A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (<Emphasis Type="Italic">GAPDH</Emphasis>) Promoter for Expressing Transgenes in the Halotolerant Alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.     

Expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit in transgenic watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. G_M1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.     

Doxorubicin and menadione decrease cell proliferation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by different mechanisms

The aim of this study was to examine the effect of doxorubicin and menadione on cell proliferation, cell cycle, glutathione concentration, the expression of ribonucleotide reductase, and the Yap1 dependent redox-sensitive pathway in Saccharomyces cerevisiae as a eukaryote cell model. Our data showed that menadione induced cell-cycle arrest in the G₁ phase, decreased intracellular GSH and GSSG concentrations, dose-dependently increased expression of ribonucleotide reductase and the activity of Yap1 pathway. Doxorubicin induced the cell-cycle arrest in G₁ and S phases, increased the GSH and GSSG concentration and the expression of ribonucleotide reductase, and modulated the Yap-dependent pathway activity.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Development of an immunofluorescence technique for detecting <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis> Lu

Prorocentrum donghaiense Lu is a key harmful algal bloom (HAB) species which is widespread along the China Sea coast. In this study, P. donghaiense-specific antiserum was developed, and the detection method, based on immunofluorescence (IF), was optimized. The antiserum was raised using 0.5% paraformaldehyde-fixed whole cells (WC) as antigen. The titer and the specificity were examined using whole-cell IF. Results showed that ethanol was an effective decolorization reagent, and 80% ethanol was able to minimize autofluorescence of cells. Samples preserved by freezing at −20°C or −80°C remained above 85% detection efficiency after 1-month storage. The antiserum against WC had a high titer (1:10000), and exhibited high specificity at species level. The antiserum showed a weak cross reaction with the closely related species P. dentatum HK, P. dentatum CCMP1517 and P. minimum HK only at very low dilution (1:5). However, it did not cross-react with the species from the same genus or other phytoplankton species when the dilution reached or exceeded 1:100. Results from different P. donghaiense samples collected at different growth phases or grown under different nutrient conditions showed no significant difference in IF intensity. In addition, the antiserum exhibited high specific binding to P. donghaiense in both mixed phytoplankton samples and field samples. The results indicate that the technique is a potentially useful tool for the rapid identification of P. donghaiense and can facilitate the analysis of various natural samples.     

Palatability and fatality of the dinoflagellate <Emphasis Type="Italic">Prorocentrum lima</Emphasis> to <Emphasis Type="Italic">Artemia salina</Emphasis>

Prorocentrum lima is a toxic alga that produces both intra-cellular and extra-cellular toxins, including okadaic acid (OA) and dinophysistoxins (DTXs). Nauplii of the brine shrimp Artemia salina were exposed to both the cell and cell-free culture medium of P. lima in order to test the hypotheses that the extra-cellular medium is toxic to brine shrimp and that the P. lima cell is palatable but fatal to it. Artemia cysts incubated in the cell-free medium hatched, but mortalities were recorded for nauplii that hatched in, and metanuaplii exposed to, test solutions (autoclaved filtered seawater + cell-free medium) that contained at least 50% of the cell-free medium. Animals exposed to cells of P. lima readily fed on the cells. Some, especially among the Day 1 nauplii, ingested only one cell before dying, while others ingested more than one cell, up to six cells in the case of Day 3 nauplii, before dying. Day 3 nauplii were readily and heavily impacted by the P. lima cells. Survival analysis was used to evaluate survivorship of Day 1 to Day 3 nauplii exposed to cells of P. lima. Estimates were made of tD50s for the different age groups. Comparisons of the tD50s showed that the tD50s for Day 1 and Day 2 nauplii did not vary significantly, but they each varied significantly from the tD50 for the Day 3 nauplii. The possible ecological implications of the findings are discussed.     

Changes in growth and composition of the marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> in response to changing sodium bicarbonate concentrations

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L^?1 NaHCO₃ added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO₃ is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly (p?<?0.05) higher in the NaHCO₃-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO₃] during the GP. During the LP, cell densities were generally higher in the NaHCO₃-treated cells compared with controls. F _V/F _M (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO₃ and significantly different values between controls and 5.0 g L^?1 NaHCO₃ at the end of the GP (p?<?0.05). F _V/F _M was variable between treatments in P. tricornutum (0.3–0.65) but less so in N. salina for (0.5–0.7) regardless of [NaHCO₃]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2?±?6.5 and 4.4?±?2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L^?1 NaHCO₃ treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L^?1 NaHCO₃. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO₃]; there was three times more crude protein in the highest NaHCO₃ (5.0 g L^?1) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03?±?0.12 and 10.16?±?0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO₃] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO₃] (t tests; p?<?0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO₃ treatments. These findings reveal the importance of considering NaHCO₃ as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels.     

Increased expression of transgene in stably transformed cells of <Emphasis Type="Italic">Dunaliella salina</Emphasis> by matrix attachment regions

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.     

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ₆₃₀ ) or a short-TIP1 fragment (ScTIP ₁₂₀ ) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.