Identification of phase relative genes in tetrasporophytes and female gametophytes of Gracilaria/Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta)

Genes differentially expressed between tetrasporophytes and female gametophytes of Gracilaria/Gracilariopsis lemaneiformis were isolated by suppression subtractive hybridization (SSH) and screened by dot-blot macro-arrays. Different expression profiles of selected clones based on the results of dot-blot macro-arrays were verified using virtual Northern blots. Totally, 14 phase relative cDNAs had been isolated and sequence identified. Among them, seven cDNAs were respectively homologous to crucial metabolic enzymes, Rab GTPase, RP42 homolog, and two hypothetical proteins, while the rest did not have significant hits in the databases examined. The results of virtual Northern blots revealed that 11 cDNAs were differentially expressed between the two samples, including 7 genes up-regulated in tetrasporophytes, 1 expressed exclusively in tetrasporophytes and 3 up-regulated in female gametophytes. By densitometric analysis relative to GAPDH, 8 cDNAs increased 1.3-4.2 fold and 3 decreased about 0.4-0.7 fold in tetrasporophytes compared to female gametophytes. The present study provides the first insight into genes that may involve in phase differentiation in G. lemaneiformis

Setting the basis for transient DNA transformation and transformant selection in the red macroalga Gracilariopsis lemaneiformis

Gracilariopsis lemaneiformis (Gp. lemaneiformis) is an economically important agar-producing red alga applicable in the food and cosmetic industries. The genetic knowledge of this species is, however, limited, and genetic tools for studying and engineering it are lacking. This has limited the understanding of its developmental genetics and hindered the development of new strains, and developing genetic tools would allow to tackle these problems. Here, transient DNA transformation via microparticle bombardment is reported for the first time in this species, as well as efficient exogenous gene expression driven by the CaMV35S promoter, the endogenous GlAct1 promoter, and the Pyropia yezoensis PyAct1 promoter in the transformed branches. Moreover, the Blue Fluorescent Protein (BFP) is demonstrated to be a suitable reporter gene for studies in Gp. lemaneiformis. Screening of antibiotic sensitivity is needed for the development of transient DNA transformation, and selection of transformants is also reported in the alga. Hygromycin B (Hyg) is determined to be the most effective antibiotic for Gp. lemaneiformis selection. The Hyg resistance gene driven by the CaMV35S promoter is shown to confer resistance to Hyg at a concentration of 1 mg.ml-1, but no transformed individual could be regenerated so far. These results are promising for future refining of the experimental conditions, for instance, by using different promoters and developing techniques for facilitating the penetration of the DNA in the cells

Boosting Heterosubtypic Neutralization Antibodies in Recipients of 2009 Pandemic H1N1 Influenza Vaccine

Our data demonstrated that the inoculation with vaccine derived from the 2009 pandemic influenza raised vigorous neutralization antibodies against both cognate H1N1 and heterotypic influenza viruses. This observation has important implication for vaccine development

Macropinocytosis in Gracilariopsis lemaneiformis (Rhodophyta)

Macropinocytosis is an endocytic process that plays an important role in animal development and disease occurrence but until now has been rarely reported in organisms with cell walls. We investigated the properties of endocytosis in a red alga, Gracilariopsis lemaneiformis. The cells non-selectively internalized extracellular fluid into large-scale endocytic vesicles (1.94 ± 0.51 μm), and this process could be inhibited by 5-(N-ethyl-N-isopropyl) amiloride, an macropinocytosis inhibitor. Moreover, endocytosis was driven by F-actin, which promotes formation of ruffles and cups from the cell surface and facilitates formation of endocytotic vesicles. After vesicle formation, endocytic vesicles could be acidified and acquire digestive function. These results indicated macropinocytosis in G. lemaneiformis. Abundant phosphatidylinositol kinase and small GTPase encoding genes were found in the genome of this alga, while PI3K, Ras, and Rab5, the important participators of traditional macropinocytosis, seem to be lacked. Such findings provide a new insight into endocytosis in organisms with cell walls and facilitate further research into the core regulatory mechanisms and evolution of macropinocytosis

Calmodulin and Its Interactive Proteins Participate in Regulating the Explosive Growth of <i>Alexandrium pacificum </i>(Dinoflagellate)

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum

Identification, characterization and expression profiles of E2 and E3 gene superfamilies during the development of tetrasporophytes in Gracilariopsis lemaneiformis (Rhodophyta)

Abstract E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases play important roles in the growth and development of plants and animals. To date, the systematic analysis of E2 and E3 genes in Rhodophyta is limited. In this study, 14 E2 genes and 51 E3 genes were identified in Gracilariopsis lemaneiformis, an economically important red alga. E2 genes were classified into four classes according to the structure of the conserved domain, UBC. E3 genes were classified into 12 subfamilies according to individual conserved domains. A phylogenetic tree of seven algae species showed that functional differentiation of RING-type E3s was the highest, and the similarity between orthologous genes was high except in Chlamydomonas reinhardtii and Chara braunii. RNA-seq data analysis showed significant differential expression levels of E2 and E3 genes under the life stages of tetraspore formation and release, especially GlUBCN and GlAPC3. According to GO and KEGG analysis of two transcriptomes, GlUBCN and GlAPC3 were involved in ubiquitin-mediated proteolysis, and other subunits of the anaphase promoting complex or cyclosome (APC/C) and its activators GlCDC20 and GlCDH1 were also enriched into this process. The CDH1 and CDC20 in 981 were down-regulated during tetraspores formation and release, with the down-regulation of CDH1 being particularly significant; CDH1 and CDC20 in WLP-1, ZC, and WT were up-regulated during tetraspores formation and release, with CDC20 being more significantly up-regulated. Therefore, GlCDH1, rather than GlCDC20, in ‘981’ might play the leading role in the activation of the APC/C, and GlCDC20 might play the leading role rather than GlCDH1 in strains WLP-1, ZC and wild type. The low fertility of cultivar 981 might be highly correlated with the inactivity of activators CDH1 and CDC20. This study provided a basic and comprehensive understanding of characteristic of E2 and E3 genes in Gp. lemaneiformis and set a foundation for further understanding of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligase in regulating tetrasporophytes development of Gp. lemaneiformis

Distinctive Nuclear Features of Dinoflagellates with A Particular Focus on Histone and Histone-Replacement Proteins

Dinoflagellates are important eukaryotic microorganisms that play critical roles as producers and grazers, and cause harmful algal blooms. The unusual nuclei of dinoflagellates &#8220;dinokaryon&#8222; have led researchers to investigate their enigmatic nuclear features. Their nuclei are unusual in terms of their permanently condensed nucleosome-less chromatin, immense genome, low protein to DNA ratio, guanine-cytosine rich methylated DNA, and unique mitosis process. Furthermore, dinoflagellates are the only known group of eukaryotes that apparently lack histone proteins. Over the course of evolution, dinoflagellates have recruited other proteins, e.g., histone-like proteins (HLPs), from bacteria and dinoflagellates/viral nucleoproteins (DVNPs) from viruses as histone substitutes. Expression diversity of these nucleoproteins has greatly influenced the chromatin structure and gene expression regulation in dinoflagellates. Histone replacement proteins (HLPs and DVNPs) are hypothesized to perform a few similar roles as histone proteins do in other eukaryotes, i.e., gene expression regulation and repairing DNA. However, their role in bulk packaging of DNA is not significant as low amounts of proteins are associated with the gigantic genome. This review intends to summarize the discoveries encompassing unique nuclear features of dinoflagellates, particularly focusing on histone and histone replacement proteins. In addition, a comprehensive view of the evolution of dinoflagellate nuclei is presented