Mouse Brn-3 family of POU transcription factors: a new aminoterminal domain is crucial for the oncogenic activity of Brn-3a

The class IV POU domain genes Brn-3a, -b and -c are differentially expressed during neural development and at least Brn-3a also in neuroectodermal tumors. In contrast to Brn-3b and Brn-3c, Brn-3a encodes two protein variants: Brn-3a(l) and Brn-3a(s). Brn-3a(s) lacks 84 aminoterminal residues but is otherwise identical to Brn-3a(l). Outside the well conserved carboxyterminal POU domains all three Brn-3 proteins (-a, -b and -c) diverge until the aminoterminal end where a new domain of about 100 amino acids is identified. This domain is conserved only between Brn-3 proteins and other class IV POU factors. Brn-3a(l) that contains the complete domain but not Brn-3a(s) that lacks 84 amino acids of it is able to tumorigenically transform primary fibroblasts. Brn-3b that lacks 40 amino acids of the new domain does itself not transform, but abolishes the oncogenic potential of Brn-3a(l) when transfected together. This demonstrates not only that Brn3-a(l) is a proto-oncogene and may well be causally involved in the generation of neuroectodermal tumors but also suggests that the intactness of the new aminoterminal domain described here is crucial for oncogenic activity. In addition, our data indicate that Brn-3b acts as an inhibitor of Brn-3a(l) activity.     

Interaction of multiple factors with a GC-rich element within the mitogen responsive region of the human transferrin receptor gene

Nuclear factors from HeLa cells were isolated by elution of DNA-cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from -34 to -79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double-stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC-rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but still contacts the GC-rich element.     

GABAA and GABAB receptors differentially regulate synaptic transmission in the auditory thalamo-amygdala pathway: an in vivo microiontophoretic study and a model

PURPOSE: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. METHODS: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. RESULTS: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. CONCLUSIONS: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.