A METHOD FOR EXTRACTION OF HIGH MOLECULAR WEIGHT DNA FROM THE MACROALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1,2

We developed a method for extraction of DNA from the red alga Porphyra yezoensis Ueda. The method consists of three preparation steps that include CsCl-gradient ultracentrifugation, cetyl trimethyl ammonium bromide treatment, and a final RNase step. The amount of DNA extracted from 1.5 g of starting material averaged 17.7 μg. The resulting DNA had a high molecular weight, was 25-166 kb in length, was digested with five common restriction enzymes, and showed no nuclease activity. It was of sufficient quality for construction of genomic libraries.     

ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH1

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d^?1 when using d,l ‐malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing d,l ‐malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.     

BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

A multimethod approach was used to characterize unicellular green algae that were traditionally assigned to the genus Chlorella Beijerinck and to resolve their phylogenetic relationships within the Chlorophyta. Biochemical, physiological, and ultrastructural characters, together with molecular data such as DNA base composition and DNA hybridization values, were compared with a molecular phylogeny based on complete 18S rRNA sequences. Our results show that Chlorella taxa are dispersed over two classes of chlorophytes, the Trebouxiophyceae and the Chlorophyceae. We propose that only four species should be kept in the genus Chlorella (Chlorophyta, Trebouxiophyceae): C. vulgaris Beijerinck, C. lobophora Andreyeva, C. sorokiniana Shih. et Krauss, and C. kessleri Fott et Nováková. Common characteristics of these taxa are glucosamine as a dominant cell wall component and the presence of a double thylakoid bisecting the pyrenoid matrix. Norspermine, norspermidine, and secondary carotenoids are never produced. Other "Chlorella" species belong to different taxa within the Trebouxiophyceae ( "C." protothecoides = Auxenochlorella protothecoides [Krüger] Kalina et Punčochářová, "C." ellipsoidea, "C." mirabilis, "C." saccharophila, and "C." luteoviridis ) and Chlorophyceae ( "C." zofingiensis and "C." homosphaera = Mychonastes homosphaera Kalina et Punčochářová). The latter taxa can easily be recognized by the production of secondary carotenoids under nitrogen-deficient conditions.     

SHORT‐TERM EFFECTS OF ACETATE AND MICROAEROBIC CONDITIONS ON PHOTOSYNTHESIS AND RESPIRATION IN CHLORELLA SOROKINIANA GXNN 01 (CHLOROPHYTA)1

The culture of microalgae using organic carbon sources decreases the cost of operation in closed systems. The effect of carbon sources on microalgae is thus an interesting problem in not only theoretical research but also practical production. The short‐term effects of acetate and microaerobic conditions on the growth, photosynthesis, and respiration of the green microalga Chlorella sorokiniana I. Shihira & R.W. Krauss GXNN 01 were described after acetate addition to autotrophic cultures. As the acetate concentration increased, cells needed a longer lag phase to grow, and 243.8 mM acetate completely inhibited growth. Acetate addition induced an immediate response in photosynthesis and respiration. The activity of PS II and PS I were impaired and declined with different rates, and then recovered compared with autotrophic cells. Carbonic anhydrase and Rubisco activities were also inhibited at the beginning, and respiration was increased. We propose that ATP consumption for acetate assimilation results in surplus NADPH, and then accumulated reducing power over‐reduces inter‐photosystem components and raises the transthylakoid proton gradient, which redistributes energy between PS I and PS II, and leads to a decrease in the PS II/PS I ratio and O₂ evolution. An apparent cyclic electron flow was also observed, which may be mainly mediated by NAD(P)H dehydrogenase‐dependent pathway since NADPH was in excess. These observations pointed to an acclimation process after acetate addition, and suggested the interaction between photosynthesis and respiration involving ATP and reducing power.     

