Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell⁻¹ was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell⁻¹.     

Flow Cytometry Estimation of Nuclear Size and Ploidy Level of Habituated Calli of Sugar Beet

A fully habituated (auxin- and cytokinin-independent) self-regenerating (organo-genic) sugar beet cell line (HO) and a fully habituated non-organogenic one (HNO) derived from the former one, were analyzed as to their nuclear size and DNA content. Flow cytometry and image analysis were used and cells of certified diploid leaves of the same sugar beet strain served as controls. The HNO cells had been shown previously to have many characteristics of cancerous cells. The analyses made on leaves and HNO cells indicated the presence of only one population of cycling cells. In HO cells, two cycling populations were detected: the first one had the same DNA content as the leaves while the second one contained two fold more DNA than the first population. HNO cells showed the higher nuclear size and DNA content. HNO cells also showed evidence of aneuploidy. Thus, nuclear size, DNA content and ploidy level increase together with the neoplasic progression to culminate in HNO cells with the loss of organogenic totipotency. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of Intraspecific DNA Content Variation in Zea mays L. by Flow Cytometry

Flow cytometry was used to determine quantitative intraspecificDNA content variation in Zea mays. Previous studies using flowcytometry had indicated that intraspecific variation in cornwas beyond the resolution of the method. The DNA content variationamong corn lines observed in this study was in agreement withthe amount of variation observed using microdensitometry. Inorder to observe intraspecific variation, the fluorochrome DAPIwas shown to be superior to mithramycin. The fluorochrome: nucleiratio was found to be critical when DAPI was used because ofself absorption of the fluorescence. Flow cytometry with thefluorochrome DAPI was found to be a rapid and reliable alternativeto microdensitometry in examining intraspecific DNA contentvariation in corn. Key words: Genome size, corn (maize), Zea mays, DAPI     

利用405nm激光激发Hoechst33342染色细胞DNA的流式细胞技术

目的:探讨流式细胞仪上405 nm激光激发Hoechst33342染色细胞DNA的效果及影响检测结果的因素。方法:SW480和A549两种细胞经Hoechst33342染色后,流式细胞仪405 nm激光激发检测DNA含量,利用软件计算出处于G0/G1期、S期和G2/M期细胞的百分比,以PI染色法结果作为对照。结果:SW480和A549细胞经Hoechst33342染色后各期的细胞百分比与PI染色法基本一致,无明显差异(P0.05)。结论:405 nm激光激发Hoechst33342染色细胞DNA结果可靠,可作为紫外检测的替代方法。     

应用双光子显微镜和流式细胞仪定性及定量确定小鼠巨噬细胞的吞噬功能

建立了流式细胞仪和双光子激光共聚焦荧光显微镜进行定性和定量检测小鼠巨噬细胞吞噬鸡红细胞的方法,并同传统光学显微镜细胞化学染色观察方法相比较,探讨其检测巨噬细胞吞噬效应的优越性。常规方法获取小鼠腹腔和脾脏巨噬细胞,制备巨噬细胞悬液。常规制备鸡红细胞,计数并调整活细胞数,用5-二醋酸羧基荧光素琥珀酸单胞菌酯(5-carboxyfluorescein diacetate succinimidyl ester,CFSE)染色,与巨噬细胞共温育一定时间后,小鼠巨噬细胞特异性荧光抗体F4/80标记巨噬细胞。应用流式细胞仪检测巨噬细胞中CFSE阳性百分率来表示巨噬细胞吞噬率;应用双光子显微镜观察被吞噬的CFSE阳性鸡红细胞动态分布情况。同时,采用传统光学显微镜吉姆萨染色观察巨噬细胞吞噬百分率。结果显示,流式细胞仪结合双光子显微镜检测巨噬细胞吞噬率与传统的显微镜计数法比较,两者有明显的正相关性。双光子显微镜和流式细胞仪可以定性与定量检测巨噬细胞吞噬功能,该方法具有灵敏、快捷、重复性好以及准确率高的特点,是进行免疫学研究的可行方法。     

