Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Structural analysis of SNARE motifs from sea perch, Lateolabrax japonicus by computerized approaches

Three cDNA sequences encoding four SNARE (N-ethylmaleimide-sensitive fusion protein attachment protein receptors) motifs were cloned from sea perch, and the deduced peptide sequences were analyzed for structural prediction by using 14 different web servers and softwares. The "ionic layer" structure, the three dimensional extension and conformational characters of the SNARE 7S core complex by using bioinformatics approaches were compared respectively with those from mammalian X-ray crystallographic investigations. The result suggested that the formation and stabilization of fish SNARE core complex might be driven by hydrophobic association, hydrogen bond among R group of core amino acids and electrostatic attraction at molecular level. This revealed that the SNARE proteins interaction of the fish may share the same molecular mechanism with that of mammal, indicating the universality and solidity of SNARE core complex theory. This work is also an attempt to get the protein 3D structural information which appears to be similar to that obtained through X-ray crystallography, only by using computerized approaches. (C) 2007 Elsevier Ltd. All rights reserved.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.

Molecular identification of 16 Porphyra lines using sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.

Loss of allele diversity in introduced populations of the hermaphroditic bay scallop Argopecten irradians

The bay scallop Argopecten irradians is a hermaphroditic bivalve native to the Atlantic coast of the United States that was introduced to China for aquaculture production in 1982. It now supports a major aquaculture industry in China. Introduced species often start with limited genetic variability, which is problematic for the further selective breeding. Bay scallop aquaculture is exclusively hatchery based and as the initial introduction consisted of only 26 scallops, there have been concerns about inbreeding and inbreeding depression in cultured populations in China. In this study, eleven simple sequence repeat (SSR) markers were used to compare genetic variation in cultured populations from China with that in a natural population from the east coast of America. Although the difference in heterozygosity was small, the Chinese populations lost 9 of the 45 alleles (20%) found in the wild population. The reduced allele diversity suggests that the Chinese bay scallop populations experienced a bottleneck in genetic diversity that remains significant despite several recent introductions of new stocks aimed at expanding the gene pool. The loss of allele diversity may affect future efforts in selective breeding and domestication, and results of this study highlight the need for additional introductions, advanced breeding programs that minimize inbreeding and continued genetic monitoring. (c) 2007 Elsevier B.V. All rights reserved.

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (Pseudosciaena crocea) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1: 100 with Ringer's solution and UV irradiated with doses ranging from 0-150 J cm (-2). The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm(-2), after fertilization, motility was < 10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm -2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 208 degrees C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures ( 28 degrees C, 38 degrees C or 48 degrees C) and five shock durations ( 4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 +/- 2.99%) were obtained at 38 degrees C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei's unbiased genetic distance (D) and fixation index (F-ST) between the populations. The Mantel test showed that two types of matrices of D and FST were highly correlated, whether from RAPD or ISSR data, r=0.9310 (P = 0.008) and 0.9313 (P=0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder. (C) 2007 Elsevier Inc. All rights reserved.

Development of the East Asian monsoon: Mineralogical and sedimentologic records in the northern South China Sea since 20 Ma

We here reconstruct the past change of the East Asian monsoon since 20 Ma using samples from Ocean Drilling Program (ODP) Site 1146 in the northern South China Sea based On a multi-proxy approach including a monomineralic quartz isolation procedure, identification of clay minerals by X-ray Diffraction (XRD) and grain-size analysis of isolated terrigenous materials. Terrigenous supply to ODP Site 1146 was dominated by changes in the strength of multiple sources and transport processes. Grain-size data modeled by an end-member modeling algorithm indicate that eolian dust from the and Asian inland and fluvial input have contributed on average 20% and 80% of total terrigenous material to ODP Site 1146, respectively. Specifically, about 40-53% of the total (quartz+feldspar) and only 6-11% of the total clay is related to eolian supply at the study site. Detailed analysis of the sedimentary environment, and clay minerals combined with previous studies shows that smectite originates mainly from Luzon, kaolinite from the Pearl River and illite and chlorite from the Pearl River, Taiwan and/or the Yangtze River. The proportion and mass accumulation rate (MAR) of the coarsest end-member EM1 (interpreted as eolian dust), ratios of (illite+chlorite)/smectite, (quartz+feldspar)% and mean grain-size of terrigenous materials at ODP Site 1146 were adopted as proxies for East Asian monsoon evolution. The consistent variation of these independent proxies since 20 Ma shows three profound shifts in the intensity of East Asian winter monsoon relative to summer monsoon, as well as aridity of the Asian continent, occurred at similar to 15 Ma, similar to 8 Ma and the youngest at about 3 Ma. In comparison, the summer monsoon intensified contemporaneously with the winter monsoon at 3 Ma. The phased uplift of the Himalaya-Tibetan plateau may have played a significant role in strengthening the Asian monsoon at similar to 15 Ma, 8 Ma and 3 Ma. (C) 2007 Elsevier B.V. All rights reserved.

