A van der Ende

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.

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.

δρ-Web, an online tool to assess composition similarity of individual nucleic acid sequences

SUMMARY Although whole-genome sequences have been analysed for the presence of anomalous DNA, no dedicated application is currently available to analyse the composition of individual sequence entries, for instance those derived by experimental techniques, such as subtractive hybridization. Since genomic dinucleotide frequency values are conserved between related species, a representative genome sequence can often be found to score for anomalous sequence composition for many of these putative horizontally transferred sequences. We developed the application deltarho-web, which enables the determination of the differences between the dinucleotide composition of an input sequence and that of a selected genome in a size-dependent manner. A feature allowing batch comparisons is included as well. In addition, deltarho-web allows the analysis of the dinucleotide composition of complete genomes. This provides complementary information for the identification of large anomalous gene clusters.

An acquisition account of genomic islands based on genome signature comparisons

BackgroundRecent analyses of prokaryotic genome sequences have demonstrated the important force horizontal gene transfer constitutes in genome evolution. Horizontally acquired sequences are detectable by, among others, their dinucleotide composition (genome signature) dissimilarity with the host genome. Genomic islands (GIs) comprise important and interesting horizontally transferred sequences, but information about acquisition events or relatedness between GIs is scarce. In Vibrio vulnificus CMCP6, 10 and 11 GIs have previously been identified in the sequenced chromosomes I and II, respectively. We assessed the compositional similarity and putative acquisition account of these GIs using the genome signature. For this analysis we developed a new algorithm, available as a web application.ResultsOf 21 GIs, VvI-1 and VvI-10 of chromosome I have similar genome signatures, and while artificially divided due to a linear annotation, they are adjacent on the circular chromosome and therefore comprise one GI. Similarly, GIs VvI-3 and VvI-4 of chromosome I together with the region between these two islands are compositionally similar, suggesting that they form one GI (making a total of 19 GIs in chromosome I + chromosome II). Cluster analysis assigned the 19 GIs to 11 different branches above our conservative threshold. This suggests a limited number of compositionally similar donors or intragenomic dispersion of ancestral acquisitions. Furthermore, 2 GIs of chromosome II cluster with chromosome I, while none of the 19 GIs group with chromosome II, suggesting an unidirectional dispersal of large anomalous gene clusters from chromosome I to chromosome II.ConclusionFrom the results, we infer 10 compositionally dissimilar donors for 19 GIs in the V. vulnificus CMCP6 genome, including chromosome I donating to chromosome II. This suggests multiple transfer events from individual donor types or from donors with similar genome signatures. Applied to other prokaryotes, this approach may elucidate the acquisition account in their genome sequences, and facilitate donor identification of GIs.

Recycling of 5'-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5′‐nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5′‐nucleotidase. Incubation of 125I‐surface‐labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5′‐nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5′‐nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5′‐nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5′‐nucleotidase is not inhibited by primaquine.

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.

Coronaviruses in polarized epithelial cells

Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supports. By inoculation from the apical or basolateral side both TGEV and MHV-A59 were found to enter the polarized cells only through the apical membrane. The release of newly synthesized TGEV from LLC-PK1 cells occurred preferentially from the apical plasma membrane domain, as evidenced by the accumulation of viral proteins and infectivity in the apical culture fluid. In contrast, MHV was released preferentially from the basolateral membrane of mTAL cells. The apical release of TGEV and the basolateral release of MHV may explain the in vivo establishment of a local and systemic infection, respectively.

Bacteriophage φX174 and G4 RF DNA replicative intermediates: A comparative study using different isolation procedures☆

We have isolated replicative intermediates of bacteriophage φX174 and the related baeteriophage G4, during RF (replicative form) DNA replication using different procedures. Biochemical and electron microscopic analysis of φX and G4 DNA replicative intermediates isolated by the same procedure, showed no significant differences. In the replication cycle of both phages rolling circles and gapped RF DNA molecules are the predominant replicative intermediates. It is concluded that G4 RF DNA also replicates according to a rolling circle model and not according to a D-looped replication model as proposed by Godson (1977b).