Kenneth C. Burtis

The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain.

The doublesex gene of Drosophila melanogaster is the final member of a well characterized hierarchy of genes that controls somatic sex determination and differentiation. The male‐specific and female‐specific doublesex polypeptides occupy a terminal position in the hierarchy, and thus regulate those genes responsible for the development of sexually dimorphic characteristics of the fly. To investigate the molecular mechanism by which these two related proteins interact with specific target genes, we have identified and characterized their DNA binding domains. Using gel mobility shift experiments with sequentially deleted polypeptides, site‐directed mutagenesis and spectrophotometric assays, we have shown that the two doublesex proteins share a common and novel zinc finger‐related DNA binding domain distinct from any reported class of zinc binding proteins. We have further shown that of 10 null dsx alleles, six encode proteins deficient in DNA binding activity, and that three of these alleles are the result of mutations that alter cysteine and histidine residues in the metal binding domain. Our results provide evidence that both the male‐specific and female‐specific doublesex proteins share and depend upon the same DNA binding domain for function in vivo, suggesting that both proteins bind to, but differentially regulate, a common set of genes in both sexes.

The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer.

The doublesex (dsx) gene of Drosophila melanogaster encodes both male‐specific and female‐specific polypeptides, whose synthesis is regulated by alternative sex‐specific splicing of the primary dsx transcript. The alternative splicing of the dsx mRNA is the last known step in a cascade of regulatory gene interactions that involves both transcriptional and post‐transcriptional mechanisms. Genetic studies have shown that the products of the dsx locus are required for correct somatic sexual differentiation of both sexes, and have suggested that each dsx product functions by repressing expression of terminal differentiation genes specific to the opposite sex. However, these studies have not shown whether the dsx gene products function directly to regulate the expression of target genes, or indirectly through another regulatory gene. We report here that the male‐ and female‐specific DSX proteins, expressed in E.coli, bind directly and specifically in vitro to three DNA sequences located in an enhancer region that regulates female‐specific expression of two target genes, the yolk protein genes 1 and 2. This result suggests strongly that dsx is a final regulatory gene in the hierarchy of regulatory genes controlling somatic sexual differentiation.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

The regulation of sex determination and sexually dimorphic differentiation in Drosophila

Sex determination and sexually dimorphic differentiation in Drosophila involve multiple regulatory mechanisms, including alternative splicing, transcriptional control, subcellular compartmentalization, and intercellular signal transduction. Regulatory interactions occur throughout the development of the fly, some requiring the continuous function of the genes involved, and others being temporally limited, but having permanent consequences. The control of sexual differentiation in Drosophila is, for the most part, subject to the continuous active control of numerous regulatory proteins operating at many levels.

A Large-Scale Screen for Mutagen-Sensitive Loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212–mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314–mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Delta factor increases promoter selectivity by Bacillus subtilis vegetative cell RNA polymerase

Summary The role of delta factor has been investigated by analyzing its effect on initiation and transcription by sigma-containing Bacillus subtilis RNA polymerase. Delta factor reduced transcription from various plasmid and chromosomal DNA templates, but had very little effect on RNA polymerase activity with phage DNA as templates. Delta factor increased the specificity of promoter selection and initiation when Hind III restriction fragments of phage 029 DNA were used as templates. These data indicate that the presence of delta factor favors transcription from efficient promoters and coordinates initiation of transcription with the sigma factor.

