共查询到20条相似文献,搜索用时 15 毫秒
1.
The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to
the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation
of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation,
(2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous
plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques
are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method,
was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means
for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique.
We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by
which to transform bacteria. 相似文献
2.
The competitive binding of anthracycline antitumour drugs, [daunomycin (DAU), doxorubicin (DOX) or nogalamycin (NOG)], with
caffeine (CAF) to a model DNA oligomer has been investigated by 500 MHz 1H NMR spectroscopy under physiological solution conditions. The method depends on the stepwise analysis of one-component (self-association),
two-component (hetero-association and DNA complexation) and three-component interactions, in order to de-convolute the overall
binding of the anthracycline antibiotic and CAF to DNA into two competing processes, viz. hetero-association of the antibiotic-CAF
(‘interceptor’ action of CAF) and CAF–DNA complexation (‘protector’ action of CAF). It is found that the complexation of DAU
with DNA in the presence of CAF is mainly affected by the CAF–DNA complexation, whereas the binding of either DOX or NOG to
DNA is affected approximately equally by both the CAF–DNA complexation and CAF-antibiotic hetero-association. Quantitative
evaluation of the three-component mixture of drug–CAF–DNA has enabled the proportion of the antibiotic displaced from DNA
on addition of CAF to be calculated over a large range of CAF concentration, which may provide a quantitative basis for the
change in anthracycline-related toxicity on addition of CAF. 相似文献
3.
Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms. 总被引:5,自引:0,他引:5
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
K M Nielsen M D van Weerelt T N Berg A M Bones A N Hagler J D van Elsas 《Applied microbiology》1997,63(5):1945-1952
A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bacterial cell density, time of residence of A. calcoaceticus or of DNA in soil before transformation, transformation period, and nutrient input were investigated. There were clear inhibitory effects of the soil matrix on transformation and DNA availability. A. calcoaceticus cells reached stationary phase and lost the ability to be transformed shortly after introduction into sterile soil. The use of an initially small number of A. calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced transformation frequencies within a limited period. The availability of introduced DNA for transformation of A. calcoaceticus cells disappeared within a few hours in soil. Differences in transformation frequencies between soils were found; A. calcoaceticus cells were transformed at a higher rate and for a longer period in a silt loam than in a loamy sand. Physical separation of DNA and A. calcoaceticus cells had a negative effect on transformation. Transformation was also detected in nonsterile soil microcosms, albeit only in the presence of added nutrients and at a reduced frequency. These results suggest that chromosomal DNA released into soil rapidly becomes unavailable for transformation of A. calcoaceticus. In addition, strain BD413 quickly loses the ability to receive, stabilize, and/or express exogenous DNA after introduction into soil. 相似文献
4.
Saturation transfer difference NMR (STD NMR) spectroscopy is one of the most powerful NMR techniques for detection and characterization
of transient (fast) receptor–ligand interactions in solution. By observing the signals of a small molecule (ligand) with spectroscopic
properties suitable for high-resolution studies, irrespective of receptor size, STD NMR enables quantitative structural and
affinity information to be obtained about the molecular recognition process under study. Approximately one decade after its
introduction, the technique has reached maturity, and is highly robust and useful. The objective of this article is to review
the current status of this powerful technique, with particular emphasis on quantitative applications, within the framework
of the (bio-)chemistry of molecular recognition. 相似文献
5.
We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra. 相似文献
6.
N. K. Notani 《Journal of biosciences》1981,3(4):431-438
InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal
DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition
of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea
that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific
biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different
clones. The reduced transformation frequencies seen inrec 1
- strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells
can increase the transformation efficiency onrec 1
+ strain. 相似文献
7.
Jana Nováková Anita Izsáková Tomáš Grivalský Christian Ottmann Marian Farkašovský 《Folia microbiologica》2014,59(1):53-61
High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7?×?108 transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source. 相似文献
8.
The introduction of exogenous DNA into Escherichia coli is a cornerstone of molecular biology. Herein, we investigate two new mechanisms for bacterial transformation involving either the use of microwave irradiation or a freeze–thaw protocol in liquid nitrogen. Ultimately, both methods afforded successful transfer of plasmid DNA into bacterial cells, with the freeze–thaw technique yielding efficiencies of ~105. More importantly, both techniques effectively eliminated the need for the preparation of competent cells. 相似文献
9.
Amyloidogenic proteins (Aβ peptide) in Alzheimer’s disease (AD) and alpha-synuclein (α-Syn) in Parkinson’s disease (PD) are typically soluble monomeric
precursors, which undergo remarkable conformational changes and culminate in the form of aggregates in diseased condition.
