Cellular aspects of pattern formation: the problem of assembly.
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Animals↗
Biomedical subjects
K J Aufderheide
Publications and source records attributed to K J Aufderheide.
Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.
Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.
Animals↗
Transformation of Paramecium by microinjection of a cloned serotype gene.
Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens). A 16-kilobase plasmid containing the gene for immobilization antigen A from Paramecium tetraurelia, stock 51, was injected into the macronucleus of deletion mutant d12, which lacks that gene. Approximately 40% of the injected cells acquired the ability to express serotype A at 34 degrees C. Expression appeared to be regulated normally. The transformed cells, like wild type, could be switched to serotype B by antiserum treatment and culture at 19 degrees C; on transfer to 34 degrees C, they switched back to serotype A expression. Many of the lines retained the ability to express serotype A until autogamy, when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.
Animals↗
Identification of the basal bodies and kinetodesmal fibers in living cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide, Daggett & Nerad, 1983.
Critical use of Nomarski DIC optics and a rotocompressor permits basal bodies and kinetodesmal fibers to be visualized in the cortices of living Paramecium tetraurelia and Paramecium sonneborni. The identification of these structures is confirmed by the correspondence of the images obtained by DIC optics of living cells and by brightfield optics of fixed cells stained by the Fernández-Galiano silver technique. Examination of cells carrying cortical inversions (portions of the cortex rotated 180 degrees) shows that inverted regions may be identified and distinguished from normal regions by the orientation of the kinetodesmal fibers of the kinetids (cortical units) within the kineties (ciliary rows). This demonstrates that both the asymmetry and the polarity of each cortical unit may be assessed in the living cell. This technique has useful applications in the study of morphogenesis and patterning in living cells and for the screening of mutations and variants conferring altered cortical phenotypes.
Animals↗
Trichocyst phenotype transformation induced by macronuclear transplantation in Paramecium tetraurelia.
A portion of the macronucleus of wild-type cells of Paramecium tetraurelia was removed and was injected into cells homozygous for the ftA mutation. The ftA mutants make defective trichocysts and are unable to perform normal trichocyst exocytosis. After injection, approx. 30% of the surviving cells show a phenotype shift from mutant to wild-type. This shift is stable during subsequent vegetative growth until clonal death. If, however, the hybrid cell lines are brought to autogamy (which discards the existing macronucleus and forms a new one from sexual products derived from a micronucleus), then the lines revert to the ftA phenotype. Since micronuclei were not transplanted, the phenotypic reversion after autogamy is to be expected, and demonstrates that the transformation affects the macronucleus only. A second series of injections involved transfer of a portion of the macronucleus from cells homozygous for the trichocyst ptA mutation into ftA host cells. These two mutations are genetically complementary, so the injection should be genetically equivalent to forming a double heterozygote. Approx. 20% of the injection survivors shift to wild-type. This shift is also vegetatively stable unless autogamy occurs; after autogamy, reversion to the ftA phenotype is seen. These results show that a portion of a macronucleus can be successfully transplanted from one cell to another and that, in the host cytoplasmic environment, normal gene expression and replication of a transplanted macronucleus does occur. The technique of macronuclear transplantation is significant to studies of the macronuclear contribution to clonal aging, and to studies on genetic control over trichocyst development.
Animals↗
Phenotypes associated with early clonal death in Paramecium tetraurelia.
The aging characteristics of various known mutations of Paramecium tetraurelia have been studied. Among the mutations which had been previously isolated on the basis of the inability of the cell to do exocytosis of its trichocysts, one group was identified which had clonal life spans which were indistinguishable from, or were somewhat shorter than, the life span of wild-type. Another group of trichocyst mutations, however, had clonal life spans which were 1/3 to 1/4 that of wild-type. Further study of the phenotypes of these various mutations revealed that members of the short-lived group all had a common phenotypic defect: a frequent inability to properly divide the macronucleus during cell division. Another mutation which has normal trichocysts, but which shows macronuclear division defects, also has an extremely short life span. Thus, the short clonal life spans seen are not associated with the inability to do trichocyst exocytosis but rather are associated with the inability to properly divide the macronucleus. An hypothesis is presented which relates macronuclear misdivision to the short clonal life spans of the mutants expressing it. By extension of the hypothesis, the significance of normal macronuclear divisions to the normal aging process in P. tetraurelia is proposed.
Aging↗
Clonal aging in Paramecium tetraurelia. Absence of evidence for a cytoplasmic factor.