A possible functional role for a new class of eukaryotic DNA polymerases

The recent Correspondence from Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1] regarding a new family of eukaryotic proteins similar to bacterial DNA polymerase I raises a number of interesting questions. Our report on the characterization of the Drosophila DNA repair gene mus308[2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2] and a related Caenorhabditis elegans gene addresses several of these questions, including the possible role of this gene family.The mus308 gene (Genbank L76559) encodes a 229 kDa polypeptide, the carboxy-terminal domain of which is closely related to the polymerase domain of the bacterial DNA polymerase I enzymes. Remarkably, the amino-terminal domain of the Mus308 polypeptide also includes the seven amino-acid sequence motifs that are characteristic of the ‘superfamily 2’ DNA and RNA helicases [3xTwo related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Gorbalenya, AE, Koonin, EV, Donchenko, AP, and Blinov, VM. Nucleic Acids Res. 1989; 17: 4713–4730Crossref | PubMed | Scopus (679)See all References[3]. This finding makes mus308 not only the first characterized gene encoding a eukaryotic homolog of the bacterial DNA polymerase I family, but also the first reported gene encoding both helicase and polymerase motifs in a single polypeptide.A gene very similar to mus308, which we have designated mus-1, encoded by genomic sequences in the cosmids R12B2 (U00066) and W03A3 (U50184), is predicted to exist in C. elegans. A combination of genomic and partial cDNA sequences indicate that the carboxy-terminal domain of the Mus-1 polypeptide is highly homologous to the polymerase domains of Mus308 and other members of the DNA polymerase I family [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2], as noted by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1]. Upon comparing the remainder of the mus308 coding sequences and C. elegans genomic sequences located upstream of the Mus-1 polymerase domain, a helicase domain closely related to the Mus308 helicase domain was immediately apparent [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2]. Further limited sequence homology between the Mus308 and predicted Mus-1 polypeptides was also seen in the region between the helicase and polymerase domains (P.H., unpublished observations). The juxtaposition of Mus308-related helicase and polymerase domains and sequence similarities in the region between the two domains strongly support the hypothesis that the mus-1 gene encodes a protein homologous to Mus308. The existence of an evolutionarily conserved enzyme family with this unique combination of helicase and polymerase motifs suggests a conserved functional role in DNA repair for these proteins. Direct evidence for such a role is available, however, only for Mus308 [4xThird-chromosome mutagen-sensitive mutants of Drosophila melanogaster. Boyd, JB, Golino, MD, Shaw, KE, Osgood, CJ, and Green, MM. Genetics. 1981; 97: 607–623PubMedSee all References[4], which is required to repair the damage caused by mutagens such as nitrogen mustard and cisplatin that result in interstrand cross-links [5xmus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease. Boyd, JB, Sakaguchi, K, and Harris, PV. Genetics. 1990; 125: 813–819PubMedSee all References[5].As reported by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1], two distinct human sequences encoding polypeptides with similarity to the polymerase domain of the bacterial DNA polymerase I family are also present in the sequence databases. We have also noted the existence of these coding sequences, which encode polypeptides related to the Mus308 polymerase domain. The sequence most closely related to mus308 is represented by human cDNA clone za38h12.r1 (Genbank W00829. Sequence information from partial cDNAs and RT-PCR products derived from this gene show additional homology to mus308 in sequences upstream of the polymerase domain (P.V.H. and K.C.B., unpublished observations), suggesting homology to the mus308/mus-1 family; however, the presence of helicase motifs remains to be established. We have also noted the sequences homologous to the Mus308 polymerase domain in the Huntingtons disease region of the genome, as reported by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1]. In this case, however, the availability of almost 2 megabases of sequence information upstream of this domain reveals the absence of any recognizable helicase motifs (P.V.H. and K.C.B., unpublished observations), suggesting that this gene is not likely to be a functional homolog of the mus308/mus-1 family.Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1] note that it is ‘tempting to consider DNA repair’ as a possible function of these DNA polymerases, but that ‘experimental verification of polymerase activity is the next step.’ Our previous report [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2] indeed confirms a role in DNA repair for at least the prototypical member of this new gene family, mus308. Furthermore, we have recently determined that the polymerase domain of the polypeptide encoded by the za38h12.r1 human cDNA possesses enzymatic polymerase activity, confirming the prediction based on amino-acid sequence (P.V.H. and K.C.B., unpublished observations). In the light of this enzymatic activity, we have termed this new human DNA polymerase gene POLH. The next step is to determine the functional role of the polymerase (and possibly helicase) domains of this new class of eukaryotic DNA polymerases in DNA repair and other cellular functions.

Are Drosophila a Useful Model for Understanding the Toxicity of Inhaled Oxidative Pollutants: A Review

Oxidative atmospheric pollutants represent a significant stress and cause injury to both vertebrate and invertebrate species. In both, the biosurfaces of their respiratory apparatus are directly exposed to oxidizing pollutant-induced stresses. Respiratory-tract surfaces contain integrated antioxidant systems that appear to provide a primary defense against environmental insults caused by inhaled atmospheric reactive oxygen species (ROS) and reactive nitrogen species (RNS), whether gaseous or particulate. When the biosurface antioxidant defenses are overwhelmed, oxidative and nitrosative stress to the acellular and cellular components of the exposed biosurfaces can ensue via direct chemical reactions that lead to the induction of inflammatory, adaptive, injurious, and reparative processes. The study of model invertebrates (e.g., Drosophila) has a long history of yielding valuable insights into both fundamental biology and pathobiology. Mutants and/or transgenic insects, with specific alterations in key components of innate and/or adaptive antioxidant defense systems and immune genes, offer opportunities to dissect the complex systems that maintain respiratory tract surface defenses against environmental oxidants and the ensuing host responses. In this article, we use a comparative absfont approach to consider interactions of atmospheric oxidant pollutants with selected biosystems. We focused primarily on ozone (O3 ) as the pollutant, vertebrate and invertebrate respiratory tracts as the exposed biosystems, and nonenzymatic micronutrient antioxidants as significant contributors to overall antioxidant defense strategies. We present parallels among these diverse organisms with regard to their protective strategies against environmental atmospheric oxidants, with particular focus given to using the invertebrate Drosophila as a potentially useful model for vertebrate respiratory-tract responses to inhaled oxidants specifically and pollutants in general. We conclude that the insect respiratory system has considerable promise toward understanding novel aspects of vertebrate respiratory tract responses to inhaled oxidative environmental challenges.

The millennium flies in

The complete genomic sequence of the flyDrosophila melanogasterwill soon be available. As a prelude to this, two groups have looked at the amount of work that will have to be done to make biological sense of the raw data. These studies include correlations between the primary DNA sequence and genetic function, and the generation of specific mutations to study the functions of particular genes.

The millennium flies in: Genomics

The complete genomic sequence of the flyDrosophila melanogasterwill soon be available. As a prelude to this, two groups have looked at the amount of work that will have to be done to make biological sense of the raw data. These studies include correlations between the primary DNA sequence and genetic function, and the generation of specific mutations to study the functions of particular genes.