Growth In Nonselective Medium Research Articles

Rate of pESC plasmid loss in Saccharomyces cerevisiae in the absence of selective pressure (VAC13P.1126)

Abstract Recombinant yeast has been proposed as a vaccine modality, especially for inducing CTL responses. The production of yeast expressing proteins of interest would be facilitated if they can be grown in less expensive non-selective medium. This would only be practical if the population of yeast retains high levels of plasmid over the duration of the growth. We tested the Saccharomyces cerevisiae multiple gene auxotroph with pESC-URA to determine the rate of plasmid loss in the population. The yeast maintain the plasmid much longer than anticipated, suggesting that growth in non-selective medium may be a viable method. Induction of protein expression increases the competitive advantage of plasmid loss. Growth rates of the yeast and of plasmid loss under these variable conditions was also determined.

Multiple gene expression by chromosomal integration and CRE-loxP-mediated marker recycling in Saccharomyces cerevisiae.

Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2 micro -based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.

Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein.

The aim of this study was to deterimine the survival of an enteric bacterium in anaerobic groundwater and effluent microcosms using the green fluorescent protein (GFP) marker gene in combination with the viability indicator propidium iodide (PI). The pEGFP vector (Clontech) was transformed into Escherichia coli DH5alpha and was stable for at least 100 generations of growth in nonselective medium at 28 degrees C and 37 degrees C. Using an epifluorescent microscope, GFP cells could be detected under blue light (450-490 nm) and the numbers of PI-positive GFPs could be detected under green light (530-560 nm). GFP-tagged E. coli could be detected for at least 132 d in sterilized water microcosms. GFP fluorescence was not lost from the culturable cell population for the duration of the experiment. However, a slow decline in the number of GFP-fluorescent cells in sterilized groundwater was observed. Escherichia coli die-off and loss of green fluorescence was more rapid in nonsterilized waters than in sterilized. Viable numbers of the GFP-tagged E. coli determined by PI counterstaining were compatible with numbers of colony-forming units. The long-term survival of E. coli and maintainance of GFP-conferred fluorescence in these cells was demonstrated in both groundwater and effluent, under sterilized conditions. However, severe starvation and/or the presence of indigenous microorganisms were found to be factors affecting the maintenance of fluorescence in dead or dying cells. This study demonstrates the successful application of PI with GFP-tagging to monitor long-term bacterial survival in nutrient-limited conditions and mixed microbial populations.

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae.

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

G418 Selection and stability of cloned genes integrated at chromosomal δ sequences of Saccharomyces cerevisiae

The chromosomal delta sequences of the yeast Saccharomyces cerevisiae were employed as recombination sites to integrate the bacterial neo(r) gene and the yeast SUC2 gene into the yeast genome. A dominate selection method employing the aminoglycoside antibiotic G418 was used. Transformation efficiencies and growth behaviors of the transformants were studied. Transformants were obtained with more than 40 integrations; the majority of insertions were tandem with a maximum of three different insertion sites utilized at one time. After 70-100 generations of growth in nonselective medium, the high copy number SUC2-neo(r) integrants were found to be unstable; only minor instability was observed for the neo(r) and low copy number SUC2-neo(r) integrants.

Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain.

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

A Novel Plasmid pIJB1 Possessing a Putative 2,4-Dichlorophenoxyacetate Degradative Transposon Tn5530inBurkholderia cepaciaStrain 2a

A 102-kb plasmid, pIJB1, was isolated fromBurkholderia cepaciastrain 2a, which is able to use 2,4-dichlorophenoxyacetate (2,4-D) as a sole carbon source, and a physical map of the plasmid has been established. It was observed that spontaneous loss of a 37-kb fragment of the plasmid after growth in nonselective medium occurred, generating a plasmid of diminished size, pIJB2. The deletion event is concomitant with the loss of the 2,4-D dissimilatory phenotype, indicating that at least some of the 2,4-D degradative genes are on the missing fragment. The missing fragment is flanked by two identical 4.3-kb insertion sequences (IS) and shows a typical composite transposon structure of 41-kb in size, designated Tn5530.The mutant plasmid pIJB2 possesses a single copy of the IS element.

Persistence of Ha-ras-induced metastatic potential of SP1 mouse mammary tumors despite loss of the Ha-ras shuttle vector.

Previous studies have shown that the SP1 mouse mammary adenocarcinoma cell line, which is tumorigenic but nonmetastatic, acquires metastatic potential when transfected with the activated human Ha-ras gene. In addition, the process of calcium phosphate-mediated DNA transfection, as well as treatment with the calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, can also result in heritable changes in the malignant behavior of SP1 cells. It was of interest, therefore, to determine whether the metastatic consequences of Ha-ras oncogene expression in SP1 cells are a primary effect of the transfected gene or whether heritable secondary changes are induced by Ha-ras oncogene expression. In the latter case, continued expression of the Ha-ras oncogene would not be required to maintain the metastatic phenotype. To test this hypothesis we introduced the Ha-ras oncogene into SP1 cells on a shuttle vector in which maintenance of the vector was dependent on selection for resistance to the antibiotic G418. Subclones which had lost the transfected Ha-ras gene were subsequently isolated following growth in nonselective medium. The Ha-ras-transfected clones and the revertant subclones were found to be equally metastatic, indicating that transfection with the Ha-ras gene does induce stable secondary changes in the metastatic phenotype of SP1 cells.

