In order to identify the murine homologue of CTNS, Ctns, a E11.5 mouse embryonic cDNA library (Clontech) was screened with a probe spanning the entire ORF of the human CTNS gene. Three overlapping cDNA clones were isolated and completely sequenced. The reconstructed sequence was homologous to a region of the human CTNS cDNA from base pair 565 to 2388. RACE (rapid amplification of cDNA ends) PCR of an E13.5 adaptor ligated-cDNA library (kindly provided by D. Weil, Institut Pasteur, Paris, France) was employed to isolate the remainder of the 5' and 3' murine cDNA sequences using the Ctns cDNA primers 1.1 (5'-ACC AAG AAC CGG ATC CTG GGG CA-3') and 2.1 (5'-GCC CTC TTT CCT ACC TCC ACT TTC TGA-3'), coupled with the adaptor primer AP1, and the nested primers 1.2 (5'-GCA TTT TTC TCT CCG CGA GGC AC-3') and 2.2 (5'-GTC AAT CAG TAA GCT GCC CTG GAT G-3'), coupled with AP2, for the amplification of the 5' and 3' ends, respectively (Marathon-Ready cDNA kit, Clontech).
The resulting consensus Ctns cDNA sequence is 2464 bp long (GenBank accession number-AJ250670) with a 19 bp 5' non-coding region, a 1344 bp 3' non-coding region and a 1101 bp ORF which, like CTNS, is predicted to encode a 367 aa protein. The Ctns translation start site, which likewise lies within a Kozak consensus sequence (GAGACCAatgA), and the polyadenylation site are predicted to be equivalent to those deduced for CTNS. All of the 5' cDNA sequences amplified by RACE-PCR began at the same position, i.e. with a sequence homologous to the beginning of the CTNS exon 3. No sequences homologous to the CTNS untranslated exons 1 and 2 were detected. Moreover, two EST clones (GenBank accession numbers gi9165096 and gi6083297) containing 69 and 126 bp of upstream sequence, respectively, preceding the beginning of the 5' non coding region that we identified, also do not show homology with the CTNS exons 1 and 2. At the nucleotide level, Ctns shows 83% homology with CTNS for the ORF and 58.8% homology over 1257 bp for the 3' untranslated region. The predicted encoded amino acid sequence shows 83.9% identity and 92.6% similarity with cystinosin (Figure ). The uncleavable signal peptide, seven amino-terminal N-glycosylation sites, seven transmembrane domains and lysosomal targetting signal of cystinosin are conserved in the murine sequence.
Figure 1 Comparison of the human and murine CTNS genes and encoded products. (A) Alignment of the human and murine cystinosin sequences. Asterisks indicate identical amino acid residues. Colons indicate residues of high similarity and fullstops residues of low (more ...)
In order to determine the exon/intron structure of Ctns, the 129/Sv Ev Tac flor DNA genomic library (Stratagene) was screened with a cDNA probe homologous to the CTNS exons 7 to 12 and four clones were isolated. Sequence analysis of their inserts demonstrated that the four overlapping clones spanned all the Ctns exons. The Ctns gene spans 14 kb is composed of ten exons and its exon/intron boundaries are identical to that of CTNS (Figure ). A combination of Long Range PCR (Expand Long Template PCR System, Boehringer Mannheim) and sequencing permitted an estimation of intron sizes. A comparison of the sizes of CTNS and Ctns introns showed that, with the exception of the human introns 3 and 5, intron sizes were similar between both species (Figure ).
expression pattern was determined by hybridisation of a probe spanning exons 4 and 5 to a Clontech multiple tissue northern blot (MTN 7762-1). A transcript with an expected size of 2.5 kb, highly expressed in kidney and liver, less expressed in heart, and weakly expressed in brain, spleen, lung and testis was detected (Figure ); a proportionally faint approximately 4.4 kb transcript could also be seen, reminiscent of the additional 4.4 kb CTNS
transcript detected in human pancreas [10
]. In contrast to the CTNS
transcript, no expression was detected in skeletal muscle (Fig. ) whereas hybridisation with a β-actin cDNA control probe confirms the loading of RNA for all tissues (Fig. ). A possible explanation for this differential pattern of expression is that the promoter situated upstream of the CTNS
exon 1 has been lost during the course of evolution and the working murine promoter does not direct the same tissue expression, as has been proposed for IGF II
]. Similarly, it is possible that sequences corresponding to the CTNS
exons 1 and 2 have also been lost during evolution or, alternatively, they exist in the form of pseudo-exons in the upstream genomic sequence. However, low stringency hybridisation of the genomic clones containing theCtns
gene with a probe corresponding to the CTNS
exons 1 and 2, did not detect any homologous sequences thus providing support for the former hypothesis. Moreover, comparison of 1.1 kb of genomic sequence immediately upstream of the murine exon 1, which is hypothesised to contain the Ctns
promoter, with the CTNS
intron 2 sequence detected a region of significant homology (66.7% over 306 bp). These results suggest the possible presence of an alternative CTNS
promoter. Such regions of homology between the regulatory sequences of other human and murine homologue, such as WT1
, have also been described [15
Figure 2 Northern blot analysis of Ctns expression. (A) A 2.5 kb transcript, accompanied by a faint 4.4 kb transcript, can be seen in all tissues except skeletal muscle upon hybridisatin with a Ctns specific probe. (B) Following hybridisation with a β-actin (more ...)
The chromosomal localisation of the Ctns
gene was determined by PCR amplification of the mouse T31 irradiation hybrid panel [17
] with the primers LOC/1 (5'-TGT GGC CCA TGG ACT TGA AG-3') and LOC/2 (5'-CAA TCT GGC AGG CAC CTC A-3') situated in the 3' non-coding region. Two point lod score analysis resulted in a significant score of 5.37 and 5.36 with the chromosome 11 markers D11Mit279 and D11Mit7, respectively. A highly significant score of 12.25 and 11.09 was obtained with the ESTs AA589579 and R74628, respectively. R74628 maps distal to D11Mit7 and both of these markers are located within two contigs which lie in the region of synteny with human chromosome 17. Hence, this leads us to assume that Ctns
also lies in the region of chromosome 11 syntenic with chromosome 17 which contains the human CTNS