In silico identification and expression of SLC30 family genes: an expressed sequence tag data mining strategy for the characterization of zinc transporters' tissue expression

Abstract

Background: Intracellular zinc concentration and localization are strictly regulated by two main protein components, metallothioneins and membrane transporters. In mammalian cells, two membrane transporters family are involved in intracellular zinc homeostasis: the uptake transporters called SLC39 or Zip family and the efflux transporters called SLC30 or ZnT family. ZnT proteins are members of the cation diffusion facilitator (CDF) family of metal ion transporters.

Results: From genomic databanks analysis, we identified the full-length sequences of two novel SLC30 genes, SLC30A8 and SLC30A10, extending the SLC30 family to ten members. We used an expressed sequence tag (EST) data mining strategy to determine the pattern of ZnT genes expression in tissues. In silico results obtained for already studied ZnT sequences were compared to experimental data, previously published. We determined an overall good correlation with expression pattern obtained by RT-PCR or immunomethods, particularly for highly tissue specific genes.

Conclusion: The method presented herein provides a useful tool to complete gene families from sequencing programs and to produce preliminary expression data to select the proper biological samples for laboratory experimentation.

Figure 1

Genomic organization of SLC30A8 and SLC30A10 genes.

Figure 2

Partial alignment of ZnT proteins A: Partial amino acid alignment of transmembrane domains V and VI from ZnT proteins identified in Homo sapiens was performed with ClustalW. Putative transmembrane domains were determined by the TMPred program [38]. Residues conserved in more than 50 % sequences are boxed in black, while semi-conservative substitutions are boxed in grey. B: Partial amino acid alignment of histidine rich-loop domain from ZnT proteins identified in Homo sapiens was performed with ClustalW. The histidine residues are boxed in black, while Serine residues are boxed in grey. For ZnT-10, the basic residues are underlined.

Figure 3

Dendrogram of ZnT proteins. Bootstrapping (2000 replicate sets) and calculation of the consensus tree by the neighbour-joining method were performed with the DAMBE program. The numbers indicate bootstrapping values as a percentage at internal nodes. The scale of the branch length is given in amino acid substitutions per site. Accession numbers in Entrez databanks are indicated for protein sequences excepted for ZnT-10 whose accession number corresponds to the cDNA sequence. Zip-2 protein sequence was used as an outgroup.

Figure 4

In silico determination of SLC30 genes tissue expression. The SLC30 sequences (ORF, 5' and 3' UTR) were used for a BLASTN search of the human EST database through NCBI BLAST web service [36]. The significant ESTs (a bit score >150 and an E-value <0.001) were sorted and information regarding each cDNA library was retrieved from either the human Unigene databank [40] or from the respective company catalogue. The calculated frequency of each mRNA transcript for a given tissue is represented by grey levels intensities ranging from no occurrence (white) to values higher than 3.1 occurrences (black).