THE MAURICEVILLE PLASMID OF NEUROSPORA SPP USES NOVEL MECHANISMS FOR INITIATING REVERSE TRANSCRIPTION IN-VIVO

The Mauriceville plasmid and the closely related Varkud plasmid of Neu rospora spp. are retroelements that propagate in mitochondria. Replica tion appears to occur by a novel mechanism in which a monomer-length p lasmid transcript having a 3' tRNA-like structure ending in CCA is rev erse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are tra nscribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site similar to 260 bp upstrea m of the 5' end of the plasmid transcript. The location of the transcr iption start site suggests that the monomer-length transcripts are gen erated by transcription around the plasmid combined with a site-specif ic RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A m ajor class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequ ence, the same site used for de novo initiation of cDNA synthesis by t he plasmid reverse transcriptase in vitro. A second class of minus-str and cDNAs begins with putative primer sequences that correspond to cDN A copies of the plasmid or mitochondrial transcripts. These findings a re consistent with the possibility that the plasmid reverse transcript ase initiates minus-strand cDNA synthesis in vivo both by de novo init iation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a n ew minus-strand cDNA directly at the 3' end of the plasmid transcript.