Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli

Summary The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the perimissive growth temperature of 30°C and only one per cell at the higher permissive growth temperature of 38°C. When the growth temperature of this mutant is changed from 30° to 38°C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.     

Effects of nitrogen delivery on chitin nanofiber production during batch cultivation of the diatom <Emphasis Type="Italic">Cyclotella</Emphasis> sp. in a bubble column photobioreactor

The photosynthetic diatom Cyclotella sp. extrudes chitin nanofibers following cell division. This diatom requires silicon for cell wall biosynthesis and division, as well as nitrogen for biosynthesis of intracellular material and extracellular chitin, an N-acetyl glucosamine biopolymer. The initial nitrogen/silicon molar ratio was the critical parameter for assessing the limits of nitrogen delivery on cell number and chitin production during batch cultivation of Cyclotella in a bubble column photobioreactor under silicon-limited growth conditions, using nitrate as the nitrogen source. The peak rate of volumetric chitin production increased linearly, from 3.0 to 46 mg chitin L^?1 day^?1, with increasing N/Si ratio over the range studied (0.82 to 8.6 mol N mol^?1 Si). However, the cell number yield and the chitin yield per cell increased asymptotically with increasing N/Si ratio, achieving a final cell number yield of 5.3?×?10⁹?±?2.6?×?10⁸ cells mol^?1 Si and chitin yield of 28.7?±?1.2 mg chitin per 10⁹ cells (1.0 S.E.). An N/Si ratio of at least 4.0 mol N mol^?1 Si achieved 90% of the asymptotic chitin yield. This study has shown that scalable cultivation systems for maximizing chitin nanofiber production will require delivery of both silicon and optimal nitrogen under silicon-limiting growth conditions to promote cell division and subsequent chitin formation.     

Adaptation of the toxic freshwater cyanobacterium Microcystis aeruginosa to salinity is achieved by the selection of spontaneous mutants

The cyanobacterium Microcystis aeruginosa causes most of the harmful toxic blooms in freshwater ecosystems. Some strains of M. aeruginosa tolerate low‐medium levels of salinity, and because salinization of freshwater aquatic systems is increasing worldwide it is relevant to know what adaptive mechanisms allow tolerance to salinity. The mechanisms involved in the adaptation of M. aeruginosa to salinity (acclimation vs. genetic adaptation) were tested by a fluctuation analysis design, and then the maximum capacity of adaptation to salinity was studied by a ratchet protocol experiment. Whereas a dose of 10 g NaCl L^?1 completely inhibited the growth of M. aeruginosa, salinity‐resistant genetic variants, capable of tolerating up to 14 g NaCl L^?1, were isolated in the fluctuation analysis experiment. The salinity‐resistant cells arose by spontaneous mutations at a rate of 7.3 × 10^?7 mutants per cell division. We observed with the ratchet protocol that three independent culture populations of M. aeruginosa were able to adapt to up to 15.1 g L^?1 of NaCl, suggesting that successive mutation‐selection processes can enhance the highest salinity level to which M. aeruginosa cells can initially adapt. We propose that increasing salinity in water reservoirs could lead to the selection of salinity‐resistant mutants of M. aeruginosa.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells.

Factors influencing the frequency of thioguanine resistant mutations were examined in Chinese hamster lung cells damaged with a carcinogen, N-acetoxy-2-acetyl aminofluorene. Factors such as inoculum density, expression time, and concentration of selective agent were found to have a profound effect on the mutation frequency.Over a range of doses, a longer expression time is required for mutant cells from a more damaged population to reach their maximum frequency. In order to investigate the elements involved in this phenomenon, the increment in the plating efficiency of treated cells as a function of expression time, spontaneous mutation rate per cell per generation, viability of mutant as well as wild type cells, and half life of HGPRTase were evaluated.There was an observed relationship between induced mutation frequency and plating efficiency of treated cells. When treated cells had recovered from effects of the treatment and arrived at the normal level of plating efficiency, they also yielded the maximum frequency of mutations.The estimated mutation rate was 5.5 × 10^?8 per cell per generation. This number is too small to account for the increment in mutation frequency with the increase in the expression time. The mutation frequency of spontaneous origin was 4 × 10^?6 and that of induction of 10^?5 M NA-AAF was 10^?4. Lower growth rates of mutant cells cannot explain this increase in the number of mutants recovered, either.Continuous diminution in the level of HGPRTase, at 35% daily, interpreted as an important factor responsible for the recovery of mutation frequency during expression time, was observed in non-dividing cells. None of a large number of mutants sampled from those isolated had HGRPT activity. This indicates that they are true mutants and are not a result of phenocopy. Only cells completely deficient in HGPRT activity are recovered in TG selection medium. It is suggested, therefore, that this cell line is suitable for mutagenicity testing in the induction of mutation at the HGPRT locus.     

