Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila

Summary We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the -amanitinresistant allele, RpII215 ^C4 , includes all sequences required to produce amanitin-resistant transformants.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.GenBank accession number: M98495     

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Host preference in Pseudacteon phorid flies: response of P. tricuspis and P. curvatus to black, red and hybrid imported Solenopsis fire ants in multiple choice bioassays

Host preferences in both sexes of Pseudacteon tricuspis Borgmeier (Jaguariuna biotype) and Pseudacteon curvatus Borgmeier (Formosa biotype) and their relative attraction to the imported fire ants (IFA), Solenopsis invicta Buren (red IFA), Solenopsis richteri Forel (black IFA) and S. invicta × S. richteri hybrids (hybrid IFA) were investigated in two separate experiments utilizing multiple choice flight bioassays. The results of both experiments clearly showed that both sexes of the Jaguariuna biotype of P. tricuspis could distinguish among the three IFA species and demonstrated greater preference for hybrid IFA and red IFA. This conclusion is supported by a variety of data collected on the number of fly visits, attack rate, and hovering duration (Experiment 1), and on the number of trapped flies (Experiment 2), which showed that black IFA is the least preferred of the three species. Similar results were recorded for the Formosan biotype of P. curvatus, although the data were not as strongly conclusive. Females of this biotype spent a significantly greater amount of time in hovering mode over red IFA and hybrid IFA compared to black IFA, but the other data were not significant. The red IFA is the natural host of both phorid fly biotypes and our results suggest that both biotypes may have evolved a specialized relationship with red IFA including an ability to discriminate it from related fire ants. These results are discussed in relation to the possible role of fire ant chemicals in mediating host preferences in phorid flies, contributions of male phorid flies to fire ant biocontrol, and the practical implications of the key findings.     

The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

The Caenorhabditis elegans gene laf-1 is critical for both embryonic development and sex determination. Laf-1 is thought to promote male cell fates by negatively regulating expression of tra-2 in both hermaphrodites and males. We cloned laf-1 and established that it encodes a putative DEAD-box RNA helicase related to Saccharomyces cerevisiae Ded1p and Drosophila Vasa. Three sequenced laf-1 mutations are missense alleles affecting a small region of the protein in or near helicase motif III. We demonstrate that the phenotypes resulting from laf-1 mutations are due to loss or reduction of laf-1 function, and that both laf-1 and a related helicase vbh-1 function in germline sex determination. Laf-1 mRNA is expressed in both males and hermaphrodites and in both the germline and soma of hermaphrodites. It is expressed at all developmental stages and is most abundant in embryos. LAF-1 is predominantly, if not exclusively, cytoplasmic and colocalizes with PGL-1 in P granules of germline precursor cells. Previous results suggest that laf-1 functions to negatively regulate expression of the sex determination protein TRA-2, and we find that the abundance of TRA-2 is modestly elevated in laf-1/+ females. We discuss potential functions of LAF-1 as a helicase and its roles in sex determination.     

马齿苋内生菌橘青霉和波兰青霉中抗青枯菌的活性物质

为了挖掘农作物病害生物防治新资源,以药用植物马齿苋(Portulaca oleracea)为材料,通过培养基种植法分离和纯化其根、茎、叶中的内生菌,以青枯菌(Ralstonia solanacearum)的抑菌试验评价其活性,采用菌落形态观察和ITS序列分析鉴定菌种。结果表明,从马齿苋筛选出2种具有抑制青枯菌的内生菌橘青霉(Penicillium citrinum)和波兰青霉(P. polonicum),采用液相与四极杆飞行时间串联质谱(UPLC-QTOF-MS)鉴定2种内生菌的主要活性物质为橘霉素,其对青枯菌的抑制效果比链霉素更好。因此,这为植物青枯病的生物防治提供科学依据。     

The Saccharomyces cerevisiae genes ( CMP1 and CMP2 ) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

Summary Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using¹²⁵I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.     

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H⁺-ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H⁺-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts

Summary The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.     

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.     

Growth dynamics of non-toxic Pseudo-nitzschia delicatissima and toxic P. seriata (Bacillariophyceae) under simulated spring and summer photoperiods

Marine planktonic diatoms of the genus Pseudo-nitzschia Peragallo have been responsible for amnesic shellfish poisoning (ASP) events worldwide through the production of the neurotoxin domoic acid (DA). The appearance and toxicity of Pseudo-nitzschia species is variable throughout the year and potentially linked to changes in environmental parameters; many ASP events occur in relatively high latitudes where day length is particularly variable with season. In UK waters, shellfish monitoring has prevented any impact on human health but has led to long-term closures of fisheries, with severe economic consequences. Laboratory experiments on two Pseudo-nitzschia species typically found in Scottish West Coast waters during spring (short photoperiod (SP)) and summer (long photoperiod (LP)) conditions were conducted to determine the influence of photoperiod on their growth and toxicity. Results indicated that non-toxic P. delicatissima (Cleve) Heiden achieved a greater cell density under SP (9-h light:15-h dark (L:D) cycle). For toxin-producing P. seriata (Cleve) H. Peragallo, a LP (18-h L:6-h D cycle) resulted in an enhanced growth rate, cell yield and total toxin production, but it decreased the toxin production per cell. A better understanding of the response of Pseudo-nitzschia species to photoperiod and other foreseeable environmental variables may help predict the appearance of toxic strains.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.