Two regions in the HO promoter are responsible for restricting HO is the URS1 and URS2 (upstream regulatory sequence). URS1 is necessary for mother cell specificity and URS2 confines HO expression to late G1. It was analyzed using two assays, the Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion. It was found that mutants defective of any SWI gene fail to express the HO gene. SWI genes function as positive regulators of HO expression. Ho expression requires at least 10 trans-acting genes called SWI1- SWI10 out of which the regulatory factors of only SW15 is correctly expressed the HO gene. The Ho endonuclease initiates this mating-type interconversion which is expressed only in mother cells. Restriction of Ho activity to mother cells is largely determined by the pattern of HO transcription. The gene is transiently transcribed in the late G1 phase of the cell cycle and is confined to mother cells because they switch mating type. The SW15 gene is required for the diploidization of homothallic yeast. During this process, individual haploid a or alpha cells switch their mating types to produce pairs of cells of opposite mating type which conjugate to produce an a or alpha diploid. Constitutive expression of SWI5 or the stabilization of Swi5p by mutation causes a certain fraction of daughter cells to switch mating type. Daughter cells switch mating type almost as efficiently as mother cells when HO transcription is driven by YCp GAL-SW16. Altering the regulation of SW15 transcription is sufficient to cause HO to be transcribed in daughter cells, without altering its cell-cycle or cell-type control. Therefore, HO is not transcribed in daughter cell because SW15 is absent when they undergo START YCp GAL-SW15 causes daughters to transcribe HO at this stage by supplying SW15 constitutively. In one study, overproduction of SW15 titrates some other factor involved in mother/ daughter control. GAL-SW15 fusion was expressed at lower levels by introducing a Gal80 mutation into the strain. The cells were grown on glucose. The induction of the GAL7 promoter by GAL4 was constitutive but was prevented from causing extensive transcription by the independent glucose-mediated catabolite repression. In glucose medium, the disruption causes the level of SW15 RNA made from YCpGAL-SW/S to rise to that the wild-type locus. This in turn causes the activity of an HO-gal promoter to rise of wild-type. Modest constitutive expression of SW15 is therefore sufficient to cause significant daughter cell switching. SW15 is segregated exclusively to mother cells at division. Although mother cells may inherit SW15, they do not have the ability to resynthesize it when blocked in Gl. Thus, if the SW15 inherited by mother cells were unstable during an a factor induced Gl arrest, then even mother cells would be unable to transcribe HO as they undergo START following release. The destruction of SW15 during a-factor-induced Gl arrest effectively turns mothers into daughters. SW15 RNA is absent in the Gl cells capable of HO transcription and is only made later in the cell cycle after cells enter S phase. Constitutive transcription of SW15 causes HO to be transcribed in daughter cells. SW15 is the only transcription factor missing in daughter cells. To test whether SW15 RNA is similarly cell cycle regulated in mother cells. a cells were arrested in G1 by growing a log phase culture in the presence of a factor for slightly over a generation time. These cells were then released from the Gl a factor block by harvesting the arrested cells by rapid filtration and suspension in fresh medium. The level of histone H2B, Ho, MATal, and SW15 RNAs at the time of the G1 block and during the three cell divisions. Three waves of H2B RNA levels detected corresponded to first, second, and third cell cycle STARTS following the release. SW15 CD RNAs are barely detectable in the G1 arrested cells and they showed three waves following release which has the same period but different phase as those of H2B. The peaks of SW15 RNA levels followed 20 min after the peaks in H2B levels in all three cycles. Therefore, that SW15 RNAs are similarly cell-cycle regulated in both mother and daughter cells. The analysis of Ho levels allows comparison of the timing of Ho and SW15 RNA fluctuations. The unbudded single cells containing nuclear SWI5 was rarely observed suggesting that the bulk of SWI5 protein inherited by both mother and daughter cells is rapidly destroyed as cells progress through G1. Tebb et al. measured level of SWI5 protein by Western blotting in a synchronous culture as cells enter GI. The SWI5 gene continued to be transcribed in cells arrested in late anaphase owing to the temperature-sensitive cdc15-2 mutation, but the SWI5 protein synthesized was mainly excluded from the nucleus. Upon return to the permissive temperature, SWI5 transcription is rapidly repressed, SWI5 protein is translocated into the nucleus and cells enter G1 with a high degree of synchrony as measured by the disappearance of anaphase spindles. Ho transcription, which is not detectable at the cdc15 block, peaks at 35 