A simple, general procedure for purifying restriction endonucleases

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.     

The actions of restriction endonucleases on lampbrush chromosomes

Lampbrush chromosomes from oocytes of Notophthalmus viridescens were dispersed in media containing restriction endonucleases isolated from Haemophilus and E. coli. These endonucleases cleave duplex DNAs at specific palindromic sequences of nucleotides, and several sensitive sites occur per micron of DNA. The overwhelming majority of the lateral loops of lampbrush chromosomes are extensively fragmented by these endonucleases, but an occasional pair of loops is refractory. A notable example of loops showing this refractory property are the giant loops on chromosome II in the presence of Hae. These loops, whose DNA-containing axes are several hundred micra long, are sensitive to other nucleases such as EcoB, endonuclease I and pancreatic DNase I; their refractory behavior towards Hae therefore indicates that the sequence sensitive to this particular endonuclease is systematically absent. This anomalous property can be comprehended if it be assumed that the axial DNA of the giant loops consists of tandem repeats of a sequence which happens not to include the sensitive site.     

Optical mapping of lambda bacteriophage clones using restriction endonucleases

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.     

Cleavage of adeno-associated virus DNA with Sali,Psti and Haeii restriction endonucleases

Duplex AAV-2 DNA was digested with SalI, PstI or HaeII restriction endonucleases and the cleavage sites were mapped. SalI cleaves AAV DNA at 0.310 map units, PstI at 0.106, 0.422 and 0.914 and the five HaeII sites were mapped at 0.110. 0.156, 0.181, 0.536 and 0.600 map units. These cleavage products will be useful for the isolation of specific regions from the AAV DNA, located outside of the stably transcribed region of the genome, and will also help to map more complex restriction enzyme cleavages.     

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.     

Mutual activation of two restriction endonucleases: interaction of EcoP1 and EcoP15

Type III restriction endonucleases recognize nonsymmetric nucleotide sequences. A necessary condition for DNA cleavage is the presence of two unmethylated recognition sites which are inversely ('head-to-head') oriented in the DNA double strand. A DNA substrate possessing one EcoP1 and one EcoP15 site in the head-to-head configuration could not be cleaved by the individual enzymes, however, it was specifically digested in the simultaneous presence of both enzymes. In agreement with the tracking-collision model for the DNA interaction of type III enzymes cleavage could be abolished by Lac repressor bound between the two sites. We conclude that two different type III enzymes can functionally cooperate in the cleavage of DNA.     

A novel equivalent circuit model for gap-connected cells

Gap junctions connect neighbouring cells, providing the intercellular communication that is essential for cell growth regulation, for example. There is some evidence that gap communication changes upon exposure to electromagnetic (EM) fields. In previous work, we performed detailed finite element method (FEM) modelling of gap junction connected cells exposed to EM fields. For cell configurations, the presence of gap junctions influences the transmembrane potential and its frequency behaviour. The relaxation frequency cannot be accurately predicted by previously developed simplified models. We present a novel equivalent circuit model (ECM) that incorporates more detailed models of the gaps, and compare results obtained with this ECM to finite element and leaky cable (LC) model results. Our ECM provides more accurate estimates of the frequency behaviour of cells than the leaky cable model. Also, our ECM results suggest limitations of the application of simple models to gap-connected cells: with higher gap resistivity, the current flow in the cell interiors becomes increasingly complex and is not well represented by simple models. In this case, techniques such as the finite element method are required to model accurately cell behaviour.     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRI, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been reduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA. Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

The resistance of the DNA of cyanophage LPP-3 to the action of different restriction endonucleases

Data on the study of structure peculiarities of cyanophage LPP-3 DNA are presented in the work. The length of cyanophage DNA calculated by means of the enzymatic hydrolysis by restrictases is 40 +/- 3.5 thou. pairs of bases. Cyanophage LPP-3 DNA was hydrolysed by more than 50 different restrictases. As a result of screening it was found out that the great number of restrictases, which recognized hexanucleotide sequences did not hydrolyze DNA of cyanophage LPP-3. A considerable deviation of the number of the observed sites of restriction from their theoretically expected number for restrictases Hae III and Cfr 131 was established. Restrictases-isoschisomeres with different sensitivity to the methylation of the recognition sites--Msp I, Hpa II and Sau 3A, MboI and DpnI were used to check the availability of methylated bases in LPP-3 DNA. Absence of methylated adenine in the site GATC and methylated cytosine in the second position of the site CCGG were established. The results obtained permit supposing that the expressed counterselection by the sites of recognition of many restriction endonucleases takes place in cyanophage LPP-3 DNA. It is supposed that apparently, this method of protection of its genome in LPP-3 is one of most important but the inconsiderable percentage of site-specific methylation of the virus DNA cannot be completely excluded.     

