Margaretha Stålhammar-Carlemalm

Plasmid analysis and restriction fragment length polymorphisms of chromosomal DNA allow a distinction between Borrelia burgdorferi strains.

We examined the relationships of the genomes of five strains of Borrelia burgdorferi isolated from ticks, two from North America, including the type strain B31, and three from Switzerland. We determined restriction fragment length polymorphisms by using eight cloned DNA fragments as hybridization probes to genomic Southern blots. Two divergent patterns were observed, represented by B31 and one Swiss strain on the one hand and the two other Swiss strains on the other. The second American strain resembled B31. One of the DNA probes allowed distinction between the closely related strains within a group. The close resemblance of one Swiss strain to the North American strains suggests the possibility of their European origin. All five strains carried a circular plasmid of about 29 kb, and three contained an additional species of about 9 kb, both of which exhibited homology between the strains. The profiles of linear plasmids revealing species of 5.1 kb to 58 kb reflected the polymorphisms of chromosomal DNAs. Linear plasmids of similar size shared DNA sequence homology. Some of the smaller plasmids tended to become lost during cultivation.

Denaturation map of bacteriophage P1 DNA

Abstract Electron microscopy of bacteriophage P1 plasmid DNA partially denatured by high pH in the presence of formaldehyde and mounted by the protein monolayer technique revealed a unique denaturation pattern. Alignment with the restriction cleavage map was obtained by comparing the melting pattern of plasmid DNA linearized by a single cut introduced by the restriction enzyme Pst I with those of the large Hind III, Bgl II, and Bam HI restriction fragments. Major A + T-rich segments center around map units 3, 21, 46, and 52. Other denatured regions are located around map units 29, 32 (within the invertible segment), 62, 88, and 96. The invertible segment of P1 DNA was precisely mapped within the Bam HI-5 and the Bgl II-5 DNA restriction fragments. The DNA segment melting first and therefore having the highest A + T content spans the P1-specific restriction modification region between map units 0 and 6 of the P1 genome. The Pl-specific recombination site, loxP , is also located in a region of low DNA helix stability. The denaturation pattern of the DNA of a hybrid P1-R plasmid revealed IS 1 -mediated transposition of the r-determinant into a site adjacent to the most A + T-rich region on Pl.

Expression of a neomycin phosphotransferase gene from Streptomyces fradiae in Escherichia coli after interplasmidic recombination

SummaryPlasmid pIJ2 carrying the neomycin phosphotransferase gene of Streptomyces fradiae was fused to E. coli plasmid pBR325 and the hybrid molecules were introduced into E. coli K12 by transformation. The neomycin phosphotransferase gene of the hybrid plasmid was not expressed in E. coli, except after interplasmidic recombination. Physical analysis of such an in vivo recombinant plasmid revealed that the recombination brought one neomycin phosphotransferase gene to a position downstream from the tet-promoter of pBR325. Subcloning experiments indicated that this is the gene copy expressed, and that transcription is initiated at the tet-promoter of pBR325.

Sequence relations among the IncY plasmid p15B, P1, and P7 prophages

Electron microscopic analysis of heteroduplex molecules between the 94-kb plasmid p15B and the 92-kb phage P1 genome revealed nine regions of nonhomology, eight substitutions, and two neighboring insertions. Overall, the homologous segments correspond to 83% of the P1 genome and 81% of p15B. Heteroduplex molecules between p15B and the 99-kb phage P7 genome showed nonhomology in eight of the same nine regions; in addition, two new nonhomologous segments are present and P7 carries a 5-kb insertion representing Tn902. The DNA homology between those two genomes amounts to 79% of P7 DNA and 83% of p15B. Plasmid p15B contains two stem-loop structures. One of them has no equivalent structure on P1 and P7 DNA. The other substitutes the invertible C segments of P1 and P7 and their flanking sequences including cin, the gene for the site-specific recombinase mediating inversion.

Repeated DNA Sequences on Circular and Linear Plasmids of Borrelia Burgdorferi Sensu Lato

We cloned a 3.8 kb EcoRI fragment from the 29 kb circular plasmid of the Borrelia burgdorferi sensu stricto type strain B31, which hybridized to several additional restriction fragments of the 29 kb circular and the 50 kb linear plasmid of this strain (1.4 kb, 6.4 kb, 7.0 kb EcoRI and 4.5 kb HindIII, respectively). Three of the latter fragments were also cloned and their crosshybridizing segments identified on subclones.

Genome fusion mediated by the site specific DNA inversion system of bacteriophage P1

SummaryThe genome of bacteriophage P1 contains a segment which is invertible by site specific recombination between sequences near the outside ends of the inverted repeats which flank it. Immediately adjacent to this C segment is the coding sequence for cin, the enzyme catalyzing inversion. We show that multicopy plasmids carrying cin and the sequences at which it acts (cix) can form dimers in the absence of the host recA function. Further, such plasmids can be cotransduced with P1 markers at high frequency from recA lysogens, indicating cointegration with the P1 genome. It is thus demonstrated that a system whose primary role is the inversion of a specific DNA segment can also mediate intermolecular recombination.

DNA of the Streptomyces phage SH10: binding sites for Streptomyces hygroscopicus RNA polymerase and in vitro transcription map

Abstract The binding sites for the Streptomyces hygroscopicus RNA polymerase on the DNA of actinophage SH10 were mapped by electron microscopy. Four major binding sites were located at map positions 52.2, 84.5, 97.9 and 99.1 very close or identical to sequences to which the Escherichia coli RNA polymerase was observed to bind. In vitro transcription activities of the two RNA polymerases were compared by analyzing transcriptional R-loops on SH10 DNA. Both enzymes transcribed almost exclusively segments in the right half of the phage genome. Most frequent initiation was found from a site at map position 98.1. Differences in efficiency of transcription between the two enzymes are discussed with respect to heterospecific gene expression.

Genotypic and phenotypic diversity among nine Swiss isolates of Borrelia burgdorferi

Nine strains of Borrelia burgdorferi isolated from ticks in the canton of Valais (Switzerland) were characterized genotypically by determining restriction fragment length polymorphisms (RFLP) and plasmid profiles. The strains were also compared with respect to presence and electrophoretic mobility of the outer surface proteins OspA and OspB, and immunoreactivity of OspA and a 12 kD antigen. By both approaches, three different patterns were observed resulting in identical grouping of the strains. However, RFLPs allowed determination of relationships among strains within a group and have shown that geographic distribution does not correlate with genotype.

DNA of the Streptomyces phage SH10: binding sites for Escherichia coli RNA polymerase and denaturation map.

SummaryEscherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.

Visualization of RNA polymerase bound to R-loop molecules improves electron microscopic analysis of in vitro transcription

An electron microscope method is described which allows improved analysis of in vitro transcription. Transcription complexes are fixed with glutaraldehyde, subjected to R-loop conditions which allow the nascent RNA chains to hybridize to the DNA templates, and mounted for electron microscopy by a protein-free preparation method. An RNA polymerase molecule (or parts of it) associated with only one end of the R-loop identifies the polarity of the transcript, thus determining the origin and direction of transcription. The method was evaluated using known in vitro promoters on the bacteriophage P1 genome and was used for mapping of additional promoters in their vicinity.