Overlap of clinical and neuropathological features of both AD and PD are observed in dementia with Lewy body (DLB) disease,
the second most common form of dementia after AD. The identification of a 35-amino acid fragment of α-Syn in the amyloid plaques
in DLB brain have raised the possibility that Aβ and α-Syn interact with each other. In this report, the molecular interaction of α-Syn with Aβ40 and/or Aβ42 are investigated using multidimensional NMR spectroscopy. NMR data in the membrane mimic environment indicate specific
sites of interaction between membrane-bound α-Syn with Aβ peptide and vice versa. These Aβ–α-Syn interactions are demonstrated by reduced amide peak intensity or change in chemical shift of amide proton of the interacting
proteins. Based on NMR results, the plausible molecular mechanism of overlapping pathocascade of AD and PD in DLB due to interactions
between α-Syn and Aβ is described. To the best of our knowledge, it is the first report using multidimensional NMR spectroscopy that elucidates
molecular interactions between Aβ and α-Syn which may lead to onset of DLB.
An erratum to this article can be found at 相似文献
10.
Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex
materials in a vacuum. It is widely applied to the discrimination of closely related microbial strains. Leaf samples from
eight cultivars (‘Apricot Delight’, ‘Cooler Grape’, ‘Cooler Peppermint’, ‘Equator Grape’, ‘Equator Rose’, ‘Equator White’,
‘Equator White Eye’, and ‘Little Bright Eye’) of Catharanthus roseus were subjected to PyMS for spectral fingerprinting. Discriminant analysis (DA) of PyMS data enabled us to assign these cultivars
to discrete clusters. A hierarchical dendrogram based on DA provided a possible relationship among them that was in general
agreement with a previously reported classification of the cultivars based on DNA fingerprints. Furthermore, those belonging
to the same ‘series’ were grouped into a single cluster, which previously could not be achieved through similar approaches
based on Fourier transform infrared spectroscopy or 1H NMR data. Overall results suggest that chemical differences (i.e., in pyrolysate composition) among cultivars, as detected
by mass spectrometry, reflect their genetic variation. 相似文献
11.
From 1988 to 1993 we assessed the variability of bacterioplankton production and biomass in Lake Xolotlán (L. Managua), Nicaragua
via [3H]thymidine incorporation into DNA and cell counting. Bacterial production ranged from 3 to 8 μg C l-1 h-1, and since
production was equal throughout the water column, areal production was high (≈ 600–1200 mg C m-2 d-1). Bacterial abundance
in Lake Managua was extremely high, 7–30 × 109 cells l-1. Thus, specific rates of bacterial production were low. There was
a strong correlation between production and number and the specific rate of bacterial production was constant. Comparable
measurements of production via [3H]leucine incorporation into proteins indicated that bacteria were experiencing ‘balanced
growth’. We conclude that bacterioplankton in Lake Xolotlán had reached its carrying capacity and a significant correlation
between bacterial production and concentration of phaeophytin implied that dead or dying algae was the limiting substrate
for bacterioplankton. The majority of bacterial number and most of bacterial production (up to 75%) were associated with particles
in the >3-μm fraction, probably lysing algal cells to which bacterioplankton were ‘attached’. Grazing on bacterioplankton
must be low and bacteria should be a ‘sink’ for organic matter in Lake Xolotlán.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
12.
Toke O Bánóczi Z Király P Heinzmann R Bürck J Ulrich AS Hudecz F 《European biophysics journal : EBJ》2011,40(4):447-462
Maximin-4 is a 27-residue cationic antimicrobial peptide exhibiting selectivity for bacterial cells. As part of the innate
defense system in the Chinese red-belly toad, its mode of action is thought to be ion channel or pore formation and dissipation
of the electrochemical gradient across the pathogenic cell membrane. Here we present the high-resolution structure of maximin-4
in two different membrane mimetics, sodium dodecyl sulfate micelles and 50% methanol, as determined by 1H solution NMR spectroscopy. In both environments, the peptide chain adopts a helix–break–helix conformation following a highly
disordered N-terminal segment. Despite the similarities in the overall topology of the two structures, major differences are
observed in terms of the interactions stabilizing the kink region and the arrangement of the four lysine residues. This has
a marked influence on the shape and charge distribution of the molecule and may have implications for the bacterial selectivity
of the peptide. The solution NMR results are complemented by CD spectroscopy and solid-state NMR experiments in lipid bilayers,
both confirming the predominantly helical conformation of the peptide. As a first step in elucidating the membrane interactions
of maximin-4, our study contributes to a better understanding of the mode of action of antimicrobial peptides and the factors
governing their selectivity. 相似文献
13.
