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G F Sprague

Publications and source records attributed to G F Sprague.

At least 55 records · Page 3Linked to original sources
PubMed1989

Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.

Cell Cycle↗
PubMed1989

The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene.

Two proteins, alpha 1 and pheromone/receptor transcription factor (PRTF), bind cooperatively to the upstream activation sequences (UAS) of yeast alpha-specific genes and thereby activate their transcription. In these protein-DNA complexes, the PRTF moiety interacts with a degenerate dyad symmetric sequence, the P box. PRTF contributes also to the regulation of a second set of cell-type-specific genes, the a-specific genes. We used two in vitro assays to show that PRTF is encoded, at least in part, by the MCM1 gene. In one assay, truncated MCM1 proteins encoded by deletion derivatives of the MCM1 gene formed protein-DNA complexes of novel mobility, demonstrated that MCM1 can bind to the P-box-containing DNA. Second, antibodies raised to a synthetic MCM1 polypeptide retard the migration of PRTF-DNA complexes in gel mobility shift assays. This result indicates that PRTF, defined as an activity that binds cooperatively with alpha 1 to alpha-specific UAS elements, shares an epitope with MCM1. In addition, we show that MCM1 deletions that remove the carboxy-terminal 129 codons of 286 total codons encode truncated MCM1 molecules that are competent to activate transcription in vivo, indicating that the carboxy-terminal residues are not required for this process.

Antibodies, Fungal↗
PubMed1989

Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants.

Saccharomyces cerevisiae haploid cells, alpha and a, mate after being appropriately stimulated by the pheromone secreted by the opposite cell type (a-factor and alpha-factor, respectively). The binding of a pheromone to its receptor is a signal that initiates a series of intracellular changes that lead to the specific physiological alterations required for mating. To identify components of the signal transduction pathway, we sought pseudorevertants that restored mating competence to receptor mutants (MAT alpha ste3::LEU2). The suppressor srm1-1 was isolated as a recessive mutation that conferred temperature-sensitive growth to all strains and mating ability to MAT alpha ste3::LEU2 strains at the nonpermissive temperature. In addition, when srm1-1 mutants were shifted to the nonpermissive temperature, they exhibited two phenotypes characteristic of pheromone response, induction of FUS1 transcription and accumulation of cells in the G1 phase of the cell cycle. The srm1-1 mutation also suppressed a deletion of the alpha-factor-receptor gene in a cells. Together, these phenotypes suggest that the wild-type SRM1 product is a component of the pheromone response pathway. Deletion of STE4 or STE5, which are required in both haploid cell types for mating and response to pheromone, was not suppressed by srm1-1, suggesting that the SRM1 product may function before the STE4 and STE5 products. SRM1 is an essential gene and is expressed in both haploid cell types as well as in the product of their mating, a/alpha diploids. Homozygous srm1-1 a/alpha diploids were temperature sensitive although they did not arrest in G1. Thus, the SRM1 product may also have a role in the vegetative life cycle of cells.

Amino Acid Sequence↗
PubMed1988

Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes.

STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone. Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS. UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes. A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression. The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2. We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types. We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes. Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha. Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box.

Base Sequence↗
PubMed1988

Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes.

Cell type specialization in yeast haploids involves the mutually exclusive expression of one of two sets of genes, the a-specific and alpha-specific genes. We demonstrated that the products of the STE7, STE11, and STE12 genes were required for the expression of both gene sets. RNA levels transcribed from these gene sets were significantly decreased but not abolished in haploids containing a null mutation in the STE7, STE11, or STE12 gene. Transcript levels from the a- and alpha-specific gene sets were not further reduced in strains harboring mutations in all three STE genes, suggesting that STE7, STE11, and STE12 are required for the same aspect of transcription. We further showed that the requirement for these products was not the same for each member of a particular gene set. However, for any given a- or alpha-specific gene, the effect on RNA levels of any of the three ste mutations was similar.

Genes↗
PubMed1987

MAT alpha 1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes.

We show by electrophoresis mobility shift and by DNAase I footprinting assays that the alpha 1 product of the yeast alpha mating-type locus binds to homologous sequences within the control regions of the three known alpha-specific genes. Binding requires both alpha 1 and a second yeast protein(s) (called PRTF) that is present in all three cell types (a, alpha, and a/alpha); neither protein binds alone. Binding and competition experiments using synthetic oligonucleotides indicate that PRTF binds to only part of the homology found at alpha-specific genes and imply that alpha 1 binds to the remainder. Our results suggest that alpha 1 renders gene expression alpha-specific by creating a binding site for PRTF. Similar experiments lead to the idea that PRTF also plays a role in transcription of a-specific genes. Perhaps a-specificity is achieved through the occlusion of the PRTF binding site by alpha 2, the negative regulator encoded by the alpha mating-type locus.

Base Sequence↗
PubMed1987

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

Amino Acid Sequence↗
PubMed1986

Yeast peptide pheromones, a-factor and alpha-factor, activate a common response mechanism in their target cells.

We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter.

Genes, Fungal↗
PubMed1986

Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor.

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer.

Base Sequence↗
PubMed1985

Control of yeast alpha-specific genes: evidence for two blocks to expression in MATa/MAT alpha diploids.

