Respiratory and desiccation constraints during encapsulated intertidal development of the marine gastropod Acanthina monodon.

Acanthina monodon commonly deposits its egg capsules in the intertidal zone. Capsule aerial exposure during low-tide can impact oxygen consumption rates (OCR) of embryos and intracapsular oxygen availability, and expose embryos to desiccation. OCR increased as embryonic development progressed, and was greater when capsules were submerged in seawater than when exposed to air. Oxygen available within the capsule was always less than that available in the immediate external environment, whether capsules were immersed or exposed. The highest internal oxygen concentrations were recorded during periods of air exposure for embryos in more advanced development stages. When exposed to air, capsules lost water the fastest when they contained early embryos, and suffered the highest mortalities following exposure. Collectively, these data suggest that, although encapsulation helps the embryos to develop across wildly fluctuating environmental conditions, the amount of stress the embryos experience will vary depending on their exact positioning within the intertidal zone.

The closest unicellular relatives of animals.

Molecular phylogenies support a common ancestry between animals (Metazoa) and Fungi, but the evolutionary descent of the Metazoa from single-celled eukaryotes (protists) and the nature and taxonomic affiliation of these ancestral protists remain elusive. We addressed this question by sequencing complete mitochondrial genomes from taxonomically diverse protists to generate a large body of molecular data for phylogenetic analyses. Trees inferred from multiple concatenated mitochondrial protein sequences demonstrate that animals are specifically affiliated with two morphologically dissimilar unicellular protist taxa: Monosiga brevicollis (Choanoflagellata), a flagellate, and Amoebidium parasiticum (Ichthyosporea), a fungus-like organism. Statistical evaluation of competing evolutionary hypotheses confirms beyond a doubt that Choanoflagellata and multicellular animals share a close sister group relationship, originally proposed more than a century ago on morphological grounds. For the first time, our trees convincingly resolve the currently controversial phylogenetic position of the Ichthyosporea, which the trees place basal to Choanoflagellata and Metazoa but after the divergence of Fungi. Considering these results, we propose the new taxonomic group Holozoa, comprising Ichthyosporea, Choanoflagellata, and Metazoa. Our findings provide insight into the nature of the animal ancestor and have broad implications for our understanding of the evolutionary transition from unicellular protists to multicellular animals.

A complex organization of the gene encoding cytochrome oxidase subunit 1 in the mitochondrial genome of the dinoflagellate, Crypthecodinium cohnii: homologous recombination generates two different cox1 open reading frames.

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3'-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15-22 base pairs) has been identified at the 5'-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists.

The origin and early evolution of mitochondria.

Complete sequences of numerous mitochondrial, many prokaryotic, and several nuclear genomes are now available. These data confirm that the mitochondrial genome originated from a eubacterial (specifically alpha-proteobacterial) ancestor but raise questions about the evolutionary antecedents of the mitochondrial proteome.

Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis.

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

Identification and structural characterization of nucleus-encoded transfer RNAs imported into wheat mitochondria.

Despite its large size (200-2400 kilobase pairs), the mitochondrial genome of angiosperms does not encode the minimal set of tRNAs required to support mitochondrial protein synthesis. Here we report the identification of cytosolic-like tRNAs in wheat mitochondria using a method involving quantitative hybridization to distinguish among three tRNA classes: (i) those encoded by mitochondrial DNA (mtDNA) and localized in mitochondria, (ii) those encoded by nuclear DNA and located in the cytosol, and (iii) those encoded by nuclear DNA and found in both the cytosol and mitochondria. The latter class comprises tRNA species that are considered to be imported into mitochondria to compensate for the deficiency of mtDNA-encoded tRNAs. In a comprehensive survey of the wheat mitochondrial tRNA population, we identified 14 such imported tRNAs, the structural characterization of which is presented here. These imported tRNAs complement 16 mtDNA-encoded tRNAs, for a total of at least 30 distinct tRNA species in wheat mitochondria. Considering differences in the set of mtDNA-encoded and imported tRNAs in the mitochondria of various land plants, the import system must be able to adapt relatively rapidly over evolutionary time with regard to the particular cytosolic-like tRNAs that are brought into mitochondria.

The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Pseudouridine in RNA: what, where, how, and why.

Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (psi) was the first modified nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a "fifth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of psi in polyribonucleotides, the biochemical mechanism of U-->psi isomerization in RNA, and the role of modification enzymes (psi synthases) and box H/ACA snoRNAs, a class of eukaryotic small nucleolar RNAs, in the site-specific biosynthesis of psi. Through its unique ability to coordinate a structural water molecule via its free N1-H, psi exerts a subtle but significant "rigidifying" influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most (but perhaps not all) of the psi residues in RNA. Certain genetic mutants lacking specific psi residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. In particular, normal growth is severely compromised in an Escherichia coli mutant deficient in a pseudouridine synthase responsible for the formation of three closely spaced psi residues in the mRNA decoding region of the 23S rRNA. Such studies demonstrate that pseudouridylation of RNA confers an important selective advantage in a natural biological context.

The complete mitochondrial DNA sequence of Scenedesmus obliquus reflects an intermediate stage in the evolution of the green algal mitochondrial genome.

Two distinct mitochondrial genome types have been described among the green algal lineages investigated to date: a reduced-derived, Chlamydomonas-like type and an ancestral, Prototheca-like type. To determine if this unexpected dichotomy is real or is due to insufficient or biased sampling and to define trends in the evolution of the green algal mitochondrial genome, we sequenced and analyzed the mitochondrial DNA (mtDNA) of Scenedesmus obliquus. This genome is 42,919 bp in size and encodes 42 conserved genes (i.e., large and small subunit rRNA genes, 27 tRNA and 13 respiratory protein-coding genes), four additional free-standing open reading frames with no known homologs, and an intronic reading frame with endonuclease/maturase similarity. No 5S rRNA or ribosomal protein-coding genes have been identified in Scenedesmus mtDNA. The standard protein-coding genes feature a deviant genetic code characterized by the use of UAG (normally a stop codon) to specify leucine, and the unprecedented use of UCA (normally a serine codon) as a signal for termination of translation. The mitochondrial genome of Scenedesmus combines features of both green algal mitochondrial genome types: the presence of a more complex set of protein-coding and tRNA genes is shared with the ancestral type, whereas the lack of 5S rRNA and ribosomal protein-coding genes as well as the presence of fragmented and scrambled rRNA genes are shared with the reduced-derived type of mitochondrial genome organization. Furthermore, the gene content and the fragmentation pattern of the rRNA genes suggest that this genome represents an intermediate stage in the evolutionary process of mitochondrial genome streamlining in green algae.

Evolutionary appearance of genes encoding proteins associated with box H/ACA snoRNAs: cbf5p in Euglena gracilis, an early diverging eukaryote, and candidate Gar1p and Nop10p homologs in archaebacteria.

A reverse transcription-polymerase chain reaction (RT-PCR) approach was used to clone a cDNA encoding the Euglena gracilis homolog of yeast Cbf5p, a protein component of the box H/ACA class of snoRNPs that mediate pseudouridine formation in eukaryotic rRNA. Cbf5p is a putative pseudouridine synthase, and the Euglena homolog is the first full-length Cbf5p sequence to be reported for an early diverging unicellular eukaryote (protist). Phylogenetic analysis of putative pseudouridine synthase sequences confirms that archaebacterial and eukaryotic (including Euglena) Cbf5p proteins are specifically related and are distinct from the TruB/Pus4p clade that is responsible for formation of pseudouridine at position 55 in eubacterial (TruB) and eukaryotic (Pus4p) tRNAs. Using a bioinformatics approach, we also identified archaebacterial genes encoding candidate homologs of yeast Gar1p and Nop10p, two additional proteins known to be associated with eukaryotic box H/ACA snoRNPs. These observations raise the possibility that pseudouridine formation in archaebacterial rRNA may be dependent on analogs of the eukaryotic box H/ACA snoRNPs, whose evolutionary origin may therefore predate the split between Archaea (archaebacteria) and Eucarya (eukaryotes). Database searches further revealed, in archaebacterial and some eukaryotic genomes, two previously unrecognized groups of genes (here designated 'PsuX' and 'PsuY') distantly related to the Cbf5p/TruB gene family.

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA.

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

Expression of mitochondrial protein-coding genes in Tetrahymena pyriformis.

