Existence of two forms of L protein of Newcastle disease virus isolates due to a compensatory mutation in Domain V.

Nucleotide sequence comparison of the L gene of the Malaysian neurotropic-viscerotropic velogenic NDV strain AF2240 with other NDV strains revealed a single nucleotide insertion at position 3870. This mutation is compensated by a nucleotide deletion downstream at position 3958 which results in two forms of the L proteins containing a 30-amino acid substitution in Domain V. This compensatory mutation does not correlate with the pathogenicity of the viral strains but it may affect the viral replication as Domain V is believed to play an important role in the replication of paramyxoviruses.

Identification of proximal sequence element nucleotides contributing to the differential expression of variant U4 small nuclear RNA genes.

The two U4 genes in the chicken genome code for distinct sequence variants of U4 small nuclear RNA that are differentially expressed during development. Whereas U4B RNA is constitutively expressed, U4X RNA is specifically down-regulated relative to U4B in a tissue-specific manner during development. To investigate mechanisms controlling the differential expression of the U4B and U4X genes, chimeric U4 genes were constructed and their transcriptional activities assayed by injection into Xenopus oocytes or by transfection of CV-1 cells. The proximal regulatory region of the U4B gene and the enhancers of both the U4B and U4X genes functioned efficiently in each expression system. However, the proximal region of the U4X gene was inactive. To localize and identify the responsible nucleotides, reciprocal point mutations were introduced into the U4X and U4B proximal regulatory regions. The results indicate that the U4X gene contains a suboptimal proximal sequence element, and that this results primarily from the identities of the nucleotides at positions -61 and -57 relative to the transcription start site.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

U4B snRNA gene enhancer activity requires functional octamer and SPH motifs.

Expression of the chicken U4B small nuclear RNA (snRNA) gene is stimulated by a transcriptional enhancer located approximately 190-227 base pairs upstream of the transcription start site. This enhancer is composed of at least two functional motifs: an octamer (binding site for Oct-1) and an SPH motif. We now report that these two motifs functionally cooperate to stimulate U4B snRNA gene expression, and both are required for the formation of a stable transcription complex. Expression in frog oocytes of 24 different point mutant constructions indicates that the functional SPH motif is at least 15 base pairs in length. It is a recognition site for a sequence specific DNA-binding protein, termed SBF, purified from chicken embryonic nuclear extracts. The ability of the mutant SPH motif constructions to be recognized by SBF in vitro correlates with their transcriptional activities, suggesting that SBF mediates the stimulatory effect of the U4B SPH motif. These results are similar to our recent findings on the chicken U1 gene enhancer, which also contains adjacent binding sites for Oct-1 and SBF. These studies, together with evolutionary considerations and sequence comparisons among snRNA gene enhancers, suggest that cooperativity between octamer and SPH motifs could be a widely-employed mechanism for generating vertebrate snRNA gene enhancer activity.