An Efficient Method for the Plating of Haploid and Diploid Emiliania huxleyi on Solid Medium1

Emiliania huxleyi is a globally important coccolithophore and one of the most successful eukaryotic organisms in the modern oceans. Despite a large body of work on this organism, including the sequencing of its genome, the tools required for forward and reverse functional genetic studies are still undeveloped. Here we present an optimized method for the clonal isolation of E. huxleyi by plating on solid medium. We demonstrate the utility of this method for a variety of strains including haploid, calcifying-diploid, and noncalcifying diploid strains. We show that, in contrast to previous studies, no changes in cell ploidy status occur when the cells are plated. Our method will greatly aid attempts to elucidate the genetic basis of the remarkable physiology of E. huxleyi by forward and reverse genetic approaches.     

Genetic analysis of replicating instabilities in yeast

Strains showing ethyl methanesulfonate (EMS)-induced replicating instability were genetically analysed to test whether within a given line, mosaics from different plating generations carry a mutation at the same site within the locus. A forward mutation system involving five loci controlling adenine biosynthesis in Schizosaccharomyces pombe was used. Genetic analysis was carried out by interallelic complementation and intragenic recombination tests. The data showed that EMS-induced instabilities are site-specific in being confined to the same recombination unit. This finding is discussed in relation to the possible mechanism(s) of replicating instabilities after different mutagenic treatments in a variety of biological systems.     

Trypanosoma cruzi: long-term sub-cultures in two different culture media do not confirm the existence of highly versatile multilocus genotypes

Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T. cruzi IIb and T. cruzi IId, respectively. Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media. We tried to reproduce this observation, but used a different cloning method of individual organisms. Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only. The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes. The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

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.     

High plating density improves Big Blue system efficiency without loss of sensitivity

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.     

Delayed chromosomal instability induced by DNA damage.

DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability.     

Mutagen screening by a simplified bacterial fluctuation test: use of microsomal preparations and whole liver cells for metabolic activation.

We describe the use of a simplified bacterial fluctuation test to detect induced mutation, either incorporating liver microsomal or whole liver cell preparations. We have evaluated both types of test using three agents. The fluctuation assay seems somewhat slower, simpler and more sensitive than a conventional plating test with microsomes. A whole cell preparation appears marginally more effective than a microsomal fraction for metabolic activation of 9,10-dimethyl-1,2-benzanthracene, but rather less effective for benz(a)pyrene and 2-acetamidofluorene. Ultimately the usefulness of activation by whole cells may depend upon whether the method can give a correlation with carcinogenicity that is more quantitative than microsome methods and better reflects organ and species specificity.     

Lethal and mutagenic effects of radiation and chemicals on cultured fish cells derived from the erythrophoroma of goldfish (Carassius auratus)

GEM 199 cells derived from an erythrophoroma of goldfish (Carassius auratus), which had a high plating efficiency, were used to investigate the lethal and mutational effects of radiations (UV and gamma-rays) and chemicals (4NQO and MNNG). The cells were more resistant to gamma-rays than mammalian cells and CAF-MM1 cells derived from the normal fin tissue of goldfish. They were also more resistant to UV-irradiation than CAF-MM1 cells. Photoreactivation after UV-irradiation was present in GEM 199 cells for both survival and mutation. The initial shoulder of the survival curve of UV-irradiated cells was reduced greatly by caffeine, suggesting a high activity of the post-replication repair. The spontaneous mutation frequency to ouabain resistance was 1-5 X 10(-6) clones per viable cell. MNNG was effective in inducing ouabain-resistant mutation, while 4NQO and gamma-rays did not induce mutation.     

High plating efficiency with plant cell cultures

In this paper we describe a simple method to improve the plating efficiency in plant cell cultures.Two-stage plating is used; in the first stage the cells are inoculated at high density in 0.2% agarized culture medium for ten days to facilitate growth; under this condition, each cell produces a single micro-colony trapped in the agar network. In the second stage the colonies are plated at different densities in 1% agarized medium.These colonies are self-sufficient and able to improve the cell growth by conditioning the medium.Abbreviations 6-BAP 6-Benzyl-aminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating.

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent Km (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while its Vmax has been reduced more than 80-fold. In mutant cell extracts with added porcine LDH-B enzyme, molecular dissociation and recombination of subunits produces two new active LDH tetramers (A1B3, A2B2). The electrophoretic mobility of at least one of the tetramers (A1B3) was different from those formed in the parental extracts. The evidence suggests the variant cell contains a mutation in the structural gene for LDH-A.     

A note on plating efficiency in fluctuation experiments

In estimating mutation rates using the Luria-Delbrück experimental protocol, it is often assumed that all mutant cells survive the plating procedure to form visible mutant colonies. This assumption of perfect plating efficiency may not hold in certain circumstances, but none of the existing estimation methods that adjust for plating efficiency is strictly based on the likelihood principle. To ameliorate this situation, we propose likelihood based algorithms for computing point and interval estimates of mutation rates.     