Mutant analysis by rescue gene excision: New tools for mosaic studies in Drosophila

A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP‐FRT technique for mitotic recombination has greatly enhanced gene loss‐of‐function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP‐FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue‐transgene loss can be induced in any tissue or cell‐type and at any time during development or in the adult using available heat‐shock‐induced or tissue‐specific flippases, or combinations of UAS‐FLP with Gal4 and Gal80^ts reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip‐out (clonal and disc‐wide) of a Ubi‐GFP‐carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.     

Genome-wide selection for increased copy number in Acinetobacter baylyi ADP1: locus and context-dependent variation in gene amplification

Renewed interest in gene amplification stems from its importance in evolution and a variety of medical problems ranging from drug resistance to cancer. However, amplified DNA segments (amplicons) are not fully characterized in any organism. Here we report a novel Acinetobacter baylyi system for genome‐wide studies. Amplification mutants that consume aromatic compounds were selected under conditions requiring high‐level expression from three promoters in a linked set of chromosomal genes. Tools were developed to relocate these catabolic genes to any non‐essential chromosomal position, and 49 amplification mutants from five genomic contexts were characterized. Amplicon size (18–271 kb) and copy number (2–105) indicated that 30% of mutants carried more than 1 Mb of amplified DNA. Amplification features depended on genomic position. For example, amplicons from one locus were similarly sized but displayed variable copy number, whereas those from another locus were differently sized but had comparable copy number. Additionally, the importance of sequence context was highlighted in one region where amplicons differed depending on the presence of a promoter mutation in the strain from which they were selected. DNA sequences at amplicon boundaries in 19 mutants reflected illegitimate recombination. Furthermore, steady‐state duplication frequencies measured under non‐selective conditions (10^?4 to 10^?5) confirmed that spontaneous gene duplication is a major source of genetic variation.     

EVOLUTION OF MICROALGAE IN HIGHLY STRESSING ENVIRONMENTS: AN EXPERIMENTAL MODEL ANALYZING THE RAPID ADAPTATION OF DICTYOSPHAERIUM CHLORELLOIDES (CHLOROPHYCEAE) FROM SENSITIVITY TO RESISTANCE AGAINST 2,4,6‐TRINITROTOLUENE BY RARE PRESELECTIVE MUTATIONS1

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6‐trinitrotoluene (TNT). When algal cultures were exposed to 30 mg·L ^{? 1} 1 Received 9 July 2001. Accepted 23 July 2002.
TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT‐resistant variants that appear after the massive lysis of the TNT‐sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT‐resistant cells; rather it was found that TNT‐resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 × 10 ^{? 5} mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT‐resistant mutants was significantly lower than that of wild‐type cells. Competition experiments between resistant mutants and wild‐type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT‐resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.     

Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.     

