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.
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- Ammermann D., Steinbrück G., von Berger L., Hennig W. The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma. 1974 May 10;45(4):401–429. doi: 10.1007/BF00283386. [DOI] [PubMed] [Google Scholar]
- Anderson R. C., Lindauer K. R., Prescott D. M. A gene-sized DNA molecule encoding heat-shock protein 70 in Oxytricha nova. Gene. 1996 Feb 2;168(1):103–107. doi: 10.1016/0378-1119(95)00735-0. [DOI] [PubMed] [Google Scholar]
- Baird S. E., Klobutcher L. A. Differential DNA amplification and copy number control in the hypotrichous ciliate Euplotes crassus. J Protozool. 1991 Mar-Apr;38(2):136–140. doi: 10.1111/j.1550-7408.1991.tb06033.x. [DOI] [PubMed] [Google Scholar]
- Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
- Bender J., Klein A. The telomere binding protein of Euplotes crassus prevents non-specific transcription initiation but has no role in positioning transcription initiation complexes. Nucleic Acids Res. 1997 Jul 15;25(14):2877–2882. doi: 10.1093/nar/25.14.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berghöfer 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 Feb;242(4):369–373. doi: 10.1007/BF00281785. [DOI] [PubMed] [Google Scholar]
- Blomberg P., Randolph C., Yao C. H., Yao M. C. Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila. Mol Cell Biol. 1997 Dec;17(12):7237–7247. doi: 10.1128/mcb.17.12.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brandt A., Klein A. Transcription rates and transcript stabilities of macronuclear genes in vegetative Euplotes crassus cells. J Eukaryot Microbiol. 1995 Nov-Dec;42(6):691–696. doi: 10.1111/j.1550-7408.1995.tb01617.x. [DOI] [PubMed] [Google Scholar]
- Chomczynski P. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem. 1992 Feb 14;201(1):134–139. doi: 10.1016/0003-2697(92)90185-a. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dickover R. E., Donovan R. M., Goldstein E., Dandekar S., Bush C. E., Carlson J. R. Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J Clin Microbiol. 1990 Sep;28(9):2130–2133. doi: 10.1128/jcm.28.9.2130-2133.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dynan W. S. Modularity in promoters and enhancers. Cell. 1989 Jul 14;58(1):1–4. doi: 10.1016/0092-8674(89)90393-0. [DOI] [PubMed] [Google Scholar]
- Gaertig J., Gorovsky M. A. Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9196–9200. doi: 10.1073/pnas.89.19.9196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gaertig J., Gu L., Hai B., Gorovsky M. A. High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic Acids Res. 1994 Dec 11;22(24):5391–5398. doi: 10.1093/nar/22.24.5391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ghosh S., Jaraczewski J. W., Klobutcher L. A., Jahn C. L. Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes. Nucleic Acids Res. 1994 Jan 25;22(2):214–221. doi: 10.1093/nar/22.2.214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godiska R., Aufderheide K. J., Gilley D., Hendrie P., Fitzwater T., Preer L. B., Polisky B., Preer J. R., Jr Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7590–7594. doi: 10.1073/pnas.84.21.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harumoto T., Hiwatashi K. Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium. Dev Genet. 1992;13(2):118–125. doi: 10.1002/dvg.1020130205. [DOI] [PubMed] [Google Scholar]
- Kahn R. W., Andersen B. H., Brunk C. F. Transformation of Tetrahymena thermophila by microinjection of a foreign gene. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9295–9299. doi: 10.1073/pnas.90.20.9295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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 Sep 11;20(17):4445–4450. doi: 10.1093/nar/20.17.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klobutcher L. A., Swanton M. T., Donini P., Prescott D. M. 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 May;78(5):3015–3019. doi: 10.1073/pnas.78.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- La Terza A., Miceli C., Luporini P. Differential amplification of pheromone genes of the ciliate Euplotes raikovi. Dev Genet. 1995;17(3):272–279. doi: 10.1002/dvg.1020170312. [DOI] [PubMed] [Google Scholar]
- Ogbourne S., Antalis T. M. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem J. 1998 Apr 1;331(Pt 1):1–14. doi: 10.1042/bj3310001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prescott D. M. The DNA of ciliated protozoa. Microbiol Rev. 1994 Jun;58(2):233–267. doi: 10.1128/mr.58.2.233-267.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Price C. M., Skopp R., Krueger J., Williams D. DNA recognition and binding by the Euplotes telomere protein. Biochemistry. 1992 Nov 10;31(44):10835–10843. doi: 10.1021/bi00159a026. [DOI] [PubMed] [Google Scholar]
- Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
- Roeder R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci. 1996 Sep;21(9):327–335. [PubMed] [Google Scholar]
- Tondravi M. M., Yao M. C. Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4369–4373. doi: 10.1073/pnas.83.12.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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 Dec 25;20(24):6621–6629. doi: 10.1093/nar/20.24.6621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wen J. P., Eder C., Lipps H. J. The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae. Nucleic Acids Res. 1995 May 25;23(10):1704–1709. doi: 10.1093/nar/23.10.1704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wünning I. U., Lipps H. J. A transformation system for the hypotrichous ciliate Stylonychia mytilus. EMBO J. 1983;2(10):1753–1757. doi: 10.1002/j.1460-2075.1983.tb01653.x. [DOI] [PMC free article] [PubMed] [Google Scholar]