is abundantly present in the whole cytoplasm of stages 1 and 2 oocytes (Fig. 5a), while in oocytes of larger size, the mRNA appears much less concentrated (Fig. 5b). However, one should take into account dilution due to cytoplasm enlargement.
4. Conclusion (1) The cDNA XL34 is 438 bp with an ORF of 351 bp and encodes a protein of 116 residues with an Mr of 13.2 kDa. (2) The nucleotide and aa deduced sequence of XL34 is conserved in prokaryotic and eukaryotic organism. (3) The XL34 messenger is accumulated at the beginning of the X. laevis oogenesis.
Acknowledgements The author would like to thank (1) Prof. C. Campanella for reading and assistance in the preparation of the manu-
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script; (2) Dr. W.J. Lennarz, SUNY at Stony Brook, New York, for the gift of the X. laevis oocytes cDNA library. References Amaldi, F., Camacho-Vanegas, O., Cardinali, B., Cecconi, F., Crosio, C., Loreni, F., Mariottini, P., Pellizzoni, L., Pierandrei-Amaldi, P., 1995. Structure and expression of ribosomal protein genes in Xenopus laevis. Biochem. Cell. Biol. 73, 969 – 977. Aoyama, Y., Chan, Y.L., Wool, I.G., 1989. The primary structure of rat ribosomal protein L34. FEBS Lett. 249, 119 – 122. Bagni, C., Mariottini, P., Annesi, F., Amaldi, F., 1990. Structure of Xenopus laevis ribosomal protein L32 and its expression during development. Nucleic Acids Res. 18, 4423 – 4426. Chen, Q.M., Mariottini, P., Bagni, C., Amaldi, F., 1992. The pyrimidine sequence encompassing the transcription start point of Xenopus laevis ribosomal-protein-encoding genes is not obligatory for activity in oocytes. Gene 119, 283 – 286. Dudov, K.P., Perry, R.P., 1984. The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell 37, 457 – 468. Dumont, J.N., 1972. Oogenesis in Xenopus laevis (Daudin) I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136, 153 – 180. Kaufer, N.F., Fried, H.M., Schwindinger, W.F., Jasin, M., Warner, J.R., 1983. Cycloheximide resistance in the yeast: the gene and its protein. Nucleic Acids Res. 11, 3123 – 3135. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283 – 292. Lan, Q., Niu, L.L., Fallon, A.M., 1994. Mosquito ribosomal protein rpL31 resembles rat rpL34 cDNA and deduced amino acid sequence. Biochim. Biophys. Acta 1218, 4460 – 4462. Levy, S., Avni, D., Hariharan, M., Perry, R.P., Meyuhas, O., 1991. Oligopyrimidine tract at the 5Vend of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad. Sci. 88, 3319 – 3323. Rosbash, M., 1974. Polyadenylic acid-containing RNA in Xenopus laevis oocytes. J. Mol. Biol. 85, 87 – 101. Steell, L.F., Jacobson, A., 1991. Sequence elements that affect mRNA translation activity in developing Dictyostelium cells. Dev. Genet. 12, 98 – 103. Ulbrich, N., Lin, A., Wool, I.G., 1979. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5 S ribosomal ribonucleic acid. J. Biol. Chem. 254, 8641 – 8645. Wiedemann, L.M., Perry, R.P., 1984. Characterization of the expressed gene and several processed pseudogenes for the mouse ribosomal protein L 30 gene family. Mol. Cell. Biol. 4, 2518 – 2528. Wool, I.G., 1979. The structure and function of eukaryotic ribosomes. Annu. Rev. Biochem. 48, 719 – 754.
