Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.     

Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.     

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication. Overproduction of DnaA protein, which in wild-type cells leads to over-replication, had no effect in the dnaN(G157C) mutant. Origins already opened by DnaA seemed to remain open for a prolonged period, with a stage of initiation involving β clamp loading, presumably limiting the initiation process. The existence of opened origins led to a moderate SOS response. Lagging strand synthesis, which also requires loading of the β clamp, was apparently unaffected. The result indicates that some aspects of β clamp activity are specific to the origin. It is possible that the origin specific activities of β contribute to regulation of initiation frequency.     

Variation in mitochondrial DNA affects locomotor activity and sleep in Drosophila melanogaster

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. Our results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we were able to quantify the among-line variance attributable to mitochondrial DNA, and we establish that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether our study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.Subject terms: Behavioural genetics, Drosophila, Genetic models     

Drosophila morphogenesis: the Newtonian revolution

Recent quantitative modeling of dorsal closure in the fruitfly Drosophila has revealed how multiple forces drive sealing of the two symmetrical epithelial sheets. A predictive model based on the new data allows gene function to be linked to the forces that drive tissue movement.     

Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function

In addition to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biology. We have recently identified a novel and conserved mitochondrial rhomboid intramembrane protease that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane. The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. Here we show that in Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. We also find that mutations in Drosophila optic atrophy 1-like (Opa1-like), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. rhomboid-7 mutant flies have severe neurological defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. rhomboid-7 mutant flies also have a greatly reduced lifespan.     

A mutation in the eln2 gene encoding a cytochrome P450 of Coprinus cinereus affects mushroom morphogenesis

A dominant mutation of the elongationless2 (eln2) gene of the mushroom Coprinus cinereus (eln2-1) affects pattern formation in the development of fruit body primordia, causing dumpy primordia which culminate in mature fruit bodies with short stipes. Phenotypic analyses revealed disturbance of tissue organization and cell morphogenesis in the primordial shaft and suggested that the defects in the primordial shaft interfere with differentiation of the stipe tissue in the mutant primordia. The eln2 locus was assigned to chromosome XII and the eln2-1 mutant gene was cloned from a chromosome XII-specific cosmid library as a genomic fragment that brings about the dominant mutant phenotype. The eln2 gene encodes a novel type of microsomal cytochrome P450 enzyme (CYP502) and is constitutively expressed. The eln2-1 mutation is a 4-bp deletion in the 3'-terminal region of the gene, leading to truncation of C-terminal 18 amino acids. We suggest that the truncation affects the activity of the CYP502 enzyme.     

Slit coordinates cardiac morphogenesis in Drosophila

Slit is a secreted guidance cue that conveys repellent or attractive signals from target and guidepost cells. In Drosophila, responsive cells express one or more of three Robo receptors. The cardial cells of the developing heart express both Slit and Robo2. This is the first report of coincident expression of a Robo and its ligand. In slit mutants, cardial cell alignment, polarization and uniform migration are disrupted. The heart phenotype of robo2 mutants is similar, with fewer migration defects. In the guidance of neuronal growth cones in Drosophila, there is a phenotypic interaction between slit and robo heterozygotes, and also with genes required for Robo signaling. In contrast, in the heart, slit has little or no phenotypic interaction with Robo-related genes, including Robo2, Nck2, and Disabled. However, there is a strong phenotypic interaction with Integrin genes and their ligands, including Laminin and Collagen, and intracellular messengers, including Talin and ILK. This indicates that Slit participates in adhesion or adhesion signaling during heart development.     

Planar signaling and morphogenesis in Drosophila

The regulatory mechanisms governing the parallel alignment of hairs, bristles, and ommatidia in Drosophila have all served as model systems for studying planar signaling and tissue level morphogenesis. Polarity in all three systems is mediated by the serpentine receptor Frizzled and a number of additional gene products. The localized accumulation of these proteins within cells plays a key role in the development of planar polarity. A comparison of the function of these gene products in the different cell types suggests cell-specific modifications of the pathway.     

Adherens junctions in Drosophila retinal morphogenesis

Adherens junctions and their core molecular components, classic cadherins, make major contributions to animal morphogenesis. Although the significance of cadherins in development is generally accepted, the mechanisms regulating adherens junction function during morphogenesis remain a subject of intense research. Adherens junctions are involved in the organization of simple cellular patterns, and more complex cell shape changes and cell movements that depend on the dynamic modulation of adherens junctions.     

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.     

A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.     

The novel Drosophila tim(blind) mutation affects behavioral rhythms but not periodic eclosion

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, tim(blind), encodes a constitutively hypophosphorylated TIM protein. The mutant protein is not properly degraded by light, and tim(blind) flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TIM(BLIND) protein, which we observed in brain pacemaker neurons and photoreceptor cells of the compound eye. tim(blind) encodes two closely spaced amino acid changes compared to the wild-type TIM protein; one of them is within a putative nuclear export signal of TIM. Under constant conditions, tim(blind) flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that TIM--in addition to its well-established role as core clock factor--functions as a clock output factor, involved in determining the period length of adult locomotor rhythms.