Essential function of nitric oxide synthase in Drosophila.
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Amino Acid Sequence↗
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A Rho GTPase signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation.
A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation . It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. We show here that in vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Galpha(12/13) family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding.
Animals↗
The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis.
MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in plants and animals (for recent reviews, see ). miRNAs are expressed in a developmentally and tissue-specific manner and regulate the translational efficiency and stability of partial or fully sequence-complementary mRNAs. miRNAs are excised in a stepwise process from double-stranded RNA precursors that are embedded in long RNA polymerase II primary transcripts (pri-miRNA). Drosha RNase III catalyzes the first excision event, the release in the nucleus of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to the cytoplasm and further processing by Dicer to mature miRNAs. Here, we characterize the human DGCR8, the DiGeorge syndrome critical region gene 8, and its Drosophila melanogaster homolog. We provide biochemical and cell-based readouts to demonstrate the requirement of DGCR8 for the maturation of miRNA primary transcripts. RNAi knockdown experiments of fly and human DGCR8 resulted in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. Our results suggest that DGCR8 and Drosha interact in human cells and reside in a functional pri-miRNA processing complex.
Animals↗
The KLP-6 kinesin is required for male mating behaviors and polycystin localization in Caenorhabditis elegans.
BACKGROUND: Male mating behavior of the nematode Caenorhabditis elegans offers an intriguing model to study the genetics of sensory behavior, cilia function, and autosomal dominant polycystic kidney disease (ADPKD). The C. elegans polycystins LOV-1 and PKD-2 act in male-specific sensory cilia required for response and vulva-location mating behaviors. RESULTS: Here, we identify and characterize a new mating mutant, sy511. sy511 behavioral phenotypes were mapped to a mutation in the klp-6 locus, a gene encoding a member of the kinesin-3 family (previously known as the UNC-104/Kif1A family). KLP-6 has a single homolog of unknown function in vertebrate genomes, including fish, chicken, mouse, rat, and human. We show that KLP-6 expresses exclusively in sensory neurons with exposed ciliated endings and colocalizes with the polycystins in cilia of male-specific neurons. Cilia of klp-6 mutants appear normal, suggesting a defect in sensory neuron function but not development. KLP-6 structure-function analysis reveals that the putative cargo binding domain directs the motor to cilia. Consistent with a motor-cargo association between KLP-6 and the polycystins, klp-6 is required for PKD-2 localization and function within cilia. Genetically, we find klp-6 regulates behavior through polycystin-dependent and -independent pathways. CONCLUSION: Multiple ciliary transport pathways dependent on kinesin-II, OSM-3, and KLP-6 may act sequentially to build cilia and localize sensory ciliary membrane proteins such as the polycystins. We propose that KLP-6 and the polycystins function as an evolutionarily conserved ciliary unit. KLP-6 promises new routes to understanding cilia function, behavior, and ADPKD.
Animals↗
DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.
Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.
Arabidopsis↗
Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans.
BACKGROUND: Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS: We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS: EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.
Amino Acid Motifs↗
Conversion of functional specificity in Qb-SNARE VTI1 homologues of Arabidopsis.
In higher multicellular eukaryotes, highly specialized membrane structures or membrane trafficking events are required for supporting various physiological functions. SNAREs (soluble NSF attachment protein receptors) play an important role in specific membrane fusions. These protein receptors are assigned to subgroubs (Qa-, Qb-, Qc-, and R-SNARE) according to their specific SNARE structural motif. A specific set of Qa-, Qb-, and Qc-SNAREs, located on the target membrane, interact with R-SNARE on the vesicle to form a tight complex, leading to membrane fusion. The zig-1 mutant of Arabidopsis lacking Qb-SNARE VTI11 shows little shoot gravitropism and abnormal stem morphology. VTI11 and its homolog VTI12 exhibit partially overlapping but distinct intracellular localization and have different biological functions in plants. Little is known about how SNAREs are targeted to specific organelles, even though their functions and specific localization are closely linked. Here, we report that a novel mutation in VTI12 (zip1) was found as a dominant suppressor of zig-1. The zip1 mutation gave VTI12 the ability to function as VTI11 by changing both the specificity of SNARE complex formation and its intracellular localization. One amino acid substitution drastically altered VTI12, allowing it to suppress abnormalities of higher order physiological functions such as gravitropism and morphology. The zip1 mutation may be an indication of the flexibility in plant cell function afforded by gene duplication, particularly among the VTI11 genes and their recently diverged orthologs.
Amino Acid Sequence↗
Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus.
Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy. The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 alpha box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species.
Amino Acid Sequence↗
MBR-1, a novel helix-turn-helix transcription factor, is required for pruning excessive neurites in Caenorhabditis elegans.
