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M Melkonian

Publications and source records attributed to M Melkonian.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of expressed sequence tags (ESTs) from the scaly green flagellate Scherffelia dubia Pascher emend. Melkonian et Preisig.

Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery, generation of molecular markers and characterization of transcription patterns. We have constructed an EST-database of the scaly green flagellate Scherffelia dubia (Chlorophyta) containing 361 sequences. cDNAs were obtained from interphase cells and from cells regenerating flagella. Analysis of the ESTs identified 138 EST-groups with significant similarity to known sequences. 134 EST-groups showed no significant similarity to any sequences in the databases. Most of the ESTs with similarity to known proteins are associated with typical interphase cell functions of a photosynthetic plant cell: assimilation of nutrients and biosynthesis of proteins. Others are related to the activation of the secretory pathway or the biogenesis of scales (e.g. kdo-synthase). Comparison of S. dubia ESTs with the genome of Arabidopsis thaliana and the EST database of Chlamydomonas reinhardtii revealed that S. dubia ESTs with similarity to known proteins were more similar to sequences in C. reinhardtii than to those of A. thaliana. Additionally, ESTs for guanylyl cyclase and cGMP phosphodiesterase are present in the two flagellates, but so far these gene products have not been found in embryophytes.

3' Untranslated Regions↗
PubMed2001

Molecular phylogeny and taxonomic revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and description of Oogamochlamys gen. nov. and Lobochlamys gen. nov.

The genus Chlamydomonas (including Chloromonas) is one of the largest green algal genera comprising more than 600 species. To initiate a comprehensive analysis of the phylogeny and systematics of the genus, we determined nuclear-encoded SSU rRNA sequences from 32 strains of Chlamydomonas, Chloromonas and Chlorogonium with emphasis on oogamous taxa and related strains, and incorporated these into global molecular phylogenetic analyses of 132 strains of Chlorophyceae. In addition, we studied the morphology and reproduction of oogamous and related strains by light microscopy. We recognize and designate 18 monophyletic lineages (clades) within the Chlorophyceae, 11 of which are confined to the CW (basal bodies displaced clockwise) subgroup. The majority of clades recognized within the Chlorophyceae do not correspond to any of the traditional classification systems, which are still largely based on the organization level. Strains assigned to Chlamydomonas and Chloromonas were found in seven different clades confirming the polyphyly of the two genera as presently conceived. To initiate the taxonomic revision of Chlamydomonas, C. reinhardtii is proposed as the conserved type of the genus. In consequence, species in clades other than the clade containing C. reinhardtii must be transferred to other genera, a process initiated in this contribution. The oogamous strains studied represent a monophyletic lineage, which is described as Oogamochlamys gen. nov. comprising three species (O. gigantea, O. zimbabwiensis and O. ettlii spec. nov.). The sister clade to Oogamochlamys consists of isogamous strains characterized by chloroplasts with incisions and is described as Lobochlamys gen. nov. with two species (L. culleus and L. segnis). Another clade is characterized by asteroid or perforated, parietal chloroplasts and contains the type species of Chloromonas (C. reticulata). Thus, the polyphyletic Chloromonas (traditionally defined as "Chlamydomonas without pyrenoids") can be legitimized as a monophyletic genus by restriction to this clade and is here emended on the basis of chloroplast characters (the clade contains strains with or without pyrenoids thus rejecting the character "absence of pyrenoids").

Animals↗
PubMed2000

Developmental regulation of the maize Zm-p60.1 gene encoding a beta-glucosidase located to plastids.

A beta-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences. The Zm-p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zmp60.1 protein was located exclusively in the plastids.

Gene Expression Regulation, Developmental↗
PubMed2000

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.

Animals↗
PubMed2000

Flagellar membrane proteins of Tetraselmis striata butcher (Chlorophyta).

Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and 57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel permeation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A polyclonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to localize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains characterized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.

