High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.     

Early Trichinella spiralis and Trichinella nativa infections induce similar gene expression profiles in rat jejunal mucosa

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ?0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella.     

Ectopic Repo suppresses expression of castor gene in Drosophila central nervous system

The ventral nerve cord (VNC) of the Drosophila embryo is derived from neuroblasts (NBs). NBs divide in a stem cell lineage to generate a series of ganglion mother cells (GMCs), each of which divides once to produce a pair of neurons or glial cells. One of the NB genes, castor (cas), is expressed in a subset of NBs and has never been identified in neurons and the peripheral nervous system; cas plays a role in axonogenesis. But its limited expression along the dorsal-ventral axis within the central nervous system has not been investigated yet. In the present study, we examined the expression patterns of both genes using confocal microscopy to determine the effects of repo mutation on cas expression. Cas was mainly expressed in layers different from repo-expressed layers during early embryogenesis: repo was expressed mostly from deep to mid layers, while cas, from mid to superficial layers. Loss-of-function of repo did not result in an ectopic expression of cas, but rather, a scattering of cas-expressing cells. However, repo gain-of-function mutation caused repression of cas. In addition, repo-expressing cells seemed to block the migration of cas-expressing cells.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

CAMBIUM, a process-based model of daily xylem development in Eucalyptus

In hardwoods such as Eucalyptus spp., xylem (wood) is a heterogeneous tissue consisting of multiple cell types. As such, xylem development involves multiple complex interactions. To describe and understand xylem development, and ultimately predict the resultant wood properties, a process-based approach to modelling wood property variation is potentially very useful. In this paper, a new model (CAMBIUM), which incorporates concepts of these processes, is described. CAMBIUM predicts how wood density and fibre and vessel anatomical properties vary from pith-to-bark at a daily time step as a function of changing environmental conditions and a set of simulated physiological processes. Simulations from an existing process-based model of stand development (CABALA) are used as inputs. A key feature of CAMBIUM is a model of the interaction between different xylem cell types. Some weaknesses were identified in the ability of the model to simulate vessel spatial patterns and frequencies, emphasizing the complexities inherent in this aspect of angiosperm wood formation. The model was, however, able to provide realistic estimates of short-term variation and temporal ranges in eucalypt fibre diameter and secondary wall development and wood density.     

ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.