Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.     

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.     

Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase

It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.     

Ancient and recent duplications of the rainbow trout Wilms' tumor gene.

The Wilms' tumor suppressor (WT1) gene plays an important role in the development and functioning of the genitourinary system, and mutations in this gene are associated with nephroblastoma formation in humans. Rainbow trout (Oncorhynchus mykiss) is one of the rare animal models that readily form nephroblastomas, yet trout express three distinct WT1 genes, one of which is duplicated and inherited tetrasomically. Sequence analyses suggest an ancient gene duplication in the common ancestor of bony fishes resulted in the formation of two WT1 gene families, that conserve the splicing variations of tetrapod WT1, and a second duplication event occurred in the trout lineage. The WT1 genes of one family map to linkage groups 6 and 27 in the trout genome map. Reverse transcribed polymerase chain reaction (RT-PCR) expression analysis demonstrated little difference in W