Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.     

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.     

Characterization of established,cell lines derived from mutagen-sensitiveDrosophila strains

Summary Six established cell lines have been generated from embryos ofDrosophila melanogaster homozygous for different X-linked mutations. Four of these mutants, confer hypersensitivity to chemical mutagens in larvae. The cell lines derived from the two mutageninsensitive stocks, serve as controls in the analyses of DNA metabolism. One cell line (UCD-Dm-mei-9-2) is uniquely identified by a strong hypersensitivity to ultraviolet radiation. Another (UCD-Dm-mus104-1) expresses an enzyme variant not found in the other lines. The population doubling time for these cultures varies between 24 and 47 h. Labeling indices of 24.4 to 37.5% were found. The duration of the S phase in one of the control cell lines is estimated to be about 9 h. Karyotype stability was monitored for five lines over a period of about 1 y. In general these cultures each, became hypotetraploid with a preferential loss of the Y and fourth chromosomes. DNA synthesis in two of the lines fails to exhibit the pattern of sensitivity to mutagens or caffeine that is observed in the corresponding primary cultures. In primary cultures three classes of cells can be identified by autoradiography. About 50% of the cells label at a moderate rate, 20% do not label within the first 1.5 d of culture, and the remaining cells exhibit a burst of labeling shortly after the cultures are initiated. This research was supported by NIH Grants GM16298 and GM22221 and by DOE Contract AT(04-3)-34 PA 210.     

Differential cytopathogenicity accompanyingMycoplasma pneumoniae infection of human lung fibroblasts maintained in newborn bovine serum or fetal bovine serum

Summary MRC-5 human lung fibroblasts maintained in Eagle's basal medium (BME) with either 10% fetal bovine serum (FBS) or 10% newborn bovine serum (NBS) did not respond identically to infection byMycoplasma pneumoniae. Fibroblasts grown in NBS did not develop any cytopathic effect (CPE) when infected withM. pneumoniae, whereas those maintained in FBS developed a pronounced CPE. There was also a difference in sensitivity to infection for fibroblasts maintained in the two sera before the infection. Fibroblasts maintained in NBS, then transferred to FBS 48 h before infection, were still less sensitive toM. pneumoniae infection than cells maintained constantly in FBS.Mycoplasma pneumoniae attached comparably to the fibroblasts grown in the two sera, so the differences in CPE development could not be attributed to differences in mycoplasma attachment. Measurements of DNA, RNA, and protein syntheses of the fibroblasts grown in NBS and FBS indicate that the cells in NBS were growing more rapidly than those in FBS. A determination of the doubling times shows that the doubling time of cells in NBS was 44 h, whereas that of cells in FBS was 51 h. Polyacrylamide gel electrophoresis of samples of NBS and FBS showed significant differences in serum protein composition. The NBS had several protein bands that were lacking in the FBS. This study demonstrates the importance of serum effects in the study ofM. pneumoniae infection. This work was supported in part by Public Health Service Grant AI 17795 from the National Institute of Allergy and Infectious Diseases, Bethesda, MD.     

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean α‐amylase inhibitor gene

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for α‐amylase inhibitor‐1 (αAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean αAI1 protein and the corresponding αAI1‐free segregating lines and non‐GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for αAI1. Proteomic analysis showed that in addition to the presence of αAI1, 33 other proteins were differentially accumulated in the αAI1‐expressing GM lines compared with their non‐GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of αAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium‐mediated transformation events. Sixteen proteins were identified after MALDI‐TOF‐TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin‐containing protein. Two proteins were uniquely expressed in the αAI1‐expressing GM lines and one new protein was present in both the αAI1‐expressing GM lines and their αAI1‐free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.     

Monoclonal antibodies specific for cell culture mycoplasmas

Summary Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody toMycoplasma pneumoniae was produced.M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis ofM. pneumoniae infection in man. These studies were supported by Grants Al-15748 from the National Institute of Allergy and Infectious Diseases, and GM20138-07 from the National Institutes of Health, Bethesda, MD.     

Elimination ofMycoplasma hyorhinis infections from four cell lines

Summary Four monolayer mammalian cell lines were cured ofMycoplasma hyorhinis infections by cloning in microtiter dishes in the presence of tetracycline and kanamycin. During cloning, cultures were refed with fresh antibiotic containing medium every 2 or 3 d for 14 d and were then cultured without effective antibiotics for at least 21 d. From the four lines we recovered 29 clones, none of which were infected after treatment as judged by the lack of extranuclear fluorescence after staining with the fluorochrome Hoechst 33258, and by normal autoradiographic labeling of the cells by tritiated nucleosides. One clone from each line was tested further by attempted culture of mycoplasmas and was also judged to be uninfected. Infection has not reappeared in any of the clones after extensive culture in the absence of the effective antibiotics. This investigation was supported by Public Health Service Research Grant GM26137 from the National Institutes of General Medical Sciences, National Institutes of Health, Bethesda, MD.     

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable.     

Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states

The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

Mycoplasma in african green monkey kidney cell cultures: Biochemical detection and effects in virus-infected cells

Summary Among a number of techniques for the detection of mycoplasmal contamination in African green monkey kidney (AGMK) cell lines, the assay of uridine phosphorylase activity is unsuitable because of the presence of high levels of endogenous enzymatic activity. A thymidine phosphorylase test, however, based on the chromatographic analysis of radiolabeled thymidine breakdown, turned out to be a simple and sensitive mycoplasma detection method. We found, using the latter technique, that 0.22-μm-filtered virus inocula could still transfer mycoplasma unless treated with diethyl ether. The effect of mycoplasmal contamination on the synthesis of simian virus 40 and adenovirus in AGMK cells was negligible under the conditions used (no depletion of arginine). Incorporation of radioactive thymidine in viral macromolecules, however, was inhibited severely by the presence of mycoplasma. This investigation was supported by a grant from theFonds voor Geneeskundig Wetenschappelijk Onderzoek (No. 20.298). F.V. R. is an Aspirant of the BelgianNationaal Fonds voor Wetenschappelijk Onderzoek.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

Decontamination of cell cultures by removing mycoplasmas: a comparison of 3 methods

Three different methods of decontamination of cell cultures from mycoplasma were compared: by macrophages in the presence of antibiotics, by ultraviolet radiation in the presence of 5-bromuridin and Hoechst-33258, by cloning of cells in the presence of antibiotics metacycline and doxicycline. Only the latter method was effective to decontaminate the cell cultures from mycoplasma. Twelve cell lines were decontaminated and tested for the presence of mycoplasma by microbiological method, by staining with the dye Hoechst-33258 and autoradiography. All the cell lines were free of mycoplasma.     

Elimination of mycoplasmas from cultured human cell lines utilizing a combination of two antibiotics, quinolone and a pleuromutilin derivative

Forty three cultured human cell lines were treated with a combination of 2 antibiotics to eliminate contaminant mycoplasmas. One course of treatment was composed of consecutive 3 or 4 cycles. Each cycle grew cells in BM-1 (pleuromutilin derivative; Boehringer Mannheim) containing medium (10 micrograms BM-1/ml culture) for 3 days, alternating with MC-210 (quinolone; Dainihon Pharmaceutical) containing medium (0.625 micrograms MC-210/ml culture) for 4 days. No treatment failure was encountered with this procedure. Before treatments, 18 (90%) of 20 cell line samples were contaminated with mycoplasma, as tested by DNA hybridization method (MYCOPLASMA T.C. RAPID DETECTION SYSTEM; Gen-Probe Inc.). Out of 43 cell lines treated, 7 were reduced in growth and dropped out. Among the other 36 cell lines, 27 became negative, 5 borderline and 4 slightly positive to the mycoplasma detection. All of the latter 9 cell lines, treated with one more similar course, found to be free from mycoplasma. Six of the dropout lines were cured of mycoplasma by a second treatment, under modified culture conditions. The last cell line (NATO) was successfully treated with another lot of FCS. Thus, the procedure proved successful even in treating promiscuously infected cell lines.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

Adenosine phosphorylase activity in a mutant Hep-2 cell line contaminated withmycoplasma hyorhinis

Summary Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor coformycin. Isopycnic separation of [³H]thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [¹⁴C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified asMycoplasma hyorhinis, a porcine mycoplasma. Following γ-irradiation (3000 rads) to block cellular mitosis, the mycoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma. Presented at the 25th Annual Meeting of the Tissue Culture Association, Philadelphia, Pa., June 1976. This work was supported by Public Health Service Grants DE 02731 from the National Institute of Dental Research and CA 16219 from the National Cancer Institute.     

Gene flow from herbicide-tolerant GM rice and the heterosis of GM rice-weed F2 progeny

Gene flow from genetically modified (GM) crops to non-GM cultivars or weedy relatives may lead to the development of more aggressive weeds. We quantified the amount of gene flow from herbicide-tolerant GM rice (Protox GM, derived from the cultivar Dongjin) to three cultivars (Dongjin, Aranghyangchal and Hwaseong) and a weedy rice line. Gene flow frequency generally decreased with increasing distance from the pollen donor. At the shortest distance (0.5 m), we observed a maximum frequency (0.039%) of gene flow. We found that the cultivar Dongjin received the greatest amount of gene flow, with the second being weedy rice. Heterosis of F2 inbred progeny was also examined between Protox GM and weedy rice. We compared growth and reproduction between F2 progeny (homozygous or hemizygous for the Protox gene) and parental rice lines (GM and weedy rice). Here, transgene-homozygous F2 progeny was significantly taller and produced more seeds than the transgene-hemizygous F2 progeny and parental lines. Although the gene flow frequency was generally low, our results suggest that F2 progeny between GM and weedy relatives may exhibit heterosis.     

5种转基因油菜转化体特异性多重PCR检测方法

【目的】全球转基因植物及其产品的数量和种类越来越多,迫切需要可同时精准高效检测多个转化载体的检测方法。【方法】针对RF1、MS8、Topas19/2、Oxy235和RF3等5个转基因油菜品系的侧翼序列及油菜内源基因cruciferin A(Cru A)序列设计多重聚合酶链式反应特异性引物,通过对转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同作物进行PCR扩增来测试所选择的引物特异性,优化多重PCR反应引物的浓度,用所建立的检测体系对不同混合比例的转基因油菜进行多重PCR扩增来测试所建立的检测方法的灵敏度。【结果】通过测试,仅在含有目标样品中检测出阳性结果,灵敏度达0.05%,表明所建立的6重PCR检测方法可同时精准检测RF1、MS8、Topas19/2、Oxy235和RF3等5种转基因油菜转化载体。【结论】所建立的6重转基因油菜转化体特异性PCR检测方法通量高、特异性好、灵敏度高,符合有关转基因产品检测的要求,可作为转基因油菜检测的有效方法。     

Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 β was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1β and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.