Highly Purified Micro‐ and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients

SALLY LYMAN ALLEN, THEODORE C. WHITE, JOHN P. LANGMORE, MARK A. SWANCUTT

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

ABSTRACT. A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from Tetrahymena thermophila. Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by electron microscopy; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC's contaminated with less than 1 MAC in 1000 MIC and MAC's contaminated with less than 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in our purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.

Original languageEnglish (US)
Pages (from-to)21-30
Number of pages10
JournalJournal of Protozoology
Volume30
Issue number1
DOIs
StatePublished - 1983

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Macronucleus
Tetrahymena thermophila
Southern Blotting
Ribosomal DNA
Light
Percoll
Microscopy
DNA
rRNA Genes

ASJC Scopus subject areas

  • Parasitology

Cite this

Highly Purified Micro‐ and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients. / ALLEN, SALLY LYMAN; WHITE, THEODORE C.; LANGMORE, JOHN P.; SWANCUTT, MARK A.

In: Journal of Protozoology, Vol. 30, No. 1, 1983, p. 21-30.

Research output: Contribution to journalArticle

ALLEN, SALLY LYMAN ; WHITE, THEODORE C. ; LANGMORE, JOHN P. ; SWANCUTT, MARK A. / Highly Purified Micro‐ and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients. In: Journal of Protozoology. 1983 ; Vol. 30, No. 1. pp. 21-30.
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AB - ABSTRACT. A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from Tetrahymena thermophila. Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by electron microscopy; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC's contaminated with less than 1 MAC in 1000 MIC and MAC's contaminated with less than 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in our purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.

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