FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

GEL PURIFICATION OF RED ALGAL GENOMIC DNA: AN INEXPENSIVE AND RAPID METHOD FOR THE ISOLATION OF POLYMERASE CHAIN REACTION-FRIENDLY DNA1

A simplified approach for the extraction of DNA from red algae in presented. Procedures are simple and fast, requiring a minimum of reagents and apparatus. The method involves an initial lysis step followed by an optional phenol/chloroform extraction. The final gel-purification step removes polymerase chain reaction-inhibiting polysaccharides from the DNA preparation. DNA is extracted as easily from dried algae as it is from snap-frozen, fresh material, thus greatly facilitating the collection and transport of algal samples.     

一种改进的鱼类线粒体DNA的快速制备方法

线粒体DNA(mitOChondrialDNA:mtDNA)是双链环状核外遗传物质,以其分子量小,易于分离,突变率高,进化速度快和母系遗传等特性,已广泛应用于分类学,种系鉴定,种群遗传学,系统发育和进化研究.       

A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.     

DNA:ATP RATIOS IN MARINE MICROALGAE AND BACTERIA: IMPLICATIONS FOR GROWTH RATE ESTIMATES BASED ON RATES OF DNA SYNTHESIS1

DNA: ATP and carbon: DNA (C:DNA) ratios were measured in a total of 14 species of marine microalgae and bacteria. Comparison of several DNA assay methods with results obtained with cultures uniformly labeled with ³³P indicated that by far the most accurate results were obtained using diaminobenzoic acid (DABA) or diphen-ylamine, with DABA having the highest precision. Both the Hoechst and DAPI methods seriously underestimated DNA concentrations in algal cultures. Average DNA: ATP ratios in the algal and bacterial cultures were I7 and 34 by weight, respectively, with almost all values lying in the range of 10–40. DNA: ATP ratios in the microalgae showed no correlation with growth conditions but varied by about a factor of 3 among species. C:DNA ratios for individual species of microalgae and bacteria ranged from 21 to 155 by weight and averaged 50 for the microalgae and bacteria taken together. Growth rates of microalgal species grown in cyclostats were estimated to within 8% of dilution rates when calculated from the uptake of ³H-adenine and the DNA: ATP ratio of the species. Use of the ³H-adenine method for estimating microalgal growth rates in the field may thus be a useful tool for investigating the physiology of microalgae in nature.     

IMPROVED METHODS FOR THE ISOLATION OF CYANOBACTERIAL DNA FROM ENVIRONMENTAL SAMPLES1

DNA isolated from environmental samples often contains enzyme inhibitors disruptive to downstream molecular applications. Most of the existing methods of cyanobacterial DNA isolation do not effectively eliminate these inhibitors from sediment samples or cells collected from freshwater ecosystems. We describe improved methods based on the xanthogenate‐SDS nucleic acid isolation (XS) method of Tillett and Neilan (2000) . Our improved methods provided high‐quality cyanobacterial DNA that could be amplified in PCR and digested with a restriction enzyme. Results were superior to several commercial kits. The DNA yield was also similar to that obtained via the standard XS method. These methods should provide valuable new tools for the expanded application of molecular genetics to limnological and oceanographic research.     

SELECTIVE CONDITIONS AND ISOLATION OF MUTANTS IN SALT-TOLERANT,LIPID-PRODUCING MICROALGAE1

Suitable conditions for the isolation and selection of generic markers were determined by testing the growth of nine axenic strains of lipid-producing algae (representatives of Bacillariophyceae, Eustigmatophyceae, and Chlorophyceae) under a variety of conditions. Mixotrophic, heterotrophic, or anaerobic growth on twenty different carbon compounds was determined. In addition, ten different carbon compounds was determined. In addition, ten different nitrogen-containing compounds were supplied as the sole nitrogen source to ascertain which of these could be utilized. Resistances to various antibiotics and herbicides were also assessed. The algae utilized a variety of compounds as their sole nitrogen source, and some species also grew heterotrophically or mixotrophically on a variety of carbon compounds. This suggests that the algae are not only able to transport these compounds into the cell but that the biochemical pathways necessary for their utilization are present and could be targeted for mutagenesis. Anaerobic growth was not possible on any of the photosystem II inhibitors diuron and atrazine and the 70S ribosome inhibitors erythromycin and streptomycin. However, the diatoms were insensitive to spectinomycin and sulfometuron methyl. Information about drug sensitivities permitted the selection of drug resistant mutants. Mutagenized cultures produced colonies when plated on media containing drug concentrations that were growth-inhibiting for wild-type cultures. Mutations were recovered in Monoraphidium, Nannochloropsis and Navicula species.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

人头发DNA的提取及其基因扩增

头发是犯罪现场最容易得到的材料之一。本文报道从人的头发中提取DNA的方法以及利用PCR(聚合酶链反应)技术,从少量人发DNA扩增大量拷贝数的基因片段。对基因扩增得到的大量基因片段进行进一步的基因分析,将为法医物证学提供客观证据,具有重要价值。     

