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Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5′ and 3′ sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5′ incision is made by a heterodimer of XPF and ERCC1 (Rad1p–Rad10p in Saccharomyces cerevisiae), and the 3′ incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the DrosophilaXPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPGDmand ERCC1Dm). XPGDmis encoded by the mus201gene; we found frameshift mutations predicted to produce truncated XPGDmproteins in each of two mus201alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1Dminteracts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210maps nearby (51E-F) but is distinct from Ercc1.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764–5771.] reported the molecular cloning of Drosophila mus308gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophilareplication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template–primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophilapolymerase classes such as α, β, γ, δ, ε and ζ and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3′–5′ exonuclease activities as shown by the chromatographies.

The transformer (tra)and doublesex (dsx)genes produce sex-specific transcripts that are generated by differential RNA processing. We have examined the effects of mutants in other regulatory genes controlling sexual differentiation on the patterns of processing of the traand dsxRNA transcripts. Our results demonstrate that the genes suggested by genetic studies to act upstream of traor dsxin the sex determination hierarchy regulate these two loci at the level of RNA processing. Our data suggest that the order of interaction of the factors controlling sex is X:A>Sxl>tra⩾tra-2>dsx⩾ix>terminal differentiation. While these results cannot preclude regulatory interactions at other levels, the regulation of RNA splicing revealed by these experiments is sufficient to account for all of the known functional interactions between the regulatory genes in this hierarchy.

The doublesex (dsx) locus encodes male-specific and female-specific polypeptides that are essential for the proper differentiation of sexually dimorphic somatic features of Drosophila melanogaster. Ectopic expression of the male-specific dsx polypeptide was obtained by P-element-mediated transformation of flies with a construct bearing a fusion between the hsp70 heat shock promoter and dsx male-specific cDNA sequences. Heat shock-induced expression of the male cDNA in either sex resulted in three novel phenotypes: transformation of bristles on all legs toward a sex comb-like morphology, pigmentation of dorsal spinules and ventral setae in third-instar larvae, and lethality. These results were not predicted by previous models of dsx function, and provide evidence that the role of the male dsx protein includes activation of some aspects of male differentiation as well as repression of female differentiation. Copyright 1993, 1999 Academic Press

The steroid hormone ecdysone initiates Drosophila metamorphosis by reprogramming gene expression during late larval and prepupal development. The ecdysone-inducible gene E74, a member of the ets proto-oncogene family, has been proposed to play a key role in this process. E74 is encoded within the 74EF early puff and consists of two overlapping transcription units, E74A and E74B. To assess the function(s) of E74 during metamorphosis, we have isolated and characterized recessive loss-of-function mutations specific to each transcription unit. We find that mutations in E74A and E74B are predominantly lethal during prepupal and pupal development, consistent with a critical role for their gene products in metamorphosis. Phenotypic analysis reveals that E74 function is required for both pupariation and pupation, and for the metamorphosis of both larval and imaginal tissues. E74B mutants are defective in puparium formation and head eversion and die as prepupae or cryptocephalic pupae, while E74A mutants pupariate normally and die either as prepupae or pharate adults. We have also investigated the effects of the E74 mutations on gene expression by examining the puffing pattern of the salivary gland polytene chromosomes in newly formed mutant prepupae. Most puffs are only modestly affected by the E74B mutation, whereas a subset of late puffs are sub-maximally induced in E74A mutant prepupae. These observations are consistent with Ashburner’s proposal that early puff proteins induce the formation of late puffs, and define E74A as a regulator of late puff activity. They also demonstrate that E74 plays a wide role in reshaping the insect during metamorphosis, affecting tissues other than the salivary gland in which it was originally identified.

BACTERIAL RNA polymerase consists of a core (α2ββ′) and a sigma (σ) factor which form the active holoenzyme (α2ββ′σ) (ref. 1). Significant differences in size of the β and β′ subunits occur in the enzymes from various bacterial species2. The exact functions of the larger subunits are still unknown. In Escherichia coli the β′ subunit is the largest polypeptide and is involved in the binding of RNA polymerase to DNA3; the β subunit is responsible for both rifampicin4and streptolydigin5,6resistance. In contrast the largest RNA polymerase polypeptide in Bacillus subtilis is responsible for rifampicin resistance and has been designated as β (ref. 7). The second largest polypeptide of B. subtilis contains the two zinc atoms associated with RNA polymerase8and has been designated as β′. We demonstrate here, using subunit reconstitution studies, that the second largest subunit of B. subtilis RNA polymerase—the β′ subunit—is responsible for streptolydigin resistance. Since the β subunit of E. coli is responsible for resistance to both antibiotics, a divergence in function has occurred between the β and β′ subunits of B. subtilis and E. coli. These and other studies9thus show clearly that function cannot be attributed to the two larger subunits purely on their relative size. Furthermore the identification of streptolydigin resistance with the β′ subunit and the map locations of rifampicin and streptolydigin resistance in B. subtilis10indicate that the genes for the β and β′ subunits are very closely linked and that streptolydigin resistance determines the rpoC locus in B. subtilis.