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PubMed1989

A plasmid to visualize and assay termination and antitermination of transcription in Escherichia coli.

To facilitate the analysis of termination and antitermination of transcription in prokaryotes, a complex operon has been assembled into the pBR322 replicon, drawing upon natural and synthetic DNA elements. This operon is initiated from a strongly inducible promoter without temperature restraints. It includes a severe transcription terminator and therefore requires antitermination of transcription to express a downstream lacZ reporter gene. Antitermination can be provided by an upstream N-utilization site from phage lambda, working in conjunction with N protein supplied in trans from a compatible plasmid. In this situation, the nusA gene of Salmonella, substituted into the Escherichia coli host, prevents lacZ function, confirming that a good facsimile of lambda's specific antitermination mechanism has been recreated. The nonessential, easily assayed product of this operon, beta-galactosidase, is also screenable by colony color on chromogenic substrate. The plasmid described will therefore serve as a tester for mutations affecting the various aspects of transcription regulation by termination.

Base Sequence↗
PubMed1989

Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces.

pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.

Amino Acid Sequence↗
PubMed1991

Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2.

In broad host-range plasmid RK2, several kil loci (kilA, kilB, kilC, kilE) and the replication initiator gene (trfA) are regulated by combination of kor determinants (korA, korB, korC, korE) in a regulatory network known as the kil-kor region. Although the kil determinants are not essential for replication, their coregulation with trfA suggests an involvement in plasmid maintenance or host-range. Plasmids carrying the cloned kilB region of RK2 cannot be maintained in the absence of korB owing to two phenotypically distinguishable, kor-regulated determinants: (1) kilB1 (kilD), which can be controlled by korA or korB, and (2) kilB2, which requires korB for control. In this study, we have determined the nature of the functions responsible for the kor-sensitive phenotypes of the kilB region. We found that insertion of transcription terminators within or downstream of the trfA operon allows plasmids carrying the kilB1 portion of the kilB region to be maintained in cells lacking korA or korB. In addition, mutants of the kilB1 region that can be maintained in the absence of korA and korB have alterations in the trfA promoter. These results show that the phenotype of the cloned kilB1 region in kor-deficient cells depends on trfA transcription but does not involve expression of any gene of the trfA operon. Therefore, the kilB1 determinant is not a structural gene. The phenotype results from entry of trfA-initiated transcription into adjacent sequences of the plasmid vector. The ability to block the kilB2 phenotype with transcriptional terminators allowed us to show conclusively that the kilB2 determinant is a host-lethal gene (klbA) whose regulation is dependent on korB. These findings have implications for the structure of the basic replicon of RK2.

Base Sequence↗
PubMed1989

DNA fork displacement rate measurements in heated Chinese hamster ovary cells.

DNA fork displacement rates (FDR) were measured in Chinese hamster ovary (CHO) cells heated at either 43.5 degrees C or 45.5 degrees C for various times. The inhibition of fork movement rate by heat was both time and temperature dependent, i.e., 10-20 min at 43.5 degrees C or 5 min at 45.5 degrees C was required to decrease the FDR to 20-30% of the control rate of 1 micron/min. Following heating, the reduced FDR was found to be constant for at least 75 min. The observed effects of heat on reduced rates of DNA replicon initiation and chain elongation and the increase in DNA with single-stranded regions could be explained by the heat sensitivity of the FDR. Any of these alterations in the DNA replication process may lead to many opportunities for abnormal DNA and/or protein interactions to occur which ultimately may lead to the observed formation of chromosomal aberrations.

Animals↗
PubMed1986

Cancer: involvement of replicative origins?

The assumption is made that movable genetic elements related to cancer are replicons and/or replicative origins. Some evidence is provided that certain mobile Alu-origins may represent candidates for precursors of chromosomal insertions. They could incorporate and interact with "fixed" chromosomal origins converting them to "procaryotic" ones. In other words, cancer is considered the result of a short and compact "re-evolution" from eucaryotic to "procaryotic" replication units.

Animals↗
PubMed1989

Detection of apramycin resistant Enterobacteriaceae in hospital isolates.

Apramycin is a recently developed aminoglycoside restricted to veterinary therapy. Production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)-IV) conferring cross-resistance to this drug and to gentamicin was detected in 1984 in France in bacteria of bovine origin. This mechanism of resistance was apparently confined to animals. We have studied 17 strains resistant to apramycin and gentamicin isolated in 5 hospitals in Belgium. Conjugative plasmids encoding an AAC(3)IV were present in 14 isolates. Comparison of the restriction fingerprints revealed 6 different plasmid patterns: 8 plasmids belonged to 2 groups sharing extensive intragroup homology and 4 were not related to the other replicons. These results indicate dissemination of plasmids within and between hospitals, but also of the gene encoding an AAC(3)IV.

Animals↗
PubMed1992

Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000.

Two plasmids were isolated as thermosensitive replicons following in vitro mutagenesis of pB4, a pAL5000 derivative mycobacteria/Escherichia coli shuttle plasmid. Plasmids pCG59 and pCG63 replicate at 30 degrees C but not at 39 degrees C. This will allow their utilisation for transposon delivery, site-specific integration, or allele exchange.

Escherichia coli↗
PubMed1992

Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis.

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.

