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Transcription of dinoflagellate chloroplast minicircle genes Dang, Yunkun

Abstract

The dinoflagellate chloroplast genome consists of a group of small circular DNA molecules (minicircles), which carry one to three genes. In Heterocapsa triquetra, most minicircles carry only one gene except petD and psbE minicircles, which were found to carry tRNA genes downstream of the protein-coding gene. I used RT-PCR to show that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA. To further understand minicircle transcription and RNA precursor processing, I used RT-PCR, primer extension and Northern analyses to show that some minicircles can produce RNAs larger than themselves. I determined with 5’ RACE the sequence of the processed 5’ ends of several long RNAs, some of which are immediately downstream of the 3’ end of mature mRNAs and tRNAs. I proposed a "rolling circle" model for the minicircle transcription in which transcription would proceed continuously around the minicircle DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs. I found multiple types of substitutional editing in rRNAs and mRNAs, with A-to-G editing predominating. The editing occurs concomitantly with RNA maturation. I used a bioinformatic approach to generate a secondary structure model of the 16S rRNA. The model suggests that 1) the A-to-G editing mechanism is different from that responsible for animal nuclear A-to-I(G) editing; 2) A-to-G editing increases the conservation of edited sites; 3) the divergent 16S rRNA is a functional component of the chloroplast ribosome. To prove the presence of a eubacteria-like RNA polymerase (encoded by rpoA, B, C1 and C2) In H. triquetra chloroplasts, I used degenerate PCR, inhibitor assay, Southern and western analyses to examine the presence of the rpoB gene in the chloroplast or nuclear genome. Surprisingly, all the tests gave negative results, suggesting that the rpoB might have been lost. On the other hand, by using RT-PCR and 5’ RACE, I cloned an rpoT gene which encodes a single subunit RNA polymerase and might be a candidate for the minicircle transcription.

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