Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

REEXAMINATION OF PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA): AN ANALYSIS OF atpB AND rbcL GENE SEQUENCES

The chloroplast-encoded atp B gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbc L gene sequence data.1128 base pairs in the coding regions of the atp B gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera— Eudorina, Pleodorina, and Volvox, excluding the section Volvox (= Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbc L gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbc L gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbc L gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atp B gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atp B and rbc L genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.     

Molecular phylogeny and evolution of the plastid and nuclear encoded cbbX genes in the unicellular red alga Cyanidioschyzon merolae

The cbbX gene is generally encoded in proteobacterial genomes and red-algal plastid genomes. In this study, we found two distinct cbbX genes of Cyanidioschyzon merolae, a unicellular red alga, one encoded in the plastid genome and the other encoded in the cell nucleus. The phylogenetic tree inferred from cbbX genes and strongly conserved gene organization (rbcLS-cbbX) suggests that the plastid-encoded cbbX gene of C. merolae came from an ancestral proteobacterium by horizontal gene transfer. On the other hand, the nuclear-encoded cbbX gene of C. merolae was classified in another cluster together with the nucleomorph-encoded cbbX gene of Guillardia theta. Furthermore, expression of the two cbbX genes were regulated differently in response to extracellular CO(2) concentration. Our results imply that cbbX gene in the plastid genome was copied and transferred to the cell nucleus after horizontal gene transfer of RuBisCo operon from ancestral beta-proteobacteria at comparatively early stage, and that each cbbX evolved in different ways.     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

Expression and localization of two SecA homologs in the unicellular red alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for Sec translocase that participates in bacterial protein export and thylakoidal import in plants. We have reported that Cyanidioschyzon merolae, a unicellular red alga, possesses a nuclear-encoded secA(nuc) and a plastid-encoded secA(pt) gene. In this study we found that the amount of SecA(nuc) protein almost quadrupled at high temperature, whereas that of the SecA(pt) protein increased far less. We were also able to determine the localization of both SecAs to the chloroplast by immunofluorescence and immunoelectron microscopy. We suggest that SecA(nuc) has an important role in the chloroplast at high temperatures.     

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.     

Isolation of the Chloroplast DNA and the Sequence of the trnK Gene from Cyanidium caldarium Strain RK-1

Nuclear, mitochondrial and chloroplast DNAs were first isolatedfrom Cyanidium caldarium strain RK-1, a eukaryote with one ofthe smallest amounts of nuclear DNA known. The chloroplast DNAincluded a trnK sequence that showed a high degree of homologyto those of chloroplasts of land plants but lacked a typicalintron. (Received December 16, 1991; Accepted May 14, 1992)     

Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae)

Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PlC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PlC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PlC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PlC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.