Mapping of BamHI and SmaI DNA restriction sites on the genome of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica

The DNA of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), has been analyzed with restriction endonucleases BamHI and SmaI. The molecular weight of the BamHI fragments, SmaI fragments, and BamHI + SmaI fragments has been determined. The molecular weight of AcNPV DNA is calculated to be about 82 million. A presumptive physical map of the BamHI and SmaI restriction sites on the AcNPV genome has been constructed.     

β-Lactamase genes of Streptomyces badius,Streptomyces cacaoi and Streptomyces fradiae: Cloning and expression in Streptomyces lividans

Genes encoding extracellular β-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The β-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi β-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The β-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the β-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three β-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi β-lactamases exhibited a 10–100-times lower activity in S. lividans, whereas the S. fradiae β-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Dissecting the Molecular Origins of Specific Protein-Nucleic Acid Recognition: Hydrostatic Pressure and Molecular Dynamics

The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

A method for isolation of a large amount of a single-stranded DNA fragment

Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.     

Isolation and characterization of R-plasmid variants with enhanced chromosomal mobilization ability in Escherichia coli K-12

Enhanced Chromosome Mobilizing (ECM) plasmids derived from the IncP-1 plasmid R68 were isolated in Escherichia coli K-12 by the same methods which have given similar plasmids such as R68.45 in Pseudomonas aeruginosa. The chromosome mobilizing properties of such plasmids in E. coli were similar to those of R68.45 but while retaining the ability to transfer to P. aeruginosa they did not mobilize the chromosome of that organism. Restriction enzyme analysis of two such plasmids, pMO163 and pMO168, showed that they both possessed an additional segment of DNA. With pMO163, an addition of 0.8 kb is located near the TnA region and is characterized by the cleavage site pattern SmaI-HpaI-PstI-BamHI. For pMO168, the additional DNA segment is located at a different site, about 4.0 kb anti-clockwise from the EcoRI site. It was also characterized by the sites SmaI-(HpaI-PstI)-BamHI. No sequence homology has been found between the additional segments of either pMO163 or pMO168 and IS21 of R68.45. However homology of these additional segments was found with the E. coli K-12 chromosome suggesting that pMO163 and pMO168 arise by the acquisition of a transposable element from the E. coli K-12 chromosome.     

Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH

Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I_d. This faster-moving form is cleaved by the restriction endonuclease BamHI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by BamHI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I_d as suggested previously.     

Spodoptera frugiperda Nuclear Polyhedrosis Virus Genome: Physical Maps for Restriction Endonucleases BamHI and HindIII

The physical map for the genome of Spodoptera frugiperda nuclear polyhedrosis virus was constructed for restriction endonucleases BamHI and HindIII. The ordering of the restriction fragments was accomplished by cross-blot hybridization of BamHI, HindIII, and EcoRI fragments. The alignment of the HindIII fragments within the BamHI map was achieved by double digestion with the two restriction endonucleases followed by cross-blot hybridization. The results showed that the viral genome consisted of mainly unique sequences. In addition, the circular nature of the viral genome was reaffirmed.     

Physical mapping of homologous segments of mitochondrial episomes from S male-sterile maize

Restriction endonuclease maps of two double-stranded plasmid-like DNAs, 6180 and 5175 bp each, isolated as linear molecules from the mitochondria of S-type cytoplasmic male-sterile maize were prepared. Twelve cleavage sites were mapped in each using HindIII, XhoI, EcoRI, SacI, XbaI, SalI, BamHI, and BstEII. BamHI does not cleave S-1 DNA and SalI does not cleave S-2 DNA. A 1150-bp homologous sequence in addition to the 200-bp terminal inverted repeats was terminally oriented on both DNAs by reciprocal hybridization and heteroduplex analysis.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

A helicase gene (helO) in Rhizobium meliloti WSM419

A 2.8 kb BamHI DNA fragment adjacent to a BamHI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced. A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with `DEAH' motif helicases. Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene. Inactivation of helO by insertion of a Ω interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc.     

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.Abbreviations Ap ampicillin - ARS autonomously replicating sequence - EtBr ethidium bromide - kb kilobase(s) - Mc. Methanococcus - R resistance - RE replication enhancer - RS replication sequence - Tc tetracycline     

Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus

A physical map of the 88 × 10⁶ dalton, circular DNA genome of Autographa californica nuclear polyhedrosis virus was constructed. The complete order of BamHI and XmaI restriction enzyme sites was determined. The EcoRI and HindIII fragments were partially ordered, and their general locations, relative to the BamHI and XmaI maps, were determined. Alterations in the restriction endonuclease fragment patterns of natural genotypic variants of A. californica nuclear polyhedrosis virus, including Trichoplusia ni MEV nuclear polyhedrosis virus, were located on the physical map. Alterations were found throughout the A. californica nuclear polyhedrosis virus DNA genome.     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.