Physical characterization of a kinetoplast DNA fragment with unusual properties

J. C. Marini, P. N. Effron, T. C. Goodman, C. K. Singleton, R. D. Wells, R. M. Wartell, P. T. Englund

Research output: Contribution to journalArticle

Abstract

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to φX174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix. In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 ° C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w)). Thermal denaturation reveals a triphasic melting curve.

Original languageEnglish (US)
Pages (from-to)8974-8979
Number of pages6
JournalJournal of Biological Chemistry
Volume259
Issue number14
StatePublished - 1984
Externally publishedYes

Fingerprint

Kinetoplast DNA
Ethanol
Gels
Circular Dichroism
Electrophoresis
Conformations
Glyoxal
Endopeptidase K
Electrophoretic mobility
Denaturation
Cloning
Leishmania
Experiments
Phenol
Sedimentation
Base Pairing
Freezing
Gel Chromatography
Organism Cloning
Digestion

ASJC Scopus subject areas

  • Biochemistry

Cite this

Marini, J. C., Effron, P. N., Goodman, T. C., Singleton, C. K., Wells, R. D., Wartell, R. M., & Englund, P. T. (1984). Physical characterization of a kinetoplast DNA fragment with unusual properties. Journal of Biological Chemistry, 259(14), 8974-8979.

Physical characterization of a kinetoplast DNA fragment with unusual properties. / Marini, J. C.; Effron, P. N.; Goodman, T. C.; Singleton, C. K.; Wells, R. D.; Wartell, R. M.; Englund, P. T.

In: Journal of Biological Chemistry, Vol. 259, No. 14, 1984, p. 8974-8979.

Research output: Contribution to journalArticle

Marini, JC, Effron, PN, Goodman, TC, Singleton, CK, Wells, RD, Wartell, RM & Englund, PT 1984, 'Physical characterization of a kinetoplast DNA fragment with unusual properties', Journal of Biological Chemistry, vol. 259, no. 14, pp. 8974-8979.
Marini JC, Effron PN, Goodman TC, Singleton CK, Wells RD, Wartell RM et al. Physical characterization of a kinetoplast DNA fragment with unusual properties. Journal of Biological Chemistry. 1984;259(14):8974-8979.
Marini, J. C. ; Effron, P. N. ; Goodman, T. C. ; Singleton, C. K. ; Wells, R. D. ; Wartell, R. M. ; Englund, P. T. / Physical characterization of a kinetoplast DNA fragment with unusual properties. In: Journal of Biological Chemistry. 1984 ; Vol. 259, No. 14. pp. 8974-8979.
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