Drosophila lacking a homologue of mammalian ALDH2 have multiple fitness defects

Little is known about the roles of aldehyde dehydrogenases in non-vertebrate animals. We recently showed that in Drosophila melanogaster, an enzyme with ～70% amino acid identity to mammalian ALDH2 is necessary for detoxification of dietary ethanol. To investigate other functions of this enzyme, DmALDH, encoded by the gene Aldh, we compared two strains homozygous for Aldh-null mutations to two closely related wild type strains in measures of fitness and stress resistance in the absence of ethanol. Aldh-null strains have lower total reproductive rate, pre-adult viability, resistance to starvation, and possibly longevity than wild-type strains. When maintained under hyperoxia, Aldh nulls die more quickly and accumulate higher levels of protein carbonyls than wild-types, thereby providing evidence that DmALDH is important for detoxifying reactive aldehydes generated by lipid peroxidation. However no effect of Aldh was seen on protein carbonyl levels in flies maintained under normoxia. It is possible that Aldh nulls experience elevated rates of protein carbonylation under normoxia, but this is compensated (at a fitness cost) by increased rates of degradation of the defective proteins. Alternatively, the fitness defects of Aldh nulls under normoxia may result from the absence of one or more other functions of DmALDH, unrelated to protection against protein carbonylation.     

Trypanosome telomeres are protected by a homologue of mammalian TRF2

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G(2)/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.     

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.     

Drosophila fibronectin: a protein that shares properties similar to those of its mammalian homologue.

This is the first report on the existence in Drosophila of a protein with properties similar to those of vertebrate fibronectin that we shall refer to as Drosophila fibronectin. Rabbit antibodies against human plasma fibronectin have allowed the detection of this molecule in Drosophila haemolymph; common epitopes are shared by the two proteins. Drosophila fibronectin with a subunit mol. wt of approximately 230 kd is a glycoprotein which binds to denatured mammalian collagen. It is present throughout development and is as abundant in embryos as in larvae and adult flies. Drosophila fibronectin is differentially expressed during embryogenesis, a small amount being present before the blastoderm stage. Its concentration increases at gastrulation and reaches a steady-state value at the end of organogenesis. Drosophila fibronectin is predominantly detected by immunofluorescence on frozen sections of 16 h embryos in the extracellular spaces lying between the different tissues and organs. In mature third instar larvae, most of the staining is concentrated in fat body and imaginal discs, and the pattern strongly supports an extracellular localization of the protein. In addition, it is shown that Drosophila embryonic cells can functionally utilize vertebrate fibronectin for their spreading and differentiation. Finally, injection of antihuman plasma fibronectin antibodies in early embryos leads to the same phenotype as injection of Arg-Gly-Asp-containing peptides. This result suggests that one of the Arg-Gly-Asp-bearing protein(s) involved in gastrulation might be fibronectin.     

Structure of the gene for human plasminogen activator inhibitor-2. The nearest mammalian homologue of chicken ovalbumin

Plasminogen activator inhibitor-2 (PAI-2) can regulate the formation of plasmin by inhibiting urokinase and tissue plasminogen activator. PAI-2 is induced in monocytes and endothelium by inflammatory mediators, and it is made in the placenta during pregnancy. PAI-2 is a member of the serine protease inhibitor gene family, and it is particularly similar to chicken ovalbumin. Like ovalbumin, PAI-2 is secreted without cleavage of a signal peptide. To determine the structure of the PAI-2 gene, two bacteriophage lambda human genomic DNA libraries were screened with PAI-2 cDNA probes. Characterization of three positive clones shows that the human PAI-2 gene spans 16.5 kilobases and has eight exons. The 5'-untranslated sequence of the PAI-2 mRNA is 77 base pairs in length as suggested by primer extension and S1 nuclease mapping. The eukaryotic consensus sequence TATAAAA is found 22 base pairs 5' of the proposed cap site. The PAI-2 gene is on chromosome 18q21-23 as determined by hybridization to flow-sorted chromosomes and by in situ hybridization. There appear to be two common PAI-2 alleles that differ by six nucleotides in exons 1, 4, and 8. The structure of the PAI-2 gene is quite different from that of PAI-1 although these two inhibitors have common target protease specificity. In contrast, the structure of the PAI-2 gene is very similar to that of the chicken ovalbumin gene. When protein sequences are aligned to obtain maximal identity, six of the seven intron positions in the PAI-2 gene are identical to those in the chicken ovalbumin gene. We conclude that PAI-2 is the closest mammalian homologue of avian ovalbumin.     

