Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Extrachromosomal deoxyribonucleic acid in different enterobacteria

Eighty-seven different enterobacteria and pseudomonas strains were examined for the presence of extrachromosomal deoxyribonucleic acid (DNA). Thirty-four strains contained closed circular DNA by the ethidium bromide CsCl density technique. Extrachromosomal DNA was most frequent in Escherichia and Klebsiella strains. The extrachromosomal DNA was isolated and characterized by analytical ultracentrifugation and electron microscopy. All the extrachromosomal DNA-containing bacteria contained circular DNA molecules of small size (0.5-4 mum). Most of these bacteria also contained larger circles (20-40 mum). The number of different size classes of circular DNA in each strain varied from one to five. The buoyant density of the extrachromosomal DNA ranged from 1.692 to 1.721 g/cm(3). Many bacteria contained extrachromosomal DNA of more than one density.     

In VivoAddition of Telomeric Repeats to Foreign DNA Generates Extrachromosomal DNAs in the Taxol-Producing FungusPestalotiopsis microspora

Transformation of the taxol-producing filamentous fungusPestalotiopsis microsporawith a plasmid containing the bacterial hygromycin resistance gene fused toAspergillusregulatory sequences resulted in thein vivoformation of extrachromosomal DNAs with telomeric repeats in the majority of transformants. Repeats of the telomeric sequence 5′-TTAGGG-3′ were appended to nontelomeric transforming DNA termini. No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs. Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs. The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection. Transformation of wild-typeP. microsporawith a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector. The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications.     

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Extrachromosomal deoxyribonucleic acid in R factor-harboring Enterobacteriaceae.

Extrachromosomal deoxyribonucleic acid (DNA) from 24 different R factor-harboring Enterobacteriaceae was isolated and characterized by analytical ultracentrifugation and electron microscopy. The R factors represented 15 different patterns of transferable drug resistance found in enterobacteria from an enclosed geographic area. All of the strains contained extrachromosomal, circular DNA molecules within the range of 0.4 to 52 mum. More than one size class of circular DNA molecules was observed in the majority of the extrachromosomal DNA preparations. The buoyant density of the extrachromosomal DNA ranged from 1.700 to 1.720 g/cm3. The majority of the bacteria contained extrachromosomal DNAs of various densities. Three-fourths of the R factors were classified as fi+. The investigation illustrates the extensive variability in the physical characteristics of plasmid DNA from R factor-harboring strains.     

热带直流型水库蓝藻季节变化特征

于2004年调查了广东省飞来峡和深圳两座直流型水库的蓝藻季节分布特征。共检到蓝藻17属,绝大多数为丝状体种类。常见属有假鱼腥藻(Pseudanabaena)、微囊藻(Microcystis)和蓝纤维藻(Dactylococcopsis)。蓝藻细胞密度为5-2052个细胞ml-1。深圳水库最高蓝藻细胞密度出现在6月,其中丝状蓝藻的相对丰度在75%以上;飞来峡水库最高蓝藻细胞密度出现在12月,丝状蓝藻的相对丰度达到90%。水动力学条件是限制蓝藻丰度和种类的主要环境因子。由于飞来峡水库的水力滞留时间变化较大,所以蓝藻季节性变化受水动力学条件的影响比深圳水库的大。假鱼腥藻等丝状蓝藻更适合在水库中生长,特别是在氮磷营养水平较高,水力滞留时间明显增加时,其细胞密度将大幅度上升。     

Evidence for a chromosomal location of the genes coding for chloramphenicol production in Streptomyces venezuelae.

Of seven chloramphenicol-producing actinomycetes examined, only Streptomyces venezuelae strain 13s contained extrachromosomal DNA detectable by agarose gel electrophoresis and cesium chloride-ethidium bromide density gradient centrifugation. The single 17-megadalton plasmid present in this strain was indistinguishable from plasmid pUC3 previously isolated from mutagenized cultures. Strains selected for their inability to produce chloramphenicol after treatment with acriflavine or ethidium bromide still contained a plasmid that had the same electrophoretic mobility as plasmid pUC3 and yielded similar fragments when digested with restriction endonucleases. By regenerating protoplasts of strain 13s and screening for isolates lacking extrachromosomal DNA, strain PC51-5 was obtained. The absence of plasmid pUC3 sequences in this strain was confirmed by Southern hybridization using 32P-labeled plasmid as a probe. Since the plasmidless strain produced as much chloramphenicol as did the parent strain, pUC3 contains neither structural nor regulatory genes for antibiotic production. Evidence from electrophoretic analysis of BamHI digests of total cellular DNA from wild-type and dye-treated nonproducing progeny indicated that acriflavine caused structural changes in the chromosome.     

