The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related

The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Secretion of invertase in mitotic yeast cells.

In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsynchronized cells. Electron microscopy of nocodazole-treated mitotic wild-type cells revealed stretches of rough endoplasmic reticulum, strongly fenestrated Golgi cisternae and clusters of vesicles with the diameter of 30-90 nm. Secretion of invertase was inhibited in mitotic sec7 cells at the restrictive temperature, but continued at the permissive temperature. Sec7 is a mutant strain where intracellular traffic is blocked in unsynchronized cells in the Golgi complex at the restrictive temperature. Thus, the elements of the mitotic Golgi complex appear to be able to support intracellular traffic.     

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.     

Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

Bovine papillomavirus mutant temperature sensitive for transformation, replication and transactivation.

The genetic analysis of the papillomaviruses has been hampered by the lack of mutants conditionally defective for viral biological activities. We report here the construction and characterization of a temperature-sensitive papillomavirus mutant. The mutation is predicted to insert the sequence Pro-Arg-Ser-Arg into the N-terminal half of the bovine papillomavirus type 1 (BPV1) ORF E2 protein, the major viral regulatory protein. The cloned mutant viral DNA displays temperature-sensitive defects in the induction of focus formation in mouse C127 cells, in its establishment as an extrachromosomal plasmid and in transactivation of a BPV1 enhancer. Genetic experiments confirm that this pleiotropic phenotype is caused by the insertion mutation in ORF E2 and that the transformation and replication defects of the mutant at 37 degrees C are corrected in trans by wild-type E2 gene activity. Most cell lines stably transformed by the mutant at 32.5 degrees C display a reduced ability to overgrow a monolayer of normal cells following temperature shift to 37 degrees C and the mutant viral DNA after temperature shift is present in decreased copy number and/or in an integrated state. These results provide strong genetic evidence that continued ORF E2 activity is required for maintenance of BPV1-induced transformation and for normal viral DNA replication.     

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.     

On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant.

Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.     

Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Electron Microscopic Characterization of the Defectiveness of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus Restricted in Assembly

The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39 degrees C) than either in cells grown at the permissive temperature (34 degrees C) or in wild-type MuLV-infected cells grown at 39 degrees C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34 degrees C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39 degrees C compared with those grown at 34 degrees C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.     

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.     

A temperature-sensitive mutation affecting cilia regeneration, nuclear development, and the cell cycle of Tetrahymena thermophila is rescued by cytoplasmic exchange.

A temperature-sensitive mutation was isolated that blocks cilia regeneration and arrests growth in Tetrahymena thermophila. Protein and RNA synthesis and ATP production appeared to be largely unaffected at the restrictive temperature, suggesting that the mutation is specific for cilia regeneration and growth. At the restrictive temperature, mutant cells arrested at a specific point in the cell cycle, after macronuclear S phase and shortly before micronuclear mitosis. Arrested cells did not undergo nuclear divisions, DNA replication, or cytokinesis, so the mutation appears to cause true cell cycle arrest. Surprisingly, the mutation does not appear to affect micronuclear mitosis directly but rather some event(s) prior to micronuclear mitosis that must be completed before cells can complete the cell cycle. The cell cycle arrest was transiently complemented by wild-type cytoplasm exchanged during conjugation with a wild-type cell. Each starved, wild-type cell apparently contained enough rescuing factor to support an average of six cell divisions. Thus, this mutation affects assembly and/or function of at least one but not all of the microtubule-based structures in T. thermophila.