The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae

Abstract

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.

Characterization of the Euplotes crassus Macronuclear rDNA and its Potential as a DNA Transformation Vehicle

“…Although higher levels of transformants are obtained when a C type rDNA is injected into a B type T. thermophila, the B type rDNA does not confer a replication advantage in B type cells and thus is comparable to the constructs used here. Our results contrast with a report of microinjection of Stylonychia anlagen at a slightly earlier developmental stage, where 30% of the injected cells carried the injected DNA [47]. Since our transformation frequency is based on the number of cells that actually showed increased resistance to anisomycin, and not the number of cells with detectable amounts of injected DNA, there may be many more cells that propagate the injected DNA but do not contain sufficient amounts to confer increased resistance to anisomycin.…”

Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae

“…All deletion constructs used in this study are based on the construct pCE5 (Figure 2a [ 25 ]). As shown previously by PCR and Southern analysis [ 25 , 27 ], the concentration of the modified 1.3 kb macronuclear DNA molecule derived from the injected construct was always very low compared with the copy number of the endogenous 1.3 kb DNA molecule [ 27 ].…”

“…All deletion constructs used in this study are based on the construct pCE5 (Figure 2a [ 25 ]). As shown previously by PCR and Southern analysis [ 25 , 27 ], the concentration of the modified 1.3 kb macronuclear DNA molecule derived from the injected construct was always very low compared with the copy number of the endogenous 1.3 kb DNA molecule [ 27 ]. This may be due to a very tight copy number control mechanism occurring during macrouclear development, which allows only a very limited amplification of the injected material, independent of the amount injected.…”

“…This may be due to a very tight copy number control mechanism occurring during macrouclear development, which allows only a very limited amplification of the injected material, independent of the amount injected. All results presented in this study are therefore based on PCR analysis as described previously [ 14 , 25 , 27 ].…”

“…We therefore decided to identify cis -acting sequences involved in DNA fragmentation and telomere addition by injecting modified macronuclear precursor sequences into the developing macronuclei. Recently, we demonstrated that injection of such a construct (pCE5) into the developing macronucleus resulted in correct fragmentation and de novo telomere addition [ 25 ]. This construct contained two macronuclear precursor sequences homologous to a 1.1 kb and a 1.3 kb macronuclear DNA molecule ([ 26 ]; GenBank accession numbers X72955 and X72956).…”