Observation of microplasmodesmata in both heterocyst-forming and non-heterocyst forming filamentous cyanobacteria by freeze-fracture electron microscopy

Structures which may establish cytoplasmic continuity between adjacent cells of filamentous cyanobacteria have been observed by freeze-fracture electron microscopy. They are visible in the septum region of the plasma membrane as pits on the E-face (EF) and corresponding protrusions on the P-face (PF). Between 100 and 250 of these structures, termed microplasmodesmata, were present between adjacent vegetative cells in all four strains of heterocyst-forming filamentous cyanobacteria, Anabaena cylindrica Lemm, A. variabilis (IUCC B377), A. variabilis Kütz. (ATCC 29413) and Nostoc muscorum, examined. Only 30–40 microplasmodesmata were observed between adjacent cells in two species, Phormidium luridum and Plectonema boryanum, that do not form heterocysts. The results suggest that in species that form heterocysts a greater degree of cytoplasmic continuity is established, presumably to facilitate the exchange of metabolites. In species capable of forming heterocysts, the number of microplasmodesmata per septum between two adjacent vegetative cells remained constant whether the filaments were grown in the presence of NH₄ and lacked heteroxysts or under N₂-fixing conditions and contained heterocysts. When a vegetative cell differentiates into a heterocyst, about 80% of the existing microplasmodesmata are destroyed as the poles of the cell become constricted into narrow necks leaving smaller areas of contact with the adjacent vegetative cells.     

Dimethyl sulfoxide affects the amount of extrachromosomal spleen focus-forming virus DNA in murine erythroleukemia cells.

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.     

贝克水蚤对水华鱼腥藻的同化速率及其对藻丝数量的影响

在实验室条件下对贝克水蚤同化利用丝状蓝藻和控制藻丝数量的能力进行了测定。当喂以水华鱼腥藻和对照食物沼泽卵形隐藻的单种培养时,三节贝克水蚤和钩状贝克水蚤的雌性成体均可同化利用水华鱼腥藻,但与隐藻相比同化速率较低。三节贝克水蚤的雌性成体能够明显地减少鱼腥藻藻丝密度,即使在有相同生物量的隐藻存在的情况下也一样。这些结果与我们关于贝克水蚤利用丝状蓝藻的能力的其他研究的结果相一致。     

Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.     

Modeling filamentous cyanobacteria reveals the advantages of long and fast trichomes for optimizing light exposure

Cyanobacteria form a very large and diverse phylum of prokaryotes that perform oxygenic photosynthesis. Many species of cyanobacteria live colonially in long trichomes of hundreds to thousands of cells. Of the filamentous species, many are also motile, gliding along their long axis, and display photomovement, by which a trichome modulates its gliding according to the incident light. The latter has been found to play an important role in guiding the trichomes to optimal lighting conditions, which can either inhibit the cells if the incident light is too weak, or damage the cells if too strong. We have developed a computational model for gliding filamentous photophobic cyanobacteria that allows us to perform simulations on the scale of a Petri dish using over 10(5) individual trichomes. Using the model, we quantify the effectiveness of one commonly observed photomovement strategy--photophobic responses--in distributing large populations of trichomes optimally over a light field. The model predicts that the typical observed length and gliding speeds of filamentous cyanobacteria are optimal for the photophobic strategy. Therefore, our results suggest that not just photomovement but also the trichome shape itself improves the ability of the cyanobacteria to optimize their light exposure.     

Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.     

A virus-like agent associated with neurofibromatosis in damselfish

Damselfish neurofibromatosis (DNF) is a transmissible disease involving neurofibromas and chromatophoromas affecting bicolor damselfish Stegastes partitus on Florida reefs. Analysis of genomic DNA by Southern blotting techniques demonstrated the presence of a group of extrachromosomal DNAs in tumors from fish affected with DNF but not in healthy individuals. Cell lines obtained from tumors contained identical DNAs and were shown to be tumorigenic in vivo, while lines established from healthy fish did not contain such DNA and were not tumorigenic. These DNA patterns were also observed in experimentally induced tumors. A DNase resistant component of this DNA was isolated from both tumor cells and conditioned media of tumor cell lines suggesting that these sequences were encapsulated in viral particles. These data support the hypothesis that one or more of these extrachromosomal DNA forms is the genome of an unusual virus and that this virus is the etiologic agent of DNF. We have tentatively termed this agent the damselfish virus-like agent (DVLA).     

