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PMID: 7910408 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 91 ·No. 10 ·1994-05-10 ·Pages 4549-53

Gaertig J, Thatcher TH, Gu L, Gorovsky MA

Abstract

Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.

Related Genes

BTU1

MeSH Terms

Animals Base Sequence Blotting, Southern Chlamydomonas/genetics DNA/analysis,genetics DNA Primers Electroporation Gene Transfer Techniques Molecular Sequence Data Mutagenesis, Site-Directed Paclitaxel/toxicity Point Mutation Polymerase Chain Reaction RNA, Messenger/analysis,biosynthesis Tetrahymena thermophila/drug effects,genetics Tubulin/genetics

Chemicals

DNA Primers RNA, Messenger Tubulin DNA Paclitaxel

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Gaertig J

Department of Biology, University of Rochester, NY 14627.

Thatcher T H

Gu L

Gorovsky M A

References (22)

22 references, click to expand

Sequence of one alpha- and two beta-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other eukaryotic tubulin genes.
J Mol Biol. 1988 Aug 5;202(3):365-82 PMID: 3139885
Beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii.
Proc Natl Acad Sci U S A. 1988 Jan;85(1):131-5 PMID: 3422409
The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.
J Cell Biol. 1991 May;113(3):605-14 PMID: 1673126
Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.
Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9493-7 PMID: 1946363
Tubulin structure and biochemistry.
Curr Opin Cell Biol. 1992 Feb;4(1):53-7 PMID: 1558754
Rate of phenotypic assortment in Tetrahymena thermophila.
Dev Genet. 1992;13(2):126-32 PMID: 1499154
Missense mutations at lysine 350 in beta 2-tubulin confer altered sensitivity to microtubule inhibitors in Chlamydomonas.
Plant Cell. 1990 Nov;2(11):1051-7 PMID: 2152107
Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants.
Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9196-200 PMID: 1409625
Characterization of the tubulin-tyrosine ligase.
J Cell Biol. 1993 Feb;120(3):725-32 PMID: 8093886
Transformation of Tetrahymena thermophila by microinjection of a foreign gene.
Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9295-9 PMID: 8415695
Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single alpha- and beta-tubulin.
Cell Motil Cytoskeleton. 1993;25(3):243-53 PMID: 8221902
Isolation of micro- and macronuclei of Tetrahymena pyriformis.
Methods Cell Biol. 1975;9(0):311-27 PMID: 805898
Two-dimensional gel electrophoresis of membrane proteins.
Biochemistry. 1976 Feb 10;15(3):616-23 PMID: 766831
Induction and isolation of mutants in Tetrahymena.
Methods Cell Biol. 1976;13:247-82 PMID: 817110
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry. 1979 Nov 27;18(24):5294-9 PMID: 518835
Genome organization and reorganization in Tetrahymena.
Annu Rev Genet. 1980;14:203-39 PMID: 6782938
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem. 1983 Jul 1;132(1):6-13 PMID: 6312838
Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.
DNA. 1984 Dec;3(6):479-88 PMID: 6096101
Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.
Proc Natl Acad Sci U S A. 1985 Oct;82(20):6908-12 PMID: 3901010
Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.
Proc Natl Acad Sci U S A. 1986 Jun;83(12):4369-73 PMID: 3459180
Preparation and properties of dynein from Tetrahymena cilia.
Methods Enzymol. 1986;134:306-17 PMID: 2950293
Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.
Mol Cell Biol. 1989 Mar;9(3):1092-9 PMID: 2725489

Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.91.10.4549

Published

1994-05-10

Pages

4549-53

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC43823

Subset

Grants

NIGMS NIH HHS · GM26973 · United States

External Links

PubMed source DOI: 10.1073/pnas.91.10.4549 PMC Full Text View journal details

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Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.

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