Algal Proteins↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Mesostigmatophyceae, a new class of streptophyte green algae revealed by SSU rRNA sequence comparisons.

Complete nuclear-encoded SSU rRNA sequences have been obtained from three taxa of streptophyte green algae (Klebsormidium nitens, Nitella capillaris, Chaetosphaeridium globosum) and two strains of the scaly green flagellate Mesostigma viride. Phylogenetic analyses of 70 taxa of Viridiplantae (Chlorophyta and Streptophyta) and 57 taxa of streptophyte green algae and embryophyte plants using distance, parsimony and likelihood methods revealed a novel monophyletic lineage among the Streptophyta comprising the genera Mesostigma and Chaetosphaeridium. This lineage is described here as the Mesostigmatophyceae classis nova. Our analyses demonstrate that (1) scaly green flagellates (prasinophytes) are polyphyletic, (2) a scaly green flagellate is a member of the Streptophyta and forms a clade with the oogamous, filamentous Chaetosphaeridium to the exclusion of all other known streptophyte green algae, (3) a previously published SSU rRNA sequence of Chaetosphaeridium (AF113506) is chimeric and contains part of a fungal SSU rRNA, and (4) the phylogenetic relationships between the Mesostigmatophyceae and other streptophyte green algae remain unresolved by SSU rRNA sequence comparisons.

Chlorophyta↗
PubMed1998

A novel 95-kD protein is located in a linker between cytoplasmic microtubules and basal bodies in a green flagellate and forms striated filaments in vitro.

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.

Amino Acid Sequence↗
PubMed1998

SF-assemblin, striated fibers, and segmented coiled coil proteins.

The flagellar basal apparatus of many flagellate green algae contains noncontractile striated microtubule-associated fibers (SMAFs). The SMAFs consist of 2-nm protofilaments and are predominantly built from striated fiber (SF)-assemblin, an acidic 33-kDa protein. In this review we summarize the present knowledge concerning the biochemical properties of SF-assemblin and the molecular architecture of the SMAFs, provide evidence for homologous proteins and similar filament systems in other eukaryotes, and, finally, discuss possible biological functions of SF-assemblin.

Algal Proteins↗
PubMed1996

Characterization of green alga, yeast, and human centrins. Specific subdomain features determine functional diversity.

Centrins are a subfamily within the superfamily of Ca2+-modulated proteins that play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems. We examined the individual molecular properties of yeast, green alga, and human centrins. Circular dichroism spectroscopy revealed a divergent influence of Ca2+ binding on the alpha-helical content of these proteins. Ca2+-free centrins were elongated in shape as determined by size exclusion chromatography. The presence of Ca2+ and binding peptide resulted in more spherical shaped centrins. In contrast to yeast calmodulin, centrins formed multimers in the Ca2+-bound state. This oligomerization was significantly reduced in the absence of Ca2+ and in the presence of binding peptide. The Ca2+-dependent polymerization of the green alga Scherffelia dubia centrin (SdCen) resulted in a filamentous network. This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen. Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae centrin Cdc31p. Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo.

Amino Acid Sequence↗
PubMed1996

Primary and secondary structure analyses of the rDNA group-I introns of the Zygnematales (Charophyta).

The Zygnematales (Charophyta) contain a group-I intron (subgroupIC1) within their nuclear-encoded small subunit ribosomal DNA (SSU rDNA) coding region. This intron, which is inserted after position 1506 (relative to the SSU rDNA of Escherichia coli), is proposed to have been vertically inherited since the origin of the Zygnematales approximately 350-400 million years ago. Primary and secondary structure analyses were carried out to model group-I intron evolution in the Zygnematales. Secondary structure analyses support genetic data regarding sequence conservation within regions known to be functionally important for in vitro self-splicing of group-I introns. Comparisons of zygnematalean group-I intron secondary structures also provided some new insights into sequences that may have important roles in in vivo RNA splicing. Sequence analyses showed that sequence divergence rates and the nucleotide compositions of introns and coding regions within any one taxon varied widely, suggesting that the "1506" group-I introns and rDNA coding regions in the Zygnematales evolve independently.

