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Transcription initiation and RNA processing in the mitochondria of the red alga Chondrus crispus: convergence in the evolution of transcription mechanisms in mitochondria.

The mitochondrial DNA (mt DNA) of the red alga Chondrus crispus is shown to be transcribed into two large RNA molecules. These primary transcripts are cleaved once, at the level of a tRNA, then the resulting products are processed via multiple maturation events into either mono- or poly-cistronic RNAs. Transcripts were detected for all genes and open reading frames, except for rps11 and orf172. For both transcription units the initiation of transcription was mapped by in vitro RNA capping and primer extension experiments within inverse repeated sequences at the north pole of the molecule. Consistent with primer extension mapping, putative promoter motifs sharing significant similarities with both chicken and Xenopus mitochondrial promoters were found in the C. crispus mitochondrial genome. Altogether C. crispus mitochondrial DNA appears to be transcribed as animal mtDNA is, suggesting that transcription mechanisms in mitochondria are dependent on the overall organization of the mitochondrial genome irrespective of the eukaryotic phylogeny.

Animals↗

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively. Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.

Amino Acid Sequence↗

Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis.

The three-dimensional structure of the vanadium bromoperoxidase protein from the marine red macroalgae Corallina officinalis has been determined by single isomorphous replacement at 2.3 A resolution. The enzyme subunit is made up of 595 amino acid residues folded into a single alpha+beta domain. There are 12 bromoperoxidase subunits, arranged with 23-point group symmetry. A cavity is formed by the N terminus of each subunit in the centre of the dodecamer. The subunit fold and dimer organisation of the Cor. officinalis vanadium bromoperoxidase are similar to those of the dimeric enzyme from the brown algae Ascophyllum nodosum, with which it shares 33 % sequence identity. The different oligomeric state of the two algal enzymes seems to reflect separate mechanisms of adaptation to harsh environmental conditions and/or to chemically active substrates and products. The residues involved in the vanadate binding are conserved between the two algal bromoperoxidases and the vanadium chloroperoxidase from the fungus Curvularia inaequalis. However, most of the other residues forming the active-site cavity are different in the three enzymes, which reflects differences in the substrate specificity and stereoselectivity of the reaction. A dimer of the Cor. officinalis enzyme partially superimposes with the two-domain monomer of the fungal enzyme.

Amino Acid Sequence↗

Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5-bisphosphate.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO(2) and O(2), respectively, with ribulose-1,5-bisphosphate. Many photosynthetic organisms have form I rubiscos comprised of eight large (L) and eight small (S) subunits. The crystal structure of the complex of activated rubisco from the green alga Chlamydomonas reinhardtii and the reaction intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate (2-CABP) has been solved at 1.84 A resolution (R(cryst) of 15.2 % and R(free) of 18.1 %). The subunit arrangement of Chlamydomonas rubisco is the same as those of the previously solved form I rubiscos. Especially, the present structure is very similar to the activated spinach structure complexed with 2-CABP in the L-subunit folding and active-site conformation, but differs in S-subunit folding. The central insertion of the Chlamydomonas S-subunit forms the longer betaA-betaB loop that protrudes deeper into the solvent channel of rubisco than higher plant, cyanobacterial, and red algal (red-like) betaA-betaB loops. The C-terminal extension of the Chlamydomonas S-subunit does not protrude into the solvent channel, unlike that of the red algal S-subunit, but lies on the protein surface anchored by interactions with the N-terminal region of the S-subunit. Further, the present high-resolution structure has revealed novel post-translational modifications. Residue 1 of the S-subunit is N(alpha)-methylmethionine, residues 104 and 151 of the L-subunit are 4-hydroxyproline, and residues 256 and 369 of the L-subunit are S(gamma)-methylcysteine. Furthermore, the unusual electron density of residue 471 of the L-subunit, which has been deduced to be threonine from the genomic DNA sequence, suggests that the residue is isoleucine produced by RNA editing or O(gamma)-methylthreonine.

Amino Acid Sequence↗

Alternatives to meiosis: the unusual genetics of red algae, microsporidia, and others.

