Morphology, taxonomy and mating‑type loci in natural populations of Volvox carteri in Taiwan

Background: Volvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the production of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a \u3e 1 Mbp linear chromosome. Fifteen sexspecific genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identified in natural populations of this species. Results: During a recent fieldwork in Taiwan, we encountered natural populations of V. carteri that had not previously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identified as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to \u3e 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specific genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specific gene HMG1f was not amplified by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain. Conclusions: Male spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii

葉緑体核様体をコンパクトに折りたたむ「DNAクリップ」の発見 --ミトコンドリアとも共通する普遍的なしくみの解明--. 京都大学プレスリリース. 2021-05-12.Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA–protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9–mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids

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

<p>Abstract</p> <p>Background</p> <p>All 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 <it>Cyanidioschyzon merolae </it>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 <it>C. merolae </it>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.</p> <p>Results</p> <p>Our 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 <it>C. merolae </it>genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.</p> <p>Conclusion</p> <p>By virtue of these attributes and others that we had discovered previously, <it>C. merolae </it>appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of <it>C. merolae </it>are extremely useful for further studies of eukaryotic cells.</p

An mt(+) gamete-specific nuclease that targets mt(−) chloroplasts during sexual reproduction in C. reinhardtii

Although the active digestion of mating-type minus (mt(−)) chloroplast DNA (cpDNA) in young zygotes is considered to be the basis for the uniparental inheritance of cpDNA in Chlamydomonas reinhardtii, little is known about the underlying molecular mechanism. One model of active digestion proposes that nucleases are either synthesized or activated to digest mt(−) cpDNA. We used a native-PAGE/in gelo assay to investigate nuclease activities in chloroplasts from young zygotes, and identified a novel Ca(2+)-dependent nuclease activity. The timing of activation (∼60–90 min after mating) and the localization of the nuclease activity (in mt(−) chloroplasts) coincided with the active digestion of mt(−) cpDNA. Furthermore, the activity of the nuclease was coregulated with the maturation of mating-type plus (mt(+)) gametes, which would enable the efficient digestion of mt(−) cpDNA. Based on these observations, we propose that the nuclease (designated as Mt(+)-specific DNase, MDN) is a developmentally controlled nuclease that is activated in mt(+) gametes and participates in the destruction of mt(−) cpDNA in young zygotes, thereby ensuring uniparental inheritance of chloroplast traits

Basal transcriptional activities of the upstream region of <i>CMJ101C orf</i> at lower temperatures in the stable transformants.

<p>A single copy of <i>sfGFP orf</i> fused with the 200 (S-200), 250 (S-250) or 345 (S-345)-bp upstream region of <i>CMJ101 orf</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111261#pone-0111261-g003" target="_blank">Figure 3A</a>) was integrated into the <i>C. merolae</i> genomic-neutral locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111261#pone.0111261-Fujiwara1" target="_blank">[11]</a>. The stable transformants were cultured at 35∼45°C for 1 day. (A) Semi-quantitative RT-PCR showing the level of the <i>sfGFP</i> mRNA. <i>CMJ101C</i> and <i>TIM13</i> (<i>CMB148C</i>) were used as a positive and quantitative control, respectively. (B) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm.</p

Duration-dependent effect of 50°C heat shock on the mRNA and protein levels in the stable transformants.

<p>The stable S-200 transformant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111261#pone-0111261-g004" target="_blank">Figure 4</a>) cultured at 34°C was shifted to 50°C and cultured for 120 min or 24 h. (A) Semi-quantitative RT-PCR showing the <i>GFP</i> mRNA level. <i>TIM13</i> (<i>CMB148C</i>) was used as a quantitative control. (B) Immunoblotting with the anti-GFP antibody showing the GFP protein level. CBB staining of the PVDF membrane is shown as a loading control. (C) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm. (D) Semi-quantitative RT-PCR showing the <i>GFP</i> mRNA level up to 24 h at 50°C. <i>TIM13</i> (<i>CMB148C</i>) was used as a quantitative control.</p