斑鳢、乌鳢及其杂种细胞核DNA流式含量分析

以斑鳢(Channa maculata)、乌鳢(C.argus)及其正交杂种斑乌鳢(斑鳢♀×乌鳢♂)和反交杂种乌斑鳢(乌鳢♀×斑鳢♂)的红细胞为材料,以鸡(Gallus gallus)血细胞为DNA标准(2.5 pg/2c,2c指2倍体),采用流式细胞仪测定了这4种鱼的细胞核DNA含量。斑鳢、乌鳢、斑乌鳢及乌斑鳢这4种鱼血细胞DNA的绝对含量分别为(1.488±0.035)pg/2c、(1.489±0.034)pg/2c、(1.522±0.077)pg/2c和(1.520±0.033)pg/2c。斑鳢和乌鳢的细胞核DNA含量差异不显著(P0.05),斑鳢和乌鳢与两种杂交鳢的DNA含量差异显著(P0.05),两种杂交鳢之间的细胞核DNA含量差异不显著(P0.05)。杂交鳢细胞核DNA含量显著高于亲本,可以作为杂种鉴定的依据。     

流式细胞术检测胃癌前病变DNA及p21，p53含量

应用流式细胞术和免疫荧光技术,对80例胃癌前病变细胞中DNA含量,rasp21和p53蛋白进行定量检测,探讨其在胃癌前病变中,作为癌变早期标志物的临床意义.检测结果发现,胃癌前病变的DNA异倍体率随不典型增生病变的分级增高而增加, rasp21和p53蛋白的表达量亦随胃癌前病变不典型增生程度的加重而增高.DNA异倍体和rasp21表达阳性的胃癌前病变出现的癌变率显著增高,提示可能是癌变早期的分子标志物.     

流式细胞术BrdU／DNA双染法测定云芝糖肽诱导的Molt-4细胞周期阻滞

目的：探究云芝糖）Ik（PSP）对人急性淋巴母细胞白血病Molt-4细胞周期的影响。方法：采用流式细胞术BrdU／DNA双染法获得各时相细胞分布状况和细胞周期的动力学参数。结果：0．1mg／mlPSP处理12h后,G2／M期细胞百分比由对照组的11．09％减少至3．69％。DNA合成时间由12．10h延长至108．40h。24h处理组中,S期细胞百分比由对照组的43.29％增加至67．26％,而G0／G1期和G2／M期细胞百分比均减少,G0／G1期细胞百分比由对照组的37．47％减少至27．43％,G2／M期细胞百分比由对照组的19．24％降低至5.31％。DNA合成时间更是由11．95h延长至114．52h。结论：PSP对人急性淋巴母细胞白血病Molt-4细胞周期的阻滞作用在于S期．该作用与DNA合成抑制有关。     

Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria

Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.     

Unexpected Inheritance Pattern of Erianthus arundinaceus Chromosomes in the Intergeneric Progeny between Saccharum spp. and Erianthus arundinaceus

Erianthus arundinaceus is a valuable source of agronomic traits for sugarcane improvement such as ratoonability, biomass, vigor, tolerance to drought and water logging, as well as resistance to pests and disease. To investigate the introgression of the E. arundinaceus genome into sugarcane, five intergeneric F₁ hybrids between S. officinarum and E. arundinaceus and 13 of their BC₁ progeny were studied using the genomic in situ hybridization (GISH) technique. In doing so, we assessed the chromosome composition and chromosome transmission in these plants. All F₁ hybrids were aneuploidy, containing either 28 or 29 E. arundinaceus chromosomes. The number of E. arundinaceus chromosomes in nine of the BC₁ progeny was less than or equal to 29. Unexpectedly, the number of E. arundinaceus chromosomes in the other four BC₁ progeny was above 29, which was more than in their F₁ female parents. This is the first cytogenetic evidence for an unexpected inheritance pattern of E. arundinaceus chromosomes in sugarcane. We pointed to several mechanisms that may be involved in generating more than 2n gametes in the BC₁ progeny. Furthermore, the implication of these results for sugarcane breeding programs was discussed.     

黄瓜属不同倍性异源多倍体的形态及生理特性分析

以黄瓜属3种不同倍性异源多倍体为试验材料,比较分析它们的形态和生理特性与基因组剂量的关系,为进一步研究黄瓜属基因组剂量效应、探讨植物多倍体进化机理奠定基础。结果表明:(1)黄瓜属异源四倍体与种间杂种F1相比,其叶片厚度、主蔓直径等性状随基因组剂量的增加而增大,而果实大小、主蔓节间长以及果瘤果刺的大小随基因组剂量的增加而减小。(2)在异源三倍体中,叶片厚度和主蔓直径等表型性状也表现出一定的基因组剂量效应。(3)基因组剂量的变化会引起黄瓜属异源多倍体中叶绿素含量、POD活性以及IAAi、PA和ZR等内源激素的变化。     