Introduction of nonselectible 2? plasmid into [cir�] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

The 2 mu DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 mu DNA therefore cannot be achieved, and the intracellular presence of 2 mu can only be assessed by molecular analysis of the DNA complement. In addition, 2 mu alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo site-specific recombination between the endogenous 2 mu DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 mu repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 mu plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 mu DNA plasmid. This system can be utilized to introduce 2 mu DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.

Cross resistance pattern towards anticancer drugs of a human carcinoma multidrug-resistant cell line.

Puromycin-resistant (PurR) mutants/variants of a human carcinoma cell line (HeLa), which show greatly reduced cellular uptake of 3H-puromycin and 3H-daunomycin have been isolated after one- and two-step selections in presence of the drug. The cross-resistance pattern of these mutant cell lines towards numerous anticancer drugs and other inhibitors has been examined. Both the first- and the second-step mutants exhibited increased resistance to a number of antimitotic drugs (viz. vinblastine, vincristine, colchicine, taxol and maytansine), several protein synthesis inhibitors (viz. chalcomycin, bruceantin, harringtonine, homoharringtonine), a large number of DNA interactive compounds (viz. aclacinomycin A, actinomycin D, adriamycin, m-AMSA, chromomycin A3, coralyne sulphoacetate, daunomycin, ellipticine, mithramycin, mitoxantrone, 5-methoxysterigmatocystin, rubidazone, variamycin, VM26 and VP16-213) and a number of other drugs acting via other mechanisms (viz. Baker's antifol, nitidine chloride and rhodamine 123). Whereas the first-step mutants showed stable resistance to these drugs, the second-step lines partially reverted upon growth in non-selective medium. Further, treatment of these mutant lines with non-cytotoxic doses of the calcium channel blocker verapamil reverted or abolished their resistance to the above drugs in a dose-dependent manner. In contrast to the above compounds, the PurR mutants showed no significant cross-resistance to a large number of other drugs which included asaley, AT-125, 5-azacytidine, azaserine, cyclocytidine, cis-platin, cytosine arabinoside, chlorambucil, chlorpromazine, alpha-difluoromethyl ornithine, 5-fluorouracil, ftorafur, gallium nitrate, hydroxyurea, ICRF-159, ICRF-187, imipramine, methotraxate, 6-methylmercaptopurine riboside, mycophenolic acid, melphalan, mitomycin C, methyl GAG, nafoxidine, reumycin, 6-selenoguanosine, 6-thioguanine, tiazofurin, tamoxifen, thalicarpine, tiapamil and verapamil). These cross-resistance data should prove useful in developing suitable drug combinations to which cellular resistance would not develop readily.

Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.

Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.

Hypersensitivity of thymidine kinase-deficient friend leukaemia cells to the induction of cytogenetic aberrations by mitomycin C

Clone 707 of the Friend leukaemia cell line was compared with the hypermutable thymidine kinase deficient subclone 707 BUF for sensitivity to the induction of cytogenetic aberrations by mitomycin C (MMC). Two 16-h doses of MMC were utilized, namely 0.1 and 0.15 μg ml −1. Following removal of MMC from the cultures, metaphase spreads were prepared after 15, 29 and 43 h growth in non-selective medium. Thirteen types of aberrations were scored. The thymidine kinase deficient subclone showed considerably increased sensitivity to the induction of aberrations, with the aberrations also persisting longer. In light of these and earlier reported results, the significance of thymidine kinase for accurate DNA repair is discussed.

A novel class of genetic variants of the L1210 cell up-regulated for folate analogue transport inward. Isolation, characterization, and degree of metabolic instability of the system.

We have isolated stable variants of the L1210 cell exhibiting increased transport inward of the folate analog, methotrexate. These variants show 3- to 14-fold increases in [3H]methotrexate influx compared to parental cells but are unaltered for [3H]methotrexate efflux. This increased influx in each variant is quantitatively reflected in corresponding elevations in intracellular exchangeable levels of drug at steady state, but there is no alteration in membrane potential. The increases in influx are associated with increased values for influx Vmax for a system normally transporting reduced folates and the same increase in the amount of a specific binding component at the cell surface. Otherwise, values for influx Km and specificity for various folate structures are unchanged. This alteration in [3H]methotrexate influx is biochemically and genetically stable, since it is expressed in isolated plasma membrane vesicles and is retained during growth in non-selective medium. Following addition of cycloheximide, the same rate of decay of this transport activity (t 1/2 = 126 +/- 24 to 137 +/- 26 min) was shown for parental and variant cells. From these results we conclude that turnover of this transport property occurs in these cells which is genetically regulated. Also, the elevated transport activity inward for this folate analog in these variant cells is probably the result of a genetic alteration up-regulating the rate of synthesis of the "putative" carrier protein itself. The absence of any effect on efflux of [3H]methotrexate in these variants in the face of evidence for increased synthesis of the carrier protein for the system mediating influx of this folate analog is construed as further evidence for the nonidentity of systems mediating each flux that we proposed on the basis of earlier kinetic studies.