Cells of Paramecium tetraurelia show clonal aging with characteristics comparable to the aging seen in tissue culture cell lines. An investigation is underway to determine the relative contributions, if any, of the cytoplasm and the macronucleus to the onset of senescence. Using a microinjection protocol, the importance of the cytoplasm to aging was tested. Cytoplasm was transferred from young cells into old cells to see if the mean age of death of the injected cell lines could be increased, compared with uninjected controls and sham injected controls. Cytoplasm from old cells was transferred into young cells to see if the mean age of death of the injected cell lines could be decreased, compared with sham injected and uninjected controls. In neither case was there any statistically significant change in mean ages of death. Furthermore, no changes can be demonstrated even with a protocol which transplanted 3 times the normal cytoplasmic volume. Thus, a cytoplasmic effect upon vegetative aging in P. tetraurelia cannot be demonstrated. Tests of a macronuclear influence are now underway.
Aging↗
Cellular aging: an overview.
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Cell Differentiation↗
Formation and positioning of surface-related structures in protozoa.
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Animals↗
Mitochondrial associations with specific microtubular components of the cortex of Tetrahymena thermophila. II. Response of the mitochondrial pattern to changes in the microtubule pattern.
Mitochondria in Tetrahymena thermophila are known to associate with the cell cortex in asymmetrical patterns corresponding to the asymmetrical organization of the microtubular components of the cortex. Specific mitochondrion-microtubule associations are seen at the light-microscope and ultrastructural levels. The hypothesis that the mitochondrial pattern is determined by the microtubular pattern was tested. Using the phenotypes of various mutations to generate changes in the organization of the cortex, the response of the mitochondrial pattern to the cortical change was assayed. The results consistently show that changes in the local organization of the cortical microtubule systems are followed by a corresponding change in the cortical mitochondrial pattern. However, the mitochondrial pattern is unaffected by a mutation which changes the 'long-distance' patterning of certain organelles, but which does not influence the local microtubular organization. Thus, the data support the hypothesis that there is a causal relationship between the arrangement of the cortical microtubules and the corresponding mitochondrial pattern.
Animals↗
The effective site of the lesion resulting from a trichocyst non-discharge stable differentiation of somatic nuclei in Paramecium tetraurelia.
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Animals↗
Motility events of trichocyst insertion in Paramecium tetraurelia.
Following electroshock-induced extrusion of its inserted trichocysts, Paramecium tetraurelia rapidly begins replacement of the population of lost organelles. Light microscopy of the cortical insertion of new trichocysts reveals a series of characteristic motility activities. An uninserted trichocyst in the cyclotic flow of the cell appears to be "captured" and removed to the noncyclotic, subcortical regions. The trichocyst then makes a series of saltatory motions which apparently serve to transport it to the cortex, with proper orientation (tip first) for insertion. Trichocyst saltations end with either cortical insertion of the organelle, or return to cyclosis. If the trichocyst is inserted, it makes a series of unique pivoting movements around the motionless tip. This form of motility, termed "wobble," continues for a short period of time. After cessation of wobble, the insertion of the trichocyst is apparently complete, since no further motility is observed. With the aid of these observations it was possible to identify saltatory motility as the means for transporting trichocysts to the cortex for insertion, and also to observe a motility of unknown significance (wobble) apparently associated with the process of cortical insertion.
Animals↗
Genetic aspects of intracellular motility: cortical localization and insertion of trichocysts in Paramecium tetraurelia.
Wild-type and selected mutants of Paramecium tetraurelia were subjected to detailed analysis of the trichocyst phenotype. The characteristics of morphology, saltatory motility, and cortical insertion of trichocysts were found to be genetically distinguishable. In mutants with defective trichocyst saltatory motility, other intracellular motility systems (i.e. mitochondrial saltatory motility, and cyclosis) appeared to be unaffected. Saltatory motility appears to be an essential intermediate step in the functional development of the trichocyst, since it serves to transport the trichocyst from its site of assembly, deep in the cytoplasm, to the cortex, where it is inserted and becomes discharge-competent. If trichocyst saltatory motility is lost, as in the mutants ndA and tam8, then trichocysts are not transported to the cortex and are therefore not inserted. However, the successful transport of a trichocyst to the cortex does not guarantee the proper insertion of that trichocyst, as in the case of the mutant am. Apparently there are independent steps involved specifically with trichocyst insertion. The gross morphology of the trichocyst appears to be independent of its saltatory motility, and to a degree, independent of cortical insertion.
Animals↗
Saltatory motility of uninserted trichocysts and mitochondria in Paramecium tetraurelia.
In the subcortical regions of the ciliate Paramecium tetraurelia, mitochondria and uninserted trichocysts each display saltatory motility with individual characteristics, making them distinguishable from each other and from cellular cyclosis. The saltatory motion of trichocysts is implicated as the means of transporting new trichocysts from the cytoplasm to their ultimate locations in the cellular cortex. Saltatory motion may also be a factor in the intracellular distribution of mitochondria.
