Ira Herskowitz

Structure of a yeast pheromone gene (MFα): A putative α-factor precursor contains four tandem copies of mature α-factor

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement (cassette insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as rules of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided (experienced cells). Spores and buds (inexperienced cells) rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis☆

Abstract We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and a α . First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of a α cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1− matα2− mutants should have the mating ability of an a cell and that matα1− matα2−/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MAT a cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

Abstract The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1−2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.

Isolation of a circular derivative of yeast chromosome III: implications for the mechanism of mating type interconversion

We describe genetic and physical characterization of rearrangements of chromosome III which result in changes of cell type in S. cerevisiae. Two types of rearrangements were obtained as rare events which caused a change at the locus controlling cell type, MAT, associated with a recessive lethal mutation, in one case from MATalpha to MATa-lethal, and in the other case from MATa to MATalpha-lethal. The MATa-lethal mutation is a deletion on the right arm of chromosome III, which we demonstrate extends to (or near) HMalpha. We suggest this deletion removes MATalpha and activates cryptic MATa information stored in HMalpha as proposed in the cassette model of mating type interconversion. The MATalpha-lethal mutation is the result of the formation of a circular chromosome III, which we interpret to remove MATa and activate the cryptic MATalpha information stored at HMa. Strains carrying the MATalpha-lethal chromosome contain a circular chromosome of length 62.6 plus or minus 5.7 mum, which is absent in related strains. This chromosome was confirmed to be chromosome III by hybridization of specific yeast DNA fragments to supercoiled DNA obtained from MATalpha-lethal strains. The isolation of a large circular derivative of chromosome III allows correlation of genetic and physical distance based on large distances-1 centimorgan corresponds to approximately 2700 base pairs.

A family of Saccharomyces cerevisiae repetitive autonomously replicating sequences that have very similar genomic environments

We have characterized a family of moderately repetitive autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Restriction mapping, deletion studies and hybridization studies suggest that these ARSs, which are probably less than 350 base-pairs in size, share one common feature: each is located close to, but not within, a repetitive sequence (131) of approximately 10(3) to approximately 1.5 X 10(3) base-pairs in length. These ARSs can be divided into two classes (X and Y) by their sequence homology and genomic environments. Each of the class X ARSs is embedded within a repetitive sequence (X) of variable length (approximately 0.3 X 10(3) to approximately 3.75 X 10(3) base-pairs); each of the class Y ARSs is embedded within a highly conserved repetitive sequence (Y) of approximately 5.2 X 10(3) base-pairs in length. Both of these sequences are located directly adjacent to the 131 sequence.

Conservation of a receptor/signal transduction system

The budding yeast Saccharomyces cerevisiae is a singlecell organism whose process of mating provides an excellent opportunity to study cell-cell interactions and response to diffusible factors. In this process, two partners of opposite type (a and a) fuse to produce a diploid cell. Each partner produces a peptide mating factor (a-factor or a-factor) that acts on the opposite cell type to prepare cells for mating by inducing expression of various genes whose products are necessary for cell and nuclear fusion (Tiueheart et al., MCB 7, 2316-2328, 1987; Rose et al., MCB 6, 3490~3497,1986) and by causing arrest in the Gl phase of the cell cycle. How do these mating factors trigger the multitude of cellular responses? The surprising answer, as the outlines of a molecular understanding become visible, seems to be that the machinery that yeast uses to respond to mating factors has similarity to the apparatus used in many other cells, including cells of the nervous system, for various signal transductions. The Receptors The receptor for yeast a-factor is coded by the STE2 gene, and the receptor for a-factor by the STE3 gene (Burkholder and Hartwell, NAR 73,8463-84751985; Nakayama et al., EMBO J. 4, 2643-2648, 1985; Hagen et al., PNAS 83, 1418-1422, 1986). The deduced amino acid sequence of the STf2 and STE3 products revealed that they are members of the rhodopsinlp-adrenergic receptor/muscarinic acetylcholine receptor family of integral membrane proteins. All of these proteins contain seven hydrophobic segments, each long enough to span a lipid bilayer membrane (see figure). These receptors are hypothesized to exhibit a transmembrane arrangement similar to that of bacteriorhodopsin, for which a partial three-dimensional structure is available. Amino acid sequence similarity among these receptors is modest; similarity between STf2 and STf3 polypeptides is virtually absent. The yeast receptors thus provide a striking example of gene products of similar function in which the topology of the protein (its arrangement with respect to the membrane), not primary sequence, has been strongly conserved (figure). The ligands for this receptor family are diverse: a skinny lipid chromophore (retinal) and small organic molecules (epinephrine and acetylcholine). The ligands for STE2 and STE3 proteins, a-factor and a-factor, respectively, are peptides of 13 and 12 amino acids (a-factor is probably a modified peptide). Intracellular Machinery Receptor switching experiments suggest that the response pathway in a and a cells may differ only in the type of receptor and that the intracellular machinery involved in subsequent transmission of the signal evoked by binding of the mating factors is the same in both cell types. It Minireview

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

Abstract The mating type locus ( MAT ) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MAT a and MAT α, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MAT α is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MAT α. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, mat α 1 mutants do not produce STE3 RNA, whereas mat α 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MAT α. Thus α1 positively regulates at least one α-specific gene by increasing the level of that genes RNA product.

Mutants of bacteriophage λ which do not require the cIII gene for efficient lysogenization

Abstract The λ c III gene is ordinarily required for the high rate, establishment mode of repressor synthesis and efficient lysogenization by phage λ We have isolated phage mutants (called can ) which no longer require c III for efficient lysogenization by selecting turbid plaque-forming pseudorevertants of phages carrying a deletion of c III. Three different kinds of can mutations have been characterized: can 10, which is located in the cro gene; can 15, which appears to be identical to cin 1 and which is located in the left part of the y region; and can 1, which is located in the right part of they region, among the clear cy mutations. Can 10 and the previously identified cro − mutation, cro 27, both improve lysogenization frequency in the absence of c III function, possibly because of overproduction of c II protein. In addition, lysogenization by λ c III + phages carrying these mutations is more efficient than by λ + at low multiplicity of infection. The cro 27 mutation also improves lysogenization by c II − phages, suggesting that Cro may prevent premature synthesis of repressor in the maintenance mode. Can 1 is dominant to can + and partially relieves the lysogenization defect of λ c III − in a cap − cya − host. The ability of λ c III − c I − can 1 to stimulate lysogenization by λ c III − c I + indicates that can 1 does not require an adjacent functional c I gene for its action and that can 1 affects the activity of a diffusible product, presumably c II protein. This proposal is supported by a cis-trans test showing that can 1 requires an adjacent functional cII gene. Although can 1 does not increase the frequency of lysogenization in the absence of cII function, it does increase lysogenization frequency when c II activity is present but limiting. These observations suggest that can 1 increases the level or activity of c II protein. Based on the ability of can 1 and can 10, to bypass the need for c III, we propose that the role of cIII protein in stimulating repressor synthesis is indirect; it acts by increasing activity of the cII protein.