In yeast alpha cells, the product encoded by the MAT alpha 1 gene of the mating-type locus is required for transcription of at least two genes, STE3 and MF alpha 1. To learn whether the lack of the MAT alpha 1 product in a and a/alpha cells is sufficient to explain the failure to express STE3 and MF alpha 1 in these cells, we have provided MAT alpha 1 product via a hybrid gene that circumvents the normal regulation of the MAT alpha 1 gene. We find by RNA blot analysis that provision of MAT alpha 1 protein permits production of STE3 and MF alpha 1 mRNA in a cells but not in a/alpha cells. These data suggest the existence of an additional regulatory mechanism that prevents expression of alpha-specific genes in a/alpha cells, even when MAT alpha 1 product is present. This regulatory mechanism appears to control expression of STE3 and MF alpha 1 at the transcriptional level rather than at the posttranscriptional level, because we show that MF alpha 1 mRNA supplied from a constitutive promoter is translated and processed in a/alpha cells to yield functional alpha-factor pheromone. This result shows further that a/alpha cells possess all the machinery necessary for pheromone maturation, even though these cells do not normally carry out these reactions.

Animals↗
PubMed1984

Induction of the yeast alpha-specific STE3 gene by the peptide pheromone a-factor.

The yeast Saccharomyces cerevisiae exhibits two mating types, a and alpha. Efficient mating of a and alpha cells requires the action of peptide pheromones secreted by each cell type. For example, a cells secrete a-factor, which alters the physiology of alpha cells, thereby preparing those cells for mating. To investigate the mechanism by which the pheromones act on the target cells, we have examined the effect of a-factor on expression of the STE3 gene, a gene which is required for mating by alpha cells and which is expressed only in alpha cells. We have monitored STE3 expression by two assays: RNA production from the chromosomal STE3 locus and beta-galactosidase activity produced from a plasmid-borne STE3-lacZ gene fusion. By both assays we show that a-factor induces a rapid increase in STE3 expression. Induction of STE3 RNA occurs even if protein synthesis is blocked by cycloheximide. Using temperature-sensitive cell division cycle mutants, we have also shown that induction occurs in cells arrested at several discrete positions in the cell cycle. These results demonstrate (1) that induction of STE3 expression by a-factor is a primary response to the pheromone, and (2) that alpha cells are capable of responding to a-factor regardless of their position in the cell cycle.

Animals↗
PubMed1983

Control of yeast cell type by the mating type locus: positive regulation of the alpha-specific STE3 gene by the MAT alpha 1 product.

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

Cloning, Molecular↗
PubMed1983

Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus.

The ultimate product of yeast mating-type interconversion is a stable a/alpha diploid cell. A haploid cell carrying the HO gene gives rise to a diploid cell in a two-step process: first, the cell switches mating type as a result of genetic rearrangement (cassette substitution) catalyzed by HO; then, cells of opposite type mate to form a/alpha diploids. Mating-type interconversion does not occur in a/alpha diploids despite the presence of the HO gene. We have identified a plasmid carrying the HO gene by screening a yeast clone bank (constructed in vector YEp13) for plasmids that allow mating-type switching by ho cells. The yeast segment responsible for mating-type interconversion integrates by homology at the ho locus, thus confirming that it carries HO. Using the HO gene as a probe, we find that strains with an active mating-type interconversion system produce HO RNA, whereas a/alpha HO/HO cells do not and that this inhibition requires products of both the MATa1 and MATa2 genes. Thus, mating-type interconversion does not occur in a/alpha HO/HO cells because the HO gene product is not synthesized. These results demonstrate the following: (i) The mating-type locus, proposed on genetic grounds to be a regulatory locus, controls expression of an unlinked gene (HO) at the level of RNA production. (ii) The HO gene is under negative feedback control: its expression is inhibited after successful completion of diploidization (formation of a/alpha diploids).

DNA, Fungal↗
PubMed1983

Cell interactions and regulation of cell type in the yeast Saccharomyces cerevisiae.

Examination of the control of cell type in yeast at the molecular level and understanding of the biochemical basis of the cell-cell interactions involved in the mating process are clearly entering an extremely productive and exciting period. The tools and opportunities are now available to answer fundamental questions with regard to the mechanism of differential gene expression in eukaryotic cells by using cloned a-specific, alpha-specific, and haploid-specific genes as the probes. Basic questions concerning eukaryotic chromosome structure and organization can be addressed by elucidating the properties of the SIR/MAR regulators and their mode of action. Furthermore, the availability both of cloned MAT, HML, and HMR regions and of the HO gene will provide the material for unravelling the enzymology of the DNA transposition that occurs during mating type interconversion. The isolation of the structural genes for the pheromones and mutations that block pheromone production will provide useful information on how extracellular hormones are synthesized, processed, and secreted by eukaryotic cells. Moreover, the apparent mode of action of the phermonones through cyclic AMP as an intracellular "second messenger," and the genetic and biochemical tractability of yeast cells, may allow tracing of the entire pathway of hormonal regulation of a eukaryotic cell division cycle. These and other studies of the developmental biology of yeast cells will provide more important insights into fundamental aspects of the genetic control of developmental processes in eukaryotic cells.

Animals↗
PubMed1981

Homology and non-homology at the yeast mating type locus.

Four mutations of the alpha mating type locus of Saccharomyces cerevisiae have been analysed to determine their relationship to the a mating type locus. Mat alpha+ recombinations are produced by mat alpha 2-/MAT but not by mat alpha 1-/MATa diploids. MAT alpha and MATa thus contain regions of homology (coding for at least part of MAT alpha 2) and regions of non-homology (coding for at least part of MAT alpha 1)-the genetic determinant for cell type is larger than the non-homologous sequence seen by DNA-DNA heteroduplexes and genetic analysis. The segment transposed in mating type interconversion includes both types of sequence.

Base Sequence↗