Sequencing and characterization of the Xenopus laevis ribosomal $ protein L34 cDNA Maria Carmela Vaccaro * Department of Evolutionary and Comparative Biology, University of Naples Federico II, via Mezzocannone n.8, 80134, Naples, Italy Received 14 February 2003; received in revised form 30 May 2003; accepted 20 June 2003 Received by M. D’Urso
Abstract In this paper, a cDNA homologous to the mammalian ribosomal protein (r-protein) L34 was isolated from a Xenopus laevis oocytes library and named XL34. It encodes a protein of 116 residues with an Mr of 13.2 kDa and a highly basic sequence. The nucleotide (nt) and deduced amino acid (aa) sequence have been compared with the L34 sequence from other species. This analysis showed that the L34 is a protein greatly conserved from prokaryotes to eukaryotes. XL34 mRNA is abundantly present in the whole cytoplasm of oocytes at stages 1 and 2. D 2003 Elsevier B.V. All rights reserved. Keywords: Oocytes; Ribosome; Conserved sequence; Evolution
1. Introduction In eukaryotes, ribosomes contain about 80 structural proteins, besides rRNA. Their biosynthesis is a complex process implying the coregulated expression of the rRNA genes and ribosomal protein genes (rp-genes). The latter are coded for by housekeeping genes, since their activity is required for the growth and maintenance of all cell kinds (Wool, 1979). The study of the ribosomal proteins (r-proteins) belonging to a variety of organisms may improve our knowledge of ribosome structure and evolution, as well as elucidate the role of r-proteins in the basic mechanisms of protein synthesis. In this regard, the experimental model utilized in this paper, Xenopus laevis oocytes, is unique with respect to other tissues. In fact, since the ribosomes formed during X. laevis oogenesis are the order of 1012 (Rosbash, 1974), high levels Abbreviations: aa, amino acid(s); nt, nucleotide(s); bp, base pair(s); 3VUTR, 3Vuntranslated region; 5VUTR, 5Vuntranslated region; rp-genes, ribosomal protein genes; r-protein(s), ribosomal protein(s); rp-mRNA, ribosomal protein mRNA. $ The nucleotide sequence data reported in this paper have been submitted to the Gene Bank database under Accession number AY079177. * Tel.: +39-081-2535173; fax: +39-081-2535035. E-mail address: [email protected] (M.C. Vaccaro). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00771-6
of ribosomal protein mRNA (rp-mRNA) are expected for their construction. In this study, we have isolated and sequenced the fulllength cDNA of the X. laevis r-protein L34. The nucleotide (nt) and deduced amino acid (aa) sequences have been compared with the homologous sequences from several living organisms. Furthermore, we have analyzed the expression of the L34 r-protein mRNA in the X. laevis previtellogenic and vitellogenic oocytes. The Northern blot analysis indicates that the messenger is highly concentrated in the previtellogenic oocytes.
2. Materials and method 2.1. Animals and gametes Adult X. laevis females was obtained from Rettili (Varese, Italy). Groups of oocytes at various stages of oogenesis were excised from the ovaries of females anaesthetized with MS222 (Sigma, St. Louis, MO, USA). The stages of X. laevis oocyte growth stages (Dumont, 1972) are st. 1 (50 – 300 Am), st. 2 (300 –450 Am), st. 3 (450 – 600 Am), st. 4 (600 –1000 Am), st. 5 (1000 – 1200 Am), st. 6 (1200 –1300 Am).
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2.2. Cloning and sequencing of XL34 cDNA Unidirectional cDNA library in pBluescript SK( F ) phagemid vector (ZAP Express cDNA cloning kit, Stratagene) constructed using poly(A) mRNA isolated from X. laevis oocytes stages 1, 2, 3, gift from dott. W.J. Lennarz, were screened by standard hybridization methods under conditions of low stringency in a solution of 15% formamide, 10 mM EDTA, 200 mM NaHPO4, pH 7.0, 7% SDS, 1% bovine serum albumin (BSA) at 37 jC. The probe was the first 700 bp of the 5V region of D. melanogaster gurken cDNA and was labeled with the random primer DNA labeled kit (Roche) and 32P-labeled dCTP (NEN). Washes were performed with 0.5 SSPE and 0.5 SDS at 50 jC, and filters were exposed to Kodak YAR5 Film. Few clones were positive and one of these clones was similar to L34 cDNA of mammals, yeast, vegetables and mosquito. The clone was sequenced in both directions manually with the Thermo Sequenase Radiolabeled terminator cycle sequencing kit (USB, Ohio) using the 33P-dNTP (Amersham). All sequence information was processed using DNA Strider software and Clustal.
