Precise mapping and characterization of the RNA primers of DNA replication for a yeast hypersuppressive petite by in vitro capping with guanylyltransferase.

The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.

Roles of rDNA spacer and transcription unit-sequences in X-Y meiotic chromosome pairing in Drosophila melanogaster males.

Meiotic pairing of the X and Y chromosomes in Drosophila melanogaster males is mediated by the rDNA repeats, which are present in two tandem clusters, one in the centric X heterochromatin and the other near the base of the short arm of the Y chromosome. Deletion of the X chromosomal rDNA cluster disrupts X-Y pairing and causes high frequences of X-Y nondisjunction. Pairing can be partly restored by insertions of cloned complete rRNA genes or by rDNA fragments that include the intergenic spacer (IGS) region. A 240 bp repeated sequence in the IGS was shown to be effective in promoting pairing when present at copy numbers above five. This study further defines the rDNA sequences involved in mediating pairing. Germline insertions of a P element construct containing most of the rDNA transcription unit but no promoter or IGS region were obtained. Two single-copy insertions and four two-copy insertions proved unable to stimulate X-Y disjunction when located on an rDNA-deficient X chromosome. In addition, three insertions of a P element construct consisting of the IGS and promoter regions of the rDNA were characterized molecularly. These three insertions had previously been shown to range in pairing ability from very weak to quite strong. Molecular analysis revealed that the three insertions also vary in copy number of the 240 bp IGS repeat and that these structural differences correlate with the differences in pairing ability. These data indicate that 240 bp repeats are considerably more effective than other regions of the rDNA in stimulating chromosome pairing.

Promoter-containing ribosomal DNA fragments function as X-Y meiotic pairing sites in D. melanogaster males.

The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X-Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X-Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X-Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X-Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P-element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X-Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter-containing rDNA fragments are autonomously capable of being transcribed and of functioning as X-Y pairing sites, but not of forming a mini-nucleolus.