一个四翼混沌系统的设计与电路实现

为提高混沌吸引子的拓扑结构的复杂性,采用双极性化z轴的方法,将一个混沌系统的双翼吸引子变为四翼吸引子,对新的系统进行了理论分析和计算机仿真,Lyapunov指数计算表明系统具有一个正的Lyapunov指数,最后用数字技术实现了这个四翼混沌系统,实验结果证明了理论分析和数值仿真的正确性.     

Inhibition of cleavage by restriction endonucleases due to modifications induced in SV40 DNA by methyl methanesulfonate

Partially decorticated rats were tested for their response to nociceptive stimulation in the formalin and tail flick tests, and for the effect of morphine on these responses and on motor activity. Undrugged rats showed vigorous responses to nociceptive stimulation in both tests, and exhibited the typical biphasic time course of pain in the formalin test. Morphine 4 and 8 mg/kg produced dose-dependent analgesia in both tests in sham operated rats, and in rats with lesions that removed all or part of the cortex from the midline to the rhinal fissure (excluding the occipital cortex). In rats with lesions that extended deep into the piriform cortex and damaged the amygdala morphine analgesia was eliminated or attenuated. These and other recent findings suggest that analgesia in the formalin test depends on ascending connections to the forebrain, probably the amygdala.     

阀门定位器阀位检测与压电阀驱动电路设计

本文主要介绍定位器两大模块:阀位检测电路模块与压电阀驱动电路模块进行详细的硬件设计,电路都是针对检测或驱动对象的特性展开设计的,通过理论分析与电路测试证明两大模块的电路设计方案可行性较好。     

Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.     

Partial purification and characterisation of Bfi57I and Bfi89I, restriction endonucleases from different strains of Butyrivibrio fibrisolvens

The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., lambda, phi 80, Mu and f1) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants (hupA hupB himA and hupA hupB himD) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.     

Characterization of two restriction endonucleases, SenPT14bI and SenPT16I, in standard phage-type strains of Salmonella enteritidis

Two restriction endonucleases (ENases) were found by screening 38 standard phage strains of Salmonella (S.) Enteritidis. An isoschizomer of SacII ENase that recognizes the sequence 5'-CCGC/GG-3' was identified in S. Enteritidis PT14b, and an isoschizomer of XmaIII ENase (5'-C/GGCCG-3') was found in S. Enteritidis PT16. It is of special interest that the recognition specificities of all known ENases in Salmonella, including those of the S. Enteritidis ENases, are very similar to each other.     

The study of responses to 'model' DNA breaks induced by restriction endonucleases in cells and cell-free systems: achievements and difficulties

The use of restriction endonucleases (RE) as a means of implicating DNA double-strand breaks (dsb) in cellular responses is reviewed. The introduction of RE into cells leads to many of the responses known to be characteristic of radiation damage--cell killing, chromosomal aberration, oncogenic transformation, gene mutation and amplification. Additionally, radiosensitive cell lines are hypersensitive to RE, including those from the human disorder ataxia-telangiectasia. However, quantitation of response and comparisons of the effectiveness of different RE are difficult, partly because of unknown activity and lifetime of RE in the cell. RE-induced dsb have also been used to reveal molecular mechanisms of repair and misrepair at specific sites in DNA. Dsb have been implicated in recombination processes including those leading to illegitimate rejoining (formation of deletions and rearrangements) at short sequence features in DNA. Also model dsb act as a signal to activate other cellular processes, which may influence or indirectly cause some responses, including cell death. In these signalling responses the detailed chemistry at the break site may not be very important, perhaps explaining why there is considerable overlap in responses to RE and to ionizing radiations.