High genome-wide mutation rates in vegetatively propagated bermudagrass

A cascade DNA amplification strategy that generates arbitrary signatures from amplification profiles (ASAP) was used to measure genome-wide mutation rates in bermudagrass (Cynodon). ASAP quantified nucleotide changes that were induced by irradiation, genetic instabilities and normal vegetative growth of cultivars and accessions of sterile interspecific hybrids. DNA sequence divergence between cultivar ‘Tifway’ and its γ radiation-induced mutant ‘Tifway II’ (0.70 ± 0.66%) was comparable to estimates in radiation-induced mutants and spontaneous sports of chrysanthemum (Chrysanthemum morifolium Ramat.). A similar divergence in sequence (0.95 ± 0.20%) was observed in the pairwise comparison of 17 nondisjunctive ‘Tifgreen’ and ‘Tifdwarf’ accessions. Mutation during normal Tifdwarf vegetative growth was evaluated by planting sprigs and sampling their offspring. Somatic sequence divergence levels (0.004 ± 0.007%) resulted in a mutation rate of 1.05 × 10^?8 per nucleotide per generation, assuming that a bermudagrass sprig constitutes a generation of growth. These rates were comparable to those found in germinal cells and individuals of either human or Drosophila melanogaster, supporting the notion that eukaryotic evolution is generation rather than time dependent. The high accumulation of somatic mutations (10 per triploid genome) is consistent with a model whereby mutation load in a population exhibiting obligate vegetative reproduction is substantially higher than in a population under sexual or asexual reproduction. These constraints could be the cause of reported genetic instabilities in the Tifgreen–Tifdwarf complex. Finally, a long-term rate measured across accessions and indicative of the accumulation of mutations in 17 Tifgreen–Tifdwarf populations (µ = 1.02 × 10^?8 per nucleotide per generation) was strikingly congruent with the bermudagrass vegetative mutation rate, suggesting absence of evolutionary constraints in the sampled genomic regions. Mutation rates calculated from across-accesions divergence estimates (5.18 ± 0.53%) indicated that plant material was evolving 100 times faster (3.8 × 10^?7 changes per nucleotide per year) than a molecular clock rate estimate for grasses, probably resulting from the compound effect of clonal growth and life span of the hybrid plant material.     

Potassium Uptake in Synchronous and Synchronized Cultures of Escherichia coli

Criteria are presented for distinguishing between synchronous and synchronized cultures (natural vs. forced synchrony) on the basis of characteristics of growth and division during a single generation. These criteria were applied in an examination of the uptake of potassium during the cell growth and division cycle in synchronous cultures and in a synchronized culture of Escherichia coli. In the synchronous cultures the uptake of ⁴²K doubled synchronously with cell number, corresponding to a constant rate of uptake per cell throughout the cell cycle. In the synchronized culture, uptake rates also remained constant during most of the cycle, but rates doubled abruptly well within the cycle. This constancy of ⁴²K uptake per cell supports an earlier interpretation for steady-state cultures that uptake is limited in each cell by a constant number of functional sites for binding, transport, or accumulation of compounds from the growth medium, and that the average number of such sites doubles late in each cell cycle. The abrupt doubling of the rate of uptake of potassium per cell in the synchronized culture appears because of partial uncoupling of cell division from activation or synthesis of these uptake sites.     

INFLUENCE OF CELL VOLUME ON THE GROWTH AND SIZE REDUCTION OF MARINE AND ESTUARINE DIATOMS1

The maximal growth rate (μ_max) of 19 marine and estuarine diatoms decreased with increasing cell volume (V). The relationship between log μ_max (Y) and log V (X) was calculated. Statistical analyses showed that the slope of the equation was not significantly different from those obtained by other researchers and that the 95% confidence intervals of mean μ_max at cell volumes of 10³–10⁵μm³ were not significantly different from those cited in most studies. A new regression line for diatoms was calculated as follows: log μ_max= 0.47–0.14 log V; r =–0.69. The rate of size reduction per generation of the 19 diatom species ranged from 0.03 to 0.87 μm per generation. The rate increased with increasing cell length and cell volume and with decreasing maximum division rate. Statistical analyses showed that the rate was closely related to the cell volume and to the reciprocal of the growth rate. The relationships between maximal growth rate and cell volume and between rate of size reduction and cell volume showed that a diatom with a large volume had a smaller maximal growth rate and a larger rate of size reduction than a diatom with a small volume. The estimates using the equation for the regression line between the rate of size reduction and the reciprocal of maximum division rate indicated that a diatom with a high maximum division rate would need more generation equivalents for a certain size reduction than a diatom with a low maximum division rate, but the periods required for reduction would be approximately equal irrespective of maximum division rate.     

