G.A. van Arkel

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.

Homology of plasmids in strains of unicellular cyanobacteria

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 106 and 32.7 × 106. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 106 and 24 × 106. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 106 and 2.0 × 106, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.

Vectors for cloning in cyanobacteria: Construction and characterization of two recombinant plasmids capable of transformation to Escherichia coli K12 and Anacystis nidulans R2

SummaryTwo plasmids were constructed consisting of the E. coli vector pACYC184 and the cyanobacterial plasmid pUC1. These recombinants, designated pUC104 and pUC105, can be transformed to E. coli K12 as well as to the cyanobacterium Anacystis nidulans R2 and in both hosts they express their antibiotic markers. pUC104 and pUC105 differ with respect to the location and the orientation of the pACYC184 segment in pUC1. pUC104 was found to be stable under all circumstances. Transformation of pUC105 to A. nidulans R2 gave intact plasmids when chloramphenicol was the selective agent, but upon ampicillin selection a deletion derivative was produced identical to pUC1. Further characteristics of pUC104 and pUC105 are described and their usefulness as cloning vectors is discussed.

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

SummarySynechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Apr gene of the E. coli plasmid pACYC177, yielding the Kmr hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Kmr shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm2 gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cmr Kmr shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.

Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described.

Improved method for the isolation of the A and A* proteins of bacteriophage φX174

The A protein (mol. wt 55 000) encoded by the gene A of bacteriophage φX174 is a multifunctional enzyme that is active in the replication of the viral DNA. This protein initiates a rolling circle type of DNA replication by introducing a break in the viral strand of RF1 DNA at the origin of replication. It is the only phage-coded protein necessary for this type of DNA replication and related phages (e.g., G4) use a similar protein.

Mapping of host range mutants of bacteriophage φX174

Abstract Three independently isolated host range mutants of phage φX174 are localized in gene G. This lends further support to the idea that the gene G product is the main protein involved in the adsorption process. The mapping was performed by hybridizing fragments of the RF DNA of the host range mutants with circular single-stranded DNA of suppressor-sensitive or temperature-sensitive mutants from seven cistrons. Different fragment lengths were used and the amount of cohybridization of the h mutation with the sus+ or ts+ allele was a measure for the distance between the two markers. It is shown that the same method can be applied to localize every marker under investigation.

The genetic map of bacteriophage ∅X174 constructed with restriction enzyme fragments

Abstract Mutants in all known ∅X174 genes were located on the fragment maps produced with restriction enzymes from Arthrobacter luteus (Alu I), Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae III). In this way physical distances between mutations were measured. The size of several genes, including gene A, could be determined. The eight gene A mutants tested show a strong asymmetric distribution within this gene, indicating a large internal region of low mutability. The relative position of many restriction enzyme fragments has been confirmed. The consequences of this genetic map for the organization of the ∅X genome are discussed.

The inactivating and mutagenic effect of hydroxylamine on bacteriophage φX174

The inactivation of bacteriophage ΦXI74 by the mutagenic agents nitrous acid and ultraviolet irradiation proceeds according to a single-hit kinetics. However, treatment of purified ΦXI74 by hydroxylamine (HA) at pH 6 and 25° results in an inactivation that is not strictly exponential. The inactivation curves contain a shoulder, which is easily detectable at a HA concentration of o.1 M. The ultimate slope of the curves at this concentration is in many cases greater than at 1 M.

Complementation experiments between conditional lethal mutants of bacteriophage ϕX174

SummaryComplementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage ϕX174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.