Activation of the U2 snRNA promoter by the octamer motif defines a new class of RNA polymerase II enhancer elements

The recent discovery that the activation domains of transcriptional activators (e.g., GAL4) from a number of species are interchangeable has led to the concept of a general mechanism for activation of RNA polymerase II genes. We have examined the different activities of the SV40 octamer motif ATGCAAAG in B cells and in HeLa cells in the context of either the beta-globin promoter, a TATA-box-containing mRNA promoter, or the U2 snRNA promoter, which contains a snRNA-specific proximal element. In the context of the beta-globin promoter, the octamer motif is a B-cell-specific enhancer element, whereas it is a ubiquitous enhancer element for the U2 snRNA promoter. The U2 promoter is unique in that it is not activated by enhancer elements that activate the beta-globin promoter, and a hybrid U2 promoter containing the upstream activating sequence UASG is not stimulated by a yeast GAL4 trans-activator. Together, these observations suggest that in the context of the U2 promoter, the octamer motif defines a new class of RNA polymerase II enhancer elements, which bind transcription factors that trans-activate gene expression by a different mechanism than the general mechanism mentioned above. These results are discussed in light of the possibility that the ubiquitous octamer binding protein Oct-1 and the B-cell-specific octamer binding protein Oct-2 are involved in the activation of the U2 and beta-globin promoters, respectively

Identification of proteins interacting with the enhancer of human U2 small nuclear RNA genes.

Protein/DNA interactions in the human U2 RNA gene enhancer have been characterized by DNase I footprint and DMS methylation protection analyses. Nuclear factors present in both HeLa and B cell extracts have been shown to protect an approximately 70 bp region from DNase I digestion. DMS and DNase I footprint competition studies demonstrated that the entire footprint can be accounted for by interactions with two previously identified transcription factors. One of these recognizes the so called octanucleotide-motif ATGCAAAT (transcription factor IgNF-A) which has been shown to be essential for transcription. The other is the transcription factor Sp1 which binds to three target sequences located adjacent to the octameric motif. The Sp1 interactions appear to be required for full transcriptional activity. No differences in the DNase I footprint patterns or in the DMS methylation protections were observed when nuclear extracts from HeLa cells, two different B cell lines, or from the adenovirus-transformed 293 cell line were compared

Molecular genetic investigations of rod cyclic GMP phosphodiesterase beta subunit in canine generalised progressive retinal atrophy

Generalised Progressive Retinal Atrophy (GPRA) is a collective term describing a number of phenotypically and genotypically heterogeneous, autosomal recessive retinal dystrophies and degenerations segregating in specific breeds of dog. All result in blindness and collectively, show much similarity to Retinitis Pigmentosa, a group of important human inherited retinal degenerations. GPRA in the Irish setter is given the locus designation rcd-1 (Rod-Cone Dysplasia Type 1) and presents as an early onset photoreceptor dystrophy with rapid, progressive loss of cells complete by approximately one year. Biochemically, the earliest detected abnormality is an accumulation of retinal cGMP, due to a defective phosphodiesterase activity. Therefore, the possibility that a mutation in the rod cGMP phosphodiesterase beta subunit (pdeb) gene causes rcd-1 in Irish setters was investigated. The normal canine pdeb cDNA, was cloned and sequenced, spanning the entire coding region to the poly-A tail. Additionally, partial intron/exon characterisation and nucleotide substitution analysis was undertaken to investigate the relationship of canine pdeb to its homologue in other species and to other members of the photoreceptor PDE family. A recently reported G to A transition in the pdeb gene described in rcd-1 setters in the USA was confirmed in affected dogs in the UK. In addition, GPRA in two other breeds, miniature longhaired dachshunds and Tibetan terriers was shown not to be due to this mutation, which creates a premature stop codon at residue 807. A rapid, non-isotopic diagnostic test was developed and used to show cosegregation of the mutation with rcd-1 phenotype in an Irish setter pedigree. Anchored PCR has been utilised to generate upstream sequence information from genomic DNA, and in tandem with mapping of the 5' end of the transcript has revealed sequence motifs in the proximal promoter of the canine pdeb gene which are conserved with the human gene. The tissue distribution of pdeb transcripts was investigated by Reverse Transcriptase-PCR and Northern analysis and the results show that transcription is not confined to the retina, but can be detected in a variety of different tissues