min. Most wild-type SWI5 protein is degraded soon after its entry into the nucleus. Thus, most SWI5 is unstable during the G1 phase of both mother and daughter cells.In situ immunofluorescence of SWI5 protein in cycling cells displayed showed the symmetric distribution of SWI5 following mitosis and its apparent destruction as cells enter G1. SWI5 mRNA is very unstable and is only made during the S, G2, and M phases of the cell cycle. If SWI5 must be present at the time of HO expression, the lack of SWI5 transcription during G1 implies that SWI5 protein used to activate HO must be made in the previous cell cycle. Ectopic expression of SWI5 during G1 causes HO to be expressed in daughter cells. Therefore, the SWI5 protein inherited by mother and daughter cells is degraded soon after entry into the nucleus. Since Swi5p is found in both mother and daughter cells and must activate transcription of genes other than HO equally well in both mother and daughter cells. Thus, some other factor, either a mother cell-specific activator or a daughter cell-specific repressor, must exist to generate the asymmetric expression of HO. From prior work, we know that SHE1–SHE5 were 5 new genes essential for HO expression in mother cells and is localized. SHE1 encodes an unconventional myosin which accumulates in the future daughter cell. she1 mutants are defective in restricting a negative regulator of HO expression to daughter cells. Therefore the inactivation of such a negative regulator should allow she1 mutants to express HO, specific known as Ash1p accumulates in the daughter cell nucleus. A myosin is required for the accumulation of Ash1p in daughter cells, which suggests that actin-based transport may play a role in the generation of HO asymmetry. Three mutant hunts led to the identification of ASH1. A daughter cell-specific repressor might be responsible for asymmetric HO expression, so that inactivation of such a repressor would lead to the expression of HO in daughter cells, allowing them to switch mating type. The cells exhibit a visible morphological change when exposed to mating pheromone secreted by cells of opposite mating type. In response to alpha factor pheromone, a cells stop dividing and differentiate into gametes that form a mating projection known as a shmoo. alpha Cells respond to a factor pheromone in the same way. A direct screen for daughter cells that can switch mating type. Switching of an alpha cell to a is scored as a change in the ability of a cell to respond to ? factor. After two cell divisions, a wild-type alpha daughter cell forms a microcolony containing two a and two alpha cells. In the presence of alpha factor, the a cells arrest and shmoo, whereas the alpha cells continue to divide. Under conditions in which wild-type cells form microcolonies containing both shmoos and budded cells, these mutants were identified because they form distinctive microcolonies containing four shmoos. A Four-Shmoo Microcolony Indicates That Both Mother and Daughter Cells Have Switched Mating Type. An HO ste3? strain (YAS131) was used to screen for mutants whose daughters switch mating type.ASH1, that is necessary to repress HO in daughters. ASH1 encodes a zinc finger protein whose preferential accumulation in daughter cell nuclei at the end of anaphase depends on She1p/Myo4p. Accumulation is higher levels in daughter nuclei. The abundance of Ash1p in daughter cells is responsible for restricting HO expression to mother cells. This asymmetry is dependent on the activity of SHE genes. Ash1p accumulates to equally high levels in mother and daughter nuclei in she mutants after She3p has become symmetrical, and this is responsible for their failure to transcribe HO. To identify the means by which Ash1p negatively regulates HO expression, in vivo repression assays were performed. The ASH1 coding sequence was fused to the LexA-DBD (LexA-ASH1) and tested for its ability to repress transcription of the synthetic pCYC1-lacZ reporter plasmid carrying multiple LexA operators. The fusion of Ash1p to LexA-DBD repressed transcription from the reporter 2- to 5-fold. Deletion derivatives of the ASH1 coding sequence were fused to the LexA-DBD to roughly map the domain structure of Ash1p. Derivatives lacking the C-terminal 90 amino acid residues (?C90) or C-terminal half of the molecule (?C288) were also able to repress the reporter construct. In contrast, a derivative lacking the N-terminal half of Ash1p (?N300) was unable to repress the reporter, indicating that the N-terminal region of Ash1p is responsible for transcriptional repression activity. These deletion derivatives were also tested for the ability to repress pHO-CAN1 in the canavanine sensitivity assay. None of the deletion derivatives conferred resistance to the test strain, suggesting that all of the deletion derivatives lacked the ability to repress. Bobola et al. performed two separate genetic selections to identify genes involved in establishing HO asymmetry. A fusion between the HO promoter and a gene essential for cell division was generated. The gene used was CDC6 gene.The HO promoter