Electron microscopy and biological properties of pBR322 DNA condensed with the trivalent cations spermidine and hexammine cobalt (III).
Electron microscopy and the biological properties of susceptibility to DNase I, genetic transcription, and transformation of pBR322 DNA compacted with spermidine or hexammine cobalt (III), were analyzed in order to characterize the association of DNA in its compacted form with these two different trivalent cations. Spermidine and hexammine cobalt (III) produced an average 4-fold reduction of the DNA perimeter in compact DNA forms, which were doughnut-shaped toroids and cylinders. Both compacted DNAs were resistant to the hydrolytic activity of DNase I. However, spermidine-condensed pBR322 DNA was 10-fold and 4 to 6-fold more active in transcription and transformation, respectively, than naked pBR322. I. Hexammine cobalt (III)-condensed pBR322 was inactive in both biological properties. An inhibitory effect of hexammine cobalt (III) on RNA polymerase and genetic transformation activities was discarded because at higher ionic strength, in which DNA is not compacted by hexammine cobalt (III), transcription and transformation were similar to those observed with naked DNA. This information showed that the interaction of hexammine cobalt (III) with the DNA converted the pBR322 DNA into an inert molecule. In contrast, pBR322 did not loose its biological properties after its interaction with the polyamine spermidine; i.e., experimental condensation of pBR322 DNA by spermidine produced compacted DNA that is more similar to compact native genomes than relaxed DNA. These experiments led us to conclude that spermidine-condensed DNA can be used to study the roll of the native supercoiling of DNA in the regulation of genetic replication and transcription, as well as to study the mechanisms that allow the accessibility of the supercoiled or condensed DNA substrate for enzymes.
Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas del IPN, México, D.F.
I
I
Baeza
L
L
Aguilar
R
R
Santiago
M
M
Ibáñez
C
C
Wong
D002412:Cations / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004269:DNA, Bacterial / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D004270:DNA, Circular / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D004274:DNA, Recombinant / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D009994:Osmolar Concentration; D013095:Spermidine / Q000494:pharmacology; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D002412:Cations; D004269:DNA, Bacterial; D004270:DNA, Circular; D004274:DNA, Recombinant; C030862:cobalt ammonium complex; D003035:Cobalt; D013095:Spermidine