At a reductional division, the two alleles at a heterozygous locus segregate into different daughter nuclei. Reductional divisions are therefore vulnerable to genetic elements that cause one cell to attack its sister. This danger can be circumvented by creating uncertainty about when reduction occurs. In conventional meiosis, this is achieved by a sequence of two divisions with crossing over. Either the first or the second division can be reductional for most loci. In some red algae, reduction appears to be spread over more than two divisions. The novel reduction sequence of microsporidia creates maximal uncertainty about the time of reduction in two divisions without crossing over.

Alleles↗

Model of the carbon concentrating mechanism in chloroplasts of eukaryotic algae.

A generic chloroplast-based model for the carbon concentrating mechanism (CCM) in eukaryotic algae is presented. The fine structure of chloroplasts is represented by separate compartments: marginal and bulk stroma, pyrenoid, girdle lamella, bulk thylakoids, and central lamella traversing the pyrenoid. The roles of the individual structural elements of the chloroplast with respect to the CCM and the effect of carbonic anhydrase activity in various compartments are analysed. Hypothetical HCO(-)(3)transport into the acidic thylakoid lumen is adjusted by imposing an optimization principle: a given [CO(2)] at the site of RuBisCO is achieved with minimum energy costs for the CCM. Our model is highly efficient in terms of saturation of RuBisCO carboxylase activity and the affinity of the chloroplast for CO(2), if either a girdle lamella or a pyrenoid is present. The highest efficiency is achieved with a pyrenoid. A eukaryotic CCM is not necessarily associated with accumulation of dissolved inorganic carbon (DIC) as in cyanobacteria. Chloroplasts are categorized into four types corresponding to morphological characteristics of all major algal classes with regard to the presence of pyrenoids, girdle lamellae, and the distribution of CA activity.

Carbon↗

The distribution of group I introns in lichen algae suggests that lichenization facilitates intron lateral transfer.

The nuclear-encoded small subunit ribosomal DNA gene of many lichen-forming green algae in the genus Trebouxia contains a group I intron at Escherichia coli genic position 1512. We studied the evolutionary history of the 1512 intron in Trebouxia spp. (Trebouxiophyceae) by analyzing intron and "host" cell phylogenies. The host trees were constructed by comparing internal transcribed spacer regions of rDNA. Maximum-likelihood, maximum-parsimony, and distance analyses suggest that the 1512 intron was present in the common ancestor of the green algal classes Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. The 1512 intron, however, was laterally transferred at least three times among later-diverging Trebouxia spp. that form lichen partnerships. Intron secondary structure analyses are consistent with this result. Our results support the hypothesis that lichenization may facilitate 1512 group I intron lateral transfer through the close cell-to-cell contact that occurs between the lichen algal and fungal symbionts in the developing lichen thallus.

Alternative Splicing↗

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↗

Hyaluronan synthesis in virus PBCV-1-infected chlorella-like green algae.

We previously reported that the chlorella virus PBCV-1 genome encodes an authentic, membrane-associated glycosyltransferase, hyaluronan synthase (HAS). Hyaluronan, a linear polysaccharide chain composed of alternating beta1,4-glucuronic acid and beta1, 3-N-acetylglucosamine groups, is present in vertebrates as well as a few pathogenic bacteria. Studies of infected cells show that the transcription of the PBCV-1 has gene begins within 10 min of virus infection and ends at 60-90 min postinfection. The hyaluronan polysaccharide begins to accumulate as hyaluronan-lyase sensitive, hair-like fibers on the outside of the chlorella cell wall by 15-30 min postinfection; by 240 min postinfection, the infected cells are coated with a dense fibrous network. This hyaluronan slightly reduces attachment of a second chlorella virus to the infected algae. An analysis of 41 additional chlorella viruses indicates that many, but not all, produce hyaluronan during infection.

Cell Wall↗

Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo.

Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction. The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5' region, but it also included the conserved regions and putative transmembrane domains. Antibodies against the regions and putative transmembrane domains. Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H. akashiwo cells. Immunostaining of H. akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.

Adenosine Triphosphatases↗

Organization and expression of a phycobiliprotein gene cluster from the unicellular red alga Cyanidium caldarium.

