Molecular characterization of a putative peroxidase gene of Drosophila melanogaster.

We have identified genomic clones and corresponding cDNAs that encode a putative peroxidase of Drosophila melanogaster. The gene (DmPO) appears as a single copy gene located on the third chromosome at position 89 D/E. It is interrupted by seven small introns and one unusually large 5' intron (about 11 kb). Sequence analysis of the cDNA showed an open reading frame of 690 amino acids resulting in a protein of 77 kDa. The deduced amino acid sequence reveals an overall homology to myeloeosinophil and thyroid peroxidase, a human superfamily of peroxidases.     

Numb suppresses the negative complementation at the Notch locus of Drosophila melanogaster, suggesting a putative mechanism for negative complementation

The mutant form of the intracellular asymmetrically localized Numb membrane-bound protein of Drosophila melanogaster suppresses the negative complementation of certain Abruptex (Ax) mutations of the Notch (N) locus encoding a transmembrane receptor protein in which the Ax mutations are mutations in the epidermal growth factor (EGF)-like repeats of the extracellular domain of the receptor. One model for how Ax mutants affect N function is that they are refractory to an antagonistic signal generated by an excess of N ligands. Genetically numb (nb) is an antagonist of N. In the absence of nb, cells follow the same fate as they would in the presence of a gain-of-function N allele, such as Ax. Numb has been shown to interact with the cytoplasmic domain of Notch. It is therefore suggested that numb counteracts the effect of Abruptex on Notch ligand binding, i.e. that Numb is an antagonist to the activation of the Notch signal generated by Notch ligands. Numb might accomplish this by interfering with the proteolytic cleavage of the Notch intracellular domain at the cell membrane. Thus, it seems possible that the mechanism of negative complementation of certain Ax mutants is the failure of this cleavage. Other possible mechanisms for negative complementation are also discussed.     

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.     

Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

Isolation and characterization of drosocrystallin, a lens crystallin gene of Drosophila melanogaster

We have cloned the drosocrystallin gene (dcy) of Drosophila melanogaster, which encodes a major protein of the corneal lens, previously described in part by Komori et al. (1992, J. Cell Sci. 102, 191-201). Synthesis of the DCY protein starts weakly in 2-day-old pupae, reaches a peak at day 3 and day 4 of pupal development, and decreases very fast in young adults. The dcy mRNA is detected in the compound eyes as well as in the ocelli. The presence of a putative signal peptide and the extracellular location of DCY suggest that DCY is a secreted protein. Interestingly, the dcy gene shows sequence similarities to some insect cuticular proteins and is detected as well in two closely related Drosophila species, D. sechellia and D. simulans, and in one more distantly related species, D. virilis. This finding supports the hypothesis that Drosophila used the same strategy as vertebrates and mollusks, namely, recruiting a multifunctional protein for refraction in the lens, by a gene-sharing mechanism. Furthermore, it supports our intercalary evolution hypothesis, which suggests that the development of an elaborate structure (for example, a compound eye) from an original primitive form (an ancestral photoreceptor organ) can be achieved by recruiting novel genes into the original developmental pathway.     

Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster

A recombinant lambda-phage DNA clone containing Drosophila melanogaster sequences encoding the gene for myosin light chain (MLC) two has been isolated from a library of randomly sheared DNA. The Drosophila MLC2 gene is located in region 99E1-3 on the right arm of chromosome 3, several bands removed from the site reported for the other myosin light chain gene at 98B. The MLC2 sequence at 99E1-3 appears to encode all of the isoforms of Drosophila MLC2. The polypeptide encoded at 99E was identified as MLC2 by the following criteria: the in vitro translation product is identical in size to MLC2 isolated from Drosophila muscle, and on two-dimensional gels the in vitro translation product can be separated into two or more peptides that co-migrate with isoforms of larval and thoracic MLC2. RNA encoding the polypeptide was detected in embryos only after the onset of muscle differentiation and was also abundant in adult thoracic muscle. The nucleotide sequence of cDNA generated from late embryonic RNA would be translated to yield a protein sequence with multiple regions of homology to vertebrate MLC2. (There are shorter regions of homology to vertebrate MLC1). Like a number of vertebrate muscle proteins, Drosophila MLC2 has an acetylated amino-terminus.     

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.     

Identification and characterization of kraken,a gene encoding a putative hydrolytic enzyme in Drosophila melanogaster

Kraken, a novel Drosophila gene isolated from a 4–8-h-old Drosophila embryo cDNA library, shows homology to a family of serine hydrolases whose common feature is that they all catalyse breakage of substrates with a carbonyl-containing group. It is a single-copy gene with at least two introns and maps to position 21D on polytene chromosomes. kraken is a member of a conserved gene family. Messenger RNA of kraken is expressed ubiquitously in early embryogenesis. Later, it is concentrated in the foregut and the posterior midgut primordium. Towards the end of embryogenesis, expression of kraken is confined to the gastric caeca. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat body. We suggest a role for Kraken in detoxification and digestion during embryogenesis and larval development.     

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.