In the ciliate protozoon, Tetrahymena pyriformis, mitochondrial protein-coding genes are highly divergent in sequence, and in a number of cases they lack AUG initiation codons. We asked whether RNA editing might be acting to generate protein sequences that are more conventional than those inferred from the corresponding gene sequences, and/or to create standard AUG initiation codons where these are absent. However, comparison of genomic and cDNA sequences (the latter generated by reverse transcriptase sequencing of T. pyriformis mitochondrial mRNAs) yielded no evidence of mitochondrial RNA editing in this organism. To delineate the 5' ends of mitochondrial protein-coding transcripts, primer extension experiments were conducted. In all cases, 5' termini were found to map within a few nucleotides of potential initiation codons, indicating that T. pyriformis mitochondrial mRNAs have little or no 5' untranslated leader sequence. The pattern of strong primer extension stops suggested that both standard (AUG) and non-standard (AUU, AUA, GUG, UUG) initiation codons are utilized by the Tetrahymena mitochondrial translation system. We also investigated expression of the nad1 gene, which in both T. pyriformis and Paramecium aurelia is split into two portions that are encoded by and transcribed from different DNA strands. Northern hybridization analysis showed that the corresponding transcripts are not trans-spliced, implying that separate N-terminal and C-terminal portions of Nad1 are made in this system. Finally, in a search for primary transcripts, we isolated from a T. pyriformis mitochondrial fraction several small RNAs that were reproducibly labeled by incubation in the presence of [alpha-(32)P]GTP and guanylyltransferase. Partial sequence information revealed that none of these cappable RNAs is encoded in the T. pyriformis mitochondrial genome.

Structural conservation and variation among U5 small nuclear RNAs from trypanosomatid protozoa.

U5 snRNAs in trypanosomatid protozoa do not contain the trimethylguanosine cap structures that are often targeted in snRNA isolation procedures. As a result, the trypanosomatids are not well represented in the database of available U5 snRNA sequences. We have isolated and determined the sequence of the U5 snRNA from Crithidia fasciculata. Comparison with previously published trypanosomatid U5 snRNA sequences allows us to deduce the pattern of structural conservation and variation among these very divergent snRNA molecules.

Evolution of organellar genomes.

Accumulating molecular data, particularly complete organellar genome sequences, continue to advance our understanding of the evolution of mitochondrial and chloroplast DNAs. Although the notion of a single primary origin for each organelle has been reinforced, new models have been proposed that tie the acquisition of mitochondria more closely to the origin of the eukaryotic cell per se than is implied by classic endosymbiont theory. The form and content of the ancestral proto-mitochondrial and proto-chloroplast genomes are becoming clearer but unusual patterns of organellar genome structure and organization continue to be discovered. The 'single-gene circle' arrangement recently reported for dinoflagellate chloroplast genomes is a notable example of a highly derived organellar genome.

Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria.

To investigate the transcriptional apparatus in wheat mitochondria, mitochondrial extracts were subjected to column chromatography and protein fractions were analyzed by in vitro transcription and mobility shift assays. Fractions eluting from DEAE-Sephacel between 0.2 and 0.3 M KCl displayed DNA-binding activity and supported specific transcription initiated from a wheat cox2 promoter. The active DEAE-Sephacel pool was further resolved by chromatography on phosphocellulose. Fractions that exhibited DNA-binding activity and that stimulated both specific and nonspecific transcription in vitro were highly enriched in a 63-kDa protein (p63). From peptide sequence obtained from purified p63, a cDNA encoding the protein was assembled. The predicted amino acid sequence (612 amino acid residues, 69 kDa) contains a basic N-terminal targeting sequence expected to direct transport of the protein into mitochondria. The p63 sequence also features an acidic domain characteristic of transcriptional activation factors, as well as sequence blocks displaying limited similarity to positionally equivalent regions in sigma factors from eubacteria related to mitochondria. Recombinant p63 possesses DNA-binding activity, exhibiting an affinity for the core cox2 promoter element and upstream regions in gel shift assays and having the ability to enhance specific transcription in vitro. Transcripts encoding p63 are expressed at an early stage in the germination of isolated wheat embryos, in a temporal pattern parallelling that of newly synthesized precursors of cox2, a mitochondrial gene. Taken together, these data suggest a role for p63 in transcription in wheat mitochondria.

Spliced leader-associated RNA from Crithidia fasciculata contains a structure resembling stem/loop II of U1 snRNA.

In contrast to earlier proposals, recent evidence suggests that trans-spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23,691-23,694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader-associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculata and propose that it may contribute a U1 snRNA-like stem/loop II to the trans-spliceosome.

Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae.

The mitochondrial DNA (mtDNA) of Porphyra purpurea, a circular-mapping genome of 36,753 bp, has been completely sequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animals and fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins and subunits of succinate:ubiquinone oxidoreductase. The mitochondrial large subunit rRNA gene contains two group II introns that are extraordinarily similar to those found in the cyanobacterium Calothrix sp, suggesting a recent lateral intron transfer between a bacterial and a mitochondrial genome. Notable features of P. purpurea mtDNA include the presence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rearrangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis of red algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs are unusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share a common ancestry with green algae and plants.

The complete mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor. Two radically different evolutionary patterns within green algae.

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.