Expression of the mitochondrial genome in HeLa cells. XII. Relationship between mitochondrial fast-sedimenting RNA components and ribosomal and 4 s RNA.
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Biomedical subjects
Publications and source records attributed to Y Aloni.
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Rapidly labeled RNA was extracted from monkey cells after infection with Simian Virus 40 (SV40) and exposure to short pulses of [5-(3)H]uridine late in infection. When this RNA was self-annealed, it became resistant to digestion with ribonuclease. The fraction of RNA that resisted the ribonuclease treatment decreased with increased labeling time, or when a short pulse of radioactivity was followed by incubation with unlabeled uridine and actinomycin D. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded by its susceptibility to ribonuclease as a function of salt concentration and temperature. This behavior was not due to RNA-DNA hybrid formation, since deoxyribonuclease had no effect upon the double-stranded molecules, even after their denaturation. The relation of the double-stranded RNA to SV40 was demonstrated by the hybridization of about 50% (corrected value, >90%) of the separated RNA strands with component I of SV40 DNA from plaque-purified virus. After self-annealing in formamide at low temperature, about 10% of the rapidly labeled, viral RNA sedimented at 13 S. This value corresponds in size to about 60% of the SV40 DNA.These observations indicate that late in infection of monkey cells, SV40 DNA is transcribed symmetrically over a considerable portion of its length, and that subsequently some sequences from one or both of the RNA strands are degraded.
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RNA.DNA hybridization experiments utilizing separated strands of HeLa mitochondrial DNA and mit-RNA from HeLa cells exposed to short pulses of [5-(3)H]uridine have shown that the labeled RNA hybridizes with both the light (L) and the heavy (H) strand, though to a different relative extent depending upon the labeling time. Thus, hybridization of pulse-labeled RNA is about equal with the two strands when the pulse is very short (1-5 min), and becomes more and more predominant with the H strand with increasing pulse length. Pulse-labeled fast-sedimenting mit-RNA forms RNase-resistant double-stranded structures up to more than 5 mum long when self-annealed or annealed with an excess of unlabeled mit-RNA. These observations and the previous evidence of complete transcription of the H strand strongly suggest that mit-DNA is transcribed in HeLa cells symmetrically over a considerable portion of its length, with the transcript of the L strand being rapidly degraded or otherwise removed from the mitochondrial fraction.
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Despite a good control of serum phosphate levels, bone disease still occurs in patients treated with chronic ambulatory peritoneal dialysis (CAPD). The purpose of this study was to determine if 25-hydroxyvitamin D (25-OH-D) penetrates through the peritoneal membrane and whether chronic peritoneal dialysis may thereby decrease the serum 25-OH-D levels. In 10 CAPD-treated patients the serum 25-OH-D was 8.8 +/- 1.1 ng/ml, which is significantly lower than in the hemodialysis-treated group (15.5 +/- 2.3 ng/ml) and lower than in healthy controls (30.3 +/- 3.6 ng/ml). The mean total daily loss of 25-OH-D in the peritoneal fluid was 1,491 +/- 260 ng/day. The serum 25-OH-D binding capacity was significantly lower in the CAPD group than in normal controls. The mean daily loss of 25-OH-D binding capacity in the dialysate was 153 +/- 28 nmol/day. We conclude that permeability of the peritoneal membrane to middle-size proteins leads to loss of 25-OH-D binding protein, and this increases loss of 25-OH-D. Low levels of 25-OH-D in plasma may aggravate the symptoms of osteodystrophy of renal failure in patients treated with CAPD.