Base Composition↗
PubMed1996

The secretory pathway of protists: spatial and functional organization and evolution.

All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.

Animals↗
PubMed1996

The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin.

Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SF-assemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SF-assemblin and related proteins in various protists are representatives of a new class of alpha-helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro.

Amino Acid Sequence↗
PubMed1995

Analyses of ribosomal RNA sequences from glaucocystophyte cyanelles provide new insights into the evolutionary relationships of plastids.

Glaucocystophyte algae (sensu Kies, Berl. Deutsch. Bot. Ges. 92, 1979) contain plastids (cyanelles) that retain the peptidoglycan wall of the putative cyano-bacterial endosymbiont; this and other ultrastructural characters (e.g., unstacked thylakoids, phycobilisomes) have suggested that cyanelles are "primitive" plastids that may represent undeveloped associations between heterotrophic "host" cells (i.e., glaucocystophytes) and cyanobacteria. To test the monophyly of glaucocystophyte cyanelles and to determine their evolutionary relationship to other plastids, complete 16S ribosomal RNA sequences were determined for Cyanophora paradoxa, Glaucocystis nostochinearum, Glaucosphaera vacuolata, and Gloeochaete wittrockiana. Plastid rRNAs were analyzed with the maximum-likelihood, maximum-parsimony, and neighbor-joining methods. The phylogenetic analyses show that the cyanelles of C. paradoxa, G. nostochinearum, and G. wittrockiana form a distinct evolutionary lineage; these cyanelles presumably share a monophyletic origin. The rDNA sequence of G. vacuolata was positioned within the nongreen plastid lineage. This result is consistent with analyses of nuclear-encoded rRNAs that identify G. vacuolata as a rhodophyte and support is removal from the Glaucocystophyta. Results of a global search with the maximum-likelihood method suggest that cyanelles are the first divergence among all plastids; this result is consistent with a single loss of the peptidoglycan wall in plastids after the divergence of the cyanelles. User-defined tree analyses with the maximum-likelihood method indicate, however, that the position of the cyanelles is not stable within the rRNA phylogenies. Both maximum-parsimony and neighbor-joining analyses showed a close evolutionary relationship between cyanelles and non-green plastids; these phylogenetic methods were sensitive to inclusion/exclusion of the G. wittrockiana cyanelle sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Comparisons of nuclear-encoded small-subunit ribosomal RNAs reveal the evolutionary position of the Glaucocystophyta.

The Glaucocystophyta (e.g., Cyanophora paradoxa) form a morphologically distinct group of photosynthetic protists that is primarily distinguished by its cyanelles (= plastids). To elucidate their evolutionary relationships, we determined nuclear-encoded small-subunit ribosomal RNA (SSU rRNA) coding regions for four taxa classified in the Glaucocystophyta (C. paradoxa, Glaucocystis nostochinearum, Glaucosphaera vacuolata, Gloeochaete wittrockiana; sensu Kies and Kremer), and these sequences were positioned within the eukaryotic phylogeny. Maximum likelihood, maximum-parsimony, and neighbor-joining phylogenetic analyses show that the Glaucocystophyta is a relatively late-diverging monophyletic assemblage within the "crown" group radiation that forms a sister group to cryptophyte algae. Glaucosphaera vacuolata is a red alga and lacks some cyanelle (e.g., bounding peptidoglycan wall) and host cell (e.g., cruciate flagellar roots) characters typical of glaucocystophytes. Our data are consistent with a monophyletic origin of the cyanelle in the glaucocystophytes. The distribution of photosynthetic taxa within the glaucocystophytes/cryptophytes and other lineages such as the filose amoebae/chlorarachniophytes and heterokont protists provide clues to the origin of plastids with four bounding membranes. We speculate that multiple, likely independent, secondary endosymbioses gave rise to these plastids.

Eukaryota↗