restricts expression of CDC6 to mother cells. Cells whose endogenous CDC6 gene had been replaced by an HO–CDC6 fusion could not colonies because daughter cells lack this essential gene product. However, mutant cells that express HO inappropriately in daughter cells should divide and form a colony. CDC6 encodes an unstable protein necessary for initiation of DNA replication, which is synthesized in two bursts during the cell cycle, at the end of mitosis and in late G1 simultaneously with HO. Experiments were done by using a strain carrying a deletion of the endogenous CDC6 locus and fusions of CDC6 to the HO and GAL1-10 promoters. When transferred to glucose medium the GAL–CDC6 is not expressed, Cdc6 synthesis only occurs from the HO promoter. Most mothers but only a few daughters were capable of this cell division. This strain grows healthily on galactose medium but due to the lack of daughter cell proliferation could not form colonies on glucose. We isolated 12 spontaneous mutants capable of growth on glucose. This ability depended on SWI5 and was therefore due to the expression of CDC6 from the HO promoter. This confirmed that in all cases the mutant strains contained mutations that allowed daughter cells to switch mating types. ASH1 is the determinant of asymmetric HO expression. ASH1 is necessary for preventing daughter cell switching. Cells deleted for ASH1 express HO in daughter cells and overexpression of ASH1 is sufficient to reduce mating-type switching in mother cells. Ash1p activity is restricted to daughter cells where protein accumulates. After completion of anaphase, Ash1p accumulates in the nucleus located in the daughter cell where it remains present during G1. The protein disappears as daughter cells enter the cell cycle and appear to be absent during S phase, G2, and mitosis. This observation might explain why ash1 mutant mother cells have increased frequency in switching mating type compared to wild-type cells. Ash1p contains a domain with homology to the zinc finger domain of the GATA-like transcription factor family. Ash1p binds to sequences within the HO promoter inhibiting transcriptional activation. Ash1p antagonizes, when SWI5 is deleted in ash1 mutants, daughter cell switching drops to minimal levels, showing that this switching is dependent on SWI5. The phenotypes associated with modulating the levels of SWI5 are exactly opposite those of ASH1. Deletion of SWI5 causes loss of HO expression in mother cells, whereas loss of ASH1 function causes both mothers and daughters to express HO. Constitutive expression of SWI5 allows daughter cells to switch whereas overexpression of ASH1 blocks HO expression and mating-type switching in mother cells. Deletion of a particular domain of Swi5p allows daughter cells to switch mating type. Ash1p may interact with this region of Swi5p and thereby inhibit Swi5p-mediated activation of the HO promoter. In vitro footprinting, experiments suggest that Ash1p does not require Swi5p to bind DNA nor does it displace Swi5p bound to HO DNA. In vivo repression assays suggest that Ash1p does not require Swi5p to repress transcription on a heterologous promoter, indicating that a conformational change induced by any Ash1p/Swi5p contact is not required for repressor activity. The binding of Swi5p to HO promoter DNA in vivo is unaffected by the inappropriate localization of Ash1p in both mother and daughter cells, suggesting that Ash1p does not prevent recognition of HO by Swi5p. The daughter cell-specific transcription factor Ash1p and its action on the HO promoter to determine the mechanism of repression of HO in daughter cells. Experiments indicate that Ash1p is a modular transcription factor that acts at the DNA level to control transcription of HO. Site-specific mutation of three Ash1p-binding sites adjacent to the two Swi5p-binding sites upstream of HO in URS1 had no demonstrable effect on the frequency of mother or daughter cell mating type switching. Repression of HO by Ash1p in daughter cells may require the occupation of many Ash1p-binding sites. Ash1p may repress transcription of HO directly by first binding to specific sequences throughout the HO promoter and then repress transcription by an active mechanism after DNA binding. Some of the selective advantages of mating type switching are that haploid yeast cells of one mating type can produce haploid cells of the other type, thereby allowing sister cells to mate and become diploid. These diploids can sporulate to produce haploid cells and offer yeast the advantages coincident with a sexual life cycle. Isolated populations also provide the opportunity to undergo sexual reproduction. Given the importance of mating type switching for reproductive survival, traits that promote switching can be positively selected over evolutionary time. Mating type switching allows strains to purge their diploid genomes of deleterious recessive mutations by going through a transient haploid phase, with subsequent homozygous diploids generated from the viable haploid segregants. They can also help with DNA repair and germinate in poorer environments.