We have sequenced a plastid gene cluster from the unicellular red alga Cyanidium caldarium which is located downstream from the psbA gene and contains, in the following order, genes for a beta-allophycocyanin-like protein (apcB'), a putative 9.5 kDa allophycocyanin linker protein (apcL9.5) and a putative 29 kDa phycocyanin linker protein (cpcL29). The apcB' and apcL9.5 genes are organized in the form of an operon. The cpcL29 gene is transcribed monocistronically from the opposite strand of DNA. Both transcription units are probably terminated at a 25 bp inverted repeat 3 and 5 bp downstream of the stop codons of the apcL9.5 and cpcL29 genes, respectively. The levels of both transcripts are greatly reduced in the dark as is the psbA transcript. Downstream from the phycobiliprotein gene cluster two open reading frames (ORFs) were found which are homologous to ORFs from plastid DNAs and cyanelle DNA of Cyanophora paradoxa. Sequence homologies between genes analysed in this study and corresponding genes from cyanobacteria, chlorophytic plastids and cyanelles point to a large phylogenetic distance between the plastids of Cyanidium and cyanobacteria and other plastid types.

Amino Acid Sequence↗

Physical map and gene organization of the mitochondrial genome from the unicellular green alga Platymonas (Tetraselmis) subcordiformis (Prasinophyceae).

The entire mitochondrial genome (mt genome) of the unicellular green alga Platymonas subcordiformis (synonym Tetraselmis subcordiformis; Prasinophyceae) was cloned and a physical map for the four restriction enzymes Hind III, Eco RI, Bg/II and Xba I was constructed. The mt genome of P. subcordiformis is a 42.8 kb circular molecule, coding for at least 23 genes. Hybridization and sequence analysis revealed the presence of a ca. 1.5 kb inverted repeat on the mt genome of P. subcordiformis. Phylogenetic analyses based on sequences of several coxI genes were carried out. Our data indicate that mitochondria from P. subcordiformis and from land plants form a natural, monophyletic group.

Chlorophyta↗

Structural organization of the chloroplast genome of the chromophytic alga Vaucheria bursata.

The chloroplast genome of the chromophytic alga Vaucheria bursata has been characterized by restriction site and gene mapping analysis. It is represented by a circular molecule 124.6 kb in size. An inverted sequence duplication (IR) not larger than 5.85 kb carries the rRNA genes and separates two single-copy regions of 64.6 kb and 48.3 kb from one another. The Vaucheria plastid genome exists in two equimolar isomers which is due to intramolecular flip-flop recombination within the IR sequences. The coding sites for 21 structural and soluble proteins have been mapped on both single-copy regions using heterologous gene sequences as probes. Although the overall gene order is found to be rearranged when compared with other chromophytic algal and land plant chloroplast genomes, most of the transcriptional units of cyanobacteria and land plant chloroplast genomes appear to be conserved. The phylogenetic implications of these findings are further discussed.

Chloroplasts↗

The mitochondrial genome of an exsymbiotic Chlorella-like green alga.

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F0-F1 ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.

Apoproteins↗

Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position.

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strainspecific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5' non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52-53% for PHYA and 57-59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under Pr conditions, phytochrome mRNA levels were tenfold higher than under Pfr conditions.

Amino Acid Sequence↗

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria.

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic alpha-helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F1 beta subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.

Animals↗

Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum.

The phytochrome photoreceptor in the green alga Mesotaenium caldariorum is encoded by a small family of highly related genes. DNA sequence analysis of two of the algal phytochrome genes indicates an atypical gene structure with numerous long introns. The two genes, termed mesphy1a and mesphy1b, encode polypeptides which differ by one amino acid in the region of overlap that was sequenced. RT-PCR studies have established the intron-exon junctions of both genes and show that both are expressed. RNA blot analysis indicates a single transcript of ca. 4.1 kb in length. The deduced amino acid sequence of the mesphy1b gene reveals that the photoreceptor consists of 1142 amino acids, with an overall structure similar to other phytochromes. Phylogenetic analyses indicate that the algal phytochrome falls into a distinct subfamily with other lower plant phytochromes. Profile analysis of an internal repeat found within the central hinge region of the phytochrome polypeptide indicates an evolutionary relatedness to the photoactive yellow protein from the purple bacterium Ectothiorhodospira halophila, to several bacterial sensor kinase family members, and to a family of eukaryotic regulatory proteins which includes the period clock (per) and single-minded (sim) gene products of Drosophila. Since mutations which alter phytochrome activity cluster within the region delimited by these direct repeats (P.H. Quail et al., Science 268 (1995): 675-680), this conserved motif may play an important role in the signal transducing function of these disparate protein families.

Amino Acid Sequence↗