| Summary: | Intracellular proteins are phosphorylated by kinases and dephosphorylated by phosphatases. Protein phosphatase inhibitor-1 (I-1) inhibits protein phosphatase-1 (PP-1), thereby increasing the phosphorylated state of proteins in the cell. The initial aim of the work reported here was to determine whether I-1 is a kidney 'stem' cell marker (Svennilson <i>et al.,</i> 1995); if so, it would be the first unique marker for these cells. The project involved characterizing the protein coding region of the mouse I-1 gene, determining the I-1 protein expression pattern in the developing mouse embryo and analysing potential promoter elements of the I-1 gene. A mouse genomic library was screened using a mouse cDNA clone homologous to the published rat I-1 mRNA and two types of clones with positive sequence homology to the rat mRNA sequence were isolated. These overlapping clones were sequenced, and analysis showed that the predicted mRNA and protein sequences contain 513 nucleotides and 171 amino acids, respectively, with both sharing over 95% homology with the known rat sequences. Further comparison of the predicted mouse protein I-1 sequence to those of rabbit, rat and human showed that there is strong homology across species, and that all share motifs which are important for inhibiting PP-1 (a KIQF sequence at amino acid positions 9-12 and a threonine at position 35). The mouse I-1 gene contains seven protein-coding exons which lie in a 7 kb region of DNA on chromosome 15, band F. Svennilson <i>et al.</i> (1995) detected I-1 expression in peripheral metanephric mesenchyme (nephron progenitor) cells in rate sections using RNA <i>in situ</i> hibridization. With this in mind, the mouse protein expression pattern was determined using wholemount tissue, a commercially available anti-I-1 antibody and fluorescent confocal microscopy. The results showed that I-1 is not a 'stem' cell marker in the developing kidney, but is restricted to the peripheral epithelial layer of cells of the kidney i.e. the mesothelium.
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