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The mitochondrial LSU rDNA of the brown alga Pylaiella littoralis reveals alpha-proteobacterial features and is split by four group IIB introns with an atypical phylogeny.

The mitochondrial DNA region coding for the large ribosomal RNA subunit from the brown alga Pylaiella littoralis (L.) Kjellm was sequenced. The LSU rRNA was folded into a secondary structure and aligned with homologous, mitochondrial and eubacterial sequences. Taking into account the primary and secondary structure levels, the mitochondrial LSU rRNA of P. littoralis shares more structural features with alpha-proteobacterial genes than do those of the green alga Prototheca wickerhamii and land plants. In phylogenetic trees, branches leading to brown algae, red algae, the protozoan Acanthamoeba castellanii and land plants, respectively, emerge approximately at the same time, as they do in nuclear-gene based phylogenies. This suggests that there is only one origin for the mitochondrial rRNA genes found in these lineages. The LSU rDNA is split by four group IIB introns. The first two introns each contain one open reading frame which encodes a reverse transcriptase-like protein. Comparison of their amino acid sequences with those of other reverse transcriptase-like genes contained in group II introns shows that these genes are more closely related to plastid and cyanobacterial homologous genes than to any known mitochondrial intronic reverse transcriptase.

Amino Acid Sequence↗

A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation.

We have investigated the reactivity of three of the seven group II introns encoded by the mitochondrial genome of the brown alga Pylaiella littoralis. While the first intron in the protein-coding cox1 gene could not be induced to self-splice under any of the conditions tested, the first two introns in the gene encoding the large ribosomal subunit are reactive in vitro and splice primarily by the standard group II two-step transesterification pathway. Intron 2 proved to be of exceptional interest, because in contrast to all group II molecules known so far, its optimal magnesium concentration is less than 10 mM and it still carries out accurate splicing at concentrations as low as 0.1 mM magnesium. Analysis of reaction products under optimal conditions showed no evidence of hydrolysis at the 5' splice site and up to 90% of precursor molecules could be converted into excised lariat intron, which migrated as a single band on non-denaturing polyacrylamide gels. Absorbance versus temperature profiles generated from the lariat form of intron 2 reveal the existence of an early melting component, the amplitude of which does not depend on the way the molecules were purified, i.e. with or without a denaturation step. This highly cooperative transition, whose position along the temperature axis changes with the concentration of magnesium, is proposed to consist of the unfolding of the tertiary structure of the molecule. We conclude that group II introns, which are the largest known ribozymes, can form conformationally homogeneous populations of molecules suitable for physical-chemical studies of higher-order structure.

Electron Transport Complex IV↗

X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 A resolution.

The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.

Amino Acid Sequence↗

Polarity determination in Fucus: from zygote to multicellular embryo.

Zygotes of fucoid algae acquire polarity de novo. The initial polarity establishes the apical-basal polarity of the multicellular embryo and the adult plant. Acquisition of polarity involves the translation of external vectorial signals into spatial information within the cell via spatial photoreceptor activation at the level of the plasma membrane. Fixation of the polar axis involves interactions between the cytoskeleton, plasma membrane and the cell wall. Recently a central role for targeted secretion in polar axis fixation has been identified. In the multicellular embryo, evidence is accumulating for roles of cell wall and intercellular communication via diffusible signals in pattern formation and control of cell fate.

Cell Polarity↗

Signalling in plant embryos during the establishment of the polar axis.

In brown algae fertilization takes place free from surrounding tissue layers. The cytoskeleton and transmembrane links to the cell wall are involved in establishing and stabilizing the polar axis and in determining the fate of cells in the early embryo. In seed plants, the egg cell and zygote exhibit apical basal polarity. Mutant studies suggest that axes of polarity of the early embryo depend on signalling between the apical and basal compartments, possibly involving auxin. Development of somatic cells into plant embryos involves extracellular matrix-derived arabinogalactan proteins. This suggests a role for the cell wall in plant embryogenesis.

Cell Polarity↗

Choosing sides: establishment of polarity in zygotes of fucoid algae.

The acquisition and expression of polarity during early embryogenesis underlies developmental pattern. In many multicellular organisms an initial asymmetric division of the zygote is critical to the determination of different cell fates of the early embryonic cells. Zygotes of the marine fucoid algae are initially apolar and become polarized in response to external cues. This results in an initial asymmetric division of the zygote. Subsequent divisions occur in a highly ordered spatial and temporal pattern. A combination of cell biological and biochemical studies is providing new details, and some controversies concerning the mechanisms by which zygotic polarity is acquired and amplified. Here, we discuss some of the more recent studies that are allowing improved understanding of polarization in this system.

