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Summary Macronuclei of Stylonychia lemnae contain millions of gene sized minichromosomes. These minichromosomes in general harbour only one gene. Their nontranscribed regions are usually less than 400 bp long and most likely carry the signals for DNA replication and transcription. Therefore, S. lemnae seems to be an ideal object to study these processes in a protist organism. By microinjecting defined linear minichromosomal DNA directly into macronuclei of vegetative cells, we have developed a suitable in vivo assay system for S. lemnae. Transfections were carried out with α1 tubulin minichromosomes whose gene was internally tagged by a 19 bp-long insertion. Although the endogenous ocl tubulin minichromosome is present in approximately 100,000 copies per cell, clonal cell lines were generated which stably contained tagged minichromosomes in up to a fivefold excess of endogenous copy numbers. As demonstrated by Southern blot analysis, the copy number of endogenous α1 tubulin minichromosomes was not reduced in these cell lines, and transfected minichromosomes were not noticeably rearranged by the cells. Moreover, tagged ocl tubulin genes were unequivocally expressed. Transcription of these genes was initiated correctly, and the amount of transcript present in steady-state RNA correlated with gene copy number. |
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