High molecular weight DNA from fixed cytogenetic preparations.

Cell pellets that have been stored in routine clinical cytogenetic fixative were studied for the presence of intact DNA. A method for the isolation of high molecular weight DNA from fixed cytogenetic preparations of human leukocytes, bone marrow, and cell hybrid cultures is presented. DNA preparations from fixed pellets were cleaved with restriction enzymes, transferred to nitrocellulose filters after agarose gel electrophoresis, and hybridized to radiolabeled probes to demonstrate that fixed cell pellets could yield DNA of sufficient quality for Southern blot hybridization analysis. This protocol may be useful for molecular analysis of DNA from fixed cell pellets of patients who are unavailable for additional sampling.     

Multisample enzyme extraction from cultured plant cell suspensions

Operational and constructional details are given of an apparatus which permits simultaneous enzyme extraction from up to 16 cell samples under standardized conditions. The main advantages of the method are: (a) individual samples remain in the same container throughout the whole operation; (b) the samples can be stirred continuously and uniformly during extraction; (c) rapid changes of extractant are facilitated by a simple draining operation; (d) the extraction temperature can be strictly controlled. The method is rapid, efficient, and reproducible in extracting a number of enzymes from cultured rose cells. Other uses of the apparatus are discussed, particularly in relation to the extraction of protein, nucleic acids, and some low molecular weight materials.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Plant regeneration from cell suspensions of spinach

Friable calluses induced from root segments of spinach (Spinacia oleracea L.) with a high amount of growth regulators (indole-3-acetic acid 48.52 μM and gibberellic acid 10 μM) were suspended in liquid medium. The cell fraction sized between 100 and 200 μm was used to establish suspension cultures. Adventitious shoots and roots were obtained from the suspensions (3.2 x 10⁵ cells per ml) by procedures comprising successive subcultures on two or three different media. In both of these procedures, the composition of the second culture medium (concentrations of plant growth regulators) had a key influence on the organogenesis of the suspensions. Regenerated shoots elongated and rooted on different solid media. Plantlets transplanted in soil grew and developed normally until flowering and produced seeds. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA extraction from crayfish exoskeleton

Crayfish exoskeleton (CE) samples are generally less invasive and easy to be collected. However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions. Specific primer pair (PPO-F, PPO-R) was used to amplify extracted DNA from CE, and compared to crayfish tail muscle DNA sample. Moreover, seven microsatellites markers were used to amplify the CE DNA samples set. Since the extracted DNA from CE is suitable for gene amplification, the results present usefulness of CE as an easy and convenient DNA source for PCR-based population genetic research.     

Embryogenesis and ribosomal DNA in carrot cell suspensions cultured in vitro

The contents of nucleic acids and rDNA were estimated during the development of carrot cell suspensions cultured under two different conditions. The cells transferred from stock culture to the medium without 2,4-dichlorophenoxyacetic acid (2,4-D) induced the embryogenesis (embryogenic culture), while the cells inoculated to the medium with 0.2 mg/l 2,4-D did not form any embryos (non-embryogenic culture). The ratio of RNA to DNA of both cultures increased in the early stage of the culture. The rise of the ratio in embryogenic culture was much higher than that in non-embryogenic culture, which showed that embryogenic culture accumulated RNA prior to the formation of embryos. The rDNA amount of non-embryogenic culture remained constant throughout the culture period. Although embryogenic culture showed a slight change in rDNA amount, the differences were at most 12% and the quantitative stability of the rDNA was demonstrated during the development of carrot cell suspension cultures.     

A novel glucosyltransferase from Catharanthus roseus cell suspensions

The potential applications of glycosyltransferases in glycoconjugate synthesis have attracted considerable interest from the biotechnology community in recent years. In this work, we present a novel glucosyltransferase from Catharanthus roseus cell cultures. The enzyme was purified to one spot in SDS-polyacrylamide gel electrophoresis, and its molecular weight was about 51 kDa. The optimum temperature was 35 °C, and the optimum pH was 7.6. Sodium ion has weak effect on enzyme activity, whereas divalent ions inhibit enzyme activity strongly. The K_m values were 0.112, 0.077, 0.064 and 1.0 mM for scopoletin, 5,7-dihydroxyflavone, 5,7-dihydroxyflavanone and UDPG, respectively. Substrate screening with the purified enzyme was performed against a range of phenolic compounds using UDPG as sugar donor. The enzyme showed activity towards a number of coumarins including umbelliferone, scopoletin, isoscopoletin and esculetin, and flavonoids including a flavone, a flavanone and chalcones. No activity was detected with compounds characterized by a single aromatic ring, i.e. simple and acidic phenols. The substrate specificity and the regioselectivity suggest enzyme structural features that are different from those of other glucosyltransferases.     