ORIGINS OF GENOTYPIC VARIATION IN NORTH AMERICAN DANDELIONS INFERRED FROM RIBOSOMAL DNA AND CHLOROPLAST DNA RESTRICTION ENZYME ANALYSIS

Restriction enzyme analysis of ribosomal DNA (rDNA) and chloroplast DNA (cpDNA) is used to assess the relative contribution of hybridization and mutation as sources of genotypic variation in weedy asexual dandelions, with focus on the dandelion flora of North America. Of 318 North American dandelions surveyed, 145 rDNA-cpDNA clones are detected. The combined rDNA-cpDNA genotypes show that most of the polymorphic rDNA and cpDNA restriction sites or lengths in these plants are also present in weedy asexual dandelions collected from natural populations in Europe and in asexual and diploid taxa (microspecies) chosen to represent diverse Eurasian members of the genus. However, of 222 combined rDNA-cpDNA genotypes found in 427 asexual plants surveyed, only 9 genotypes are found in both North American and Eurasian dandelions. Two rDNA and three cpDNA characters are unique to individual plants in North America and are consistent with mutational origins of genotypic variation in asexual lineages. But the array of genotypic diversity, characterized by different combinations of the rDNA and cpDNA characters, show that multiple hybridization events are a more important source of genotypic variation than mutation in the asexual polyploids. The rDNA and cpDNA data also indicate polyphyletic origin of several asexual Taraxacum taxa.     

CHARACTERIZATION OF TWO CHLORELLA VULGARIS (CHLOROPHYCEAE) STRAINS ISOLATED FROM WASTEWATER OXIDATION PONDS1

We report here on the characterization and isolation of two ecotypes of Chlorella vulgaris Beyerinck that coexist in wastewater reservoirs. One ecotype (C1) contains high amounts of chlorophyll b, is capable of autotrophic growth, and can utilize only a few organic solutes for growth. The second ecotype (C2) contains low amounts of chlorophyll b, requires vitamin B₁₂, and can support its growth with a broad range of organic compounds. Of the two ecotypes, the latter showed slower growth rates when light was the sole source of energy. Cells of C2-type Chlorella attained higher photosynthetic activities than C1-type cells at saturating irradiances. However, their low chlorophyll b content and lower light utilization efficiency suggest that C2-type Chlorella contains relatively low amounts of light-harvesting antennae, a disadvantage in severely light-limited ecosystems like wastewater reservoirs. We hypothesize that the two Chlorella types coexist by adopting different lifestyles: C1-type cells rely largely on their photosynthetic potential for energy conservation and growth, whereas C2-type cells may exploit their heterotrophic properties for this purpose.     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)1

Effects of CO₂ concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low-CO₂ cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO₂ conditions (high-CO₂ cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO₂ cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low-CO₂ cells of C. vulgaris 11h, while a grana-like structure was found in high-CO₂ cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low-CO₂ cells than in high-CO₂ cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO₂ conditions, whereas CO₂-effects on lamellar stacking are species dependent.     

RESTRICTION ENDONUCLEASE ANALYSIS OF RIBOSOMAL DNA SEQUENCE VARIATION IN LAMINARIA (PHAEOPHYTA)1

The taxonomy and evolutionary relationships of species in the genus Laminaria are poorly understood. Previous studies have demonstrated significant plasticity of morphological characters used to describe taxa, and interfertility has been reported among putative species. We analyzed nuclear ribosomal DNA (rDNA) sequence variation in eight species of Laminaria (L. agardhii Kjell., L. digitata (Huds.) Lamour., L. groenlandica Rosenv. [sensu Druehl 1968], L. longicruris De la Pyl., L. longipes Bory, L. saccharina (L.) Lamour., L. setchellii Silva, and L. yezoensis Miyabe) to elucidate evolutionary relationships in this genus. Restriction maps were constructed using a small subunit rDNA probe from Costaria costata (Turn.) Saunders, an rDNA repeat from the nematode Caenorhabditis elegans, and 11 hexameric restriction endonucleases in an annealing analysis of genomic DNA. Laminaria rDNA restriction maps were compared to each other and to that of the outgroup taxon, C. costata. rDNA restriction maps of Laminaria species and C. costata were similar. Restriction fragment length polymorphisms mapped to both the coding regions and the nontranscribed spacer of rDNA. Laminaria species were distinguished with this method. The restriction maps of L. agardhii, L. saccharina, and L. longicruris were identical, supporting a previous hypothesis that these species are conspecific. Comparison of restriction maps of Laminaria species suggested that the generic subdivision of Sections Simplices and Digitatae may be invalid.     

GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μ g genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A ₂₆₀/ A ₂₈₀ ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum . The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.     

马来熊脑源性神经营养因子基因编码区的克隆与序列分析

参照人脑源性神经神经生长因子（ＢＤＮＦ）基因序列设计出一对引物，利用聚合酶链式反应，首次从马来熊基因组ＤＮＡ中扩增和克隆到ＢＤＮＦ基因的编码区。对该基因所作的序列分析表明，马来熊和人ＢＤＮＦ基因编码区的核苷酸序列同源性为９４％，而与大熊猫ＢＤＮＦ基因的核苷酸序列同源性高达９９％。在推导的多肽序列中，除在前导肽区有两个氨基酸的差异外，马来熊ＢＤＮＦ的成熟区与人和其他已报道的哺乳动物ＢＤＮＦ成熟区的氨     

溶藻弧菌HY9901 ahr基因克隆与序列分析

TouchdownPCR扩增溶藻弧菌依赖ATP的DNA解旋酶rep基因部分序列,得一1．2kb片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由2016bp组成,共编码671aa的完整基因。该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与副溶血弧菌RIMD2210633、溶藻弧菌12G01、Vibriosp．Ex25、创伤弧菌YJ016、霍乱弧菌RC385、灿烂弧菌12801、费氏弧菌ES114及矿angustumS14分别为96％、95%、95%、94%、92％、91%、89％、86％。     

PLASTID DNA RESTRICTION ANALYSIS LINKS THE HETEROMORPHIC PHASES OF AN APOMICTIC RED ALGAL LIFE HISTORY1

Gymnogongrus sp. (Phyllophoraceae) from Nova Scotia, Canada, identified tentatively as G. devoniensis (Greville) Schotter, grows in association with an Erythrodermis-like that forms chains of tetrasporangia or bisporangia. The crust resembles tetrasporophytic phases of other Gymnogongrus species, but in culture both it and the G. devoniensis gametophytes cycle independently by apomictic reproduction. A method was developed for extracting organelle DNA from this carrageenophyte genus involving purification of nucleic acids by binding to hydroxylapatite. Plastid DNA from G. devoniensis and bisporangial Erythrodermis-like crusts was compared with that of G. devoniensis and G. crenulatus (Turner) J. Agardh from France and of G. furcellatus (C. Agardh) J. Agardh from Chile. Plastid genomes of all Gymnogongrus species and the Erythrodermis-like crust were approximately 175 kb long. A single 3.5-kb plasmid DNA species was found in G. devoniensis and the Erythrodermis-like bisporophyte but not in other samples. Digestion of plasted DNA with several restriction endonucleases produced identical patterns in G. devoniensis and the Erythrodermis-like bisporophyte from the same location, indicating clearly that these entities represent two phases of an uncoupled life history. These results were confirmed with heteologous probes. A restriction fragment length polymorphism was identified between two Nova Scotian G. devoniensis populations. There was no similarity in restriction patterns between G. devoniensis from Nova Scotia, G. devoniensis from France. G. crenulatus or G. furcellatus, suggesting that molecular taxonomic methods could be important in delineating members of this morphologically variable genus. Further study is necessary to determine whether either Nova Scotian G. devoniensis or French G. devoniensis corresponds to type populations of G. devoniensis from Devon, England.     

