The anterior determinant bicoid of Drosophila is a derived Hox class 3 gene

The Drosophila gene bicoid functions as the anterior body pattern organizer of Drosophila. Embryos lacking maternally expressed bicoid fail to develop anterior segments including head and thorax. In wild-type eggs, bicoid mRNA is localized in the anterior pole region and the bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal caudal mRNA in the anterior region of the embryo. caudal genes as well as other homeobox genes or members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila. This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior–posterior polarity. We have cloned bicoid from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza), and show that the gene originated from a recent duplication of the direct homolog of the vertebrate gene Hox3, termed zerknüllt, which specifies extraembryonic tissues in insects.

Molecular coevolution within a Drosophila clock gene

The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock’s temperature compensation.

The gastrulation defective gene of Drosophila melanogaster is a member of the serine protease superfamily

The establishment of dorsal–ventral polarity in the oocyte involves two sets of genes. One set belongs to the gurken-torpedo signaling pathway and affects the development of the egg chorion as well as the polarity of the embryo. The second set of genes affects only the dorsal–ventral polarity of the embryo but not the eggshell. gastrulation defective is one of the earliest acting of this second set of maternally required genes. We have cloned and characterized the gastrulation defective gene and determined that it encodes a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spätzle activity) during establishment of dorsal–ventral polarity in the Drosophila embryo.

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.

Su(UR)ES: A gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34.8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form “weak points,” underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally β-heterochromatic, acquire a distinct banding pattern, i.e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional α-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic stripe expression in the developing Drosophila epidermis

The egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that sr encodes a transcriptional activator which acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Sex-specific quantitative trait loci affecting longevity in Drosophila melanogaster

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of Drosophila spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and age-specific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late age-of-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.

A screen for fast evolving genes from Drosophila

In an attempt to quantify the rates of protein sequence divergence in Drosophila, we have devised a screen to differentiate between slow and fast evolving genes. We find that over one-third of randomly drawn cDNAs from a Drosophila melanogaster library do not cross-hybridize with Drosophila virilis DNA, indicating that they evolve with a very high rate. To determine the evolutionary characteristics of such protein sequences, we sequenced their homologs from a more closely related species (Drosophila yakuba). The amino acid substitution rates among these cDNAs are among the fastest known and several are only about 2-fold lower than the corresponding values for silent substitutions. An analysis of within-species polymorphisms for one of these sequences reveals an exceptionally high number of polymorphic amino acid positions, indicating that the protein is not under strong negative selection. We conclude that the Drosophila genome harbors a substantial proportion of genes with a very high divergence rate.

Calx, a Na-Ca exchanger gene of Drosophila melanogaster

We have cloned Calx, a gene that encodes a Na-Ca exchanger of Drosophila melanogaster. Calx encodes two repeated motifs, Calx-α and Calx-β, that overlap domains required for exchanger activity and regulation. Calx has multiple transcripts in adults, including at least one expressed in the retina. The Calx genomic locus comprises ≥35 kb between the Atpα and rudimentary-like genes in chromosomal region 93B. In Xenopus oocytes, microinjected Calx cRNA induces calcium uptake like that of its homolog, the 3Na+-1Ca2+ exchanger of mammalian heart. Implications of Calx-α motifs for the mechanism of Na-Ca exchange are discussed.

Proximo–distal specification in the wing disc of Drosophila by the nubbin gene

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing’s proximo–distal axis and in the formation of compartment boundaries.

Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death

Wolbachia, a maternally transmitted microorganism of the Rickettsial family, is known to cause cytoplasmic incompatibility, parthenogenesis, or feminization in various insect species. The bacterium–host relationship is usually symbiotic: incompatibility between infected males and uninfected females can enhance reproductive isolation and evolution, whereas the other mechanisms enhance progeny production. We have discovered a variant Wolbachia carried by Drosophila melanogaster in which this cozy relationship is abrogated. Although quiescent during the fly’s development, it begins massive proliferation in the adult, causing widespread degeneration of tissues, including brain, retina, and muscle, culminating in early death. Tetracycline treatment of carrier flies eliminates both the bacteria and the degeneration, restoring normal life-span. The 16s rDNA sequence is over 98% identical to Wolbachia known from other insects. Examination of laboratory strains of D. melanogaster commonly used in genetic experiments reveals that a large proportion actually carry Wolbachia in a nonvirulent form, which might affect their longevity and behavior.

A phylogenetic perspective on P transposable element evolution in Drosophila

The P element, originally described in Drosophila melanogaster, is one of the best-studied eukaryotic transposable elements. In an attempt to understand the evolutionary dynamics of the P element family, an extensive phylogenetic analysis of 239 partial P element sequences has been completed. These sequences were obtained from 40 species in the Drosophila subgenus Sophophora. The phylogeny of the P element family is examined in the context of a phylogeny of the species in which these elements are found. An interesting feature of many of the species examined is the coexistence in the same genome of P sequences belonging to two or more divergent subfamilies. In general, P elements in Drosophila have been transmitted vertically from generation to generation over evolutionary time. However, four unequivocal cases of horizontal transfer, in which the element was transferred between species, have been identified. In addition, the P element phylogeny is best explained in numerous instances by horizontal transfer at various times in the past. These observations suggest that, as with some other transposable elements, horizontal transfer may play an important role in the maintenance of P elements in natural populations.