High sensitive approach for point mutation detection based on electrochemiluminescence

An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for point mutation detection has been developed. The target is amplified using a tris (bipyridine) ruthenium (TBR)-labeled forward and a biotinylated reverse primer. The amplification products are digested with specific restriction enzyme, then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. The established technique was further applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the system has a low detection limit of 100 fmol and a linear range of more than 3 orders of magnitude for H-ras amplicon; the two genotypes can be reliably discriminated. In summary, the mutant specific ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in single nucleotide polymorphism (SNP) and mutation detection due to its safety, high sensitivity and simplicity.     

Mutation in continuous cultures of Schizosaccharomyces pombe II. Effect of amino acid starvation on mutational response and DNA concentration

In agreement with the results obtained in Escherichia coli by other workers and our own previous data, the kinetics with which spontaneous mutations to resistance to the 12,13-epoxytrichothecene trichodermin accumulate in a lysine auxotroph of Schizosaccharomyces pombe are dependent upon the nutrilite used to limit the growth of the population. Under conditions of glucose-limitation mutation accumulation is proportional to generation time, while under lysine-limitation it becomes proportional to chronological time. In contrast to observations made in bacterial system, however, no significant change in the DNA content per cell is noted in slow growing cultures grown under amino acid starvation. These findings help to eliminate some of the theories put forward to explain the differential mutational responses observed under different growth limiting regimes.     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

Somatic embryogenesis in protoplast derived calli of cultivated jute,Corchorus capsularis L.

Protoplasts were isolated from cotyledon, hypocotyl and mesophyll cells of Corchorus capsularis L., a major fibre crop, by one step enzyme digestion method. They were further cultured successfully on modified KM-8p (Kao and Michayluk, 1975) medium to form microcalli. The required cultural conditions could be used to achieve 34% to 78% plating efficiency, depending upon the source of protoplasts. Hypocotyl protoplasts gave the highest plating efficiency. On transfer to regeneration medium, somatic embryos developed at high frequency. The present success is a significant step forward in the development of meaningful plant cell culture methods for application in jute.Abbreviations B₅ Gamborg et al., 1968 - MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - Ad-SO₄ Adenine sulphate - BAP 6-benzylaminopurine - NAA 1-naphthalene acetic acid - GA₃ Gibberellic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - Kn Kinetin - IBA Indole-3-butyric acid     

Enhanced mutability at the tk locus in the radiosensitive double-strand break repair mutant xrs5

The thymidine kinase locus (tk) has been utilised as the target locus to measure the induced mutation frequency following X-irradiation in the X-ray-sensitive xrs5 mutant and its parent CHO K1 line of Chinese hamster cells. Mutations to tk- cells were measured by plating cells in selective medium containing trifluorothymidine after a post-irradiation expression time of 4 days. Our results show that the mutation frequency was 3-4 times higher in the xrs5 mutant than in the CHO K1 cell line. This enhanced mutation frequency in xrs5 is though to result from the deficiency in DNA double-strand break repair in this cell line which also results in the enhanced cell killing and higher frequencies of chromosomal aberrations in response to X-irradiation. The findings of the present study suggest that DNA double-strand break is a critical lesion leading to mutations in irradiated cells.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

Mutagenesis of yeast by hydrazine: dependence upon post-treatment cell division.

Hydrazine was found to be mutagenic for yeast (Saccharomyces cerevisiae) at exposures (concentration × time) ranging over nearly three orders of magnitude. Little or no forward mutation from CAN1 to can1 was detectable upon immediate plating following treatment in neutral buffer suspension. Post-treatment cell division in yeast extract peptone dextrose complex growth medium was required for expression of induced mutation to canavanine resistance. Frequencies of induced mutation rose to levels approximately 10-fold higher than spontaneous levels for exposures between 0.1 and 12.0 min mol/l. Survival remained at 100%. For exposures greater than 80 min mol/l viability and mutation frequency began to decrease sharply. By contrast, single treatments of ethyl methanesulfonate, methyl methanesulfonate, N-methyl-N′-nitro-N-nitro-soguanidine, nitrous acid, hydroxylamine, and ultraviolet light were able to increase mutation frequency with this system upon immediate assay. Further growth-dependent increases in mutation frequency were not observed with HA and UV.Expression of HZ-induced mutation was detectable after treated cells had undergone less than one population doubling in YEPD. Such mutation expression could be blocked by the inhibitors cycloheximide and hydroxyurea, which block protein synthesis and DNA synthesis respectively. Results were similar to those obtained previously with Haemophilus influenzae and similarly suggest that, in this eukaryote, HZ-induced lesions lead to mutation by causing base mispairing at DNA replication rather than by means of an error-prone repair mechanism.     

Isolation and characterization of temperature-sensitive mutants of Chinese hamster ovary cells after treatment with UV and x-irradiation.

The isolation of ten conditionally lethal temperature-sensitive mutants of the Chinese hamster ovary cell (CHO-Kl, pro-) by the BUdR-visible light selection procedure described. Treatment with radiation at doses known to cause single gene mutation in mammalian cells increases the mutation frequency by a factor of at least 14. These mutants will grow with normal plating efficiency at 34.5 degrees but will not grow at 39.5 degrees. Complementation analysis by two independent methods indicates that all mutants are recessive and allows the assignment of the mutants to six genetically independent complementation groups. Reversion analysis indicates that the TS-mutants are stable, spontaneous revertants arising at a frequency of less than 10(-6). Preliminary chromosome analysis revealed no systematic chromasomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5 degrees and 39.5 degrees. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.