Isolation and cloning of a Drosophila homolog to the mammalian RACK1 gene,implicated in PKC-mediated signalling

The mammalian RACK1 protein binds activated protein kinase C, acting as an intracellular receptor to anchor the activated PKC to the cytoskeleton. Genes encoding RACK1-like proteins have been isolated from a wide range of eucaryotic organisms; we report the isolation of a Drosophila member of this family. This Drosophila RACK1-like protein shows 76% identity to the mammalian RACK1 proteins, but only about 60% identity to related proteins from plants and fungi. The Drosophila rack1 gene has a dynamic pattern of expression during early embryogenesis with the highest expression in the mesodermal and endodermal lineages.     

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non‐professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.     

The Drosophila Toll-9 activates a constitutive antimicrobial defense

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. We analyzed the nine Drosophila Toll proteins and found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms.     

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis

MCM (minichromosome maintenance) gene family of Saccharomyces cerevisiae encodes essential DNA replication factors that participate in the initiation of DNA replication. In addition, their localization to the nucleus in a mitosis-dependent manner fueled the hypothesis that MCMs also act to couple DNA replication to mitosis. We report the identification of a Drosophila gene family with extensive sequence identity to the MCM genes. Results from antibody injection experiments suggest that MCMs play an essential role in DNA replication during embryogenesis. Evolutionary conservation of MCM sequences and function in Drosophila could potentially facilitate studies of how initiation of DNA replication is regulated and coupled to mitosis during metazoan development.     

Characterization of an HSP70 Cognate Gene Family in Arabidopsis

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5′ regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.     

Novel phenotypes of <Emphasis Type="Italic">Drosophila spinster</Emphasis> locus on the head formation during embryogenesis

spinster (spin) is a late endosome/lysosome membrane protein with the amino acid sequence of a lysosomal sugar carrier and expressed in the glial cells. Spin is required for autophagy and lysosome reformation by releasing lysosomal degradation products of autolysosome into the cytosol in Drosophila larvae and adults. However, such kind of function has not been investigated in embryos yet. In this study, for the first time, we examined the effects of spin mutation on the endocytic pathway and autophagy during embryogenesis. Loss-of-function spin mutation led to the abnormal process of early endosome/recycling endosome and the accumulation of enlarged autophagosome/autolysosome. These abnormal endocytic pathway and autophagy subsequently caused the malformation of head at embryonic stages. These results show that Spin is involved in the endocytic pathway and autophagy during embryogenesis as well as larval and adult stages.     

Cloning of a novel homeobox (NK-7.1) containing gene,DmHboxNK-7.1, from Drosophila melanogaster

We have cloned a novel Drosophila melanogaster homeobox (Hbox) containing gene, NK-7.1 (Dm.HboxNK-7.1), which is located at 88B3 on the chromosome map, and is 1.5 kb downstream of the spn-B gene. The newly identified gene is expressed at high levels in the embryo, is switched off during larval and pupal stages, and is expressed again in the adult. The Hbox is highly similar to NK-1/S59 (Drosophila) and NK-3/bap (Drosophila). The amino acid (aa) identity ratios (%) were 58 between NK-7.1 and NK-1/S59, and between NK-7.1 and NK-3/bap. The other characteristic structures are the presence of homopolymeric aa stretches consisting of Q, N, and E.     

Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval PNS. We report a role of scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis we find that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an “off-centred” manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality.     

The Him gene inhibits the development of Drosophila flight muscles during metamorphosis

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.     

Mutation in<Emphasis Type="Italic"> slowmo</Emphasis> causes defects in<Emphasis Type="Italic"> Drosophila</Emphasis> larval locomotor behaviour

We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions. Cloning of the slowmo gene identifies a product related to a family of proteins of unknown function. We show that Slowmo is associated with mitochondria, indicative of it being a mitochondrial protein, and that during embryogenesis and early larval development is restricted to the nervous system in a subset of cells. The enhancer-trap marks a cellular component of the CNS that is seemingly required to regulate peristaltic movement.     

A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins,wasp venom antigen 5, and plant group 1 pathogenesis-related proteins

The CAP protein family is made up of a group of secreted proteins that share sequence similarity. Members of this family are found in animals, plants, and fungi, and their shared sequence similarity suggests that members share a common, but as yet unknown, molecular function. As a first step in defining the function of CAP family proteins, an 878 bp partial cDNA encoding a novel member of the CAP family was cloned by the polymerase chain reaction (PCR) from total RNA of adult Drosophila. The cDNA contained the complete coding sequence for a protein 256 amino acids in length, as well as the complete 3′ untranslated region (UTR) and a portion of the 5′ UTR. The protein, named Antigen 5-related (Agr), was most similar in sequence to antigen 5 (Ag5), a CAP family member found in social wasps and ants. The corresponding Agr RNA is about 1 kb in length and is present at all stages of development, with highest levels observed in adults. Agr RNA is transcribed from a single gene that is located within region 12F of the X chromosome. The identification of Agr in Drosophila expands the number of known CAP family members to well over four dozen. Further studies of Agr and the gene which encodes this protein using the Drosophila model system may help provide important insight into the molecular functioning of this little known, but increasingly significant protein family.