A PRELIMINARY COMPARISON OF RESTRICTION FRAGMENT PATTERNS IN THE GENUS CAULERPA (CHLOROPHYTA) AND THE UNIQUE STRUCTURE OF THE CHLOROPLAST GENOME OF CAULERPA SERTULARIOIDES1

Comparisons of chloroplast DNA restriction fragments in four species of Caulerpa revealed that patterns between the species were different, with few and possibly no homologous bands. Two forms of Caulerpa sertularioides also revealed different patterns, and it is possible that the forms are separate species. The chloroplast genome in Caulerpa sertularioides f sertularioides (S. G. Gmelin) Howe is 131.4 kb in size and lacks large repeat units. The discovery of another green-algal chloroplast genome that lacks an inverted repeat indicates that this feature is either not ancestral to the Chlorophyceae or has been lost several times. Several gene clusters commonly found in chloroplast DNAs were found to occur in Caulerpa chloroplast DNA, for example, psbD/C, atpF/H, and psaA/B. The 16S and 23s rRNA, which are typically adjacent, contained in an inverted repeat, and cotranscribed, are over 40 kb apart. Genes rps12 and tufA, members of the str operon in eubacteria, are over 50 kb in distance from each other in Caulerpa. The gene order in Caulerpa is unlike any other chloroplast genome characterized to date.     

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES,CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.     

POPULATION STRUCTURE OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) AS DETERMINED BY RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA

Abstract: Restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) were used to test for population subdivision in the bottlenose dolphin (Tursiops truncatus). Atlantic and Pacific dolphin mtDNA samples exhibited distinctly different haplotypes (approximately 2.4% sequence divergence), indicating a lack of gene exchange. Within the Atlantic Ocean, mtDNA samples from the Gulf of Mexico and the Atlantic Coast were also found to be distinct, with a sequence divergence of approximately 0.6%. The Atlantic Coast–Gulf of Mexico dichotomy is consistent with patterns of genetic variation from other marine and coastal organisms from this region, and supports the hypothesized role of bio-geographic events in promoting the divergence of these and other forms. Regional differentiation was identified along the Atlantic Coast, whereas low sequence divergences among haplotypes and consistent haplotype frequencies across populations suggested considerable gene exchange among Gulf of Mexico populations. A highly divergent haplotype found in two individuals from two localities in the Gulf of Mexico is best explained by dispersal from either a distinct offshore Gulf stock or an unsampled Atlantic Coast stock. Additional samples are required to test for the existence of a distinct offshore race and, if it exists, to identify its distribution and contribution to population structure.     

ISOZYME ANALYSIS OF MATING POPULATIONS OF GONIUM PECTORALE (CHLOROPHYTA)1

Intraspecific variation among 36 strains of the freshwater alga Gonium pectorale Müller (Chlorophyceae) isolated from three geographically different locations in Tibet, Nepal, and Japan was investigated by isozyme analysis. Variation in isozyme patterns of eight enzyme systems (malate dehydrogenase, glutamate dehydrogenase, tetrazolium oxidase, lactate dehydrogenase, octanol dehydrogenase, xanthine dehydrogenase, phosphoglucomutase, and malic enzyme) of axenic and clonal cultures was revealed by polyacrylamide gel electrophoresis. Unweighted average linkage clustering, based on Jaccard's similarity coefficient, illustrated the high similarity between most strains from Nepal and all strains from the Ryukyu Islands (Japan). However, there was relatively low similarity between strains from Tibet and those from Nepal and Japan. Strains from the Ryukyu Islands (Japan) grouped into two clusters, and most Nepalese strains formed a single cluster, but Tibetan strains were heterogenous.     

中国牛朊病毒基因的克隆和序列分析

从中国牛外周血中分离淋巴细胞,提取基因组DNA.用所设计引物以聚合酶链式反应扩增出不致病的朊病毒蛋白(PrP~c)基因,并克隆到pGEM-Teasy Vector.序列分析表明所克隆的牛PrP~c的片段大小为795bp,包含了牛朊病毒基因的完整编码区序列.该基因无内含子,同国外报道的已知序列完全相同.