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived ‘MgPar’ donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10^?4 events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption‐deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C–MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.     

Physiological characterization of the sex pheromone protoplast-release-inducing protein from the Closterium peracerosum-strigosum-littorale complex (Charophyta)

In the unicellular charophycean alga Closterium peracerosum‐strigosum‐littorale complex, the protoplast‐release‐inducing protein (PR‐IP), a sex pheromone responsible for gametic protoplast release from mating‐type minus (mt^–) cells, was found to stimulate secretion of mucilage from the cells. Induction of sexual cell division by PR‐IP was also confirmed. Bioassays were used to determine the minimum doses required to induce these functions, revealing that 5 · 10^?16 M of PR‐IP stimulated mucilage secretion, and that 5 · 10^?10 M of PR‐IP were required for protoplast release. Exposure of the cells to 5 · 10^?11 M of PR‐IP resulted in the induction of sexual cell division as well as mucilage secretion. These results strongly suggest that PR‐IP is a multifunctional pheromone that independently promotes multiple steps in conjugation at the appropriate times through different induction mechanisms.     

Changes in Chloroplast DNA Levels during Growth of Spinach Leaves

In young spinach leaves, 1–4 mm long, 7–10% of thetotal DNA of the leaf was chloroplast (pt) DNA. Growth in theseleaves was mainly by cell division with plastid division keepingpace with cell division and maintaining about 10 plastids percell. About 1% of the leaf cells were formed in 4.0 mm leaves.Both cell division and cell expansion contribute to the nextstage of leaf growth, which was quantitatively the major periodof new cell formation, nuclear DNA synthesis and ptDNA synthesis.Relative to the nuclear DNA level ptDNA levels rose to 21% ofthe total DNA and chloroplast.plastome copy numbers from 1500to 5000 per cell while chloroplast numbers rose from 10 to 30per cell. In the final period of leaf growth, cell expansionwas the main determinant of growth and chloroplast number percell rose to 180. In contrast to young leaves, newly emergedcotyledons contained 20% of their DNA as ptDNA and, during cellexpansion, cell number per cotyledon doubled. On average, thecells became octoploid, and chloroplast numbers and plastomecopy numbers rose to 500 and 22 000 per cell respectively. Similarlevels of nuclear ploidy, chloroplast number and plastome copynumber were induced in the first leaf pair of spinach followingdecapitation. When senescence was induced in mature leaves byshading, no loss of nuclear or ptDNA occurred. Following theonset of leaf yellowing and a form of senescence induced bynitrogen deficiency in leaves which had not fully expanded,there was preferential loss of ptDNA which fell from 8200 to3700 plastome copies per cell over an 11 d period. Key words: Spinach, Chloroplast, DNA, Ploidy     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

A Thy-1 − mutant defining a gene acting in trans position to regulate cell-surface Thy-1 glycoprotein expression and Thy-1 messenger RNA content

The Thy-1 glycoprotein is a differentiation antigen which exhibits tissue-specific regulation. A mutant of a Thy-1.1⁺ T-cell lymphoma has been isolated which does not express Thy-1 glycoprotein on the cell surface and does not accumulate Thy-1 mRNA in the cytoplasm. Hybrids between the mutant and a Thy-1.2⁺ T-cell lymphoma express 20–30-fold lower levels of Thy-1 glycoprotein on their cell surface compared to wild-type T-cell lymphomas, and they have correspondingly low levels of cytoplasmic Thy-1 mRNA. A revertant of one hybrid was isolated which expressed wild-type levels of both Thy-1 alleles on its surface and contained correspondingly increased levels of Thy-1 mRNA. A Thy-1⁺ revertant of the Thy-1 ^– mutant was isolated by cell sorting. A second generation Thy-1 ^– mutant could be isolated from this revertant which also did not accumulate Thy-1 mRNA and which behaved in a way similar to the first generation mutant when hybridized to a Thy-1.2⁺ lymphoma. No changes in the structure or copy number of the Thy-1 structural gene could be detected in this series of mutants and revertants. These properties are consistent with a mutation in one (or more) gene(s) which acts in trans position to regulate Thy-1 glycoprotein expression.