Macronuclear control of DNA processing in Paramecium tetraurelia

Yun You, Purdue University

Abstract

Paramecium tetraurelia contains two types of functionally and morphologically distinct nuclei: two micronuclei and a macronucleus within a cell. The transcriptionally inert micronuclei are diploid and possess the germline DNA. During sexual reproduction micronuclear DNA produces the new micronuclei as well as macronuclei. Macronuclear DNA is a highly edited version of micronuclear DNA in which each gene has been amplified to approximately 1700 copies. It is transcriptionally active and therefore determines the phenotype of the cell. A non-Mendelian mutant, d48, lacks the A surface protein gene in the macronucleus and therefore A surface protein can not be expressed, but the A gene in the micronuclei remains intact. Microinjection of the entire A gene into the macronucleus of d48 resulted in a wild type cell after autogamy. My work has focused on the analysis of the A gene regions responsible for this d48 rescue activity. By injecting different non-overlapping portions of the A gene, it has been shown that a plasmid with the A gene internal tandem repeats and surrounding sequence can rescue about 50% of the transformants while the 5$\sp\prime$ portion of the A gene has poor activity (2 out of 19 transformants rescued). Interestingly, when the two plasmids were coinjected into the macronucleus of d48, 32 out of 33 transformants were rescued. Injecting the highly related B gene (73% nucleotide sequence identity) did not rescue d48. The injected DNA did not function as template. A Sst II restriction site, created within the coding region of injected A gene, disappeared after autogamy and wild type DNA was incorporated into the rescued cell. The A gene with a central region inverted could rescue d48 at the same level as the intact A gene. In general, shorter fragments of the A gene rescued d48 with a lower efficiency than larger fragments. Analysis of subclone lines showed that the difference in rescue efficiency between short and long fragments could not be explained by a difference in the distribution of injected DNA among the vegetative descendants of a transformed cell. A recombinant clone containing one of the wild type A gene telomeres was transformed into d48, after autogamy the sequence near the telomere was incorporated into the macronuclear DNA as shown by Southern blot hybridization. However, these d48 cells lacked the A gene in the macronuclear DNA. It is concluded that the DNA in the old macronucleus affects the micronuclear DNA processing during macronuclear development.

Degree

Ph.D.

Advisors

Forney, Purdue University.

Subject Area

Molecular biology|Genetics

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