Yeast cell biology: the wave of the present. Yeast Cell Biology sponsored by the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, August 15-20, 1989.
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Cell Cycle↗
Biomedical subjects
J R Pringle
Publications and source records attributed to J R Pringle.
Fluorescence microscopy methods for yeast.
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Fluorescent Antibody Technique↗
Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1.
Genes CDC24, CDC42, and CDC43 are required for the establishment of cell polarity and the localization of secretion in Saccharomyces cerevisiae; mutants defective in these genes fail to form buds and display isotropic expansion of the cell surface. To identify other genes that may be involved in these processes, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress a temperature-sensitive cdc24 mutation. We identified four such genes. One of these proved to be CDC42, which has previously been shown to be a member of the rho (ras-homologous) family of genes, and a second is a newly identified ras-related gene that we named RSR1. RSR1 maps between CDC62 and ADE3 on the right arm of chromosome VII; its predicted product is approximately 50% identical to other proteins in the ras family. Deletion of RSR1 is nonlethal but disrupts the normal pattern of bud site selection. Although both CDC42 and RSR1 can suppress cdc24 and both appear to encode GTP-binding proteins, these genes do not themselves appear to be functionally interchangeable. However, one of the other genes that was isolated by virtue of its ability to suppress cdc24 can also suppress cdc42. This gene, named MSB1, maps between ADE9 and HIS3 on the right arm of chromosome XV.
Amino Acid Sequence↗
Functions of microtubules in the Saccharomyces cerevisiae cell cycle.
We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.
Actins↗
Mapping of the Saccharomyces cerevisiae CDC3, CDC25, and CDC42 genes to chromosome XII by chromosome blotting and tetrad analysis.
CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome. The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5. The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.
Blotting, Southern↗
Concurrent radiation and chemotherapy for carcinoma of the cervix recurrent after radical surgery.
Results of salvage therapy in patients with carcinoma of the cervix, recurrent after primary surgery, have been dismal even when disease was apparently confined to the pelvis. Further surgery or radiation therapy cured only some with central pelvic disease alone who had recurred at intervals longer than 6 months after primary therapy. To try to improve the results of salvage therapy, we used a combination of concurrent chemotherapy, 5-Fluorouracil with or without Mitomycin-C, and radiation therapy. Seventeen patients were treated. Recurrent disease was present in the pelvis or pelvis and paraaortic nodes after radical surgery for Stage IB carcinoma of the cervix. Eight of seventeen (47%) are alive, disease-free, 21 to 58 months after therapy. Seven of the eight had biopsy proven recurrence. Five of eight had recurred within 9 months of primary surgery and 7/8 had a component of pelvic side wall disease. Thus the survivors had unfavorable prognostic features. Nevertheless, the use of concurrent radiation and chemotherapy produced an exceptionally high proportion of sustained complete remissions and possible cures.
Combined Modality Therapy↗
Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.
Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.
Cell Compartmentation↗
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.
Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.
Chromosome Mapping↗
Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae.
The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochrome-labeled phallotoxins after fixing and permeabilizing the cells by several methods. The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers. Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope. The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud. Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds. These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle.
Actins↗
Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes.
A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine and screened for temperature-sensitive (Ts-) mutants capable of growth on rich, glucose-containing medium at 25 degrees but not at 37 degrees. Recessive mutations induced on chromosome I are expressed whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely.--Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss.--The available information about the size of chromosome I suggests that it should contain approximately 60-100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts--lethal phenotype.--During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.
Chromosome Mapping↗
Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle.
Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.
Cell Wall↗
Effect of growth temperature upon heat sensitivity in Saccharomyces cerevisiae.
The resistance of exponentially growing yeast cells to killing by exposure to 52 degrees C increase markedly as the growth temperature was increased. Identical killing curves were obtained for cells suspended in growth medium or in 0.9% saline. Cells resistant to killing at 52 degrees C were quite sensitive to killing at slightly higher temperatures. These results suggest a primary role for membrane damage in the mechanism of heat killing.
Hot Temperature↗
Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.
The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.
Culture Media↗
Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae.
We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.
Actins↗
A mutant of yeast defective in cellular morphogenesis.
In the budding yeast Saccharomyces cerevisiae, each bud appears within a ring of chitin formed in the cell wall of the mother cell. Temperature-sensitive mutants defective in gene cdc24 synthesize chitin at restrictive temperatures, but do not organize it into the discrete rings found in normal cells, nor do they form buds. The chitin ring or an annular precursor structure may play an essential role in reinforcing the region of the cell wall involved in budding.
Cell Wall↗
The use of conditional lethal cell cycle mutants for temporal and functional sequence mapping of cell cycle events.
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Cell Cycle↗
Coordination of growth with cell division in the yeast Saccharomyces cerevisiae.
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Cell Division↗
Methods for monitoring the growth of yeast cultures and for dealing with the clumping problem.
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Animals↗