A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes.     

A combined in vitro / in vivo selection for polymerases with novel promoter specificities

Background  

The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro / in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10³ – 10⁶) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendent mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.     

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression

Background  

RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.     

Controlled expression of functional miR-122 with a ligand inducible expression system

Background  

To study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression.     

An optimization framework for unsupervised identification of rare copy number variation from SNP array data

Background

Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation.

Results

To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation.

Conclusions

These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation.