Nuclear factors specifically bind to upstream sequences of aXenopus laevisribosomal protein gene promoter

Abstract

The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position.

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

“…1, we analyzed, by methylation interference and DNase footprinting, the interaction of the ABF1 factor with both the coding (probe 2) and the noncoding (probes 1 and 3) strands of the L2A and L2B promoters. For methylation interference, 60,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 2 jig of proteins from fraction 80 and treated as described by Carnevali et al (5); for DNase I footprinting, 10,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 3 jig of proteins from fraction 80 and treated as described by Huet et al. (14).…”

The bidirectional promoter of the divergently transcribed mouse Surf-1 and Surf-2 genes.

“…Sites Su2 and Su3 were found to bind the same or similar factor(s) that was originally identified for two ribosomal protein genes, RPGa in mouse rpL32 (1) and XrpFl in Xenopus rpL14 (5). Surf-1 and Surf-2 are unique genes (32) and are transcribed at a low level (34), and thus they most likely do not encode ribosomal proteins, which are members of multigene families containing processed pseudogenes and are transcribed at a high level (14).…”

“…3. The following oligonucleotides contain known factor binding sites: rpL32 RPGcx, 5'-GATCCACCCAGAGCCGGAAG TGCTTCCCTG-3'; rpL32 RPG5, 5'-AATTCCGTAAAACA GATGGCAGCCACCTCG-3'; adenovirus E4.ATF, 5'-AAGC TTCTAAAAAATGACGTAACGG-3'; and Ad2MLP.UEF, 5'-GATCCGTAGGCCACGTGACCGG-3'. The sequence of the neomycin sequencing primer was 5'-ACCTGCGTGCA ATCCATCTTGTTCAATCATGCGAA-3'; the sequence of the HindIII-BamHI adaptor was 5'-AGCTTCCTG-3'.…”

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

“…Amino acid sequence comparison of the encoded 188 amino acid protein to the GenBank database revealed a high level of homology to other L18 sequences : 85.5% identity with salmon (GenBank No. CAC36993), 85.7% with rat [25] and 85.7% with L14a and 85.1% with L14b (L18 equivalents) from X. laevis [16] (Fig. 2A).…”

“…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).…”