glycine buffer. The sections were hybridized overnight at 60 jC in 40% formamide, 1 Denhardt’s solution, 5 SSC, 200 Ag/ml tRNA (Sigma) and 100 ng of sense or antisense digoxigenin-labeled RNA on each slide. The slides were exposed to 0.5 SSC, 20% formamide for 1 h at 60 jC and to the following treatments: (1) NTE (10 mM Tris –HCl, pH 7.0, 0.5 M NaCl, 5 mM EDTA) at 37 jC for 15 min; (2) NTE containing RNAse A (10 Ag/ml) at 37 jC for 30 min; (3) NTE at 37 jC for 15 min; (4) 0.5% SSC, 20% formamide at 60 jC for 30 min; (6) 2% Roche blocking solution in 100 mM maleic acid, 150 mM NaCl, pH 7.5 for 30 min. Sections were incubated overnight in anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche), diluted 1:2000 in 2% Roche blocking solution in 100 mM maleic acid, 150 mM NaCl, pH 7.5. Following washing in 0.1% Tween-20, 0.5 mg/ml levamisole (Sigma), sections were incubated at room temperature in the developing solution, BM purple color substrate (Roche) in 2% Tween-20 and 1 mM levamisole. After stopping the reaction in 1 mM EDTA in PBS, sections were mounted in PBS-glycerol and observed on a Leitz Photomicroscope. Photographs were taken using a Kodacolor ASA 100 film.
2.3. Northern blot analysis 3. Results and discussion For analysis of transcripts, total RNA from X. laevis previtellogenic and vitellogenic oocytes was isolated using a Trireagent solution (Sigma). Twenty micrograms of total RNA from each one was separated on 1% formaldehyde agarose gel and blotted on a nylon membrane (Roche). The XL34 cDNA was labeled with the random primer DNA labeled kit (Roche) and 32P-labeled dCTP (NEN), and the RNA blots were hybridized in a mixture of 0.5 M NaHPO4, 7% SDS, 1% BSA, 1 mM EDTA at 60 jC. The filter was washed at 60 jC in 2 SSC, 2% SDS, 0.2 SSC, 0.2% SDS before exposure to X-ray film (Kodak). 2.4. In situ hybridization on sections The XL34 cDNA was digested with EcoRI and XhoI and used as template to synthesize digoxigenin-labeled antisense and sense, with digoxigenin-labeled UTP and, respectively, T3 and T7 RNA polymerase according to the manufacturer recommendations (RNA T3-SP6 transcription and labeling kit, Roche). X. laevis oocytes were fixed in 2% flavin adenine dinucleotide (FAD), 250 mM NaCl, 5% acetic acid for 1 h at 4 jC and processed for embedding in paraffin according to standard techniques. Following attachment onto Superfrost Plus slides (BDH), the 7-Am-thick sections were deparaffinized in hystolemon and rehydrated in graded ethanol concentration. Sections were then fixed in 4% paraformaldehyde in PBS, washed in PBS and digested with proteinase K (Roche) (10 Ag/ml in 20 mM Tris/HCl, pH 7.0, containing 1 mM EDTA, pH 7.2) at 37 jC. After refixing with 4% paraformaldehyde, the sections were rinsed in PBS and incubated in 0.1 M Tris/
3.1. Structural and evolutive analysis A clone of 441 bp was isolated from a cDNA library of X. laevis oocytes at stages 1, 2, 3 and the DNA sequenced (Fig. 1). Through a Gene Bank search, the clone was identified as an rp-cDNA homologous to L34 rp-cDNA of Homo sapiens (84% nucleotide sequence identity) and Rattus rattus (82% nucleotide sequence identity) (Aoyama et al., 1989). In consideration of this, the clone was named XL34. The size is similar to that of the corresponding mRNA as estimated by Northern blot analysis (see Fig. 4). XL34 cDNA has an open