Modifications of the high reversion frequency in the nic-2 mutant of Coprinus radiatus. 1. Evidence for the existence of three strain types with more or less elevated reversion frequencies

Summary In Coprinus radiatus, a cross homozygous for nic-2 mutation yields two types of nic-strains. The first one (nic-2F) retains the character of the original mutant, reverting at meiosis with a frequency of 20 to 30%. All crosses with the second type (nic-2 fa) show a sharply reduced reversion rate.The analysis of the progeny of crosses involving a nic-2 fa strain show the appearance of a third type of strains (nic-2 fn). The reversion of nic-2 fn strain is low, but this character is not imposed to the partner in a cross with a nic-2F strain.     

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.     

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

Cytoplasmic reversion to fertility in cms-S maize need not involve loss of linear mitochondrial plasmids

Cytoplasmic male sterility of the S type (cms-S) in maize is characterized by the presence of two autonomously replicating plasmid-like elements, S1 and S2. These plasmids have not been found in the mitochondrial genomes of normal (male-fertile) maize nor previously in male-fertile cytoplasmic revertants. This paper reports the discovery of spontaneous cytoplasmic reversion to fertility in cms-S maize not involving the loss of S1 and S2 plasmids. Data are presented showing that loss of the plasmids during cytoplasmic reversion is under nuclear influence and is not a characteristic of the S cytoplasm itself.     

Substoichiometric shifting in the fertility reversion of cytoplasmic male sterile pearl millet

Cytoplasmic male sterility (CMS) represents an important agricultural trait in pearl millet [Pennisetum glaucum (L.) R. Br.] with a value to the seed industry in facilitating economical hybrid seed production. Among the CMS systems available in millet, the A1 source is the most commonly used for hybrid production, but it can undergo low frequency reversion to fertility. Plant mitochondrial genomes are highly recombinogenic, becoming unstable and prone to ectopic recombination under conditions of tissue culture, somatic hybridization, or interspecific crossing. Similarly, CMS systems prone to spontaneous fertility reversion experience sporadic mitochondrial genome instability. We compared mitochondrial genome configurations between the male-sterile A1 line and fertile revertants of pearl millet to develop a model for millet mitochondrial genome reorganization upon reversion. Relative copy number of a subgenomic molecule containing the CoxI-1-2 junction region, a component of the recombination process for reversion, is amplified tenfold following reversion, relative to the CMS A1 line. We propose that increased copy number of this molecule in a small number of cells or at low frequency triggers a recombination cascade, likely during reproductive development. The proposed recombination process initiates with ectopic recombination through a 7-bp repeat to produce a novel CoxI-3-2 junction molecule and an unstable recombination intermediate. Subsequent intra-molecular recombination stabilizes the intermediate to form a new copy of CoxI accompanied by a deletion. This study furthers the argument that substoichiometric shifting within the plant mitochondrial genome plays an important role in the evolution of the mitochondrial genome and plant reproductive dynamics.     

Potentials and conditions for the reversion of pathogenic properties of the causative agent of cholera in an experiment

The passage of V. cholerae noncholerigenic strains and their mutants, both in vitro and in vivo, has demonstrated that strains in which one of such properties as mobility, viability, adhesive, lecithinase and neuraminidase activities, is sharply decreased or lost, are still capable of reversion to cholerigenic forms. V. cholerae strains which have lost two or more of these properties, as well as strains having stable hemolytic activity determined by Greig's test, seem to be incapable of such reversion.     

Quantitative proteomic analysis of tumor reversion in multiple myeloma cells

Tumor reversion is defined as the process by which cancer cells lose their malignant phenotype. However, relatively little is known about the cellular proteome changes that occur during the reversion process. A biological model of multiple myeloma (MM) reversion was established by using the H-1 parvovirus as a tool to select for revertant cells from MM cells. Isolated revertant cells displayed a strongly suppressed malignant phenotype both in vitro and in vivo. To explore possible mechanisms of MM reversion, the protein profiles of the revertant and parental MM cells were compared using a quantitative proteomic strategy termed SILAC-MS. Our results revealed that 379 proteins were either activated or inhibited during the reversion process, with a much greater proportion of the proteins, including STAT3, TCTP, CDC2, BAG2, and PCNA, being inhibited. Of these, STAT3, which is significantly down regulated, was selected for further functional studies. Inhibition of STAT3 expression by RNA interference resulted in suppression of the malignant phenotype and concomitant down regulation of TCTP expression, suggesting that myeloma reversion operates, at least in part, through inhibition of STAT3. Our results provide novel insights into the mechanisms of tumor reversion and suggest new alternative approaches for MM treatment.     

