Early metabolic changes in regenerating microfragments of the liverwort Riella helicophylla (Bory et Mont.) Mont.: Protein and RNA synthesis,free α-amino concentration

Microfragments of constant size were isolated from the thallus of Riella by a rapid punching procedure. Thus it was possible to study various metabolic parameters especially during the first hours after fragment isolation. Protein synthesis increases rapidly after a slight decrease at 30 min. The earliest significant increase in RNA synthesis occurs at 8 h, indicating a different activation pattern. The concentration of -amino compounds drops at 30 min and then increases continuously, thus exhibiting a close relationship with the measured alterations in protein synthesis. Another indication of metabolic conversions during regeneration is provided by the changing level of free radioactive leucine, which shows a marked turning point at 24 h after fragmentation. Analysis of free -amino concentrations in small regions of larger fragments also indicates the establishment of new intercellular correlations only a few hours after isolation of the cells from the meristem. Up to the 8th h after fragmentation, the contents of both the adaxial and peripheral fragment regions increase. Thereafter only the adaxial (regenerating) cells continue accumulating; the peripheral (nonregenerating) ones remain at the same level.     

Kinetics of rapid RNA evolution in vitro

Summary A rapidly acquired partial resistance to the replicase antagonist, ethidium bromide (EB), seen by Spiegelman and coresearchers in Q RNA variants competitively replicating under defined conditions in vitro, reflected existence of a pool of mutant RNA molecules, preadapted to EB, and their cross-propagation from the pre-EB optimum species, MDV-1, and from other kindred variants, some of which remained undetected, according to this quantitative analysis of midivariant RNA replication kinetics. DNAlike features of their evolution, such as the cloning of variants from an MDV-1 subtype and a complicance with the fundamental theorem of natural selection, resulted from the suppression, both real and apparent, of intrinsic RNA heterogeneity through sampling and detection methods, and also by the ascendency of self-propagation over cross-propagation with advancement of a superior variant. The deficit in mean polymer fitness, compared with optimum levels, determines the lower limit of this heterogeneity. Stability conditions for frequency equilibrium and strategies for counteracting viral drug resistance have been considered.     

Antibiotics and the search for new principles

Originally presented as an Invited Lecture at the 1990 Society for Industrial Microbiology Annual Meeting in Orlando, Florida.     

On the Stability of Messenger RNA and Ribosomal RNA in the Brains of Control Human Subjects and Patients with Alzheimer''s Disease

The levels of the mRNAs encoding the G protein subunits GS alpha, G beta 1, and G beta 2 were measured by northern blotting in the frontal cortex and hippocampus of control subjects and of patients with a clinical and histopathological diagnosis of dementia of the Alzheimer type (DAT). There was no significant difference, in either brain region, between the control and DAT groups for any of the G protein mRNAs measured. The degree of intersubject variability was very high, e.g., GS alpha mRNA in the frontal cortex (mean optical density +/- SD) was 405 +/- 342 in the control group versus 305 +/- 207 in the DAT group. The extent of generalised RNA degradation was assessed by detecting the breakdown products of 28S rRNA. RNA degradation was present in tissue samples from every human subject studied. The extent of 28S rRNA degradation in each subject was found to be related to the levels of G protein mRNA detected. The degree of RNA degradation in human subjects was found to be very variable and unaffected by the presence of DAT. RNA degradation correlated poorly with postmortem interval and this was confirmed by a controlled study of postmortem degradation in rat tissue. The possibility that the relative hypoxia and ischaemia in patients immediately before death could influence RNA degradation is discussed. The variable extent of RNA degradation means that great care must be taken to ensure the validity of RNA analyses undertaken in human postmortem brain, particularly when techniques are employed (such as in situ hybridisation) that themselves give no indication of RNA integrity.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Sequence complexity of poly(A) RNA fromPhysarum polycephalum

Summary Poly(A) RNA from S phase, G₂ phase and starved macroplasmodia of Physarum contain mRNA sequences which when translated in vitro, yield similar patterns of polypeptides after fluorography.Reassociation of nick-translated DNA (Cot) allows the isolation of highly labeled single copy DNA which, after saturation hybridization with poly(A) RNA, gives values of 23% for growth and 17% for starvation.Homologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that 22–28% of the genome is transcribed during growth and 12% during starvation and that about half of the cDNA reacts with 0.1% of the genome and could represent 50–80 RNA species, each present in about 1,000 copies per nucleus. Up to 25,000 different RNA species, 1–5 copies each per nucleus, are estimated to be present during growth, and about 15,000 during starvation. Heterologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that the RNA sequences in S and G₂ phase of the cell cycle are similar, with RNA sequences being more abundant in G₂ phase.During starvation about 25% of the sequences present during growth cannot be detected and those sequences present during growth have become diluted during starvation.     

Storage of embryonically transcribed poly(A) RNA and its utilization during metamorphosis of the hydroidHydractinia echinata

Summary The formation of tentacles and stolons during metamorphosis is severely disturbed if inhibitors of mRNA metabolism are applied during certain phases of development. The periods of sensitivity to -amanitin are late gastrulation and the disk stage of metamorphosis. A cordycepin sensitive phase exists during the first hour of metamorphosis. In all drug sensitive phases an enhanced poly(A) synthesis is found indicating increased mRNA metabolism in these stages. Pulse-chase experiments show that planula larvae store a poly(A)-rich RNA population sedimenting between 28–18s. These long living molecules are of embryonic origin, are located in RNP particles and are degraded during metamorphosis. The particles in question appear to be stored mainly in interstitial cells. In early metamorphosis no uridine is incorporated but labelled poly(A) is added to preexisting molecules.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.