《Trends in biotechnology》1988,6(12):303-309
The introduction of DNA into bacteria by transformation is an essential step in the construction of recombinant strains. Recently, electroporation, or electropermeabilization, in which a brief high voltage electric discharge is used to render cells permeable to DNA, has revolutionized the transformation of bacteria. The technique is fast, simple, reproducible, frequently gives very high transformation frequency and appears to be applicable to a wide range of bacterial types previously thought untransformable. The technique can also offer advantages for transformable bacteria such as Escherichia coli. 相似文献
14.
In the past decade several methods have been developed for the introduction of foreign DNA into plant cells to obtain transgenic
plants. In some of these methods, purified DNA is directly introduced into protoplasts that for some species can be regenerated
into mature plants. The more commonly used protocols, however, employ the natural capacity ofAgrobacterium tumefaciens to transfer a defined peice of DNa, called T-DNA, to the nucleus of plant cells that are more easy to regenerate than protoplasts.
In plant cells, like in animal cells, foreign DNA (including T-DNA) is readily inserted into the genome via illegitimates
recombination. In contrast, targeted integration via homologous recombination, referred to as ‘gene targeting’, can only be
obtained at relatively low frequencies. Nevertheless, gene targeting has become a standard strategy for reverse genetics studies
in animals. In plants, the occurrence of gene targeting was only reported recently. This review focuses on the use of theAgrobacterium vector system to achieve gene targeting in plants. Recent experimental data concerning gene targeting in plants are presented
and the overall suitability ofAgrobacterium T-DNA transfer for this purpose is assessed in light of contemporary views on the mechanism of T-DNA transfer. 相似文献
15.
Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2). 相似文献
16.
Pontes B Viana NB Campanati L Farina M Neto VM Nussenzveig HM 《European biophysics journal : EBJ》2008,37(2):121-129
We investigate properties of a reported new mechanism for cell–cell interactions, tunneling nanotubes (TNT’s). TNT’s mediate
actin-based transfer of vesicles and organelles and they allow signal transmission between cells. The effects of lateral pulling
with polystyrene beads trapped by optical tweezers on TNT’s linking separate U-87 MG human glioblastoma cells in culture are
described. This cell line was chosen for handling ease and possible pathology implications of TNT persistence in communication
between cancerous cells. Observed nanotubes are shown to have the characteristic features of TNT’s. We find that pulling induces
two different types of TNT bifurcations. In one of them, termed V-Y bifurcation, the TNT is first distorted into a V-shaped
form, following which a new branch emerges from the apex. In the other one, termed I-D bifurcation, the pulled TNT is bent
into a curved arc of increasingly broader span. Curves showing the variation of pulling force with displacement are obtained.
Results yield information on TNT structure and elastic properties.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome
(ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells
of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with
ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and
treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells
with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as
C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and
elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species
revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA.
Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7–20% and and HC2:11–25%) they represented the
bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C
(1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA
flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by
analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms
in germ cell proportions observed following treatment, fore.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple
steps in germ cell transformation 相似文献
18.
Doogab Yi 《Journal of the history of biology》2008,41(4):589-636
The existing literature on the development of recombinant DNA technology and genetic engineering tends to focus on Stanley
Cohen and Herbert Boyer’s recombinant DNA cloning technology and its commercialization starting in the mid-1970s. Historians
of science, however, have pointedly noted that experimental procedures for making recombinant DNA molecules were initially
developed by Stanford biochemist Paul Berg and his colleagues, Peter Lobban and A. Dale Kaiser in the early 1970s. This paper,
recognizing the uneasy disjuncture between scientific authorship and legal invention in the history of recombinant DNA technology,
investigates the development of recombinant DNA technology in its full scientific context. I do so by focusing on Stanford
biochemist Berg’s research on the genetic regulation of higher organisms. As I hope to demonstrate, Berg’s new venture reflected
a mass migration of biomedical researchers as they shifted from studying prokaryotic organisms like bacteria to studying eukaryotic
organisms like mammalian and human cells. It was out of this boundary crossing from prokaryotic to eukaryotic systems through
virus model systems that recombinant DNA technology and other significant new research techniques and agendas emerged. Indeed,
in their attempt to reconstitute ‹life’ as a research technology, Stanford biochemists’ recombinant DNA research recast genes
as a sequence that could be rewritten thorough biochemical operations. The last part of this paper shifts focus from recombinant
DNA technology’s academic origins to its transformation into a genetic engineering technology by examining the wide range
of experimental hybridizations which occurred as techniques and knowledge circulated between Stanford biochemists and the
Bay Area’s experimentalists. Situating their interchange in a dense research network based at Stanford’s biochemistry department,
this paper helps to revise the canonized history of genetic engineering’s origins that emerged during the patenting of Cohen–Boyer’s
recombinant DNA cloning procedures. 相似文献
19.