Integration Pattern of Human JC Virus Sequences in Two Clones of a Cell Line Established from a JC Virus-Induced Hamster Brain Tumor

The physical state of the JC virus (JCV) genome was studied in two clonal cell lines (clones 2 and 7) derived from a tissue culture cell line (HJC-15) established from a hamster brain tumor induced by JCV. Saturation-hybridization and reassociation kinetic analyses, using in vitro (32)P-labeled JCV DNA, indicated that clone 7 and 2 cells contain 9 to 10 and 4 to 5 copies per cell, respectively, of all or most of the viral genome. Both cell DNAs were analyzed by using the Southern blotting procedure with three restriction endonucleases: XhoI, which does not cleave JCV DNA; EcoRI, which cleaves once; and HindIII, which cleaves three times. With each DNA, a variety of JCV-specific DNA fragments were detected. The following conclusions are possible: (i) JCV DNA is integrated into cell DNA in both clonal lines; (ii) both clonal lines contain multiple copies of the viral genome integrated in a tandem head-to-tail orientation; (iii) neither clonal line contains detectable free-form I, II, or III JCV DNA; (iv) each clonal line contains multiple independent sites of JCV DNA integration; and (v) most or all of the sites of integration on the cellular or the viral genome, or both, are different in clone 7 DNA than in clone 2 DNA. Thus, although both clone 7 and clone 2 cells were established from the HJC-15 tumor cell line, they differ in the copy number and integration pattern of JCV DNA.     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.     

克隆生长对被子植物传粉过程的影响

克隆植物与其传粉者的相互作用是植物繁殖生态学的重要研究领域之一。植物克隆生长与有性繁殖通常相伴进行, 往往产生较大的花展示与复杂的克隆空间结构, 通过传粉过程对有性繁殖过程产生影响, 共同决定植物的适合度。本文回顾了克隆生长对被子植物传粉过程影响的国内外研究进展, 从植物克隆大小、花资源空间配置、克隆构型与种群遗传结构四个方面讨论了克隆生长对传粉过程的影响及其生态学与进化生物学意义。早期研究预期, 随着克隆增大, 同株异花授粉水平增加, 因而通过增大自交率或花粉阻塞效应降低植物的适合度。但是, 后来的一些模拟与野外实验研究发现, 传粉者在同一克隆内访问的花数量并不会随克隆增大而一直增加, 访花行为也主要发生在分株内; 而且分子标记的自交率组分分析也表明自交主要发生在分株内。另一方面, 人工模型模拟以及传粉者访问行为研究表明, 当花朵数量相同时, 与所有花集中生长在同一分株上相比, 将花朵分散在多个分株上的克隆生长方式不会增加, 反而降低了同株异花授粉的发生水平。如果花序内花雌雄同熟, 花朵同时提供与接收花粉, 克隆生长会使植物接收到更高比例的异交花粉, 在提高后代质量的同时不增加同株异花授粉概率。这是从传粉生物学角度对植物克隆生长习性进化的一个全新的解释。今后, 克隆植物传粉生物学研究需要针对传粉者与克隆生长之间的相互作用建立理论模型, 探究克隆大小、克隆构型、花资源空间配置模式对传粉者访问频率和行为、花粉散布、交配格局的影响。同时, 需要在自然种群中, 尤其是克隆与非克隆的近缘类群、同一物种克隆与非克隆种群开展比较研究, 利用更高效的分子标记来研究克隆生长的生态与进化意义。     

Tumorigenic,invasive, karyotypic,and immunocytochemical characteristics of clonal cell lines derived from a spontaneous canine anaplastic astrocytoma

Summary Tumor cells from a spontaneously arising canine astrocytoma were isolated and cloned. Three clonally derived cell lines (DL3580 clone 1, DL3580 clone 2, and DL3580 clone 3) were developed and found to express glial fibrillary acidic protein (GFAP) as well as epidermal growth factor receptor (EGFR/c-erbB₁). The cell lines were tumorigenic as subcutaneous xenografts or as intracranial implants in athymic mice, or both. Both the monolayer astrocytoma cells and the xenograft tumor cells from clone 2 were aneuploid, with a modal number of 84 chromosomes per metaphase; clones 1 and 3 were also aneuploid with modal numbers of 82 and 75/79, respectively. The histology of both the initial spontaneously occurring tumor in the dog and the intracranial astrocytoma in athymic mice demonstrated features of diffuse infiltration into normal brain. These newly developed canine glioma cell lines are karyotypically stable for 1 yr in culture and carry the same marker chromosomes as the parental lines. These glioma cell lines may serve as models for investigating mechanisms of glioma invasion into brain. Additionally, clonal cell lines with divergent properties isolated from the same tumor may assist in studies of the molecular basis of astrocytoma progression and heterogeneity.     