In the developing brain, excessive neurites are actively pruned in the construction and remodeling of neural circuits. We demonstrate for the first time that the pruning of neurites occurs in the simple neural circuit of Caenorhabditis elegans and that a novel transcription factor, MBR-1, is involved in this process. We identified MBR-1 as a C. elegans ortholog of Mblk-1, a transcription factor that is expressed preferentially in the mushroom bodies of the honeybee brain. Although Mblk-1 homologs are conserved among animal species, their roles in the nervous system have never been analyzed. We used C. elegans as an ideal model animal for analysis of neuronal development. mbr-1 is expressed in various neurons in the head and tail ganglia. A comparison of the morphology of mbr-1-expressing neurons revealed that excessive neurites connecting the left and right AIM interneurons are eliminated during larval stages in wild-type but are sustained through the adult stage in the mbr-1 mutant. In addition, mbr-1 expression is regulated by UNC-86, a POU domain transcription factor, and the pruning of the excessive AIM connection is impaired in the unc-86 mutant. These findings provide an important clue for further genetic dissection of neurite pruning.
Amino Acid Sequence↗
Evolution in action: following function in duplicated floral homeotic genes.
Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.
AGAMOUS Protein, Arabidopsis↗
Exposure to host resistance mechanisms drives evolution of bacterial virulence in plants.
Bacterial pathogenicity to plants and animals has evolved through an arms race of attack and defense. Key players are bacterial effector proteins, which are delivered through the type III secretion system and suppress basal defenses . In plants, varietal resistance to disease is based on recognition of effectors by the products of resistance (R) genes . When recognized, the effector or in this scenario, avirulence (Avr) protein triggers the hypersensitive resistance reaction (HR), which generates antimicrobial conditions . Unfortunately, such gene-for-gene-based resistance commonly fails because of the emergence of virulent strains of the pathogen that no longer trigger the HR . We have followed the emergence of a new virulent pathotype of the halo-blight pathogen Pseudomonas syringae pv. phaseolicola within leaves of a resistant variety of bean. Exposure to the HR led to the selection of strains lacking the avirulence (effector) gene avrPphB (or hopAR1), which triggers defense in varieties with the matching R3 resistance gene. Loss of avrPphB was through deletion of a 106 kb genomic island (PPHGI-1) that shares features with integrative and conjugative elements (ICElands) and also pathogenicity islands (PAIs) in diverse bacteria . We provide a molecular explanation of how exposure to resistance mechanisms in plants drives the evolution of new virulent forms of pathogens.
Bacterial Proteins↗
Mating type gene analysis in apparently asexual Cercospora species is suggestive of cryptic sex.
The genus Cercospora consists of numerous important, apparently asexual plant pathogens. We designed degenerate primers from homologous sequences in related species to amplify part of the C. apii, C. apiicola, C. beticola, C. zeae-maydis and C. zeina mating type genes. Chromosome walking was used to determine the full length mating type genes of these species. Primers were developed to amplify and sequence homologous portions of the mating type genes of additional species. Phylogenetic analyses of these sequences revealed little variation among members of the C. apii complex, whereas C. zeae-maydis and C. zeina were found to be dissimilar. The presence of both mating types in approximately even proportions in C. beticola, C. zeae-maydis and C. zeina populations, in contrast to single mating types in C. apii (MAT1) and C. apiicola (MAT2), suggests that a sexual cycle may be active in some of these species.
Amino Acid Sequence↗
Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis.
A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal sequence (1-19) followed by a mature peptide (20-71). The sequence identity with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The mature peptide, with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp, and that Ch-penaeidin was constitutively expressed mainly in haemocytes.
Amino Acid Sequence↗
Presence of multiple isoforms of TNF alpha in carp (Cyprinus carpio L.): genomic and expression analysis.
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Amino Acid Sequence↗
Cloning and characterisation of natural resistance associated macrophage protein (Nramp) cDNA from red sea bream (Pagrus major).
Nramp (natural resistance associated macrophage protein) controls aspects of innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from spleen of red sea bream (Pagrus major). The full-length cDNA of the Nramp is 4709 bp in length, including 197 bp 5'-terminal untranslated region (UTR), 1662 bp encoding region and 2850 bp 3'-terminal UTR. The 1662 nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of the amino acid sequence indicated that red sea bream Nramp consists of 12 transmembrane region (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of red sea bream Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5% and 69.5% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, mouse Nramp 1 and 2, and human Nramp1 and 2, respectively. Reverse transcription-polymerase chain reaction indicated that levels of Nramp expression were similar among head kidney, spleen, intestine and liver in non-challenged red sea bream, and that challenge of red sea bream with the pathogenic bacterium, Vibrio anguillarum, significantly elevated Nramp mRNA levels in liver and spleen in a time-dependent fashion.
Amino Acid Motifs↗
Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) interleukin-10 cDNAs.
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Amino Acid Sequence↗
Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus).
Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paralichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization.
Amino Acid Sequence↗