Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Chandrananimycins A-C: Production of novel anticancer antibiotics from a marine Actinomadura sp isolate M048 by variation of medium composition and growth conditions

In our screening of marine actinomycetes for bioactive principles, three novel antibiotics designated as chandrananimycin A (3c), B (3d) and C (4) were isolated from the culture broth of a marine Actinomadura sp. isolate M045. The structures of the new antibiotics were determined by detailed interpretation of mass, 1 D and 2 D NMR spectra.

Biopotentials of marine sponges from China oceans: past and future

An extensive literature survey of over 17 Journals was carried out on Chinese sponges and their natural products in the period from 1980 to 2001. This review is thus intended to provide the first thorough overview of research on marine sponges from China Ocean territories. Information is provided about the rather-limited taxonomic study of Chinese marine sponges, with an analysis on their distribution and diversity. Research findings on the natural products and their bioactivity screening from Chinese sponges are summarized. The weaknesses, gaps and problems in the past R&D program of Chinese sponges are identified, which point to the future opportunities in exploiting these huge untapped sponge resources. The report is expected to serve as an entry point for understanding Chinese sponges and for furthering R&D on their bioactive compounds for new drug development. (C) 2003 Elsevier Science B.V. All rights reserved.

Analysis of expressed sequence tags of a marine red alga, Gracilaria lemaneiformis

The expressed sequence tags (EST) has been proved to be a useful tool for discovering and identifying functional genes, especially in some species whose genetic information is unavailable. A total of 180 ESTs have been generated from a cDNA library of gametophytic Gracilaria lemaneiformis in this study. These clones are clustered into 151 groups, among which 8 groups are highly homologous to chloroplast genes and are abundant in the library. After searching for matches in the EST database of red alga, 22 groups are found to match with the registered ESTs of Rhadophyta and 6 with Gracilaria. Searching in the protein database reveal that 73 non-redundant clones have significant similarity to some known sequences, the majority of which are involved in photosynthesis, DNA transcription or translation, and 6, 4 and 3 clones are associated with growth or development, signal transduction and stress or defense response, respectively.

In vitro assembly of R-phycoerythrin from marine red alga Polysiphonia urceolata

Scanning tunneling microscope was used to investigate the in vitro assembly of R-phycoerythrin (R-PE) from the marine red alga Polysiphonia urceolata. The results showed that R-PE molecules assembled together by disc-to-disc while absorbing on HOPG surface, which just looked like the rods in the phycobilisomes. When the water-soluble R-PE was dissolved in 2% ethanol/water spreading solution, they could form monolayer film at the air/water interface. Similar disc-to-disc array of R-PE was constituted in the two-dimensional Langmuir-Blodgett film by the external force. It could be concluded that, apart from the key role of time linker polypeptides, the in vivo assembly of phycobiliproteins into phycobilisomes is also dependent on the endogenous properties of phycobiliprotein themselves.

Isolation of PSI from a siphonous marine green alga, Codium fragile

Three different forms of PS I complexes were isolated from a siphonous marine green alga, Codium fragile, by Triton X-100 sucrose gradient centrifugation. Zone III had a Chl a/b>20, and designated as PS I. core complex CC I because it created only CP I band in mild PAGE. Zone IV and V had absorption at 436 and 674 nm, 467 and 650 nm, and 540 nm, suggesting the presence of Chl a, Chl b, siphonaxanthin and siphonein, Chl a/b were 3.23 and 2.4, respectively. Both CP I and CP I a bands were observed when they were subjected to mild PAGE. Therefore, Zone IV and V were different forms of PS I complexes that consisted of CC I and different amount of light-harvesting complex LHC I. Zone III contained only 66 and 56 ku peptides in SDS-PAGE, while Zone IV and V had 4 different LHC I peptides of 25, 26, 26.2 and 27.5 ku in addition to 66, 56 ku peptides. Fluorescence emission spectra showed that efficient energy transfer were kept among pigments in isolated PS I complexes. Excitation energy absorbed by Chl b, siphonaxanthin and siphonein can be transferred to Chl a.

Analysis of three marine fish cell lines by RAPD assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG. SPH, and RSBK and as a possible tool to detect cross-contamination. Sixth commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical Patterns produced by 35-48% of the primers tested. the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851. indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic hand pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

Study on the concentration and seasonal variation of inorganic elements in 35 species of marine algae

The concentrations of five major and 28 trace elements in 35 marine algae collected along the coast of China were determined by instrumental neutron activation analysis. The concentrations of halogens, rare earth elements and many transition metal elements in marine algae are remarkably higher than those in terrestrial plants. The concentration factors for 31 elements in all collected algae were calculated, those for tri- and tetra-valent elements were higher than those of the mono- and di-valent elements in marine algae. The biogeochemical characteristics of inorganic elements in marine algae were investigated. In addition, the seasonal variation of inorganic elements in Sargassum kjellmanianum was also studied. (C) 1998 Elsevier Science B.V. All rights reserved.

MARINE ORGANISMS ATTACHED TO SEAWEED SURFACES IN JIAOZHOU BAY, CHINA

Scanning electron microscopy of the surfaces of the seaweeds Laminaria japonica, haploid Porphyra yezoensis, Ulva pertusa and the diploid conchocelis of P. yezoensis and P. haitanensis revealed Vibrio and Micrococcus to be abundant on the surfaces of U. pertusa and P. yezoensis. Vibrio, Flavobacterium, Pseudomonas, Staphylococcus, Bacillus, Corynebacterium and other genera were isolated from the surfaces of L. japonica.

New antibiotic with typical plant anthraquinone structure obtained studying terrestrial and marine Streptomycetes

In our screening for new antibiotics from bacteria, the streptomycete isolate M097 from Jiaozhou Bay in China was found to produce aloesaponarin II (1a) and 1,6-dihydroxy-8-hydroxymethyl-anthraquinone (2). Similarly, a terrestrial streptomycete GW24/1694 produced 1a and its methyl ether, the new compound 1-hydroxy-6-methoxy-8-methyl-anthraquinone (1b). All structures were derived by spectrochemical analysis and by comparison with reference data. The results showed that the marine streptomycete isolate M097 and the terrestrial streptomycete GW24/1694 could be a promising material for studying the biosynthetic pathway of polyketides.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Intergeneric conjugation in holomycin-producing marine Streptomyces sp strain M095

Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isotated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9 +/- 0.13 x 10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively. (c) 2006 Elsevier GmbH. All rights reserved.