Results

2.1. Cloning and Tissue Expression of Bis

A partial clone of Bis, the product of the Bag-3 gene, a member of the Bcl-2 associated athanogene family of Bcl-2-binding proteins (36), was originally isolated from a cDNA library obtained from HeLa cells grown for 10 h in the presence of 250 |M ZnCl2 (37). Comparison by Blast analysis with the Bag-3 gene sequence present in GenBank confirmed that the nucleotide sequence of this clone corresponded to Bis sequences in the region between nucleotides 915 and 1728. The full-length 2.-kb Bis cDNA was obtained through reverse transcription-polymerase chain reaction (RT-PCR), using primers deduced from the published sequence (33) and as template RNA from zinc-induced HeLa cells (37).

Domain analysis of Bis reveals the previously reported Bag domain and a WW region (Fig. 1). The WW domain is a small globular module composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction (38). Functionally, it is similar to the SH3 domain in the binding to polyproline ligands (39).

The tissue expression profile of the Bis gene was obtained by Northern blot and showed that a single specie of 2.6-kb mRNA was present in a variety of adult human tissues, with high levels in skeletal muscle, heart, and lung (Fig. 2).

2.2 Regulation of the Expression of the Bis Gene by Copper

Because our clone of Bis was isolated from a library made from RNA of zinc-induced HeLa cells, we next examined if copper regulated the expression of this gene. Thus, HeLa cells were exposed for different times to 250 ||M and 500 ||M CuSO4 and experiments of Northern blot were performed

Fig. 2. Tissue expression of the Bis gene. The tissue distribution of Bis mRNA was examined in Northern blot experiments using RNAs from human adult tissues (CLONTECH) and a 32P-labeled human Bis probe, nucleotide region 915-1728 (GenBank). Levels of RNA were normalized for equal amounts of P-actin mRNA.

Fig. 2. Tissue expression of the Bis gene. The tissue distribution of Bis mRNA was examined in Northern blot experiments using RNAs from human adult tissues (CLONTECH) and a 32P-labeled human Bis probe, nucleotide region 915-1728 (GenBank). Levels of RNA were normalized for equal amounts of P-actin mRNA.

using as probe the Bis cDNA region between nucleotides 915 and 1728. In Fig. 3 are shown the results: Bis probe hybridized to a single mRNA species, which was accumulated with time-dependent kinetics following exposure to the cited concentrations of copper (Fig. 3A). Bis mRNA was highly accumulated between 1 and 6 h after exposure to 250 |M CuSO4 (Fig. 3A: lanes 1, 3, 5, 7, and 9) or 500 |M CuSO4 (Fig. 3A: lanes 2, 4, 6, 8, and 10) with respect to the controls (Fig. 3A-C). Because copper is a known positive regulator of Hsp synthesis (23), we next examined if in the same experimental conditions Hsp70 expression was activated also. Northern blot experiments were performed on the same samples, using as a probe the human Hsp70 cDNA. We found that Hsp70 mRNA was highly accumulated in cells grown in the presence of 250 |M and 500 |M CuSO4, with a kinetic very similar to Bis (Fig. 3B). Finally, a probe of the human MTI-e cDNA, which hybridizes to all the different MT mRNA isoforms, was used to determine the activation of MT genes in these experimental conditions. The kinetic of expression of MTs (Fig. 3A) overlapped with the copper-mediated responses of Bis (Fig. 3C) and Hsp70 genes (Fig. 3B). Therefore copper appear to regulate with similar time-dependent kinetics the expression of Bis together with two other well characterized metal-regulated genes. The lower panel of the figure shows the hybridization of a human ribosomal 28S RNA probe as internal control (Fig. 3D).

2.3 Subcellular Localization of Bis Protein Cells Grown in Absence or in the Presence of High Concentrations of Copper

To examine the intracellular localization of the Bis protein, we used indirect immunofluorescence technique. A polyclonal rabbit antiserum was generated against the last 16 amino acids of the carboxy-

0 0

Post a comment