Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

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.     

Cloning of a 1.1-kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant

The srnB⁺ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1-kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild-type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region

Summary The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of -lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that 1.9x10⁶ daltons of the 6.0x10⁶ dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Construction of recombinant K88 DNA with Ptac promoter

The gene encoding K88ab was localized on the 11.6 kbHindIII-HindIII fragment of 74 kb plasmid DNA ofE. coli 7301. The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kbEcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vectro. The size of the smallest fragment was 6.5 kb. Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector. Substitution of promoter P_tac for promoter P1 made it possible to achieve expression of the K88ab antigen byE. coli MT. Substitution of promoter P_L for promoter P1 failed to achieve expression of the K88 ab antigen in the recipient strains used.     

Cloning of bacteriophage T5 promoters

Summary Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonuclease PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Ap^s-Tc^r-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments.Two PstI/HindIII fragment, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tc^r levels for these plasmids were as high as 18 g/ml and 75 g/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.Abbreviations Ap ampicillin - Tc tetracycline - bp base pairs - NTPs nucleoside triphosphates - PBB polymerase binding buffer     

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.     

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

Summary Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.Abbreviations kop kilobase pairs - OcTase ornithine carbamoyl transferase - CPSase carbamoyl phosphate synthetase     

Recombinant plasmids capable to replication in B. subtilis and E. coli.

Summary The plasmid pBC16 (4.25 kbases), originally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBC16 (pBC16-1), 2,7 kb) which has lost an EcoRI fragment of pBC16 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS1, a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et al., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBS161 (8.2 kb) and the smaller plasmid pBS162 (2.1 kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindIII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-1 is generated, which can be used as a HindIII and EcoRI cloning vector in Bacillus subtilis.Hybrid plasmids consisting of the E. coli plasmids pBR322, pWL7 or pAC184 and different HindIII fragments of pBS161 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindIII fragment of pBS161 can replicate in E. coli and B. subtilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. subtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. subtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.     

A plasmid cloning vector for Kpnl-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.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

Mapping of regions on cloned Saccharomyces cerevisiae 2-mum DNA coding for polypeptides synthesized in Escherichia coli minicells.

Summary Saccharomyces cerevisiae 2-m DNA and some of its restriction fragments were integrated in vector pCR1, pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2-m DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322, promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-m DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons repectively. The DNA sequence coding for the 37,000 dalton polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the sites RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 19,000 dalton polypeptides were mapped to the S-loop region.The integrated inverted repeat sequence of yeast 2-m DNA did not induce any detectable insert-specific polypeptide synthesis.     

Cloning of mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in the bacterial plasmid pBR322

Summary The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.     

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     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA) · poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA) · poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA) · poly(dT) fragment of 10–20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.     

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

Summary The construction of a plasmid carrying the right end PstI·B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI·C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrate that Mu-specific functions can be analyzed from cloned fragments.     

Genomic Fingerprinting of Virulent and Avirulent Strains of Clavibacter michiganensis subspecies sepedonicus

Genomic fingerprints of C. michiganensis subsp. sepedonicus were generated by CHEF gel electrophoresis of restriction digested high-molecular weight DNA. Low levels of intra-subspecific variation were detected by cluster analysis of the fingerprints. Four haplotypes were identified by genomic fingerprinting with HindIII, and eight were identified with EcoRI. Haplotypes generated with HindIII were less similar than those generated by EcoRI. Haplotypes generated with HindIII formed groups that corresponded well with plant reactions of the strains, but similar types of groupings were less apparent with haplotypes generated with EcoRI. When disease severity in eggplant and potato, population size in potato, and ability to induce a hypersensitive response (HR) in tobacco were overlaid onto dendograms of genetic similarity, avirulent HR-negative strains clustered separately from virulent HR-positive strains in both EcoRI and HindIII profiles. Avirulent HR-positive strains that lack pCS1 clustered with avirulent HR-negative strains in a EcoRI dendogram, but clustered with virulent HR-positive strains in a HindIII dendogram. Genomic fingerprinting of high-molecular weight DNA fragments provided a means for detecting genomic variability associated with virulence in C. michiganensis subsp. sepedonicus. Received: 1 March 2001 / Accepted: 7 June 2001