Satellite DNA and cytogenetic evolution

The genusDipodomys (kangaroo rats) exhibits major interspecies variations in the proportions of highly reiterated satellite DNA sequences in the genome as well as in the chromosome number and the proportions of uni-armed and bi-armed chromosomes. For nearly all of the approximately 22 species of the genus and several subspecies, liver DNA was distributed in neutral CsCl buoyant density gradients into four fractions: principal DNA (1.698 g/ml), intermediate-density DNA (1.702 g/ml), MS satellite (1.707 g/ml) and HS (heavy) satellites (1.713 g/ml). The total nuclear DNA content of diploid liver cells measured in eleven species by quantitative cytophotometry, ranged from 6.9 to 10.9 pg. These data were correlated with known features of the karyotypes of individual species. The salient findings were: (1) that interspecies variations in diploid chromosome number cluster at 52–54, 60–64 and 70–72 (2) that high total nuclear DNA was associated with high chromosome number, and with relatively large amounts of satellite DNA (3) that a high ratio of HS satellites to intermediate-density DNA was generally correlated with a predominance of metacentric and submetacentric chromosomes (high fundamental number). The relationships of satellite DNA to karyotype structure reveal a new level of hierarchy in the genome that appears capable of exerting global control over environmental adaptation and the evolution of new species. This mechanism is consistent with recent hypotheses that changes in the macro-structure of the genome are more important than point mutations in facilitating the rapid phases of animal evolution.     

Microbial DNA extraction from intestinal biopsies is improved by avoiding mechanical cell disruption

Currently, standard protocols for microbial DNA extraction from intestinal tissues do not exist. We assessed the efficiency of a commercial kit with and without mechanical disruption. Better quality DNA was obtained without mechanical disruption. Thus, it appears that bead-beating is not required for efficient microbial DNA extraction from intestinal biopsies.     

陈旧标本DNA提取方法

对于自然环境中的或长期保存的动物标本,由于保存环境不良或保存时间过长,DNA提取的难度较大。受标本保存时间和损害程度等因素影响,导致实验结果的不稳定性加强,对于同一标本需要反复实验。为了提高DNA提取效率,节省实验成本,现对陈旧损坏标本的DNA提取方法进行综述。     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

High-throughput DNA extraction from forest trees

It is difficult to extract pure high-quality DNA from trees, which may not be amenable to advances in extraction methods suitable for other plants. A new commercial high-throughput DNA extraction system, using a silica binding matrix for purification and a multisample mixer mill for tissue disruption, was evaluated for its suitability withEucalyptus spp.,Pinus spp., andAraucaria cunninghamii (hoop pine). DNA suitable for a range of molecular biology applications was successfully extracted from all genera. The method was highly reliable when tested in more than 500 preparations and could be adapted to different tree species with relatively minor modifications.     

石蜡包埋组织中DNA的提取

从石蜡包埋组织中提取DNA,是进一步研究抑癌基因的基础,而石蜡包埋组织中提取到的DNA往往已被降解成小分子且量微,作者经多次摸索试验,改进了一些步骤,取得了较好的抽提效果。     

Rapidly-labelled RNA species isolated from cell suspensions ofDaucus carota

Summary Carrot cells in suspension culture were incubated during the log-phase of the culture transfer cycle for different periods with one of the following precursors of nucleic acid synthesis: [^32P]-orthophosphate, [5,6-³H]-uridine, and [2-¹⁴C]-uridine. Cells were gently broken by a short period of sonication, and the total RNA of the cells was extracted by a phenoldetergent method at pH 9.0. Subsequently, crude RNA was purified from contaminating substances like carbohydrates and nucleotides, and the pure RNA preparations were characterized by MAK-chromatography and constant velocity sedimentation in isokinetic sucrose gradients.Rapidly-labelled RNA-fractions were detected in the radioactive profiles obtained with both separation methods. These RNA-fractions showed a high specific incorporation rate, but almost no detectable UV-absorbance,i.e., they are RNA species with a high turnover rate and represent only a small part of the total RNA of the cell. With increasing periods of labelling and in a series of pulse-chase experiments high molecular weight RNA-fractions released by high-salt washing of MAK-columns exhibited a shift of the incorporated radioactivity from fractions with higher to those of lower molecular weights. Furthermore, in sucrose gradients a similar shift was observed for RNA-fractions with estimated sedimentation coefficients of 50 S, 40 S, 34 S and 22 S; the radioactivity was converted from these high to the low S-values of the 26 S and 18 S rRNAs, respectively. This parallel in the behaviour of the high molecular weight RNA-fractions from both separation methods indicates their putative role as precursors of rRNA-synthesis. Moreover, there is evidence that the high molecular weight RNA-fractions from the MAK-columns which were eluted after the 26 S rRNA consist not only of the precursors of rRNAs, but also of polydisperse RNA-fractions with S-values smaller than 18 S. These probably contain fractions of HnRNA and mRNA.     

Photosynthetic studies with leaf cell suspensions from higher plants

A technique for obtaining intact mesophyll cell suspensions derived from higher plant leaves is described. A large number of taxonomically unrelated plants were found suitable for cell `extraction' including several plant species from monocotyledonous group.