首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.  相似文献   

2.
Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.  相似文献   

3.
We have generated temperature-sensitive (ts) mutants for steroid-regulated anchorage-independent cell growth. Androgen-responsive S115+A mouse mammary tumor cells were mutagenized with ethyl methane sulfonate and the variants which were growth-arrested in suspension at the nonpermissive temperature of 41 degrees C were selected by killing dividing wild-type cells with the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine or cytosine arabinoside. Fifteen clones were isolated and characterized for morphology and growth properties. Three (ts21, ts27, ts33) of the phenotypic variants were ts for androgen-maintained anchorage-independent growth, two of them (ts27 and ts33) also for growth in monolayer. Growth arrest at 41 degrees C was not due to a defect in androgen receptor function in any of the mutant cell lines as shown by steroid binding assays and by the androgen-stimulated expression of both endogenous MMTV RNA and the transiently transfected LTR-CAT gene at the nonpermissive temperature. It remains to be determined for clone ts33 whether the defect is in postreceptor events of steroid action or in genes affecting general mechanisms of cell growth. However, since in clones ts21 and ts27 general cell growth remains functional at 41 degrees C under serum stimulation, defects may be in postreceptor steroid-related pathways. It is hoped that these mutants will provide a useful tool for study of steroid regulation of cell growth and in particular of the property of anchorage-independent growth.  相似文献   

4.
Conditional "cycle-blocked" (cb) mutants of Chlamydomonas reinhardtii have been detected and isolated. These mutants exhibit normal vegetative growth at permissive temperature but are unable to complete a cell cycle (or a specified number of cell cycles) at restrictive temperature. A simple technique has been devised to determine the cell cycle stage in each mutant when the defective gene product, which ultimately affects cell division, completes its function. This stage is called the "block point", and is determined by scoring the residual cell division in an exponentially growing population after shift to temperature restrictive conditions. In the cb mutants isolated so far, block points representing many stages throughout the cell cycle have been found. Two categories of cb mutants are described here: one set which prevents the subsequent cell division when the cell encounters the block point after a shift to restrictive temperature, and another set which permits an additional round of cell division after the block point is encountered. The general applicability of block point analysis to other cell systems is presented.  相似文献   

5.
A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.  相似文献   

6.
Summary A protocol has been developed for the negative selection of plant auxotrophs using the nucleoside analogues BUdR and FUdR. The protocol was optimised using nitrogen-starved protoplast-derived cells of Nicotiana plumbaginifolia to simulate auxotrophy. The present results represent a significant improvement over previous reports in that: 1) The background of colonies escaping BUdR/FUdR kill is low and reproducible. 2) The protocol was improved to the point where background survival was 0.03% for non-starved cultures and 0.09% for auxin-starved cultures. 3) It was shown that UV irradiation decreases BUdR sensitivity of dividing cells and that this is overcome by increased exposure to BUdR. 4) Application of the method to auxin-starved haploid protoplast-derived cell suspensions resulted, for the first time, in the selection of temperature-sensitive (ts) auxin auxotrophs. 5) It could be demonstrated, for the first time, that the method in practice enriches for auxotrophs, in this case by a factor of 10 for auxin auxotrophs and at least 60 for ts auxin auxotrophs.Abbreviations BUdR 5-bromodeoxyuridine - FUdR 5-fluoro-deoxyuridine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthaleneacetic acid - BAP 6-benzylamino-purine - CFE colony forming efficiency - PE plating efficiency - ts temperature sensitive  相似文献   

7.
The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.  相似文献   

8.
A number of cell-cycle-specific temperature-sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK-ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7:1683-1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5' end have been replaced by SV40 sequences. The wild-type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK-ts13 cells undergoing temporary complementation.  相似文献   

9.
A method for killing dividing cells (Puck and Kao, '67) was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10(-4) M for 3 days and then irradiated with long UV light. The selective elimination of dividing cells led to a loss of myosin Ca2+-activated ATPase in the cultures. This indicates the presence of dividing cells which contain myosin. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages.  相似文献   

10.
Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.  相似文献   

11.
To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.  相似文献   

12.
Temperature-sensitive (ts) mutants of the cyanobacteriumAnabaena variabilis ATCC 29413 were isolated following mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and post-treatment with metronidazole at 40°C. Of the 8000 clones isolated and tested, six mutants were conditionally lethal at the restrictive temperature (40°C). All the ts mutants exhibited differences in their rates of growth, chlorophyll content, pigment (phycocyanin and/or chlorophyll) ratios, heterocyst frequency, oxygen evolution and nitrogenase activity at the permissive temperature (28°C). A gradual loss of all the above features occurred after a period of 3 d at 40°C, followed by lysis of the cultures. Cessation of nitrogenase activity was found to be different in the different ts mutants. The temperature-sensitive nature of the mutants is suggested to be due to an impairment in iron metabolism since addition of ferric citrate to cultures at 40°C restored the ability to grow, produce heterocysts and fix nitrogen.  相似文献   

13.
Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.  相似文献   

14.
Stocks of Rous sarcoma virus Bryan strain were mutagenized using a bromodeoxyuridine treatment immediately after infection. Thirty temperature-sensitive (ts) mutants defective in transformation (td) were isolated by a replica plating technique. Twenty of these mutants were preliminarily characterized and found to be defective in late functions related to transformation. These mutants were used in experiments of cooperative transformation with four Prague strain td ts mutants of different co-transformation group. A small number of Bryan ts mutants were found to cooperate with some of the Prague mutants in transforming chicken embryo cells at the nonpermissive temperature. However, the amount of co-transformation observed was lower than that observed with cooperating Prague ts mutants and no clear-cut pattern of cotransformation was obtained in Prague and Bryan crosses. Indirect evidence indicates that cooperative transformation is the result of recombination events.  相似文献   

15.
A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.  相似文献   

16.
We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.  相似文献   

17.
Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.  相似文献   

18.
The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation. cdc40-1 mutants shown only slight sensitivity to UV irradiation. Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS. rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures. An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature. UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.  相似文献   

19.
The use of a temperature switch to control the growth and productivity of temperature-sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39 degrees C to select mutagenized CHO-K1 cells, which resulted in the isolation of 31 temperature-sensitive mutants that were growth inhibited at 39 degrees C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1-B1) that maintains the ts phenotype.Continuous exposure to the nonpermissive temperature (npt) of 39 degrees C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34 degrees C and 39 degrees C arrested the 5G1-B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth-arrested cells was 3-4 times that of control cultures maintained at a constant 34 degrees C over the crucial 72-130-h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1-B1 cells arrested in the G(1) or G(0) phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime.These results demonstrate that growth-arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. (c) 1993 John Wiley & Sons, Inc.  相似文献   

20.
A high frequency of morphogenetic mutants of Dictyostelium discoideum can be induced by treatment with MNNG under conditions which result in relatively low cell killing. Six temperature-sensitive growth mutants induced by this treatment were isolated by replica plating. Among these, five showed spontaneous reversion rates of 10(-4) to 10(-5). The mutagenic activity of ems, measured for the induction of both morphogenetic and temperature-sensitive mutants, was weaker than that of MNNG and UV radiation. High frequencies of morphogenetic mutants were obtained only with doses of UV irradiation that resulted in high killing of cells or spores. Caffeine, at concentrations that slightly decreased the growth rate of amoebae in axenic medium, induced morphogenetic defects and also enhanced the mutagenic effect of UV irradiation. However, all the aggregateless clones derived from caffeine treatment that were studied reverted to the wild-type phenotype after a variable number of clonal re-isolations.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号