Actins↗

Genome structure of a virus infecting the marine brown alga Ectocarpus siliculosus.

We describe a procedure for the isolation of virus particles from the marine brown alga Ectocarpus siliculosus. Virus particles are composed of at least 13 different polypeptides, including two glycoproteins, and double-stranded DNA. A typical virus DNA preparation contains three fractions, namely linear DNA and circular DNA, each composed of about 320 kilobase pairs, as well as DNA fragments, 10 to 60 kilobase pairs in size. The large linear and the circular DNA contain single-stranded regions (average length: 2.9 kilobase). We propose that the native Ectocarpus virus genome is a circular DNA molecule whose double strand is interrupted by single-stranded regions. During the preparation procedure, the DNA circles tend to break at the single-stranded sites producing large linear as well as fragmented DNA.

DNA, Viral↗

Single-stranded regions in the genome of the Ectocarpus siliculosus virus.

The double-stranded DNA genome of the Ectocarpus siliculosus virus, EsV, is interrupted by numerous single-stranded gaps. We have investigated whether single-stranded regions occur at random or at specific sites. A brief treatment with a single-strand-specific endonuclease dissected the genome into two large fragments, but more extensive treatment produced a spectrum of nuclease-resistant fragments, mainly 10 to 70 kb in size. Native EsV DNA was also used as a substrate for gap-filling DNA synthesis. Restriction analysis revealed that essentially all of the 60 or more SacI restriction fragments became labeled but a few fragments were more intensely labeled than others. The EsV genome may contain a few extended single-stranded regions at fixed sites in addition to numerous single-stranded regions probably occurring at random and varying sites.

Chromosome Mapping↗

Characterization of a repetitive DNA element in a brown algal virus.

We describe a family of repetitive sequences found in viruses infecting the brown alga Feldmannia sp. Previously we have demonstrated that the dsDNA genomes of viruses infecting one Feldmannia sp. isolate exist as two size classes of 160 and 179 kb. Repetitive sequences within these genomes were first demonstrated based on the anomalous hybridization among five BamHI fragments from digested virus DNA. Sequence analysis of one of those fragments, B2.4, revealed the presence of 173-bp direct repeats. The restriction maps of the cross-hybridizing BamHI fragments in the two FsV (Feldmannia sp. virus) genome size classes show that these repeats are not widely dispersed, rather they are confined to a small region of each virus genome. We estimate the number of these repeats in the 179-kb genome to be about 109 and in the 169-kb genome to be about 41. In the 179-kb genome, the repeats are contained within a 22-kb region, about 12% of that virus genome, and in the 160-kb genome the repeats are contained within a 10-kb region, about 6% of the genome. The difference in repeat numbers can account for 62% of the size difference between the two size classes of the FsV genome.

Base Sequence↗

A brown algal virus genome contains a "RING" zinc finger motif.

The brown filamentous alga Feldmannia sp. contains a large icosahedral dsDNA virus, FsV, of which there are multiple variants. A 4.5-kb SstI-HindIII fragment (SH4.5) that is conserved among all genome variants was sequenced. Three open reading frames (ORF-1, -2, and -3, containing 555, 2022, and 411 bp, respectively) were shown to be transcriptionally active by ribonuclease protection assay. A "RING" zinc finger motif and a nucleotide binding site motif were identified in ORF-2.

Amino Acid Sequence↗

A Feldmannia algal virus has two genome size-classes.

Persistent viruses occur intracellularly in brown algae, specifically the Ectocarpales, and as reported here in the genus Feldmannia. Feldmannia species are small (1 mm-several cm), filamentous forms with single-celled meiotic sporangia that normally produce haploid zoospores. In the isolate reported here, spores were not observed in the sporangia but rather numerous (approximately 10(6) per cell) polyhedral viruses are formed in their place. Two dsDNA genome classes of 158 and 178 kbp, with two restriction site variants of each, are described. The individual abundance of each genome in viral preparations is affected by culture temperature. A cosmid library was used to generate circular restriction enzyme (BamHi, Noti, and Psti) site maps.

DNA, Circular↗

Repetitive DNA insertion in a protein kinase ORF of a latent FSV (Feldmannia sp. virus) genome.

