V-ATPase is a major component of the Golgi complex in the scaly green flagellate Scherffelia dubia

Highly purified membranes isolated from the Golgi complex of the scaly green flagellate Scherffelia dubia (Chlorophyta) were subjected to Triton X-114 two-phase partitioning. Proteins in the detergent phase were analyzed by 2D gel electrophoresis and a major protein of 66 kD (p66) was N-terminally sequenced. The complete cDNA sequence of p66 was obtained by 3' RACE-PCR and screening of a cDNA library of S. dubia with a PCR probe derived from the 3' RACE. Sequence analysis of the cDNA clone identified p66 as subunit A of V-ATPase. Other major proteins in the isolated Golgi complex were immunoreactive to heterologous antibodies raised against subunit B or the holoenzyme of V-ATPase. A polyclonal (anti-p66) antibody raised against a recombinant, bacterially expressed p66 fusion protein recognized p66 in the isolated Golgi complex in western blots and localized the antigen by immunogold electron microscopy mostly to the scale reticulum but also to the Golgi stack within the Golgi complex. Concanamycin A-sensitive (but bafilomycin A1-insensitive) ATPase activity was present in the isolated Golgi complex, and monensin at 0.5-1 microM reversibly inhibited flagellar regeneration and resulted in swelling of Golgi cisternae. It is concluded that a functional V-ATPase is a major protein of the Golgi complex in S. dubia and is presumably associated with sorting processes at the endocytotic/exocytotic boundary of the Golgi complex.     

Morphological and molecular characterization of a new terrestrial allogromiid species: Edaphoallogromia australica gen. et spec. nov. (Foraminifera) from Northern Queensland (Australia).

The foraminiferal family Allogromiidae occurs mainly in marine environments, although some genera are described from brackish and freshwater habitats. We report here the occurrence of a terrestrial allogromiid foraminiferan. Phylogenetic relationships were investigated by sequencing part of the SSU rDNA. DNA sequence analysis confirms a close relationship of the new species to the genus Allogromia. Morphological studies corroborate the affiliation to the family Allogromiidae but the lack of an entosolenian tube and an internal septum as well as the different ecology do not allow a classification into a known genus of this family. Taking the molecular, morphological and ecological differences into account, a new genus Edaphoallogromia with the type species E. australica is erected.     

Analysis of expressed sequence tags (ESTs) from Lentinula edodes

The 1,031 expressed sequence tags (ESTs) from the basidiomycete Lentinula edodes were generated as a pilot experiment to see distribution of genes expressed in L. edodes. Among them, genes for hydrophobin, which are specifically found in filamentous fungi, were the most frequently obtained ESTs (33 times), suggesting that they are highly expressed in L. edodes. In addition to known hydrophobin 1 and 2 types, our analysis revealed the existence of novel types of hydrophobin, which we named hydrophobin 3, 4, and 5. The second and the third most highly obtained ESTs were phosphatidylserine decarboxylase and formate dehydrogenase, which were obtained eight and seven times, respectively. It should be noted that two important genes (argonaute and RNA-dependent RNA polymerase) involved in the RNAi pathway were found, suggesting a future application for gene knock-down by RNA interference. The 53 ESTs were identical with the sequences already reported in L. edodes. The 433 ESTs were found to show significant sequence similarity (E value <1 x 10(-5)) with the proteins reported (or predicted) in other species. In total, 387,952 bp were sequenced and registered in DDBJ/GenBank (accession number BJ998097-BJ999127).     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila

To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein. Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis.     

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs)

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.     

A light and electron microscopic study of Scherffelia dubia, a new member of the scaly green flagellates (Prasinophyceae)

In clonal culture Scherffelia dubia (Perty) Pascher shows a considerable range of morphological variation, with different cell shapes being produced including those typical for three other species of Scherffelia Pascher ( S. opisthostigma Skuja, S. ovata Pascher, S. pelagica Skuja) and one species of Tetraselmis Stein ( T. bichlora Ettl & Ettl). It is concluded these taxa should be reduced to the synonymy of S. dubia , the type species of Scherffelia. The general cell morphology of S. dubia is described in detail, including a serial section and absolute configuration analysis of the flagellar apparatus. S. dubia is compared with species of Tetraselmis of which ultrastructural details are known and it is shown that both genera are very similar but the generic distinction is nevertheless justified. Main features of distinction include the pyrenoid (present in Tetraselmis but lacking in Scherffelia ) and the chloroplast (single and more or less cup-shaped in Tetraselmis , two separate lateral chloroplasts in Scherffelia ). Both genera are accommodated in the same family, the correct name being Chlorodendraceae and not Tetraselmidaceae or Platymonadaceae which have often been used.     

Analysis of expressed sequence tags (ESTs) from cocoa (Theobroma cacao L) upon infection with Phytophthora megakarya

Phytophthora megakarya, the causative agent of cacao black pod disease in West African countries causes an extensive loss of yield. In this study we have analyzed 4 libraries of ESTs derived from Phytophthora megakarya infected cocoa leaf and pod tissues. Totally 6379 redundant sequences were retrieved from ESTtik database and EST processing was performed using seqclean tool. Clustering and assembling using CAP3 generated 3333 non-redundant (907 contigs and 2426 singletons) sequences. The primary sequence analysis of 3333 non-redundant sequences showed that the GC percentage was 42.7 and the sequence length ranged from 101 - 2576 nucleotides. Further, functional analysis (Blast, Interproscan, Gene ontology and KEGG search) were executed and 1230 orthologous genes were annotated. Totally 272 enzymes corresponding to 114 metabolic pathways were identified. Functional annotation revealed that most of the sequences are related to molecular function, stress response and biological processes. The annotated enzymes are aldehyde dehydrogenase (E.C: 1.2.1.3), catalase (E.C: 1.11.1.6), acetyl-CoA C-acetyltransferase (E.C: 2.3.1.9), threonine ammonia-lyase (E.C: 4.3.1.19), acetolactate synthase (E.C: 2.2.1.6), O-methyltransferase (E.C: 2.1.1.68) which play an important role in amino acid biosynthesis and phenyl propanoid biosynthesis. All this information was stored in MySQL database management system to be used in future for reconstruction of biotic stress response pathway in cocoa.     

Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.