The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

Abstract

A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The complex multimeric nature of RNA polymerases has been recognized for many years. RNA polymerases in eukaryotes consist of 9 to 14 subunits (for reviews, see references 43, 45, and 53). Some of these subunits are similar or identical in all three forms of polymerase in all species, while others are form and species specific. The largest subunits of RNA polymerase forms I, II, and III of the yeast Saccharomyces cerevisiae are sufficiently similar to allow detection of all three proteins by using antibodies generated to any one of the peptides (18). Furthermore, the fifth-, sixth-, and eighth-largest subunits (RPB5, RPB6, and RPB8) of RNA polymerase II are not just similar to those of the other polymerases but are shared between all three forms in yeast cells (51). This sequence conservation is not restricted to S. cerevisiae. The largest subunit of RNA polymerase II in yeast cells has regions with a high degree of interspecies homology, including similarity to Drosophila, mammalian, and even prokaryotic RNA polymerases (2,3,7,11,15 MATERUILS AND METHODSIsolation and maintenance of Drosophila strains. Fly stocks were maintained at 22.5°C and 65% relative humidity. The RpII15Z23 allele was generated as a lethal mutation in combination with Df(3R) red"52. Males homozygous for red, ebony were fed 0.024 M ethyl methanesulfonate (25) and mated to doubly balanced TM6B/TM3 females. Progeny were mated to Df(3R) red"52/TM6B flies. Mutations which resulted in no Df(3R) red 52/red, ebony flies thus delineated lethal complementation groups within the red"52 deficiency.Isolation and enzymology of nucleic acids. Isolation of plasmid DNA, screening of lambda libraries, and DNA labeling and enzymology were carried out by standard procedures (27). Genomic DNA from Drosophila adults was prepared as described by Parkhurst et al. (35). Total RNA was isolated by homogenization in 10 mM Tris hydrochloride (pH 7.4)-0.1 M NaCl-1 mM EDTA-0.5% sodium dodecyl sulfate followed by phenol extraction and ethanol precipitation. Poly(A)+ RNA was selected by chromatography on oligo(dT)-cellulose (4). Southern and Northern (RNA) analyses were done as described by Parkhurst et al. (35).

An endogenous Suppressor of Hairy-wing insulator separates regulatory domains in Drosophila

“…For this reason, we only consider differences between scores of a unit or more to be significant (26). (30,31). Both su(Hw) mutant backgrounds completely reverse gypsy insulator effects.…”

The DNA-Binding and Enhancer-Blocking Domains of the Drosophila suppressor of Hairy-wing Protein

“…Homozygous su (Hw) V and the su(Hw) V /su(Hw) f heterozygous combination both strongly suppress the gypsy insertion alleles. The su(Hw) V allele is a deletion and is usually homozygously lethal because an RNA polymerase subunit gene (RpII215) just upstream of su(Hw) is also affected (25). However, when RpII215 is supplied transgenically, su (Hw) V flies are viable and female sterile (25).…”

“…The su(Hw) V allele is a deletion and is usually homozygously lethal because an RNA polymerase subunit gene (RpII215) just upstream of su(Hw) is also affected (25). However, when RpII215 is supplied transgenically, su (Hw) V flies are viable and female sterile (25). The P element used to express mutant su(Hw) cDNAs in these experiments contains RpII215, allowing the ability of the mutant SUHW proteins to restore fertility of homozygous su(Hw) V females to be determined.…”

RPII15 codes for the Mr 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II

“…Bound antibodies were detected using diaminobenzidine/H,O, as substrate. The coding region of RPIZl5 was amplified from genomic DNA of adult flies by polymerase chain reaction using primers corresponding to the 5' and 3' terminal regions of the RPIIl5 coding sequence reported by Harrison and co-workers [6]. The polymerase chain reaction resulted in a single product of 430 bp.…”

“…Sequence analysis showed that this subunit is similar to a Drosophila protein encoded by a gene upstream of the suppressor of Hairy wing (su(Hw)). cDNA of this gene (ZWZZZ5) was isolated which codes for 129 amino acids with a predicted molecular weight of 15,100 Da [6]. Detailed analysis of RPZZl.…”