Origin of replication, oriC, of the Escherichia coli chromosome: mapping of genes relative to R.EcoRI cleavage sites in the oriC region.

Summary A precise genetic-physical map of the tnailv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F carrying the genes between aroE and ilv.A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30–45 kb counterclockwise of ilv.The pattern of R.EcolRI cleavage sites in the het region is identical with the pattern obtained by Marsh and worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.Abbreviations CCC covalently closed circular - kb kilobases - MD 10⁶ Daltons - mw molecular weight - R.EcoRI restriction endonuclease EcoRI New Genetic Symbols het heterogeneity of cell size distribution (this study) - maf maintenance of F (Wada et al., 1977) - oriC chromosomal origin of replication (Hiraga, 1976) - oriV origin of vegetative replication of F (Guyer et al., 1976) - oriT origin of transfer replication of F (Guyer et al., 1976) - poh permissive on Hfr (Hiraga, 1976)     

Induced expression of <Emphasis Type="Italic">Eco</Emphasis>RI endonuclease as an active maltose-binding fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.     

The Role of the Fast Motion of the Spin Label in the Interpretation of EPR Spectra for Spin-Labeled Macromolecules

Abstract

The spin label method was used to observe the nature of the fast motions of side chains in protein monocrystals. The EPR spectra of spin-labeled lysozyme monocrystals (with different orientations of the tetragonal protein crystal in relation to the direction of the magnetic field) were interpreted using the method of molecular dynamics (MD). Within the proposed simple model, MD calculations of the spin label motion trajectories are performed in a reasonable real time. The model regards the protein molecule as frozen as a whole and the spin labeled amino acid residue as unfrozen. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was assembled, and the parameters of the force fields were specified for atoms of the protein molecule, including the spin label. The calculations show that the protein environment sterically limits the area of the possible angular reorientations for the NO reporter group of the nitroxide (within the spin label), and this, in turn, affects the shape of the EPR spectrum. However, it turned out that the spread in the positions of the reporter group in the angle space strictly adheres to the Gaussian distribution. Using the coordinates of the spin label atoms obtained by the MD method within a selected time range and considering the distribution of the spin label states over the ensemble of spin-labeled macro- molecules in a crystal, the EPR spectra of spin-labeled lysozyme monocrystals were simulated. The resultant theoretical EPR spectra appeared to be similar to experimental ones.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore

Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg²⁺ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

Mini-chromosomes: plasmids which carry the E. coli replication origin.

Summary We have isolated plasmids by linking the 5.9 MD EcoRI fragment of E. coli that carries the origin of replication to an EcoRI fragment that carries an ampicillin resistance determinant, but lacks an origin of replication. 3 plasmids of this type, pOC1, pOC2, and pOC3, are described in detail in this report. Although the plasmids have some adverse effect on the growth properties of the host strain, their existence shows that two functioning chromosomal origins can coexist in one cell.Deletions generated from this type of plasmids allow an allocation of the origin of replication of E. coli within a DNA segment less than 0.4 MD in size.     

Construction and properties of recombinant plasmids containing the rII genes of bacteriophage T4.

Summary The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6x10⁶ Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.Abbreviations used Ap ampicillin - Tc tetracycline - Mdal 10⁶ Daltons - bp base pairs     

Characterisation of plasmids coding for the restriction endonuclease EcoRI

Summary The properties of two plasmids coding for the EcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6x10⁶ daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor R1–19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7x10⁶ daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1–19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Isolation and characterization of a genomic sequence of maize coding for a zein gene

Summary An EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage gt WES. The zein gene was identified by the electron microscopic analysis of RNA-DNA hybrids (R-loops) and DNA-DNA hybrids (D-loops). The R-loops were formed with poly(rA)-containing RNA isolated from 18 days post-pollination maize endosperm and showed no intervening non-hybridizing sequences (introns) within their 800 base length. A cDNA clone specific for the 19,000 dalton zein protein formed D-loops in the same position and orientation as the R-loops. The cloned fragment measured 4.4 kilobases (kb), the same size as an EcoRI fragment of maize DNA revealed by Southern analysis.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

The control region of the F sex factor DNA transfer cistrons: Restriction mapping and DNA cloning

Summary A restriction endonuclease map of EcoRI fragment f6 of F sex factor DNA was constructed and aligned with pre-existing physical and genetic maps. Results of genetic complementation tests and analysis of proteins synthesized in minicells from PstI and BglI₁ sub-fragment clones, or from a specific BglII fragment deletion, have allowed mapping of the locations of the origin of DNA transfer and many of the transfer genes known to lie on f6. The proteins detected account for 78% of the coding capacity of fragment f6.     

Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the p_R promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.Correspondence to: K. Schügerl     

DNA methylation and AFLP marker distribution in the soybean genome

Amplified fragment length polymorphisms (AFLPs) have become important markers for genetic mapping because of their ability to reliably detect variation at a large number of loci. We report here the dissimilar distribution of two types of AFLP markers generated using restriction enzymes with varying sensitivities to cytosine methylation in the soybean genome. Initially, AFLP markers were placed on a scaffold map of 165 RFLP markers mapped in 42 recombinant inbred (F_6:7) lines. These markers were selected from a map of over 500 RFLPs analyzed in 300 recombinant inbred (F_6:7) lines generated by crossing BSR101×PI437.654. The randomness of AFLP marker map position was tested using a Poisson-model distribution. We found that AFLP markers generated using EcoRI/MseI deviated significantly from a random distribution, with 34% of the markers displaying dense clustering. In contrast to the EcoRI/MseI AFLP markers, PstI/MseI-generated AFLP markers did not cluster and were under represented in the EcoRI/MseI marker clusters. The restriction enzyme PstI is notably sensitive to cytosine methylation, and these results suggest that this sensitivity affected the distribution of the AFLP markers generated using this enzyme in the soybean genome. The common presence of one EcoRI/MseI AFLP cluster per linkage group and the infrequent presence of markers sensitive to methylation in these clusters are consistent with the low recombination frequency and the high level of cytosine methylation observed in the heterochromatic regions surrounding centromeres. Thus, the dense EcoRI/MseI AFLP marker clusters may be revealing structural features of the soybean genome, including the genetic locations of centromeres. Received: 5 November 1998 / Accepted: 20 February 1999