In vitro phytochrome dark reversion process

Thermal reversion of the far-red absorbing form of phytochrome to the red absorbing form in darkness has been investigated in crude and partially purified isolates from a number of etiolated and light grown higher plants. The influence of temperature, aging and urea on the rate of reversion was also determined.

Phytochrome isolated from all higher plants underwent reversion. The reversion proceeded in at least 2 distinct stages; a short rapid initial phase being followed a slow phase which continued for many hours. Reversion rate was highest in phytochrome isolated from green leaves of parsnip (Pastinacea sativa) and lowest in that isolated from etiolated oats (Avena sativa). Although the rate of reversion could be changed by modifying the tertiary structure of the protein component, the large differences in rate appeared to be characteristic of the plant source. Observed in vitro rates of reversion are slower than those occurring in vivo. Removal of other buffer solubilized material during purification had little effect on the rate of reversion of phytochrome isolated from etiolated material.

     

Research on meiotic chromosome pairing in <Emphasis Type="Italic">Roegneria sinica</Emphasis> var. <Emphasis Type="Italic">media</Emphasis> evaluated using genomic in situ hybridization

The analysis of chromosome pairing during meiosis is important for understanding the relationships between different genomes. To evaluate the diversity of chromosome pairing behavior in the wild species of Roegneria sinica var. media Keng with St and H genomes in Triticeae (Poaceae), differences and similarities in the meiotic chromosome pairing behaviors of the two genomes in two populations of R. sinica var. media, were analyzed using genomic in situ hybridization. Chromosome pairing at meiotic metaphase I in the two populations of R. sinica var. media mainly formed bivalents, although several univalents, trivalents and quadrivalents also occurred. Chromosome pairings occurred mainly between homologous chromosomes. However, some non-homologous pairings were observed under natural conditions. No significant differences in karyotype were found between the St and H genomes. Chromosome pairing behaviors differed between and within the two populations. Genetic variation occurred mainly within populations (94.04 %), and variation was more abundant in one population than the other. The genomes St and H differed, but there was some relationship between the two genomes. These findings suggest that homoeologous pairing of chromosomes or exchanges occurred between different genomes of the wild species in Triticeae during evolution. The findings also provide conclusive cytological evidence for genetic variation within the wild species, which forms the basis of their genetic diversity.     

Preparation,reversion, mutagenesis and intra-species fusion of protoplasts ofOudemansiella mucida

A technique was developed for preparation and reversion of protoplasts of the mucidin-producing fungusOudemansiella mucida. The protoplasts can be obtained in sufficient amounts from an exponentially growing young mycelium treated with snail digestive juice in the presence of an osmotic stabilizer. The resulting protoplasts readily regenerate their cell walls and, at a frequency of 10-20%, reverse into mycelium. InO. mucida, which does not form asexual spores, protoplasts are a suitable starting material for mutagenesis. Auxotrophic and higher-producing mutants were obtained in this way. Fusion of protoplasts of two compatible monokaryotic auxotrophic isolates, induced by polyethylene glycol, yielded nutritionally complemented dikaryotes forming clamp connections and producing mucidin.     