Grain-size records at ODP site 1146 from the northern South China sea: Implications on the east Asian monsoon evolution since 20 Ma

273 samples from Ocean Drilling Program (ODP) Site 1146 in the northern South China Sea (SCS) were analyzed for grain-size distributions using grain-size class vs. standard deviation method and end-member modeling algorithm (EMMA) in order to investigate the evolution of the East Asian mon-soon since about 20 Ma. 10-19 mu m/1.3-2.4 mu m, the ratio of two grain-size populations with the highest variability through time was used to indicate East Asian winter monsoon intensity relative to summer monsoon. The mass accumulation rate of the coarsest end member EM1 (eolian), resulting from EMMA, can be used as a proxy of winter monsoon strength and Asian inland aridity, and the ratio of EM1/(EM2+EM3) as a proxy of winter monsoon intensity relative to summer monsoon. The combined proxies show that a profound enhancement of East Asian winter monsoon strength and winter monsoon intensity relative to summer monsoon occurred at about 8 Ma, and it is possible that the summer monsoon simultaneously intensified with winter monsoon at 3 Ma. Our results are well consistent with the previous studies in loess, eolian deposion in the Pacifc, radiolarians and planktonic foraminifera in the SCS. The phased uplift of the Himalaya-Tibetan Plateau may have played a significant role in strengthening the Asian monsoon at 8 Ma and 3 Ma.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 degrees C to 11 degrees C, 23 degrees C and 28 degrees C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 degrees C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 degrees C to 28 degrees C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 degrees C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. (C) 2007 Elsevier B.V. All rights reserved.

Phyllophorus (Phyllophorus) maculatus, a new species of sea cucumber from the Yellow Sea (Echinodermata : Holothuroidea : Dendrochirotida)

Phyllophorus (Phyllophorus) maculatus new species is described from a depth of 22-45 meters near the western edge of the Yellow Sea. The body is cylindrical, with body wall ossicles present only in the anal region. Ossicles are four-pillared tables with low spires and indented disc margin. A revised key to the eleven phyllophorine (Family Phyllophoridae, Subfamily Phyllophorinae) species now known from China is presented.

Yellow perch strain evaluation I: Genetic variance of six broodstock populations

As a prelude to strain selection for domestication and future marker assisted selection, genetic variation revealed by microsatellite DNA was evaluated in yellow perch, Perca flavescens, from four wild North American populations collected in 2003-2004 (Maine, New York, North Carolina, and Pennsylvania,), and two captive populations (Michigan and Ohio). For the loci examined, levels of heterozygosity ranged from H-e=0.04 to 0.88, genetic differentiation was highly significant among all population pairs, and effective migration ranged from low (N(e)m=0.3) to high (N(e)m=4.5). Deviation from Hardy-Weinberg equilibrium was regularly observed indicating significant departures from random mating. Instantaneous measures of inbreeding within these populations ranged from near zero to moderate (median F=0.16) and overall inbreeding levels averaged F-IS=0.18. Estimates of genetic diversity, Phi(ST), and genetic distance were highest between Michigan and all other broodstock groups and lowest between New York and Ohio. Genetic differentiation among groups did not correlate with geographic distance. Overall, the patterns of variation exhibited by the captive (Michigan and Ohio) populations were similar to patterns exhibited by the other wild populations, indicating that spawning and management practices to date have not significantly reduced levels of genetic variation. (C) 2007 Elsevier B.V. All rights reserved.