The glioma ampli®ed sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently ampli®ed in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, speci®cally at the Nterminus, and identi®ed the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain. After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immuno¯uores-cence microscopy. The nuclear localization was con®rmed for several human tumors including gliomas of dierent grades of malignancy. In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm. Yeast two-hybrid screening of the TX3868 cell line identi®ed the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41. We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA.
Nuclear DEAD box protein p68 is immunologically related to SV40 large tumour antigen and both proteins possess RNA helicase activity. In this report, we describe the structural organisation of the human p68 gene and aspects of the regulation of its expression. Northern blot and primer extension analyses indicate that, although its level is variable, the p68 RNA helicase appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis revealed that the p68 promoter harbours a 'TATA', a 'CAAT' and an initiator element and contains high affinity binding sites for Sp1, AP-2, CRE and Myc. This and functional promoter analyses in transient expression assays suggest that transcriptional regulation of the p68 gene is complex. Furthermore, there are indications that p68 expression is also regulated post-transcriptionally. Steady-state pools of poly(A)(+)RNA from human cells contain completely spliced p68 mRNA and alternatively spliced forms that contain introns 8-11 or 8-12 (from a total of 12 introns) and are not translated. Analysis of a conditionally p68-overproducing HeLa cell line points to negative autoregulation at the level of splicing, which is confirmed by a recently reported association of p68 with spliceosomes in human cells.
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