John R. Pringle

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one‐step PCR‐mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C‐ or N‐terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N‐ or C‐terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR‐based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418‐resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C‐ or N‐terminal protein tagging (with HA, Myc, GST, or GFP), and partial C‐ or N‐terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

Immunofluorescence methods for yeast

Publisher Summary This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.

Coordination of growth with cell division in the yeast Saccharomyces cerevisiae

Abstract Yeast cell growth and cell division are normally coordinated. The mechanism of this coordination can be examined by attempting to dissociate the two processes. We have arrested division (with the use of temperature-sensitive cell division cycle mutants) and observed the effect of this arrest on growth, and we have limited growth (by nutritional deprivation) and observed the effect of this limitation on division. Mutants blocked at various stages of the cell cycle were able to continue growth, as evidenced by increases in cell volume, mass, and protein content. Cells that had initiated cell cycles were able to complete their cycles and arrest in G1 even when growth was very severely restricted. Under these conditions the daughter cells produced were abnormally small. Such abnormally small cells did not initiate new cell cycles (i.e., did not bud or complete any of the three known gene-controlled steps ( cdc 28, cdc 4, cdc 7) in G1) after nutrients were restored until growth to a critical size had occurred. We have also prepared abnormally large cells (by arresting division temporarily with the appropriate mating pheromone); when such cells were allowed to bud, the buds produced were much smaller than the mother cells when cytokinesis occurred. We propose that the normal coordination of cell growth with cell division is a consequence of the following two relationships. (1) Growth, rather than progress through the DNA-division cycle, is normally rate-limiting for cell proliferation. (2) A specific early event in G1, at or before the event controlled by the cdc 28 gene product, cannot be completed until a critical size is attained.

The septins: roles in cytokinesis and other processes

The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.

The Saccharomyces cerevisiae Cell Cycle

INTRODUCTION The cell cycle is the process of vegetative (asexual) cellular reproduction; in a normal cell cycle, one cell gives rise to two cells that are genetically identical to the original cell. Questions about the cell cycle can be conveniently divided into two categories. First, one can ask how a cell carries out a cell cycle, once it has undertaken to do so. Into this category fall questions about the morphological and biochemical aspects of cell-cycle events and about the mechanisms that ensure their temporal and functional coordination. Second, one can ask what determines when a cell will undertake a cell cycle, or how the overall control of cell proliferation is achieved. Into this category fall questions about the coordination of successive cell cycles, the coordination of growth with division, the coordination of cell proliferation with the availability of essential nutrients, and the selection of developmental alternatives. In the text that follows, we consider these two categories of questions in turn. Our bibliography is intended more as a guide to the literature than as a historically accurate record of the development of the field; we apologize to the earlier workers whose contributions thus get less explicit credit than they deserve. HOW DOES A CELL CARRY OUT A CELL CYCLE? As has often been noted, successful completion of a cell cycle requires a cell to integrate the processes that duplicate the cellular material with the processes that partition the duplicated material into two viable daughter cells. Another useful formulation of the...

Fluorescence microscopy methods for yeast.

Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae.

Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations. We have now sequenced MSB1 and constructed a deletion of this gene. The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype. However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence. Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability. The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1. In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C. In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C. In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence. This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J. Chant, K. Corrado, J. Pringle, and I. Herskowitz, submitted for publication). BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V.

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

The septin cortex at the yeast mother-bud neck.

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.