Rapid small-scale DNA isolation from filamentous cyanobacteria

Abstract A rapid small-scale DNA isolation procedure is described for the filamentous cyanobacteria, which yields enough chromosomal and plasmid DNA for restriction endonuclease digestions, Souther hybridizations, and electroelution from gels for further manipulation. DNA from seven strains of cyanobacteria were isolated and analyzed on agarose gels.     

Stable carbon isotope variability associated with taxonomic composition of lotic benthic algae

We examined the relationship between the δ¹³C and taxonomic composition of benthic algae collected from a riffle (fast current habitat) of a non‐shaded mountain stream, which is a tributary of the Kiso River, Japan. The benthic algal δ¹³C ranged from ?20.6 to ?14.2‰ and tended to be ¹³C‐depleted with increasing relative abundance of upright filamentous cyanobacteria and ¹³C‐enriched with increasing relative abundance of prostrate filamentous cyanobacteria. Using isotopic mass balance equations, the relative abundance of the dominant taxa, upright and prostrate filamentous cyanobacteria, small diatoms and others, explained 74% of δ¹³C variability. This study shows a case where the difference in taxonomic composition is a possible source of the isotopic variability of benthic algae, which is a mixture of taxa with distinct isotopic signatures.     

Fine-structural analysis of black band disease-infected coral reveals boring cyanobacteria and novel bacteria

Examination of coral fragments infected with black band disease (BBD) at the fine- and ultrastructural levels using scanning (SEM) and transmission electron microscopy (TEM) revealed novel features of the disease. SEM images of the skeleton from the host coral investigated (Montastraea annularis species complex) revealed extensive boring underneath the BBD mat, with cyanobacterial filaments present within some of the bore holes. Cyanobacteria were observed to penetrate into the overlying coral tissue from within the skeleton and were present throughout the mesoglea between tissue layers (coral epidermis and gastrodermis). A population of novel, as yet unidentified, small filamentous bacteria was found at the leading edge of the migrating band. This population increased in number within the band and was present within degrading coral epithelium, suggesting a role in disease etiology. In coral tissue in front of the leading edge of the band, cyanobacterial filaments were observed to be emerging from bundles of sloughed-off epidermal tissue. Degraded gastrodermis that contained actively dividing zooxanthellae was observed using both TEM and SEM. The BBD mat contained cyanobacterial filaments that were twisted, characteristic of negative-tactic responses. Some evidence of boring was found in apparently healthy control coral fragments; however, unlike in BBD-infected fragments, there were no associated cyanobacteria. These results suggest the coral skeleton as a possible source of pathogenic BBD cyanobacteria. Additionally, SEM revealed the presence of a potentially important group of small, filamentous BBD-associated bacteria yet to be identified.     

Effects of grazers' species identity on cyanobacteria in bitrophic and tritrophic food webs

Using laboratory microcosms, we studied direct and indirect interactions among different components of bi- and tritrophic communities. Filamentous cyanobacteria ( Phormidium sp.) and autotrophic flagellates ( Chlorogonium elongatum ) were primary producers. The second trophic level was represented by ciliates Furgasonia blochmanni and Pseudomicrothorax dubius grazing on the filamentous cyanobacteria and two filter feeders, Euplotes octocarinatus and Stylonychia pustulata , feeding on the autotrophic flagellates. An oligochaete, Chaetogaster sp., was used as the top predator. An experiment was carried out for all combinations of two factors: (1) the identity of the cyanobacteria consumer ( Furgasonia or Pseudomicrothorax ) and (2) the presence or absence of the top predator. Significant effects of the treatments on both the abundance of cyanobacteria and filter feeding ciliates and predator-induced defense in Phormidium and Euplotes were observed in a 36-day experiment. The experiment showed that the substitution of one species ( Furgasonia ) for another ( Pseudomicrothorax ) seemingly playing the same ecological role may lead to significant changes in the whole community.