reading frame of 354 bp. The start codon ATG is enclosed in the AAAATGG sequence, where purine (A) is three nt upstream from the start codon, a position that is highly conserved in the mRNA of several eukaryotes, and guanine (G) is four nt downstream from AUG. This sequence pattern is considered to be optimal for efficient recognition and initiation of translation by eukaryotic ribosomes (Kozak, 1986). The 3Vuntranslated region (3VUTR) is made of 60 nt and has the polyadenylation signal (AATAAA) 22 nt downstream from the stop codon TAA (Fig. 1). The 5Vuntranslated region (5VUTR) consists of 27 nt with a brief repeat of four pyrimidines (TTTC). As the cDNA 5V start site was not determined, the repeat of pyrimidines might be of longer extension. A stretch of 8 –20 pyrimidines is typical of the mRNA 5VUTR of several eukaryotic rproteins and of the corresponding genes. Studies conducted both in X. laevis (Amaldi et al., 1995) and in other organisms, such as mouse (Levy et al., 1991) and Dictyostelium (Steell and Jacobson, 1991), indicate that the 5VUTR of the r-
M.C. Vaccaro / Gene 318 (2003) 163–167
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Fig. 1. Nucleotide and deduced amino acid sequence of cDNA XL34. The sequence AAAATGG that encloses the start codon ATG is underlined. The pyrimidine repeat in the 5Vuntranslated region, the stop codon TAA and the polyadenylation signal are in boldface. The basic residues are marked by the symbol z. The three acidic residues are marked by the symbol ..
proteins mRNA sequence has elements responsible for controlling the translation of these proteins. The pyrimidine stretch may play a key role in regulating the expression of rpgenes. However, Chen et al. (1992) have shown that the presence of the pyrimidine stretch in the 5V region of rpgenes is not necessary for their transcription and translation in Xenopus oocytes. Yet, this finding does not eliminate a possible regulatory control confined to specific stages of embryonic development. XL34 cDNA encodes a protein of 117 residues with an Mr of 13.29 kDa, quite similar to the size of other L34 proteins, as isolated from a variety of living organisms, with two exceptions, i.e. a bacterial L34-like protein of only 88 aa, and the insect Aedes albopictus (130 aa), where an additional
sequence, absent from other L34-like proteins, is present in the C-terminus of the protein (Lan et al., 1994) (Fig. 2). The analysis of the deduced aa sequence indicates that XL34 is rather hydrophobic (42 hydrophobic aa over 117 aa) and has a high percentage of basic aa (16 arginines, 16 lysines, 1 histidine) scattered in the sequence. Acidic aa are poorly represented (one aspartic acid and two glutamic acids) and are located only in the C-terminus half of the deduced protein sequence (Fig. 1). The strong basic character of L34 is a characteristic shared with other mouse r-proteins (Dudov and Perry, 1984; Wiedemann and Perry, 1984), with L29, a yeast r-protein (Kaufer et al., 1983), and with several histones, where basic aa are generally organized in clusters. It has been hypothesized that the high basicity of this protein
Fig. 2. Sequence comparison of X. laevis ribosomal protein L34 with homologous protein of the six organism. The position in the alignment perfectly conserved is indicated by an asterisk (*) and in boldface.
