Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties.
(17/4849)
Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae. (+info)
Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis.
(18/4849)
Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence. (+info)
Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.
(19/4849)
The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves. (+info)
A novel follicle-cell-dependent dominant female sterile allele, StarKojak, alters receptor tyrosine kinase signaling in Drosophila.
(20/4849)
We describe a new dominant allele, StarKojak, that alters receptor tyrosine kinase signaling in the follicle cells and in the eyes in Drosophila. We isolated StarKojak in a screen for follicle-cell-dependent dominant female sterile mutations. We show that StarKojak and revertants of StarKojak do not complement Star loss-of-function mutations. We propose that StarKojak is a novel type of allele of Star that has both dominant gain-of-function phenotypes early in development and dominant loss-of-function phenotypes later in development. Star encodes a putative transmembrane protein that has previously been shown to be a critical component of the epidermal growth factor receptor tyrosine kinase signaling pathway. Early in oogenesis, Star mRNA expression is higher in StarKojak egg chambers than in wild-type egg chambers, consistent with its gain-of-function phenotype. Later in oogenesis, Star mRNA expression is lower in StarKojak follicle cells than in wild-type follicle cells, consistent with its loss-of-function phenotype. By genetically analyzing StarKojak and its revertants, we present evidence that Star is involved in anterior-posterior axis formation both in the female germline cells and in the somatic follicle cells. We also demonstrate that at least part of the dominant female sterile phenotype of StarKojak is restricted to the posterior-pole follicle cells. We propose that Star functions by processing pro-Gurken to mature Gurken, which is thereby released in the region between the oocyte and the follicle cells and binds to the epidermal growth factor receptor in the follicle cells. (+info)
adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS.
(21/4849)
Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve. (+info)
Kinetic analysis of segmentation gene interactions in Drosophila embryos.
(22/4849)
A major challenge for developmental biologists in coming years will be to place the vast number of newly identified genes into precisely ordered genetic and molecular pathways. This will require efficient methods to determine which genes interact directly and indirectly. One of the most comprehensive pathways currently under study is the genetic hierarchy that controls Drosophila segmentation. Yet, many of the potential interactions within this pathway remain untested or unverified. Here, we look at one of the best-characterized components of this pathway, the homeodomain-containing transcription factor Fushi tarazu (Ftz), and analyze the response kinetics of known and putative target genes. This is achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, we find that both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (<10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes are analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy. (+info)
The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart.
(23/4849)
In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also "Gellon et al., 1997"), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heart tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages. (+info)
The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.
(24/4849)
Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide. (+info)