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly.     

The Ketel gene encodes a Drosophila homologue of importin-beta

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.     

Characterization of mouse Dach2, a homologue of Drosophila dachshund

The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea.     

Drosophila Ana2 is a conserved centriole duplication factor

In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP—orthologues of ZYG-1, SAS-6, and SAS-4, respectively—are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.     

The atypical mammalian ligand Delta-like homologue 1 (Dlk1) can regulate Notch signalling in Drosophila

Background

Mammalian Delta-like 1 (Dlk-1) protein shares homology with Notch ligands but lacks a critical receptor-binding domain. Thus it is unclear whether it is able to interact with Notch in vivo. Unlike mammals, Drosophila have a single Notch receptor allowing a simple in vivo assay for mammalian Dlk1 function.

Results

Here we show that membrane-bound DLK1 can regulate Notch leading to altered cellular distribution of Notch itself and inhibiting expression of Notch target genes. The resulting adult phenotypes are indicative of reduced Notch function and are enhanced by Notch mutations, confirming that DLK1 action is antagonistic. In addition, cells expressing an alternative Dlk1 isoform exhibit alterations in cell size, functions previously not attributed to Notch suggesting that DLK1 might also act via an alternative target.

Conclusion

Our results demonstrate that DLK1 can regulate the Notch receptor despite its atypical structure.     

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.     

Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes.

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.     

Identification of the Drosophila melanogaster homologue of the mammalian signal transducer protein, Vav

Mammalian Vav signal transducer protein couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, thus leading to cell differentiation and/or proliferation. We have isolated and characterized the DroVav gene, the homologue of hVav in Drosophila melanogaster. DroVav encodes a protein (793 residues) whose similarity with hVav is 47% and with hVav2 and hVav3 is 45%. DroVav preserves the unique, complex structure of hVav proteins, including the 'calponin homology', dbl homology, pleckstrin homology; SH2 and SH3 domains in addition to regions that are acidic rich, proline rich and cysteine rich. DroVav is located on the X chromosome in polytene interval 18A5;18B and is expressed in all stages of development and in all tissues. In mammalian cells, DroVav is tyrosine-phosphorylated in response to epidermal growth factor receptor (EGFR) induction; in vitro, the DroVav SH2 region is associated with tyrosine-phosphorylated EGFR. Thus, DroVav probably plays a pivotal role as a signal transducer protein during fruit fly development.     

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.     

The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Drosophila glial cells missing (gcm) is a key gene that determines the fate of stem cells within the nervous system. Two mouse gcm homologs have been identified, but their function in the nervous system remains to be elucidated. To investigate their function, we constructed retroviral vectors harboring Drosophila gcm and two mouse Gcm genes. Expression of these genes appeared to influence fibroblast features. In particular, mouse Gcm1 induced the expression of astrocyte-specific Ca(2+)-binding protein, S100beta, in those cells. Introduction of the mouse Gcm1 gene in cultured cells from embryonic brains resulted in the induction of an astrocyte lineage. This effect was also observed by in utero injection of retrovirus harboring mouse Gcm1 into the embryonic brain. However, cultures from mouse Gcm1-deficient mouse brains did not exhibit significant reductions in the number of astrocytes. Furthermore, in situ hybridization analysis of mouse Gcm1 mRNA revealed distinct patterns of expression in comparison with other well-known glial markers. The mammalian homolog of Drosophila gcm, mouse Gcm1, exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells.     

Analysis of a swallow homologue from Drosophila pseudoobscura

We analyzed a functional homologue of the swallow gene from Drosophila pseudoobscura. The swallow gene of D. melanogaster plays an essential role in localizing bicoid mRNA in oocytes, and swallow mutant embryos show anterior pattern defects that result from the lack of localization of the bicoid morphogen. The pseudoobscura homologue rescues the function of swallow mutants when introduced into the genome of D. melanogaster, and its expression is similar to that of the melanogaster gene. The predicted pseudoobscura and melanogaster proteins are 49% identical and 69% conserved. The coiled-coil domain previously identified in the melanogaster swallow protein is strongly conserved in the pseudoobscura homologue, but the weak similarity of the melanogaster swallow protein to the RNP class of RNA-binding proteins is not conserved in the pseudoobscura homologue. These and other observations suggest a structural role for swallow in localizing bicoid mRNA, perhaps as part of the egg cytoskeleton. Received: 3 August 1999 / Accepted: 29 September 1999     

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.