Bacillus thuringiensis↗
PubMed1992

Cloning and characterization of an amplified DNA sequence in chromosomal DNA of Streptomyces aureofaciens 2201.

In strain 2201 of Streptomyces aureofaciens, a high copy number amplified DNA sequence (ADS-Sa2201) was found and characterized. The amplified sequence in the chromosomal DNA of this strain forms a stretch of about 10 kb tandemly repeated 100-500 times. In this strain also, extrachromosomal copies of the repeated unit of the ADS-Sa2201 were found. In cloning experiments any autonomous replicon was found on ADS-Sa2201 and it thus can be presumed that the presence of the extrachromosomal copies of the repeated unit of ADS-Sa2201 is only a result of its excision from the chromosome. A part of the repeated unit of ADS-Sa2201 was also found in chromosomal DNA of strain 13 of S. aureofaciens. In this strain no part of ADS-Sa2201 is present extrachromosomally.

Cloning, Molecular↗
PubMed1982

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction.

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.

Acetyltransferases↗
PubMed1983

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli.

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.

Cloning, Molecular↗
PubMed1985

ColD-derived cloning vectors that autoamplify in the stationary phase of bacterial growth.

The construction of cloning vectors based on the replicon of plasmid ColD-CA23 is reported. These vectors, like ColD itself, autoamplify when cultures of host bacteria enter the stationary phase of growth, thereby resulting in a substantial increase in the expression of cloned genes as a consequence of the increase in gene dosage. The principal advantage of these vectors is that, unlike the situation pertaining to other expression vectors, the increase in expression of genes cloned in ColD vectors does not require any experimental intervention (i.e., occurs naturally), and takes place at high cell densities. The vectors show high stability in Escherichia coli strains and are compatible with ColE1-type cloning vectors.

Catechol 2,3-Dioxygenase↗
PubMed1985

Versatile mercury-resistant cloning and expression vectors.

Cloning vectors have been constructed employing two diverse replicons, IncQ and P15A. Both vectors confer resistance to kanamycin (Km) and mercuric ions (Hg2+). One of these vectors, pDG105, is a broad-host-range, nonconjugative, oligocopy IncQ plasmid, which is capable of transforming Escherichia coli, Acinetobacter calcoaceticus, and Pseudomonas putida. The second vector, pDG106, is a narrow-host-range, multicopy cloning vector compatible with pBR322. Both vectors contain unique cloning sites in the Km-resistance gene for HindIII, SmaI, and XhoI, as well as unique EcoRI and ScaI sites in the mer operon. Cloning into the EcoRI site in the mer operon results in the mercury "supersensitive" phenotype, easily detectable by replica plating. Insertion of the galK gene into the EcoRI site in the mer operon results in Hg2+-inducible galactokinase activity, demonstrating the application of these plasmids as regulated expression vectors.

Biological Transport↗
PubMed1987

Design and construction of a versatile system for the expression of foreign genes in plants.

We have built a series of vectors to allow the constitutive or light-regulated expression of foreign genes in plants. These vectors carry expression cassettes consisting of either the cauliflower mosaic virus 35S promoter or the pea rbcS-E9 promoter, a multiple cloning site derived from M13um20, and the rbcS-E9 polyadenylation site. These cassettes have been incorporated into pBR322-based or RK2-based replicons to facilitate direct DNA uptake or Agrobacterium tumefaciens-mediated gene transfer. Their application for the expression of a bacterial gene is described.

Base Sequence↗
PubMed1987

A cos-mini-Mu vector for in vivo DNA cloning.

Construction of a mini-Mu plasmid vector containing a cosmid replicon is described. Upon derepression of mini-Mu transposition, bacterial DNA sequences can be flanked by the integrated mini-Mu. These sequences can then be packaged into lambda heads by superinfection with a lambda helper phage. Cosmid clones carrying particular bacterial genes can be recovered by selection after infection of appropriate strains with the cosmid transducing lambda lysate. We report here the successful in vivo cloning of several Escherichia coli genes using the transposoncosmid vector.

Bacteriophage lambda↗
PubMed1988

Smart2, a cosmid vector with a phage lambda origin for both systematic chromosome walking and P-element-mediated gene transfer in Drosophila.

We describe a new phage-lambda-replicon-based cosmid vector suitable for both chromosome walking and P-element-mediated transformation in Drosophila. Its unique BamHI cloning site is flanked by the promoters for the SP6 and T7-encoded RNA polymerases, permitting the synthesis of probes complementary to the ends of the cloned inserts for library screening. The selectable marker is tet for bacterial cell transformation and neo for Drosophila transformation expressed under the control of the Drosophila hsp70 promoter.

Animals↗
PubMed1989

Chromosomal transfer and in vivo cloning of genes in Vibrio cholerae using RP4::mini-Mu.

The RP4::mini-Mu replicon has been used to transfer chromosomal genes by conjugation and to clone in vivo metabolic, toxin and flagellar genes of Vibrio cholerae. RP4::mini-Mu was introduced into several strains of V. cholerae and these strains were mated with V. cholerae or Escherichia coli K-12 recipients. R'-episomes carrying the respective genes were maintained in recA recipients and were detected by complementation of auxotrophic, nontoxinogenic and aflagellate mutations in V. cholerae.

Cloning, Molecular↗
PubMed1989

Vectors permitting visual monitoring of simple transposition events.

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.

Bacterial Proteins↗