INITIATION OF TRANSCRIPTION OF RIBOSOMAL DEOXYRIBONUCLEIC-ACID SEQUENCES IN ISOLATED-NUCLEI OF PHYSARUM-POLYCEPHALUM - STUDIES USING NUCLEOSIDE 5'-[GAMMA-S]TRIPHOSPHATES AND LABELED PRECURSORS

Nuclei of the plasmodial stage of the lower eucaryote Physarum polycephalum contain 100-200 copies of an extrachromosomal, linear, palindromic rDNA molecule of 61 kilobase pairs (kb), which includes duplicate coding regions for 5.8S, 19S, and 26S rRNA species. We have used incorporation of nucleoside 5'-[γ-S] triphosphates and nucleoside 5'-[α-32P] triphosphates to study initiation of ribosomal gene transcription in isolated nuclei. Sulfur-containing, labeled RNA, isolated by organomercurial column chromatography, contains newly initiated rDNA transcripts with sulfur-deriva-tized 5'-triphosphate termini. Following a 25-min pulse with sulfur-derivatized adenosine 5'-triphosphate (ATP) and 3H-labeled uridine 5'-triphosphate in the presence of α-amanitin, 5.5 ± 2.8% of the incorporated 3H label can be detected in newly initiated chains. Initiation of transcription of RNA complementary to the rDNA preferentially utilizes 5'-[γ-S]-ATP over 5'-[7-S]GTP by a factor of nearly 15-fold, suggesting that pppA- is the primary intiating group in rDNA transcription. Sequences comprising the rDNA transcription unit (positioned from about 17 to 5 kb from the ends of the palindrome) are selectively transcribed while a segment of at least 27 kb in the central spacer is not transcribed, as determined by hybridizing labeled nuclear transcripts to rDNA restriction fragments. Most rDNA transcripts in nuclei initiate near an Xhol cleavage site at 17.0 kb as revealed by hybridization of newly initiated transcripts to restriction fragments. These results are in accord with those obtained using in vivo labeled nuclear rRNA precursor. A significant percentage of rDNA transcripts initiated in nuclei elongates to a size greater than 11 kb. © 1979, American Chemical Society. All rights reserved.