Clonal variation and aging of diploid fibroblasts : Cinematographic studies of cell pedigrees

Time-lapse cinematographic (TLC) analysis of clones of human diploid fibroblasts indicate heterogeneity in clonal division behaviour. Variations are noted in interdivision time, clone size and generations per clone. Correlation coefficients for interdivision times of sister pairs are high in young clones and generally low in aged clones. A consistent division pattern at all population doubling levels is one of low average interdivision time for early and late generations of a clone and high average interdivision time for the middle range of generations of a clone. The clonal division patterns observed experimentally have been duplicated in computer simulated pedigrees. The computer model is based on an oscillating system which allows for flux of regulator substances. The critical concentrations of regulator substances determine the clonal division pattern for a given progenitor cell.     

Influence of Cell Size and DNA Content on Growth Rate and Photosystem II Function in Cryptic Species of Ditylum brightwellii

DNA content and cell volume have both been hypothesized as controls on metabolic rate and other physiological traits. We use cultures of two cryptic species of Ditylum brightwellii (West) Grunow with an approximately two-fold difference in genome size and a small and large culture of each clone obtained by isolating small and large cells to compare the physiological consequences of size changes due to differences in DNA content and reduction in cell size following many generations of asexual reproduction. We quantified the growth rate, the functional absorption cross-section of photosystem II (PSII), susceptibility of PSII to photoinactivation, PSII repair capacity, and PSII reaction center proteins D1 (PsbA) and D2 (PsbD) for each culture at a range of irradiances. The species with the smaller genome has a higher growth rate and, when acclimated to growth-limiting irradiance, has higher PSII repair rate capacity, PSII functional optical absorption cross-section, and PsbA per unit protein, relative to the species with the larger genome. By contrast, cell division rates vary little within clonal cultures of the same species despite significant differences in average cell volume. Given the similarity in cell division rates within species, larger cells within species have a higher demand for biosynthetic reductant. As a consequence, larger cells within species have higher numbers of PSII per unit protein (PsbA), since PSII photochemically generates the reductant to support biosynthesis. These results suggest that DNA content, as opposed to cell volume, has a key role in setting the differences in maximum growth rate across diatom species of different size while PSII content and related photophysiological traits are influenced by both growth rate and cell size.     

The Use of Simulated Annealing in Chromosome Reconstruction Experiments Based on Binary Scoring

We present a method of combinatorial optimization, simulated annealing, to order clones in a library with respect to their position along a chromosome. This ordering method relies on scoring each clone for the presence or absence of specific target sequences, thereby assigning a digital signature to each clone. Specifically, we consider the hybridization of oligonucleotide probes to a clone to constitute the signature. In that the degree of clonal overlap is reflected in the similarity of their signatures, it is possible to construct maps based on the minimization of the differences in signatures across a reconstructed chromosome. Our simulations show that with as few as 30 probes and a clonal density of 4.5 genome equivalents, it is possible to assemble a small eukaryotic chromosome into 33 contiguous blocks of clones (contigs). With higher clonal densities and more probes, this number can be reduced to less than 5 contigs per chromosome.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Clonal Variation in Growth and Flavour Production in Tissue Cultures of Allium cepa L.

Callus cultures of onion were initiated from seedling root tissueof three varieties, Rijnsburger, White Lisbon and Red Italian.Twenty separate clones of each variety were sub-cultured fornine passages and estimates made of growth, friability, sliminessand pigmentation at each sub-culture; measurement of alliinaseactivity and precursor level was made at the ninth sub-culture.A number of clones showing a uniformity in one or more charactersappeared but clonal characteristics could not be correlatedwith alliinase activity or flavour precursor levels. The alliinaseactivity was comparable to the normal plant but the precursorlevel was 2–10 per cent of that in the plant. The workshows that despite the large number of separate cultures establishedand the development of stable clonal lines, it was not possibleto select out a clone able to produce a high level of the onionflavour compounds.     

