Lingyun Zhu

Analysis of the human neurofibromatosis type 2 gene promoter and its expression

OBJECTIVE: It is hypothesized that transcriptional regulation plays an important role for neurofibromatosis type 2 (NF2) expression in Schwann cells and other cell types. The objective of this study is the isolation and characterization of the transcriptional regulatory elements of the NF2 gene. STUDY DESIGN AND SETTING: A bacterial artificial chromosome library and a partial genomic DNA library were used to isolate the human NF2 gene; NF2 promoter-luciferase constructs were generated, and promoter activities were assayed. This study was carried out in a molecular biology laboratory. RESULTS: A bacterial artificial chromosome clone with an approximately 100-kilobase insert containing nearly the entire human NF2 gene has been isolated. An additional 5’ NF2 sequence has also been cloned. Transient transfection experiments demonstrate strong promoter activity from the NF2 5’ flanking DNA. CONCLUSIONS: The NF2 gene is approximately 100 kilobases long. Both positive and negative regulatory elements are present in NF2 5’ flanking regions. SIGNIFICANCE: Better understanding of the NF2 gene and its regulation will improve molecular diagnostics and ultimately treatment of patients with NF2. (Otolaryngol Head Neck Surg 2000; 123:413-8.)

Induction of apoptosis by the p53-related p73 and partial inhibition by the baculovirus-encoded p35 in neuronal cells

To better understand whether the p53-related p73 gene could induce neuronal apoptosis, we tested whether p73 induced cell killing in three neuronal cell lines and whether apoptosis could be inhibited by p35, a baculovirus-encoded protein that blocks caspase 3. Recombinant adenoviruses carrying the hemagglutinin (HA)-tagged p73beta or p35, or the green fluorescent protein gene driven by the cytomegalovirus immediate-early promoter were constructed, and used to infect human SK-N-AS and SK-N-SH neuroblastoma, and rat PC12 pheochromocytoma cells. Infection with Adp73beta virus resulted in p73beta over-expression and substantial reduction of cell viability due to apoptosis in all three neuronal cell lines as compared with the control AdGFP virus. These results indicate that p73beta over-expression in neuronal cells could induce apoptotic cell death regardless of the endogenous expression of p73. The p73 effect was partially blocked by co-expression of the wild-type p35, suggesting caspase-mediated cell killing. Insertion of a hemagglutinin (HA) tag at the N-terminus of p35 markedly reduced its ability to inhibit the p73 effect compared with the wild-type p35, while insertion of an HA tag to the C-terminus of p35 had no appreciable effect. Taken together, our results suggest that the N-terminal structure of p35 is critical for its anti-apoptotic activity on p73-induced apoptosis in neuronal cells.