ENDOGENOUS CYTOKININS IN THREE GENERA OF MICROALGAE FROM THE CHLOROPHYTA1

Nine axenic microalgal (Chlorophyta) strains from three genera (Protococcus, Chlorella, and Scenedesmus) were analyzed for endogenous cytokinins. Cytokinin‐like activity was detected using the excised cucumber cotyledon bioassay. Five strains showed no cytokinin‐like activity and four strains, low cytokinin‐like activity. Ethanolic extracts of the microalgae containing a mixture of deuterium‐labeled standards were purified using a combined DEAE‐Sephadex octadecysilica column and immunoaffinity column based on wide‐range specific mon‐oclonal antibodies and analyzed by HPLC linked to a micromass single quadrupole mass spectrometer with an electrospray interface and a photodiode array detector. There were similar trends in cytokinin profiles for the nine microalgal strains investigated, although concentrations did vary. Both isopentenyladenine and isopentenyladenosine were detected in all nine strains. cis‐Zeatin and cis‐zeatin riboside occurred at higher concentrations than the trans isomers, whereas trans‐zeatin‐O‐glucoside and trans‐zeatin riboside‐O‐glucoside were dominant over the cis isomers. Dihydrozeatin and its conjugates were not detected in any significant amounts. The aromatic benzyladenine always occurred at higher concentrations than benzyladenosine. The topolins were well represented with all three isomers (ortho, meta, and para) being detected, with ortho‐topolin and ortho‐topolin riboside occurring at higher concentrations than the other isomers. However, for the O‐glucosides, the meta isomers (meta‐topolin‐O‐glucoside and meta‐topolin riboside‐O‐glucoside) occurred at higher concentrations than the other isomers. No N‐glucosides were detected (isopentenyladenine‐9‐glucoside, zeatin‐9‐glucoside, dihydrozeatin‐9‐glucoside, benzyladenine‐9‐glucoside, ortho‐topolin‐9‐glucoside, and meta‐topolin‐9‐glucoside). Generally, zeatin and topolin conjugates were the dominant forms of isoprenoid and aromatic cytokinins, respectively. There was no distinct trend in the proportions of isoprenoid to aromatic cytokinins.     

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.     

大鼠,豚鼠心肌细胞的简单,快速分离

本文介绍了一种简单、快速分离成年大鼠、豚鼠心肌细胞的方法。所分得的细胞形态结构完整,具有良好的钙耐受性,膜片钳上易于形成高阻抗接封,因而适于作各种电生理记录。     

A RAPID SIMPLE TECHNIQUE UTILIZING CALCOFLUOR WHITE M2R FOR THE VISUALIZATION OF DINOFLAGELLATE THECAL PLATES1

The fluorescent stain Calcofluor White M2R readily binds to cellulose and other β-linked glucans (Hughs and McCully 1975). We have found the stain to readily bind to the thecal plates of armored dinoflagellates. Considerable detail is revealed about plate structure in both living and preserved specimens at the light microscopic level. This simple rapid technique should prove useful for the tabulation and study of dinoflagellate thecae.     

DEVELOPMENT OF IMMUNOASSAYS FOR THE IRON‐REGULATED PROTEINS FERREDOXIN AND FLAVODOXIN IN POLAR MICROALGAE1

While the growth of Southern Ocean phytoplankton is often limited by iron availability, there are no comparable experiments on sea‐ice algae. Here we assess the use of ferredoxin and flavodoxin to investigate the iron nutritional status of sea‐ice algae and describe the development of a quantitative immunoassay for both proteins in marine diatoms. High‐affinity monoclonal antibodies toward both proteins were produced from Cylindrotheca closterium (Ehrenb.) J. M. Lewin et Reimann, and these were used to develop Western blots. Western blots run on whole protein extracts detected both proteins with little cross‐reactivity toward other proteins. The two proteins could be successfully quantitated when applied to gels at between 5 and 50 ng in a volume of 25 μL (0.2–2 μg · mL^?1). Flavodoxin and ferrodoxin expression was examined in the Antarctic diatoms Entomoneis kjellmannii (Cleve) Poulin et Cardinal, Navicula directa (W. Sm.) Ralfs, Fragilariopsis curta (Van Heurck) Hust., Pseudo‐nitzschia sp., Porosira glacialis (Grunow) E. G. Jørg., Fragilariopsis cylindrus (Grunow) Willi Krieg., Fragilariopsis sublinearis (Van Heurck) Heiden et Kolbe, C. closterium, Nitzschia lecointei Van Heurck, and the dinoflagellate Polarella glacialis Montresor, Procaccini et Stoecker. Two Arctic isolates were also examined, Nitzschia frigida (Grunow) and Fragilariopsis oceanica (Cleve) Hasle. Significant heterogeneity of protein expression was observed despite all cultures being grown in iron‐replete f/2 medium. Only one species, F. cylindrus, displayed the expected expression of ferredoxin only in iron‐replete medium. Four were observed to produce both proteins under iron‐replete conditions. Ferredoxin was not detected at all in F. curta and Pseudo‐nitzschia sp., but distinct flavodoxin bands were observed in both of these organisms. All species examined were observed to express either flavodoxin or ferredoxin or both of the proteins as determined by Western immunoblotting.