Density-dependent growth responses in two clonal herbs: regulation of shoot density

Summary It has been shown in clonal perennial herbs that shoot natality decreases, and shoot mortality increases, in stands of increasing density. In a two-year garden experiment, we have tested Hutchings' (1979) hypothesis that these responses are the result of physiological integration, i.e. the exchange of resources and growth substances between shoots of a single clone. Dense monocultures of two rhizomatous graminoids, Brachypodium pinnatum and Carex flacca, were created that differed more than 10-fold in the density of clones (genets), but that had similar densities of shoots. A more effective shoot density control was expected in stands with the smaller clone densities (larger clones) due to more extensive clonal connections. Shoot turnover was evaluated by counting living and dead shoots at different times. In the summer of the second year, when shoot densities and stand structure were similar between treatments, shoot natality (the number of shoots born per plot) and shoot mortality (the number of shoots that died per plot) were usually unrelated to clone density in either species. If there was a significant treatment effect, it could be attributed to (small) differences in shoot density. Over the whole range of shoot densities, natality was negatively density-dependent. The number of shoots that died in a given growth period was proportional to the number of shoots present, suggesting that mortality rates were density independent. In Carex, however, there were some indications that mortality rate increased with increasing density. Our study confirms that clonal herbaceous species can effectively prevent an overproduction of shoots, but in contrast to Hutchings' (1979) propositions, we found no evidence that physiological integration may be the responsible mechanism. An alternative explanation for the observed patterns is proposed.     

The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells

Epstein-Barr virus (EBV) is an exceptionally successful human viral pathogen maintained as a licensed, plasmid replicon in proliferating cells. We have measured the distributions of EBV-derived plasmids in single live cells throughout the cell cycle in the absence of selection and confirmed the measured rates of duplication and partitioning computationally and experimentally. These analyses have uncovered a striking, non-random partitioning for this minimalist plasmid replicon and revealed additional properties of it and its host cells: (1) 84% of the plasmids duplicate during each S phase; (2) all duplicated plasmids are spatially colocalized as pairs, a positioning that is coupled to their non-random partitioning; (3) each clone of cells requires a certain threshold number of plasmids per cell for its optimal growth under selection; (4) defects in plasmid synthesis and partitioning are balanced to yield wide distributions of plasmids in clonal populations of cells for which the plasmids provide a selective advantage. These properties of its plasmid replicon underlie EBV's success as a human pathogen.     

松嫩平原不同生育期虎尾草无性系构件生长与生物量分配

以松嫩平原不同生育期的虎尾草无性系为研究对象,分别在虎尾草无性系拔节期和完熟期进行大样本取样,并对其地上、地下各构件的数量性状及生物量分配进行统计分析,研究虎尾草在不同生育期的生长特性及生长策略.结果表明:虎尾草株高、总根长、总根表面积、根体积、地上生物量、地下生物量、总生物量在两个生育期之间均存在显著差异.在两个生育期,地上、地下生物量分配与总生物量呈显著幂函数异速生长关系.在拔节期,总生物量与分株数、总根长、总根表面积和根体积呈显著线性同速生长关系;而在完熟期,均呈显著幂函数异速生长关系.虎尾草无性系在不同生育期存在着不同的生长策略,在拔节期主要采取的是无性系外部空间的优先扩展策略,而在完熟期主要采取的是无性系内、外部空间兼顾的补充和扩展策略.     

EFFECTS OF FIRE ON CLONAL GROWTH AND DYNAMICS OF PITYOPSIS GRAMINIFOLIA (ASTERACEAE)

The rhizomatous perennial Pityopsis graminifolia was studied in a Florida sandhill community in an annually burned site, a periodically burned site, and a site that has been protected from fire since 1965. These different fire regimes significantly affected the demography and life histories of both plants and plant parts in this clonal species. Fires resulted in reductions in ramet biomass and height, and an increase in the (root + rhizome)/shoot biomass ratio. Burning also decreased the total number of flower heads and new rhizomes produced per ramet. However, the survivorship of initiated rhizomes was greater in burned sites and resulted in a larger number of established daughter ramets per clone. As a result, in burned sites there was a shift in clone structure toward larger numbers of smaller ramets, but there were no significant reductions in seed or rhizome production on a per genet basis. The results showed that the responses to fire in P. graminifolia are different when measured at the genet vs. ramet level and that the effects of fire on clones can be explained by demographic responses of plant parts. Population regeneration in the study sites was dependent on successful clonal ramet production because no seedling recruitment was observed. This suggests that disturbances other than fire are important for new genet recruitment in these clonal populations.     

松嫩平原贝加尔针茅无性系构件的结构及生长规律

采用整个分蘖丛挖掘的取样方法,对松嫩平原栽培条件下贝加尔针茅无性系构件的结构,以及生长与生产规律进行了定量分析.结果表明,在9月末停止生长期,经过2个生长季的营养繁殖,贝加尔针茅无性系的丛径为9.4±3.24 cm.无性系的全体构件数为161.±8.2个.其中,生殖分蘖株为14.6±11.48个,占9.2%;营养分蘖株为146.9±78.70个,占90.7%.全体构件总生物量为3.8±34.22 g,其中生殖分蘖株生物量为2.0±20.34 g,占43.7%;营养分蘖株为28.8±19.43 g,占6.2%.随着丛径的增加,不同构件的数量均具有线性同速生长规律,而不同构件的生物量均具有幂函数异速增长规律.不同构件生物量与无性系全体构件的数量和生物量之间均呈显著(P<0.0)或极显著(P<0.01)的幂函数正相关关系.平均单个生殖分蘖株的生产力约为营养分蘖株的10倍.生殖分蘖株的数量和生物量的表型可塑性普遍大于营养分蘖株.     

