Transcriptional activation by the parvoviral nonstructural protein NS-1 is mediated via a direct interaction with Sp1.

The nonstructural protein NS-1, encoded by the parvovirus minute virus of mice, is a potent regulator of viral gene expression. NS-1 does not bind DNA in a sequence-specific manner, and the mechanism by which it modulates viral promoter function is unclear. We have used Gal4-NS-1 fusion protein constructs to identify and characterize an activating domain encoded within the C-terminal 88 amino acids of NS-1 which competes effectively with the acidic activator domain of the herpes simplex virus VP16 protein. DNA affinity chromatography and immunoprecipitation experiments demonstrate that protein-protein interactions between the transcription factor Sp1 and NS-1 are required to bind NS-1 to promoter DNA in vitro. Cotransfection of Gal4-NS-1 and Sp1-VP16 acidic activator constructs into Drosophila melanogaster Schneider cells, which lack endogenous Sp1, stimulates transcription from a minimal promoter containing five Gal4 binding sites, while single-construct transfections do not. Cotransfection of Schneider cells with wild-type NS-1 and Sp1 constructs activates transcription from a simian virus 40 promoter 10- to 30-fold over that of either construct alone. Thus, Sp1-NS-1 interactions in vivo can stimulate transcription from a heterologous promoter containing Sp1 binding sites.

Use of avidin-biotin subtractive hybridization to characterize mRNA common to neurons destroyed by the selective neurotoxicant trimethyltin.

Trimethyltin (TMT), a selective neurotoxicant, destroys a distinct subpopulation of neurons which possess no known biochemical or anatomic linkage. However, TMT-sensitive neurons may share common gene products related to susceptibility. In an effort to isolate mRNAs common to TMT-sensitive neurons, avidin/biotin based-subtractive hybridization was used to generate a cDNA library specifically related to TMT-toxicity. Out of 50 cDNAs, two clones hybridized only to poly(A+) mRNA isolated from the brains of saline-treated rats. Two of these cDNAs, p9T10 and p9T19, were used for in situ hybridization; both hybridized to hippocampus, limbic cortex, amygdala and other regions destroyed by TMT, suggesting that these probes identified mRNA enriched in TMT-sensitive neurons. The patterns of in situ hybridization coupled with the loss of p9T10 and p9T19 hybridization to mRNA isolated from the brains of TMT-treated rats suggests that one or both of these two clones may represent mRNA found in neurons damaged by TMT. The combination of selective neurotoxic lesions followed by cDNA subtractive hybridization should prove to be a useful strategy for the isolation of gene products from specific neuronal populations.

Localization and characterization of stannin: relationship to cellular sensitivity to organotin compounds.

The cDNA encoding the protein stannin was isolated previously via subtractive hybridization, using differential expression after trimethyltin (TMT) intoxication, as a basis for isolating mRNA which may be expressed in TMT-sensitive cells. Initial characterization revealed a novel gene product which was differentially expressed in several tissues sensitive to TMT. In the current study, biochemical and molecular techniques were used to quantitate stannin expression at the cellular and subcellular levels. Northern blot analysis showed that the stannin 3.0 kb mRNA transcript was present, in decreasing amounts, in: spleen, hippocampus, neocortex, cerebellum, striatum, midbrain, kidney and lung. Liver, heart, skeletal muscle and testis showed no detectable expression of stannin mRNA. Immunoblot analysis using antipeptide antisera raised against stannin indicated a high level of expression in spleen, followed by brain and kidney. Stannin mRNA was present during early brain development and consolidated by post-natal day (PND) 20 to patterns and levels seen in adults. In situ hybridization studies showed widespread neuronal expression of stannin mRNA at PND 1, which shifted to a restricted pattern of expression in specific regions by PND 20. Stannin was partially purified from rodent brain and spleen using cation exchange, sizing and hydrophobic interaction chromatography. It behaved as a monomer throughout purification. Stannin was also expressed in a baculovirus system, using a series of constructs containing the entire cDNA, 1.0 kb of DNA flanking the open reading frame, and a 400 bp open reading frame minimal construct. While all constructs expressed stannin, the best expression was seen with the entire cDNA. Based on current findings, we suggest that stannin expression is necessary but not sufficient for TMT toxicity.

Molecular neurotoxicology of trimethyltin: identification of stannin, a novel protein expressed in trimethyltin-sensitive cells.

The molecular basis of selective vulnerability of specific neuronal populations to neurotoxicants remains a key focus in neurotoxicology. Trimethyltin (TMT) selectively damages neurons in rodent and human central nervous system after a single exposure. By coupling subtractive hybridization with molecular cloning techniques, we isolated a cDNA specifically localized in TMT-sensitive cells. This 2.9-kilobase cDNA encodes a putative 10-kDa peptide of 88 amino acids, termed "stannin." In immunocytochemical experiments, antisera raised against the amino terminus of stannin exhibited strong immunoreactivity in TMT-sensitive neurons in the hippocampus and entorhinal cortex, areas previously identified by in situ hybridization. Northern blot and in situ hybridization experiments detected a 3.0-kilobase stannin mRNA in brain, spleen, and kidney; expression occurred as early as embryonic day 15 in rat brain and thymus. In situ hybridization in human hippocampus demonstrated a stannin mRNA in pyramidal and dentate gyrus neurons. High stringency Southern blot analysis of genomic DNA identified stannin homologs in rabbit, Drosophila, and human. These findings indicate that stannin is present in TMT-sensitive cells and may play a role in the selective toxicity of organotin compounds.