We describe the structure of a Feldmannia sp. virus (FsV) genome integrated in the brown alga, Feldmannia. This integrated FsV genome appears to be permanently inactivated and lost its ability to excise and replicate. Unlike the replicated form of FsV, this integrated FsV genome contains a large (>50 kb) repeat region inserted in a protein kinase open reading frame. While related to the 173-bp repeats previously characterized in the FsV genome (Lee et al., 1995), Southern blot analysis indicates that the repeats in the inactive, integrated FsV genome are distinct from those previously characterized. Fine structural analysis of the repeat-insertion sites in the protein kinase gene indicates that there are 8- and 10-bp palindromic sequences present in multiple locations located near the repeat-insertion site. The translated protein kinase contains all of the catalytic motifs conserved in most serine/threonine protein kinases and a potential autophosphorylation site. This protein kinase gene is expressed as RNA in sporophyte plants where virus production is active but not in gametophyte plants where the virus genome is latent. The structure of the integrated virus genome is discussed.

Amino Acid Sequence↗

Characterization and immunolocalization of major structural proteins in the brown algal virus EsV-1.

The Ectocarpus siliculosus virus (EsV-1) is endemic in all populations of the cosmopolitan filamentous brown alga Ectocarpus siliculosus. EsV-1 has a large circular double-stranded DNA genome of about 320 kilobase pairs, and a complex virion structure with a central nucleoprotein core surrounded by several proteinaceous layers. To investigate the protein composition of the virion, we screened an expression library of EsV-1 with antibodies raised against purified detergent-disrupted viral particles. We isolated several clones encoding novel structural proteins and investigated two of them in detail. These clones encode viral proteins vp55 and vp74. Electron microscopy reveals that vp55 is most likely a component of the surface of the viral core, whereas vp74 may be part of an inner core structure. To initiate a genetic analysis, we sequenced regions of the EsV-1 genome encoding vp55 and vp74 and found several adjacent open reading frames with the potential to code for several interesting viral proteins including a putative calcium-binding protein, a collagen-like protein, and a RING finger protein.

Amino Acid Sequence↗

The brown algal virus EsV-1 particle contains a putative hybrid histidine kinase.

The Ectocarpus siliculosus virus, EsV-1, occurs worldwide in all populations of the filamentous marine brown alga E. siliculosus. We have screened an expression library of EsV-1 restriction fragments and identified a DNA clone with the potential to code for a 52-kDa histidine protein kinase. The derived amino acid sequence includes all homology boxes diagnostic for histidine protein kinases and, in addition, amino acid motifs that are commonly found in response regulators of bacterial two-component signal transduction proteins. Thus, the novel viral protein can be classified as a hybrid histidine protein kinase of a type that has previously been detected in fungi, slime molds, and plants. By using purified antibodies, we found that the protein with its potential kinase activity is located on the outer shell of viral particles. This is the first report on a two-component regulator-like protein in viruses and could provide the basis for speculations with regard to the evolution of EsV-1 and related viruses.

Amino Acid Sequence↗

Secondary structure and phylogeny of the chloroplast 23S rRNA gene from the brown alga Pylaiella littoralis.

The entire nucleotide sequence of a 23S rRNA gene from the brown alga Pylaiella littoralis (L.) Kjellm has been determined. The predicted length of the 23S rRNA is 2948 nucleotides, including the 4.5S rRNA-like region at the 3' end of the molecule. The putative transcript has been folded into a secondary structure by comparison to existing structure models, and the predicted helical regions were inspected by identifying compensatory downstream base changes. The 23S rRNA secondary structure presented here has features that are unique to P. littoralis (no other chromophyte or red algal 23S rRNA sequences are yet available), but has none of the features specific to the chloroplast rRNAs of green plants and green algae. The Pylaiella sequence was aligned with analogous plastidial and eubacterial gene sequences, and the alignment was used to construct a phylogenetic tree. The plastidial sequences formed a coherent cluster closely associated with the 23S rRNA of the cyanobacterium Anacystis nidulans. Within the plastid group, the P. littoralis sequence was most closely related to that of Euglena gracilis confirming earlier analyses based upon 16S rRNA sequences.

Base Sequence↗

Nucleotide sequence and phylogenetic implication of the ATPase subunits beta and epsilon encoded in the chloroplast genome of the brown alga Dictyota dichotoma.

We have cloned and sequenced the genes atpB and atpE, coding for CF1 subunits beta and epsilon, respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3' to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit epsilon and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota epsilon subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74-79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits alpha and beta. A phylogram predicted from F1/CF1-beta subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.

Amino Acid Sequence↗

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm.

The nucleotide sequence and the 5' flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria, the P. littoralis rbcL gene is more closely related to that of a beta-purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996-4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599-608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.

Amino Acid Sequence↗