Promotion of initiated cells by radiation-induced cell inactivation

Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.     

Clonal diversity and structure of the invasive aquatic plant Eichhornia crassipes in China

The information on diversity and spatial distribution of clones of an invasive clonal plant is crucial for the understanding of its clonal structure and invasive history. In this paper, random amplified polymorphic DNA (RAPD) markers were used to explore the clonal diversity and clonal structure of Eichhornia crassipes (Mart.) Solms in natural populations, and their possible effects on the plant success as an invader are also discussed. Five populations covering the entire distribution area in China were studied, sampling 43 individuals per population at an interval of 1 m in a sampling plot. Twelve RAPD primers produced 69 reproducible bands, with 22 being polymorphic. Only five RAPD phenotypes (clones) were detected in these five populations, but each population consisted of at least three clones, contrary to the traditional expectations that E. crassipes populations should be monoclonal. The diversity of clones within populations is thought to be mainly resulted from multiple introductions by humans. The evenness of distribution of clones varied slightly and most clones were widespread, suggesting clonal growth is the predominant mode of regeneration in all the populations. A single clone dominated each population and this clone might be the first one introduced into China or the genotype with a higher phenotypic plasticity, which could survive and reproduce via clonal growth in various habitats. The clones in each population were highly intermixed, especially in river populations, suggesting this species has a guerilla clonal structure which can be facilitated by water current.     

An isozyme study of clone diversity and relative importance of sexual and vegetative recruitment in the grass Brachypodium pinnatum

We asked whether differences in abundance and seed prodtiction of Brachypodium pinnatum after 16 yr of four different experimental land use regimes were reflected in differences in vegetative dispersal distance, clone diversity, clone area, and the proportions of sexual and vegetative recruitment. Mean vegetative dispersal distance was 5.5 mm yr'. Electrophoresis of 5 polymorphic isozyme loci of 20 tillers sampled at defined positions in each of twelve 1 × 6 m sampling areas (3 per treatment) revealed considerable clonal diversity. Per sampling area we found on average 9.98 enzyme phenotypes (clones), mean Simpson index was 0.825. and mean Shannon index 0.801. The mean ratio of sexual vs vegetative recruitment was about 1:32000. Despite this low ratio, clonal diversity within the population of" B. pinnatum was higher than reported for other clonal plant populations, possibly because of its high ramet densities. Moan clone area was 5.73 m²-. i.e. mean clone radius was 1.35 m. None of the 10 pairwise correlations between abundance and seed production on the one hand, and number of clones per plot sample, plot Simpson index, plot Shannon index, ratio of vegetative vs sexual recruitment, and clone area on the other, was significant. Mean clone radius was 245 times larger than the mean distance of yearly vegetative dispersal which suggests old ages and low turnover rates of clones. The time scale of the inert response of clonal diversity of B. pinnatum to changes in land use appears to largely exceed the experimental period of 16 yr.     

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepato-cellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chro-mosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome hetero-geneity and evolution.     

In situ hybridization analysis of polyoma DNA replication in an inducible line of polyoma-transformed cells.

In situ hybridization has been used to study polyoma DNA replication in a clonal derivative of the inducible LPT line of polyoma-transformed cells designated as clone 1A. This study has shown that in clone 1A cultures maintained under normal growth conditions, 4–25 in 10,000 cells are spontaneously induced to synthesize polyoma DNA at an enhanced rate. In cultures exposed to mitomycin C (MMC), the percentage of induced cells remains approximately equal to the spontaneous level for 9 hr, and then increases for at least 24 hr up to 30–57% as more and more cells are asynchronously recruited to replicate the virus DNA.DNA reassociation kinetics and in situ hybridization have been used to determine the amount and distribution of polyoma DNA accumulated within clone 1A cells. These measurements have shown that a single induced cell in an MMCtreated culture produces 24,500 genome-equivalents of the virus DNA; second, that the average yield of virus DNA in a normally growing culture is only 41.7 genome-equivalents per cell; however, a single spontaneously induced cell in this culture produces as much virus DNA as an MMC-induced cell; third, that all the virus DNA molecules are found within the nuclei and many are clustered in aggregates containing up to 2000 genome-equivalents. We discuss the implications of these findings regarding the regulation of polyoma DNA replication in the LPT line.