Faithful Expression of a Heterologous Gene Carried on an Artificial Macronuclear Chromosome in Euplotes Crassus

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1999 Aug 24

PMID 10454614

Citations 3

Authors

J Bender

M Kampfer

A Klein

Affiliations

Soon will be listed here.

Abstract

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.

Citing Articles

Transformation with Artificial Chromosomes in and Their Applications.

Clay D, Kim H, Landweber L G3 (Bethesda). 2019; 9(10):3119-3127.

PMID: 31506318 PMC: 6778790. DOI: 10.1534/g3.119.400298.

RNA-dependent control of gene amplification.

Heyse G, Jonsson F, Chang W, Lipps H Proc Natl Acad Sci U S A. 2010; 107(51):22134-9.

PMID: 20974970 PMC: 3009782. DOI: 10.1073/pnas.1009284107.

Intron Evolution and Information processing in the DNA polymerase alpha gene in spirotrichous ciliates: a hypothesis for interconversion between DNA and RNA deletion.

Chang W, Addis V, Li A, Axelsson E, Ardell D, Landweber L Biol Direct. 2007; 2:6.

PMID: 17270054 PMC: 1805493. DOI: 10.1186/1745-6150-2-6.

References

Berghofer Y, Agha-Amiri K, Klein A . Selenium is involved in the negative regulation of the expression of selenium-free [NiFe] hydrogenases in Methanococcus voltae. Mol Gen Genet. 1994; 242(4):369-73. DOI: 10.1007/BF00281785. View

Wang W, Skopp R, Scofield M, Price C . Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs. Nucleic Acids Res. 1992; 20(24):6621-9. PMC: 334579. DOI: 10.1093/nar/20.24.6621. View

Blomberg P, Randolph C, Yao C, Yao M . Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila. Mol Cell Biol. 1997; 17(12):7237-47. PMC: 232581. DOI: 10.1128/MCB.17.12.7237. View

Anderson R, Lindauer K, Prescott D . A gene-sized DNA molecule encoding heat-shock protein 70 in Oxytricha nova. Gene. 1996; 168(1):103-7. DOI: 10.1016/0378-1119(95)00735-0. View

Wen J, Eder C, Lipps H . The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae. Nucleic Acids Res. 1995; 23(10):1704-9. PMC: 306925. DOI: 10.1093/nar/23.10.1704. View

Harumoto T, Hiwatashi K . Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium. Dev Genet. 1992; 13(2):118-25. DOI: 10.1002/dvg.1020130205. View

Church G, Gilbert W . Genomic sequencing. Proc Natl Acad Sci U S A. 1984; 81(7):1991-5. PMC: 345422. DOI: 10.1073/pnas.81.7.1991. View

Godiska R, Aufderheide K, Gilley D, Hendrie P, Fitzwater T, Preer L . Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci U S A. 1987; 84(21):7590-4. PMC: 299345. DOI: 10.1073/pnas.84.21.7590. View

Klobutcher L, Swanton M, Donini P, Prescott D . All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A. 1981; 78(5):3015-9. PMC: 319490. DOI: 10.1073/pnas.78.5.3015. View

10.

Ghosh S, Jaraczewski J, Klobutcher L, Jahn C . Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes. Nucleic Acids Res. 1994; 22(2):214-21. PMC: 307774. DOI: 10.1093/nar/22.2.214. View

11.

Baird S, Klobutcher L . Differential DNA amplification and copy number control in the hypotrichous ciliate Euplotes crassus. J Protozool. 1991; 38(2):136-40. DOI: 10.1111/j.1550-7408.1991.tb06033.x. View

12.

Brandt A, Klein A . Transcription rates and transcript stabilities of macronuclear genes in vegetative Euplotes crassus cells. J Eukaryot Microbiol. 1995; 42(6):691-6. DOI: 10.1111/j.1550-7408.1995.tb01617.x. View

13.

Prescott D . The DNA of ciliated protozoa. Microbiol Rev. 1994; 58(2):233-67. PMC: 372963. DOI: 10.1128/mr.58.2.233-267.1994. View

14.

Roeder R . The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci. 1996; 21(9):327-35. View

15.

KAHN R, Andersen B, Brunk C . Transformation of Tetrahymena thermophila by microinjection of a foreign gene. Proc Natl Acad Sci U S A. 1993; 90(20):9295-9. PMC: 47554. DOI: 10.1073/pnas.90.20.9295. View

16.

Kaufmann J, Klein A . Gene dosage as a possible major determinant for equal expression levels of genes encoding RNA polymerase subunits in the hypotrichous ciliate Euplotes octocarinatus. Nucleic Acids Res. 1992; 20(17):4445-50. PMC: 334170. DOI: 10.1093/nar/20.17.4445. View

17.

Ptashne M . How eukaryotic transcriptional activators work. Nature. 1988; 335(6192):683-9. DOI: 10.1038/335683a0. View

18.

Price C, Skopp R, Krueger J, Williams D . DNA recognition and binding by the Euplotes telomere protein. Biochemistry. 1992; 31(44):10835-43. DOI: 10.1021/bi00159a026. View

19.

Wunning I, Lipps H . A transformation system for the hypotrichous ciliate Stylonychia mytilus. EMBO J. 1983; 2(10):1753-7. PMC: 555354. DOI: 10.1002/j.1460-2075.1983.tb01653.x. View

20.

Gaertig J, Gorovsky M . Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc Natl Acad Sci U S A. 1992; 89(19):9196-200. PMC: 50092. DOI: 10.1073/pnas.89.19.9196. View