Heterozygous C3ΔRFB/B strains undergo stochastic excision of the rDNA in the developing macronucleus

<p><b>Copyright information:</b></p><p>Taken from "Deletion of the rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome"</p><p>Nucleic Acids Research 2006;34(2):620-634.</p><p>Published online 30 Jan 2006</p><p>PMCID:PMC1356531.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Restriction map and fragment sizes for products following digestion with EcoRV (downward arrowheads) or XbaI (upward arrowheads) for micronuclear rDNA locus (Mic), excised non-palindromic macronuclear rDNA (NP) and palindromic (P) rDNA minichromosomes derived from wild-type B rDNA (WT) or C3ΔRFB deletion alleles. Hybridization probes A and B: thick black lines in micronuclear rDNA diagram. () Stochastic appearance and structure of macronuclear C3 rDNA in clonal C3ΔRFB transformants. Left panel: Southern blot analysis of EcoRV digested genomic DNA with probe B. Wild-type B rDNA strain; CU428, heterozygous C3ΔRFB/B germline transformant strains; TX607(1), TX610(11) and TX611(1), and wild-type C3 rDNA transformant strain; TX614(1). The position of wild-type B and wild-type C3 rDNA monomers (WT non-pal) and palindromes (WT pal), predicted C3ΔRFB species (ΔRFB pal, ΔRFB non-pal), and unexpected products (C3-long and X) are indicated. The corresponding restriction fragments for expected products are symbol coded (see panel A). TX611(C1) and TX611(C3) are clonal progeny derived from a cross between TX611(1) and amicronucleate strains A* III and A* V, respectively, and have generated a new macronucleus by round II genomic exclusion (see text). Note the presence of the C3ΔRFB rDNA palindrome in TX611(C1) and absence of this species in its parent, TX611(1). Right panel: Southern blot analysis of genomic DNA with probe B from mating progeny harvested 24 h after mixing strains of opposite mating type. Crosses: TX611(C1) or TX611(C3) were mated with tester strains, CU427 (wild B rDNA in micronucleus) or SB1934 (wild-type C3 rDNA in micronucleus). Control mating: CU427 × SB1934. X indicates rDNA species that were not predicted by conventional processing at the Cbs element upstream of the rDNA 5′ NTS. () The C3-long rDNA species is fragmented at a novel site upstream of the 5′ Cbs element. Southern blot of XbaI digested genomic DNA hybridized with probe B (left) or probe A (right). Numbers correspond to clonal lines established from each heterozygous C3ΔRFB/B rDNA germline transformant