斑茅两个看家基因片段的克隆及其在基因芯片中的应用

根据已发表的同源基因序列,利用RT-PCR技术分离了斑茅(Erianthus arundinaceus)的GAPDH和APRT两个看家基因片段,用它们作为cDNA芯片阳性参照,以未经聚乙二醇(PEG)胁迫处理的斑茅叶片为对照,和PEG胁迫的4组材料同cDNA芯片进行杂交分析。杂交结果显示,GAPDH杂交的Cy5与Cy3平均信噪比(Signal/Noise,S/N)分别为56.12和60.8,APRT杂交的Cy5与Cy3平均信噪比分别为51.06和47.25,信噪比均很高;同时两个看家基因的杂交都显示出极强的信号,其中GAPDH的杂交信号值大于10000,APRT也在8000以上,杂交结果可靠。分析了PEG胁迫4个时段BADH与两个看家基因的表达,BADH的表达有明显变化,而看家基因表达均较稳定。上述结果表明所克隆的两个看家基因在斑茅中表达量高,且PEG胁迫下表达较为稳定,是基因芯片理想的阳性参照。     

Identification and characterisation of sugarcane intergeneric hybrids,Saccharum officinarum x Erianthus arundinaceus,with molecular markers and DNA in situ hybridisation

Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.     

基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。     

Differentiation of Phytophthora infestans Sporangia from Other Airborne Biological Particles by Flow Cytometry

The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen Phytophthora infestans from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of Alternaria or Botrytis spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between P. infestans sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed.     

Determination of Total Protein Content of Bacterial Cells by SYPRO Staining and Flow Cytometry

An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell⁻¹ (r² = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell⁻¹.     

流式细胞术BrdU/DNA 双染法测定云芝糖肽 诱导的Molt-4 细胞周期阻滞

目的:探究云芝糖肽(PSP)对人急性淋巴母细胞白血病Molt-4细胞周期的影响。方法:采用流式细胞术BrdU/DNA双染法获得各时相细胞分布状况和细胞周期的动力学参数。结果:0.1 mg/mlPSP处理12 h后,G2/M期细胞百分比由对照组的11.09%减少至3.69%。DNA合成时间由12.10 h延长至108.40 h。24 h处理组中,S期细胞百分比由对照组的43.29%增加至67.26%,而G0/G1期和G2/M期细胞百分比均减少,G0/G1期细胞百分比由对照组的37.47%减少至27.43%,G2/M期细胞百分比由对照组的19.24%降低至5.31%。DNA合成时间更是由11.95 h延长至114.52 h。结论:PSP对人急性淋巴母细胞白血病Molt-4细胞周期的阻滞作用在于S期,该作用与DNA合成抑制有关。     

Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus.

Erianthus arundinaceus has great potential as a germplasm source for better ratoonability, vigour, tolerance to environmental stresses, and disease resistance in sugarcane. Many unsuccessful attempts have been made to introduce these characters into modern sugarcane cultivars. We report on significant progress made since molecular tools were implemented. Sequence-tagged PCR, revealing size variation in the 5S rDNA cluster, was performed on intact leaf tissue to identify genuine hybrids six weeks after germination. This early screening of seedlings avoids the loss of genuine hybrids due to competition with selfed progeny. Of 96 crosses made involving female Saccharum officinarum or sugarcane cultivars (Saccharum spp.) and male E. arundinaceus, 26 were fertile producing 1328 seedlings. Thirty-seven genuine hybrids were unequivocally identified but only 19 have survived. Genuine hybrids were produced from only three crosses, all involving S. officinarum as the female parent. Chromosome elimination was observed in all seven hybrids analyzed using genomic in situ hybridization (GISH). Very little cross-hybridization was observed between the genomes of the two species after GISH, confirming recent molecular studies which showed that E. arundinaceus is quite distant from the genus Saccharum. The major limitation in the introgression of E. arundinaceus resides now in the apparent sterility of the hybrids.     

17种金线鲃核DNA含量及倍性的研究

采用血涂片、Feulgen染色和显微分光光度技术 ,测定了金线属 (Sinocyclocheilus) 17个种的核DNA含量。结果显示 ,除侧条金线 (S .lateristritus)中采自云南沾益炎方山那边的样本之 2C值为 7 79pg外 ,其余的核DNA 2C值都集中分布在 4 19～ 4 86pg范围 ,大体与其近缘四倍体种 2C值相同或相近 ,是其近缘二倍体种 2C值的大约 2倍。据此我们推断 ,这 17种金线很可能都是四倍体 ,个别种还含有八倍体的类型 ,金线属可能是整属的四倍体起源。     

Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC₄(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.