Selection, Generalized Transmission and the Evolution of Modifier Genes. I. the Reduction Principle

Modifier gene models are used to explore the evolution of features of organisms, such as the genetic system, that are not directly involved in the determination of fitness. Recent work has shown that a general "reduction principle" holds in models of selectively neutral modifiers of recombination, mutation, and migration. Here we present a framework for models of modifier genes that shows these reduction results to be part of a more general theory, for which recombination and mutation are special cases. The deterministic forces that affect the genetic composition of a population can be partitioned into two categories: selection and transmission. Selection includes differential viabilities, fertilities, and mating success. Imperfect transmission occurs as a result of such phenomena as recombination, mutation and migration, meiosis, gene conversion, and meiotic drive. Selectively neutral modifier genes affect transmission, and a neutral modifier gene can evolve only by generating association with selected genes whose transmission it affects. We show that, in randomly mating populations at equilibrium, imperfect transmission of selected genes allows a variance in their marginal fitnesses to be maintained. This variance in the marginal fitnesses of selected genes is what drives the evolution of neutral modifier genes. Populations with a variance in marginal fitnesses at equilibrium are always subject to invasion by modifier genes that bring about perfect transmission of the selected genes. It is also found, within certain constraints, that for modifier genes producing what we call "linear variation" in the transmission processes, a new modifier allele can invade a population at equilibrium if it reduces the level of imperfect transmission acting on the selected genes, and will be expelled if it increases the level of imperfect transmission. Moreover, the strength of the induced selection on the modifier gene is shown to range up to the order of the departure of the genetic system from perfect transmission.     

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells.Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.     

Effects of various chemical compounds on spontaneous and hydrogen peroxide-induced reversion in strain TA104 of Salmonella typhimurium.

In experiments designed to determine which active oxygen species contribute to hydrogen peroxide (HP)-induced reversion in strain TA104 of Salmonella typhimurium, 1,10-phenanthroline (an iron chelator, which prevents the formation of hydroxyl radicals from HP and DNA-bound iron by the Fenton reaction), sodium azide (a singlet oxygen scavenger), and potassium iodide (an hydroxyl radical scavenger) inhibited HP-induced reversion. These results indicate that hydroxyl radicals generated from HP by the Fenton reaction, and perhaps singlet oxygen, contribute to HP-induced reversion in TA104. However, reduced glutathione (reduces Fe3+ to Fe2+ and/or HP to water), diethyldithiocarbamic acid (an inhibitor of superoxide dismutase), diethyl maleate (a glutathione scavenger), and 3-amino-1,2,4-triazole (an inhibitor of catalase) did not inhibit HP-induced reversion in TA104. Thus, superoxide radical anions and HP itself do not appear to be the cause of HP-induced reversion in this strain. In experiments on the effect of 5 common dietary compounds (beta-carotene, retinoic acid, and vitamins A, C and E), chlorophyllin (CHL), and ergothioneine, the frequency of revertants in TA104 increased above the spontaneous frequency in the presence of beta-carotene or vitamin C (about 2-fold) or vitamin A (about 3-fold). The 5 dietary antimutagens and CHL did not inhibit HP-induced reversion in TA104. However, L-ergothioneine inhibited HP-induced reversion in this strain. Therefore, it is likely that L-ergothioneine is a scavenger of hydroxyl radicals or an inhibitor of their formation, and perhaps of singlet oxygen, at the concentrations tested in TA104.     

Floral bud reversion in Impatiens balsamina under non-inductive photoperiods

Summary All floral buds of Impatiens balsamina plants exposed to 4 short-day (SD) cycles and then returned to long days reverted to vegetative growth. The same happened with the upper buds of plants receiving a larger number of SDs, even as many as 90 cycles. The reversal proceeded in a basipetal order. The number of floral buds and flowers increased, and their reversion to vegetative growth was delayed with increasing numbers of SD cycles. Depending upon the stage attained by the floral bud before the transfer of the plant to noninductive photoperiods one or more inner whorls of the flower were replaced by a vegetative apex. The tip of the placenta was able to resume vegetative growth even after the formation of fertile anthers and an ovary with abortive ovules, showing that the potentiality for reversion is maintained till quite late stages in floral bud development. Continuous exposure to SD cycles is required not only for the continued production of floral buds, but also for their development to mature flowers, indicating that the floral stimulus in this plant is not self-perpetuating.     

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.     

Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations.

F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells. This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer. Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source. We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ. Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation. Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele. However, a direct causal relationship between transfer and reversion remains to be established. These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination. Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.