Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle.

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences.

Mapping of replication initiation sites in the mouse ribosomal gene cluster.

We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification-promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found.

Medicine as interpretation: the uses of literary metaphors and methods.

Theorists at the interface of medicine and the humanities have recently suggested that interpretation as a literary activity can be applied to the practice of clinical medicine. This article reviews such theories and their literary metaphors and methods. In pushing these ideas further, it is proposed that a number of guidelines can be applied to interpretation as a practical activity for clinical medicine.

Probenecid inhibits the secretion of nephrogenous adenosine 3',5'-monophosphate in normal man.

Nephrogenous cAMP, that fraction of urinary cAMP secreted by the kidney, is PTH dependent and is used clinically as a measure of parathyroid function. To clarify the nature of secretion of nephrogenous cAMP, probenecid, an inhibitor of organic anion transport, was administered to eight healthy young males. Nephrogenous cAMP decreased after probenecid treatment (P less than 0.001), and plasma cAMP increased reciprocally (P less than 0.01) so that urinary cAMP did not change. There was no change in serum PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, or urinary clearance of calcium or phosphate. These results suggest that secretion of nephrogenous cAMP may be a carrier-mediated process in normal man. The effect of probenecid to increase plasma cAMP is consistent with other observations suggesting that cAMP is cleared from plasma in part by a carrier-mediated process.

Cushing's disease in a patient with a 'nonfunctioning' pituitary tumor. Spontaneous development and remission.

A 40-year-old woman had visual loss and a large nonfunctioning pituitary tumor. After partial surgical resection and radiation treatment, clinical and biochemical evidence of Cushing's disease developed. The pituitary source of her adrenocorticotropic hormone hypersecretion was documented on selective venous sampling. After 18 months of medical therapy with metyrapone and aminoglutethimide, the patient experienced a spontaneous remission of her hypercortisolism. A "nonfunctioning" pituitary tumor has a hypersecretory potential.