Herpes simplex virus thymidine kinase gene is stably maintained and expressed in cells transformed by protoplast fusion.

We examined a series of transformed cell lines resulting from transfer of the herpes simplex virus type 1 thymidine kinase gene to Ltk- cells by protoplast fusion gene transfer. We show that multiple copies of the transforming plasmid DNA, ranging from a minimum of two to greater than 20, were present in one or at most a few integration sites in each cell line. The TK+ phenotype was stable in five independent transformed cell lines after growth in nonselective medium for over a year. Transforming plasmid DNA was stable in one cell line containing from two to five copies after a year of growth in nonselective medium. In another cell line initially containing about 20 copies, the transforming DNA became rearranged soon after growth to mass culture, resulting in a decrease to two to five copies which then remained stably maintained. This suggests that TK+ transformants resulting from protoplast fusion are stable when the input DNA has integrated in a relatively low copy number.

Optimal conditions for transformation of Azotobacter vinelandii.

Optimal transformation of Azotobacter vinelandii OP required a 20-min incubation of the competent cells with deoxyribonucleic acid at 30 degrees C in buffer (pH 6.0 to 8.0) containing 8 mM magnesium sulfate. Nitrogen-fixing transformants of nitrogen fixation-deficient recipients could be plated immediately on selective medium, but transformants acquiring rifampin and streptomycin resistance required preincubation in nonselective medium. The three phenotypes achieved an approximately equal and stable frequency after 17 h (six generations) of growth in nonselective medium.

Transfer of the Herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.

Genetic and biochemical characterization of mutants of CHO cells resistant to the protein synthesis inhibitor trichodermin.

Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Trir) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunits from Trir cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-14C]TRICHODERMIN. The lesion in Trir mutants therefore seems to have affected this ribosomal subunit. Trir X Tris hybrids are sensitive to trichodermin indicating that the Trir mutation behaves recessively to Tris in hybrids. The Emtr and Trir markers segregate independently from hybrid cells showing that the Trir mutation is probably not linked to the Emtr locus, which as we have shown earlier affects the 40S ribosomal subunit.

The induction of thioguanine-resistant mutants of Chinese hamster cells by γ-rays

The induction of mutation to purine analogue resistance was assessed in Chinese hamster V79-4 cells exposed to gamma-radiation. After irradiation, the cells were grown in non-selective medium for different time intervals before respreading into medium containing 0.5-0.7 mug/ml thioguanine. In some experiments colonies arising in thioguanine-medium were counter-selected in medium containing the glutamine analogue azaserine, which distinguishes mutants with very little activity of the enzyme hypoxanthine-guanine phosphoribosyl transferase. Only these mutants were increased in frequency by radiation, the maximum measured frequencies occuring in cells respread after 2 days growth in non-selective medium. With longer intervals of post-irradiation growth in non-selective medium a fraction of the induced mutants was lost, and after large doses of radiation it is doubtful if the maximum frequency observed after 2 days post-irradiation growth represents the true induced frequency. The detection of freshly-induced mutants seemed to depend upon the dilution and decay of products formed from the genes prior to their mutation by radiation, since (a) selection of mutants in a higher concentration of thioguanine (2 mug/ml) increased the post-irradiation growth interval required to detect the maximum frequency of induced mutants, and (b) with increasing duration of post-irradiation growth in the non-selective medium, induced mutant cells were able progressively to overcome the growth-limiting effects of the analogue, to give large colonies when respread in thioguanine-medium. The radiosensitivities of 7 isolated mutant lines were indistinguishable from that of wild type cells, but the mutants were at a slight disadvantage when grown in competition with wild type cells. This disadvantage was consistent with the expected fitness of mutants relative to wild type cells calculated from estimates of the spontaneous mutation rate and the mean spontaneous mutation frequency. The induction of mutation to thioguanine resistance was non-linear with dose, yielding induced frequencies per rad of 5-10(-8) to 3-10(-7), but a plot of induced mutation frequency against log surviving fraction gave an approximately linear relationship. The same linear relationship holds for recently-published data on human and mouse cell cultures, so that all three mammalian cell types exhibit the same fixed probability of mutation induction relative to the extent of inactivation caused by ionising radiation.

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.