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ABSTRACTThe origin of replication complex subunit ORC1is important for DNA replication. The gene is known to encode a meiotic transcript isoform (mORC1) with an extended 5′-untranslated region (5′-UTR), which was predicted to inhibit protein translation. However, the regulatory mechanism that controls the mORC1transcript isoform is unknown and no molecular biological evidence for a role of mORC1in negatively regulating Orc1 protein during gametogenesis is available. By interpreting RNA profiling data obtained with growing and sporulating diploid cells, mitotic haploid cells, and a starving diploid control strain, we determined that mORC1is a middle meiotic transcript isoform. Regulatory motif predictions and genetic experiments reveal that the activator Ndt80 and its middle sporulation element (MSE) target motif are required for the full induction of mORC1and the divergently transcribed meiotic SMA2locus. Furthermore, we find that the MSE-binding negative regulator Sum1 represses both mORC1and SMA2during mitotic growth. Finally, we demonstrate that an MSE deletion strain, which cannot induce mORC1, contains abnormally high Orc1 levels during post-meiotic stages of gametogenesis. Our results reveal the regulatory mechanism that controls mORC1, highlighting a novel developmental stage-specific role for the MSE element in bi-directional mORC1/SMA2gene activation, and correlating mORC1induction with declining Orc1 protein levels. Because eukaryotic genes frequently encode multiple transcripts possessing 5′-UTRs of variable length, our results are likely relevant for gene expression during development and disease in higher eukaryotes.

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p–Cbk1p, Cbk1p–Kic1p, Kic1p–Tao3p, and Kic1p–Hym1p interactions, in addition to the previously documented Cbk1p–Mob2p and Cbk1p–Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p–Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p–Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiaemitotic exit network and Schizosaccharomyces pombeseptation initiation network and is likely conserved among eukaryotes.

The yeast “two-component” osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with acis-acting element identified upstream ofOCH1that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.

The Saccharomyces cerevisiae OCH1 gene encodes an α-1,6-mannosyltransferase that initiates the polymannose outer chain elongation of N-linked glycans. Transcription of OCH1 is regulated by two transcription factors, Swi4 and Skn7. To learn more about the signals that feed into the Swi4 regulation, we isolated a mutant, bon1-1 (bypass of Skn7), that activates OCH1–HIS3 and OCH1–lacZ reporters in a strain deleted for SKN7. bon1-1 is an allele of CDC4 (cdc4^bon) and leads to the additional phenotypes of temperature sensitivity and abnormal cell morphology. The cdc4^bon mutant is partially suppressed by CLB5 overexpression and accumulates Sic1 protein, indicating that OCH1 transcription is controlled by the ubiquitin-dependent degradation pathway through the Skp1–Cdc53–F box^Cdc4 protein complex. We suggest that transcriptional control of OCH1 by cdc4^bon is through Swi4, because cdc4^bon cannot activate the OCH1–lacZ reporter in a strain deleted for SWI4. Interestingly, cdc4^bon and Δswi4 show a synthetic growth defect when combined. Copyright © 2002 John Wiley & Sons, Ltd.

The Saccharomyces cerevisiae OCH1gene encodes an α‐1,6‐mannosyltransferase that initiates the polymannose outer chain elongation of N‐linked glycans. Transcription of OCH1is regulated by two transcription factors, Swi4 and Skn7. To learn more about the signals that feed into the Swi4 regulation, we isolated a mutant, bon1‐1(bypass of Skn7), that activates OCH1–HIS3and OCH1–lacZreporters in a strain deleted for SKN7. bon1‐1is an allele of CDC4 (cdc4bon)and leads to the additional phenotypes of temperature sensitivity and abnormal cell morphology. The cdc4bonmutant is partially suppressed by CLB5overexpression and accumulates Sic1 protein, indicating that OCH1transcription is controlled by the ubiquitin‐dependent degradation pathway through the Skp1–Cdc53–F boxCdc4protein complex. We suggest that transcriptional control of OCH1by cdc4bonis through Swi4, because cdc4boncannot activate the OCH1–lacZreporter in a strain deleted for SWI4. Interestingly, cdc4bonand Δswi4show a synthetic growth defect when combined. Copyright © 2002 John Wiley & Sons, Ltd.

Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects. Copyright © 2000 John Wiley & Sons, Ltd.

Tagged transposons are powerful tools for large‐scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time‐consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon‐specific primer, BigDye‐labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon‐based projects. Copyright © 2000 John Wiley & Sons, Ltd.

PCR‐based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae. The techniques work best when the host strain lacks sequence homology to the PCR‐amplified selectable markers. One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6and HIS3MX6) and homologous (TRP1) markers. Here we describe the trp1‐ΔFAdesigner deletion allele that removes precisely from the genome TRP1sequences carried in the pFA vectors. The trp1‐ΔFAallele can be introduced easily into TRP1and most trp1starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1‐based constructs. Unlike trp1‐Δ1, trp1‐ΔFAdoes not remove neighbouring GAL3upstream activating sequences and therefore does not interfere with GALgene induction. Copyright © 1999 John Wiley & Sons, Ltd.

PCR-based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae. The techniques work best when the host strain lacks sequence homology to the PCR-amplified selectable markers. One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6 and HIS3MX6) and homologous (TRP1) markers. Here we describe the trp1-ΔFA designer deletion allele that removes precisely from the genome TRP1 sequences carried in the pFA vectors. The trp1-ΔFA allele can be introduced easily into TRP1 and most trp1 starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1-based constructs. Unlike trp1-Δ1, trp1-ΔFA does not remove neighbouring GAL3 upstream activating sequences and therefore does not interfere with GAL gene induction. Copyright © 1999 John Wiley & Sons, Ltd.