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     

Two-Fold Symmetry of Crystalline DNA-ECORI Endonuclease Recognition Complexes

Abstract

Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding V_M values of 3.1 A³/dal and 2.8 A³/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

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     

FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease

We found unusual reactivity of a heterochiral dodecadeoxynucleotide toward EcoRI that contains an unnatural l-nucleotide residue at the 3′-flanking site of the hexameric EcoRI recognition sequence. In this study, the kinetic parameters for the reaction were determined by means of a heterochiral molecular beacon that contains the EcoRI recognition sequence in the stem region and possesses the fluorescent and quenching dyes at the 5′- and 3′-termini, respectively. We found that the heterochiral molecular beacon showed large V_max and small K_m values compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.     

Characterisation of three Alphabaculovirus isolates from the gypsy moth,Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170?kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 10³ and 10⁶?OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC₅₀ and LC₉₅ values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC₅₀ value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.     

Complete Sequence and Enhancer Function of the Homologous DNA Regions of Autographa californica Nuclear Polyhedrosis Virus

The nucleotide sequence of the five regions of homologous DNA in the genome of Autographa californica nuclear polyhedrosis virus DNA was determined. The homology of repeated sequences within a region was 65 to 87%, and the consensus sequences for each region were 88% homologous to each other. Sequences proximal to the EcoRI sites were most conserved, while the distal sequences were least conserved. The EcoRI sites formed the core of a 28-base-pair imperfect inverted repeat. All homologous regions functioned as enhancers in a transient expression assay. A single EcoRI minifragment located between EcoRI-Q and -L enhanced the expression of 39CAT as efficiently as the regions containing numerous EcoRI repeats did.     

Two different satellite DNAs in Beta vulgaris L.: evolution,quantification and distribution in the genus

Summary Two highly repeated EcoRI (0.45 × 10⁶) and BamHI (0.17 × 10⁶) fragments per haploid genome were found in sugar beet genomic DNA. Both fragments were located by 6% acrylamide-gel electrophoresis, purified and cloned in pUC18. Four of the inserts corresponding to each family were chosen for further study. Both fragment families display the main characteristics of the satellite DNA of animals and plants. The EcoRI and BamHI fragment families are arranged in long tandem arrays. Fragments of the EcoRI family (pBVE) were analyzed. They vary both in sequence and in length (158–160 nt) in comparison with the consensus sequence of 159nt. Both families are A-T rich; pBVE is 59% rich while pBVB is 69% rich. The BVESAT family is present in all the members of the section Vulgares. It is conserved in the section Procumbentes with 80% homology and the same length, but is not detectable in the Corollinae. The sequence variation rate and the variation in length (330±5 nt) are of the same order in comparison with those of the BVESAT family. However, the BVBSAT family is present in species of the section Vulgares only. As regards other plant satellite DNAs, the BVESAT family shares homology with Allium cepa satellite DNA, with three of the yeast centromeric sequences, and with three Arabidopsis thaliana sequences. The BVBSAT family is unique to the Vulgares and does not share any homology with other plant or animal satellite DNAs sequences so far.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

A plasmid cloning vector for KpnI-cleaved DNA

A plasmid cloning vector containing a single site for KpnI has been generated by insertion of a 3.5-kb EcoRI/HindIII fragment of pCR1 into the EcoRI/HindIII sites of pBR322. KpnI cleavage yields 3′ rather than 5′ “sticky ends” which allows reconstitution of the recognition site after cloning by a homopolymer joining procedure. This is an advantage shared with only one or two other commercially available restriction enzymes.     

Structure–function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg²⁺: but in binding to DNA in the absence of Mg²⁺, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg²⁺ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg²⁺, while the complexes at other DNA sequences have low affinities for Mg²⁺. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg²⁺. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg²⁺ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.     

DNA Duplexes Containing Altered Sugar Residues as Probes of EcoRII and MvaI Endonuclease Interactions with Sugar-Phosphate Backbone

Abstract

Oligonucleotides containing 1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(β-D-2′-deoxy-threo-pentofuranosyl)thymine (dT_x) in place of dC and dT residues in the EcoRII and MvaI recognition site CC^A/_TGG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2′-deoxyxylosyl moieties of dC_x and dT_x, 3′-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2′-deoxyxylo-syl moiety into a dC·dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA·dT pair the effect of a 2′-deoxyxylose incorporation was much more pronounced. Multiple dC_x modifications and their combination with dT_x did not enhance the destabilization effect. Hydrolysis of dC_x-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dT_x residue was cleaved by the enzyme, but k_cat/K_M was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases

The SinI and EcoRII DNA methyltransferases recognize sequences (GG^A/_TCC and CC^A/_TGG, respectively), which are characterized by an ^A/_T ambiguity. Recognition of the A·T and T·A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine·C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G·C or a C·G base pair in the center of the recognition sequence (GG^G/_CCC and CC^G/_CGG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG^A/_TCC and GG^G/_CCC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that ^A/_T versus ^G/_C discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

Single Turnover Kinetics of Methylation by T4 DNA-(N6-Adenine)-Methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k ₁ = 0.21 s^–1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k ₂ = 0.023 s^–1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme–substrate complexes is usually sharply reduced. The flipping of the adenosine residue to be modified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly²⁶⁸-Met³⁹¹ of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.     

Solid-Phase Synthesis of Oligodeoxyribonucleotide Analogues Containing 5, 6-Dihydroimidazo [1, 2-c] Pyrimidin-5-One as a Base Moiety

Abstract

The synthesis on a polyacrylamide resin of oligonucleotides containing 5,6-dihydroimidazo [1, 2-c] pyrimidin-5-one (X) as a base residue is described. Perliminary results on enzymatic hydrolysis of the modified octanucleotide d(TXAATTCA) (21), containing. the recognition sequence for the endonuclease -ECO RI where dG is replaced by dX (1), are reported.