Animals↗
Cytoplasmic inheritance in Saccharomyces cerevisiae: comparison of zygotic mitochondrial inheritance patterns.
Mitochondrial movements in Saccharomyces cerevisiae (Sc) zygotes were monitored with phase-contrast microscopy and compared to known mitochondrial inheritance systems. The mitochondria of Sc were convincingly identified by integrated use of phase-contrast, cytochemical and electron microscopic observations. Mitochondria in Sc appear to move by saltatory jumps, which appear to be oriented towards movement of mitochondria into developing buds. Tracking of mitochondria of different genotypes was made possible by positive identification of each mitochondrial population before zygosis, and by the low degree of mixing (less than 10%) of mitochondrial populations before first bud septation. A grande by grande cross demonstrated equal numbers of mitochondria from each haploid moving into the first zygotic bud. A grande by neutral petite cross gave a 2:1 ratio of grande to petite mitochondria. However, a grande by suppressive petite cross gave equal numbers of grande and petite mitochondria. Using drug resistance systems, a comparison was made of highly biased (97%) and moderately biased (71%) chloramphenicol resistant inheritance patterns. In both cases, the ratios of drug resistant to sensitive mitochondria were 1:1. When numbers of mitochondria moving into an individual bud were compared to the phenotypic content of the clone of that bud, no model could be constructed which could predict the latter from the former. The data indicate (with the exception of the neutral petite by grande cross) that the numbers of each mitochondrial type "inserted" into the first zygotic bud are equal, regardless of the degree of asymmetry of inheritance of mitochondrial markers.
Chloramphenicol↗
Cytoplasmic inheritance in Saccharomyces cerevisiae: comparison of first zygotic budsite to mitochondrial inheritance patterns.
Zygotic first budsite in Saccharomyces cerevisiae was studied in relation to defined mitochondrial inheritance systems: both petite and drug resistance. It was hypothesized that a highly asymmetric inheritance pattern would be correlated to a high frequency of first budsites on the petite or drug resistant end of the zygote (i.e., that portion of the zygote which was originally the drug resistant or petite haploid before zygote formation). The data collected did not support the hypothesis. For drug resistance, the budsite pattern is identical for a highly biased and a moderately biased inheritance pattern. In a grande by grande cross there is a high probability of the first bud appearing on the conjugation bridge, with lower but equal probabilities of the first bud appearing on one end or the other of the zygote. A grande by petite cross changes this pattern to a high probability of the first bud appearing on the grade end of the zygote, with a lesser probability of the first bud appearing on the conjugation bridge and virtually no budding of the petite end. This phenomenon is independent of degree of neutrality or suppressiveness of the petite strain used, however. The difference between a grande and a grande by petite pattern may be due to the relative functional ability of the mitochondria in each end of the zygote. Tests using antimitochondrial drugs suggest that selection of first budsite on a zygote is a complex phenomenon, not simply dependent upon mitochondrial phenotype. In conclusion, selection of the first zygotic budsite appears to be independent of mitochondrial inheritance patterns.
Cell Division↗
Clonal aging in Paramecium tetraurelia. II. Evidence of functional changes in the macronucleus with age.
The contribution of the macronucleus of Paramecium tetraurelia to the long term proliferation potential of a cell line was tested using a nuclear transplantation protocol. Macronuclei from young or old wild-type cells were injected into genetically marked host cells of a standard clonal age. The subsequent proliferation to clonal death of successfully and stably transformed hybrids was compared to the proliferation of injected but untransformed lines (injection controls). Young macronuclear donor material significantly prolonged the proliferation of the hybrid cell lines over that of the injection controls, but old donor material caused only a slight increase in post-injection proliferation of the hybrids. Total cell proliferation was also considered. Comparison of the total life spans of injected, non-transformed lines with uninjected host controls demonstrates that the injection itself has no significant effect upon proliferation potential. The mean life spans of uninjected donor controls and the mean total life spans of the transplanted macronuclear material (donor age at injection plus subsequent hybrid proliferation) are similar, regardless of the age of the donor at the time of transplantation. These results suggest that there is an age-related decrease in the ability of a macronucleus to support subsequent cell growth and division. The results also show that the proliferation potential of the donor macronucleus does not appear to be changed to any great degree by transplantation into a host cell of different clonal age. The macronucleus thus "remembers its age" after transplantation. Coupled with an absence of any detectable cytoplasmic effects upon aging during vegetative growth, these results argue in favor of a macronuclear determination of the proliferation potential of a cell line. The identification of a macronuclear basis for clonal aging in P. tetraurelia should permit a better directed approach for further research in this area.
Cell Division↗
Directed positioning of micronuclei in Paramecium tetraurelia with laser tweezers: absence of detectible damage after manipulation.
Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of "slow growth mutations" after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.
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