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N. tabacum) and at the C-terminus region from position 89 to 101. A marked difference is present in position 30 –88 at the centre of the sequence (46%, 40% and 30%, respectively). The dendogram of the alignment (Fig. 3) confirms the clustal analysis and indicates that the sequence of L34 protein
Fig. 3. Sequence dendogram of X. laevis ribosomal protein L34 with homologous protein of the six organisms.
may be instrumental for its binding to rRNA in the 60S subunit of eukaryotic ribosomes (Dudov and Perry, 1984; Wiedemann and Perry, 1984; Ulbrich et al., 1979). Further search of the Gene Bank showed that the deduced aa sequence of XL34 has a high identity percentage to the L34-like proteins in the following species: H. sapiens (95% identity) and R. rattus (94%), R. norvegicus (93%), Sus scrofa (88%), Schizosaccaromyces pombe and D. melanogaster (59%), Aedes albopticus (54%); Arabidopsis thaliana, Nicotiana tabacum and Pisum sativum (47 – 49%). Furthermore, XL34 shows a 35% identity to the L34 protein of the bacterial 50S subunits. The high conservativeness of the L34 aa sequence from prokaryotes to eukaryotes suggests that this protein might have been subjected to a strong selective pressure during evolution, and that its role might have a substantial biological meaning from prokaryotes to eukaryotes. XL34 was aligned with six protein sequences, namely H. sapiens, R. rattus, A. albopticus, S. pombe and Methanothermobacter thermautotrophicus. The results indicate high homology of XL34 with the sequences of H. sapiens and R. rattus. By contrast, for the rest of the species compared, the homology is high in the first 30 residues of the N-terminus region (74% identity to S. pombe and A. albopictus, 50% to
Fig. 4. The Northern blot analysis on total RNA forms stages 1 and 2 previtellogenic oocytes (lane A) and vitellogenic oocytes (lane B) with the cDNA XL34 as probe (upper panel). The same filter was reprobed with histone H2 cDNA as control (lower panel). The size was determined with 0.24 – 9.5 Kb RNA ladder (Invitrogen).
Fig. 5. In situ hybridization on sections of paraffin-embedded oocytes with XL34 antisense and sense riboprobe. The signal is evident in stages 1 and 2 oocytes (a). In oocytes at stage 3, the signal decreases (b). Section hybridized with sense riboprobe as negative control (c). The bar indicates the unit length: 100 Am.
M.C. Vaccaro / Gene 318 (2003) 163–167
from X. laevis and mammals is very similar. The similarity between the mosquito A. albopictus and the vegetable N. tabacum, two organisms from different phyla, probably results from event of convergence. 3.2. Gene expression in the amphibian oogenesis In X. laevis, as in mammals, gene expression regulation has been reported to occur at transcriptional, posttranscriptional and translational levels (Amaldi et al., 1995). In particular, during X. laevis oogenesis, there is a typical modulation of transcription that is common to all the rpgenes. At the beginning of oogenesis, there is a relatively rapid accumulation of transcripts that peaks at stage 2 followed by a similarly rapid decrease of the transcription activity (Bagni et al., 1990). In this paper, the Northern blot analysis showed that the messenger is abundantly present in the previtellogenic oocytes (stages 1 and 2), in contrast, the signal is lower in the vitellogenic oocytes (stages 3, 4, 5, 6) (Fig. 4). The ‘‘in situ’’ hybridization on paraffin sections, utilizing XL34 antisense and sense riboprobe, indicates that messenger is abundantly present in the whole cytoplasm of stages 1 and 2 oocytes (Fig. 5a), while in oocytes of larger size, the mRNA appears much less concentrated (Fig. 5b). However, one should take into account dilution due to cytoplasm enlargement.
4. Conclusion (1) The cDNA XL34 is 438 bp with an ORF of 351 bp and encodes a protein of 116 residues with an Mr of 13.2 kDa. (2) The nucleotide and aa deduced sequence of XL34 is conserved in prokaryotic and eukaryotic organism. (3) The XL34 messenger is accumulated at the beginning of the X. laevis oogenesis.
Acknowledgements The author would like to thank (1) Prof. C. Campanella for reading and assistance in the preparation of the manu-
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