MEASUREMENT OF THE TRANSCRIPTION OF NUCLEAR SINGLE-COPY DEOXYRIBONUCLEIC-ACID DURING CHLOROPLAST DEVELOPMENT IN EUGLENA-GRACILIS

The fraction of nuclear single-copy deoxyribonucleic acid (DNA) transcribed at different stages of chloroplast development in Euglena gracilis (Z strain) was measured by RNA-DNA hybridization. Euglena cells were grown in a heterotrophic medium in the dark to stationary phase and transferred to the light. Total cell RNA was isolated at various stages of chloroplast development and hybridized in a vast excess to 125I-labeled single-copy DNA. The fraction of 125I-labeled single-copy DNA in the form of a duplex was measured by using S1 nuclease. The amount of RNA-DNA hybrid in the duplex mixture was determined by correcting for the contribution of DNA-DNA renaturation. The fraction of single-copy DNA transcribed was calculated by multiplying by 2 the amount of DNA in the form of an RNA-DNA hybrid and correcting for the reactivity of the single-copy DNA probe with total DNA. In dark-grown cells (i.e., prior to the initiation of chloroplast development), the complexity of total cell RNA derived from single-copy DNA was 8.0ËŸ107 nucleotides. After initiation of chloroplast development, the complexity of the total cell RNA derived from single-copy DNA first increased slightly to 8.9ËŸ107 nucleotides and then progressively decreased to 7.4ËŸ107 and 6.4ËŸ107 nucleotides after 12, 48, and 72 h of exposure to light, respectively. Total cell RNA isolated from cells which had never been cultured in the dark had a complexity of 6.5ËŸ107 nucleotides. © 1979, American Chemical Society. All rights reserved.