Merolae Genome Research Articles

Study of Dispersed Repeats in the Cyanidioschyzon merolae Genome.

In this study, we applied the iterative procedure (IP) method to search for families of highly diverged dispersed repeats in the genome of Cyanidioschyzon merolae, which contains over 16 million bases. The algorithm included the construction of position weight matrices (PWMs) for repeat families and the identification of more dispersed repeats based on the PWMs using dynamic programming. The results showed that the C. merolae genome contained 20 repeat families comprising a total of 33,938 dispersed repeats, which is significantly more than has been previously found using other methods. The repeats varied in length from 108 to 600 bp (522.54 bp in average) and occupied more than 72% of the C. merolae genome, whereas previously identified repeats, including tandem repeats, have been shown to constitute only about 28%. The high genomic content of dispersed repeats and their location in the coding regions suggest a significant role in the regulation of the functional activity of the genome.

Cis-acting elements involved in the G2/M-phase-specific transcription of the cyclin B gene in the unicellular alga Cyanidioschyzon merolae.

M-specific activator (MSA) cis-acting elements have been determined to be involved in the regulation of G2/M-phase-specific transcription in spermatophytes. In this study, the involvement of MSA-core elements in G2/M-phase-specific transcription was examined in the unicellular red alga Cyanidioschyzon merolae. In the C. merolae genome, MSA-core elements do not accumulate specifically in the upstream of mitosis-specific transcriptional genes. Mutations of the four MSA-core elements of the cyclin B gene, which encodes a central factor of the G2-to-M-phase transition, have resulted in the abolishment of transcription or permission of transcription even in the G1 phase. These results suggest that all four MSA-core elements located in the upstream region of cyclin B are involved in G2/M-phase-specific transcription in C. merolae; however, the nature of the involvement of MSA-core elements in G2/M-phase-specific transcription differed among the four elements. Thus, MSA-core-element-mediated G2/M-phase-specific transcription in C. merolae seems to be regulated by a complex mechanism.

Subcellular distribution of central carbohydrate metabolism pathways in the red alga Cyanidioschyzon merolae.

Comprehensive subcellular localization analysis revealed that the subcellular distribution of carbohydrate metabolic pathways in the red alga Cyanidioschyzon is essentially identical with that in Arabidopsis , except the lack of transaldolase. In plants, the glycolysis and oxidative pentose phosphate pathways (oxPPP) are located in both cytosol and plastids. However, in algae, particularly red algae, the subcellular localization of enzymes involved in carbon metabolism is unclear. Here, we identified and examined the localization of enzymes related to glycolysis, oxPPP, and tricarboxylic acid (TCA) and Calvin-Benson cycles in the red alga Cyanidioschyzon merolae. A gene encoding transaldolase of the oxPPP was not found in the C. merolae genome, and no transaldolase activity was detected in cellular extracts. The subcellular localization of 65 carbon metabolic enzymes tagged with green fluorescent protein or hemagglutinin was examined in C. merolae cells. As expected, TCA and Calvin-Benson cycle enzymes were localized to mitochondria and plastids, respectively. The analyses also revealed that the cytosol contains the entire glycolytic pathway and partial oxPPP, whereas the plastid contains a partial glycolytic pathway and complete oxPPP, with the exception of transaldolase. Together, these results suggest that the subcellular distribution of carbohydrate metabolic pathways in C. merolae is essentially identical with that reported in the photosynthetic tissue of Arabidopsis thaliana; however, it appears that substrates typically utilized by transaldolase are consumed by glycolytic enzymes in the plastidic oxPPP of C. merolae.

Proteomic comparison between interphase and metaphase of isolated chloroplasts of Cyanidioschyzon merolae (Cyanidiophyceae, Rhodophyta)

SUMMARYWith the completion of the Cyanidioschyzon merolae genome project, detailed analysis of organelle proteins with mass spectrometry has now become possible. Chloroplasts of the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei et Varano (Rhodophyta, Cyanidiophyceae) were isolated from synchronized culture and the chloroplast lysates of both interphase and metaphase cells were prepared and subjected to 2D‐polyacrylamide gel electrophoresis. A total of 355 spots (170 identical spots) were recognized and quantified and then analyzed using mass spectrometry. A total of 105 proteins were identified, including 18 proteins for posttranslational functions, 17 photosynthesis‐related proteins, 10 carbohydrate‐related proteins, 15 proteins of unknown functions, and eight proteins predicted to be contaminated from other organelles. On the basis of spot quantity, photosynthesis‐related proteins were most dominant (45.3% in interphase and 56.4% in metaphase). In particular, the proteins forming phycobilisomal complexes were abundant. Comparison of interphase with metaphase revealed that CMG086C (aminomethyltransferase) notably increased in interphase. CMN235C (similar to chlorophyll a/b‐binding protein, CP24) increased in metaphase in agreement with a previously performed microarray analysis. Both CMQ295C (cell division protein FtsH) and CMS004C (plastid division protein FtsZ) increased in interphase. Seven proteins were detected to be interphase‐specific, and 12 proteins were metaphase‐specific. Proteins of unknown functions were poorly characterized by homology search, although thioredoxin‐like domains were predicted in several proteins.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Centromere structures highlighted by the 100%-complete Cyanidioschyzon merolae Genome

Centromere dynamics are largely unknown in lower plants (algae). We have recently identified the centromere-specific histone H3 variant (CENH3) and clarified the dynamic centromere rearrangement at mitosis in the primitive red alga Cyanidioschyzon merolae. We also showed that the CENH3-containing nucleosomes constituted the kinetochore closely interacting with the nuclear envelope. CENH3 visualization during the whole cell cycle suggests that C. merolae centromeres are monocentric and confined to specific loci. We completed 100% no-gap telomere-to-telomere sequencing of the C. merolae genome. Interestingly, a single A+T-rich region has been identified on each fully sequenced chromosome. No centromere-like A+T rich repetitive sequence have been found within these regions, implying that the C. merolae centromeres may be ‘point’ centromeres, or be comprised of non-repetitive heterogeneous DNA sequences.

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae

BackgroundAll previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.ResultsOur present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.ConclusionBy virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae.

Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae (CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of alpha-Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results of Western analysis suggest that CmFtsZ1 is localized in mitochondria.

Delta-9 fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae

A delta-9 fatty acid desaturase gene, homologous to animal and fungal acyl-coenzyme A (CoA) desaturases, was isolated from the red alga Cyanidioschyzon merolae using a degenerate PCR strategy. This gene, designated as CmFAD9, has no intron. The encoded delta-9 fatty acid desaturase (CmFad9p) consists of 476 amino acids and has an estimated molecular mass of 55.4 kDa. CmFad9p is a unique delta-9 fatty acid desaturase among plants, in that it is fused with the cytochrome b5 domain at its carboxyl terminus. This is characteristic of yeast acyl-CoA desaturase. Genomic Southern hybridization suggested that the C. merolae genome contains a single gene for delta-9 fatty acid desaturase of the animal and fungal type. Southern hybridization combined with pulsed-field gel electrophoresis revealed that CmFAD9 is probably located on chromosome XI of the 17 C. merolae chromosomes. A 1.6-kb product of this gene was transcribed throughout a light/dark synchronization culture. The discovery of CmFAD9 indicates the existence of a novel type of plant delta-9 fatty acid desaturase that may function in the endoplasmic reticulum, but not in the plastid.

Isolation, characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae.

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has been suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42 003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.