Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene

https://doi.org/10.1016/S0167-4781(01)00272-XGet rights and content

Abstract

We have cloned and characterized a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene, including the complete transcribed region and 488 bp of upstream regulatory sequences. We have also isolated two L18 cDNAs from another tilapia (Oreochromis niloticus) with a few conservative nucleotide differences. Our results suggest the presence of two genes in both species. Reporter constructs were tested for transient expression in CV1 cells and in microinjected zebrafish and tilapia embryos. The tilapia L18 promoter was able to drive expression of the reporter gene in all three experiments, with no apparent preference for a particular tissue. The tilapia L18 promoter is therefore likely to be a powerful tool to drive tissue-independent gene expression in fish.

Introduction

Ribosomes are composed of 3–4 rRNA molecules and about 60–80 protein subunits. Ribosomal protein (r-protein) genes have been considered housekeeping genes, with a strongly coordinated constitutive expression that is closely linked to that of the different ribosomal RNAs to form the entire ribosome. R-protein expression is adjusted according to the needs of the cell for protein biosynthesis [1].

The coordinated expression of the r-protein genes is controlled at various levels. In prokaryotes, most of them are organized in a comparatively small number of operons [2], which encode a repressor of both transcription and translation that binds to a specific site on the polycistronic RNA [3], [4]. In eukaryotes, r-protein gene-specific regulatory elements have been identified in the different promoter sequences, consistent with a coordinated expression. Most eukaryotic r-protein genes have special short promoters devoid of a TATA box. The transcription start site (mostly a C) is part of an oligopyrimidine tract flanked by GC-rich sequences [5], [6], [7], [8]. The mRNA thus starts with a 5′-terminal oligopyrimidine tract (5′-TOP) that is required for translational control of r-proteins [9]. Such a 5′-TOP tract was recently described in the mRNA coding for the medaka (Oryzias latipes) r-protein gene S3a [10]. Control of expression at the level of RNA splicing and degradation has also been described (for a review, see [11]).

The r-protein L18 in concert with L5 and L25 interacts with the 5S rRNA, which is a component of the large ribosomal subunit [12]. Only a few genomic sequences have been reported in higher eukaryotes (human [13], Xenopus [14], Caenorhabditis elegans [15]) and only one promoter study in Xenopus laevis [16]. Here we report the molecular cloning of a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene (tiL18). We analyzed its expression and performed promoter studies, using a reporter gene strategy, by transient assays in cultured cells or in microinjected zebrafish (Danio rerio) and tilapia (Oreochromis niloticus) embryos. We observed high levels of expression under ideal metabolic conditions suggesting that the L18 regulatory sequences are likely to drive efficient and constitutive expression of a transgene in tilapia.

Section snippets

Oligonucleotides

Oligonucleotide primers (Eurogentec, Seraing, Belgium) used for PCR procedures were: L18pf1: TCCCTTTTCGCTCTGAGTCC; L18pr1: TTGGTCCTGCTCATGAACAG; T12MG, T12MA, T12MT and T12MC (M represents G, A, or C); nL18pr1: TTCTTGTAGCCGCAGCTGGCTC; fL18pr1: TTGGTCCTGCTCATGAACAG; pL18f1: AGCCATGGCCCGATTAGAATGCTTGGTG; pL18r1: GGGAATTCTTGGACTCAGAGCGAAAAGGG.

All RT-PCR or PCR products were cloned into the pCRII vector and sequenced.

Library screening

Using a salmon L18 cDNA (GenBank No. CAC36993) as a probe, 2×106 plaques

Genomic library screening

A salmon ribosomal protein L18 cDNA clone was used as a probe to screen 2.0×106 λ-phage plaques from a tilapia (O. mossambicus) genomic library. One out of 94 positive clones containing approx. 13 kb of insert, λ5jtiL18, was chosen for restriction mapping and Southern blot analysis (data not shown). A 4.5 kb XhoI fragment hybridizing to the L18 probe was subcloned (pB4.5-tiL18) and sequenced. Primers from the 5′- and 3′-ends of the pB4.5-tiL18 insert were used to identify flanking regions by

Discussion

Although heterologous regulatory sequences are often used to express transgenes in fish with success, a number of researchers have recommended the use of fish-derived sequences for appropriately regulated and high levels of expression. In addition, the unacceptability to the food market of transgenic fish containing heterologous sequences leads to a demand for new fish promoters [26]. We present a new, strong fish promoter that can be used to drive ubiquitous expression of a gene of interest.

Acknowledgments

This work was supported by the ‘Région Wallone (ULg 1815); the ‘Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles’ (PAI P4/30 and ‘Actions de Recherche Concertées’: 95/00-193); the Fonds National de la Recherche Scientifique (FNRS) (−3.4537.93 and −9.4569.95), the EU (No. BIO4-CT97-0554). M.M. is a ‘Chercheur qualifié’ at the Fonds National de la Recherche Scientifique (FNRS). A.M. held fellowships from ‘CGRI’ and ‘ULg patrimoine’. L.F.M held a scholarship from CAPES,

References (36)

  • R.R. Genuario et al.

    The GA-binding protein can serve as both an activator and repressor of ribosomal protein gene transcription

    J. Biol. Chem.

    (1996)
  • R.S. Carter et al.

    The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors

    J. Biol. Chem.

    (1992)
  • M. Nomura et al.

    Regulation of the synthesis of ribosomes and ribosomal components

    Annu. Rev. Biochem.

    (1984)
  • M. Nomura

    Regulation of ribosome biosynthesis in Escherichia coli and Saccharomyces cerevisiae: diversity and common principles

    J. Bacteriol.

    (1999)
  • T. Allen et al.

    Phylogenetic analysis of L4-mediated autogenous control of the S10 ribosomal protein operon

    J. Bacteriol.

    (1999)
  • L.M. Wiedemann et al.

    Characterization of the expressed gene and several processed pseudogenes for the mouse ribosomal protein L30 gene family

    Mol. Cell. Biol.

    (1984)
  • M. Wagner et al.

    Characterization of the multigene family encoding the mouse S16 ribosomal protein: strategy for distinguishing an expressed gene from its processed pseudogene counterparts by an analysis of total genomic DNA

    Mol. Cell. Biol.

    (1985)
  • N. Hariharan et al.

    Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region

    Proc. Natl. Acad. Sci. USA

    (1990)
  • Cited by (6)

    • Application of comparative genomics in fish endocrinology

      2002, International Review of Cytology
    • Gene structure of the carp fish ribosomal protein L41: Seasonally regulated expression

      2002, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Conversely, in other r-protein genes, this orthodox cis-acting sequence is replaced by the γ element that can bind TBP, as is the case with the mouse L32 r-protein promoter [36,37]. The cL41 transcription start site is separated from the ATG by one small intron and the 5′ region comprises two T-rich regions (−468 and −246) similar to those described for yeast [38] and tilapia r-protein promoters [39]. In higher eukaryotes, the promoter region of the r-proteins comprises four elements recognizable by the ubiquitous transcription factors α (RFX-1), β (GABP), γ (γ-factor), and δ (NF-E1) [37].

    View full text