MAT Alpha Cells Research Articles

Special type of pheromone-induced invasive growth in Saccharomyces cerevisiae

The ability to invade a solid substrate is an important phenomenon due to its connection with pathogenic activity of fungi. We report here on invasion displayed by MATalpha cells of Saccharomyces cerevisiae lacking Isw2p, a subunit of the ISW2 chromatin remodelling complex. We found that on minimal medium, where the isw2Delta MATalpha mutant is not invasive, additional absence of another ISW2 complex subunit, Dls1p or Dpb4p, promoted invasion. Our microarray data showed that derepression of MAT a-specific genes caused by absence of Isw2p is very low. Their expression is increased only by the autocrine activation of the mating pathway. Invasion of isw2Delta MATalpha cells thus resembles the pheromone-induced invasion, including dependence on Fig2p. We show here that another pheromone-induced protein, mating agglutinin Aga1p, can play a role in the agar adhesion necessary for invasion. In contrast with MAT a-cells invading agar under low alpha-pheromone concentration, the invasive growth of isw2Delta cells specifically requires Fus3 kinase. Its function in the invasion of isw2Delta MATalpha cells cannot be completely substituted by Kss1 kinase, which plays a basic role in invasive growth signalling. We suggest that partial dependence of the isw2Delta MATalpha invasion on Fus3p and Aga1p corresponds to a weaker pheromone response of this mutant.

The KlSTE2 and KlSTE3 genes encode MATα‐ and MATa‐specific G‐protein‐coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae alpha-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATalpha and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MATa cells and binds the alpha-pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATalpha cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATalpha cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Galpha) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Galpha subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Galpha subunit.

Mating-type locus control of killer toxins fromKluyveromyces lactisandPichia acaciae

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.

TUP1 disruption reveals biological differences between MATa and MATα strains of Cryptococcus neoformans

Cryptococcus neoformans exists in two mating types MATa and MATalpha. Although the morphology, growth characteristics and genetic segregation patterns among MATa and MATalpha strains are indistinguishable in the laboratory, the predominance of MATalpha strains in nature suggests that MATalpha strains are better suited for survival in nature. We disrupted the TUP1 gene, a global repressor, to find the possible biological differences in congenic MATalpha and MATa cells of C. neoformans. Disruption of TUP1 affected neither the yeast nor the hyphal cell morphology but resulted in a similar reduction of mating frequencies in both MATalpha and MATa cells. Disruption of TUP1, however, functionally manifested itself in several mating type-dependent phenotypes: (i) MATalpha cells became more sensitive to 0.8 M KCl while MATa cells showed no change in sensitivity, (ii) a temperature-dependent growth reduction was exhibited at both 30 degrees C and 25 degrees C in MATa but a similar growth reduction was not observed in MATalpha cells until the temperature was lowered to 25 degrees C and (iii) the transcriptional level of genes in several different biological pathways was markedly altered in a mating type-dependent manner. This work is the first case in which non-mating-related biological differences are observed between two congenic mating partners in yeast.

The Saccharomyces cerevisiae TRT2 tRNAThr gene upstream of STE6 is a barrier to repression in MATalpha cells and exerts a potential tRNA position effect in MATa cells.

A growing body of evidence suggests that genes transcribed by RNA polymerase III exhibit multiple functions within a chromosome. While the predominant function of these genes is the synthesis of RNA molecules, certain RNA polymerase III genes also function as genomic landmarks. Transfer RNA genes are known to exhibit extra-transcriptional activities such as directing Ty element integration, pausing of replication forks, overriding nucleosome positioning sequences, repressing neighboring genes (tRNA position effect), and acting as a barrier to the spread of repressive chromatin. This study was designed to identify other tRNA loci that may act as barriers to chromatin-mediated repression, and focused on TRT2, a tRNA(Thr) adjacent to the STE6 alpha2 operator. We show that TRT2 acts as a barrier to repression, protecting the upstream CBT1 gene from the influence of the STE6 alpha2 operator in MATalpha cells. Interestingly, deletion of TRT2 results in an increase in CBT1 mRNA levels in MATa cells, indicating a potential tRNA position effect. The transcription of TRT2 itself is unaffected by the presence of the alpha2 operator, suggesting a hierarchy that favors assembly of the RNA polymerase III complex versus assembly of adjacent alpha2 operator-mediated repressed chromatin structures. This proposed hierarchy could explain how tRNA genes function as barriers to the propagation of repressive chromatin.

Mutation of the regulator of G protein signaling Crg1 increases virulence in Cryptococcus neoformans.

The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans. A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MATalpha strains of the fungus. Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains. In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12alpha but not on the mitogen-activated protein kinase homolog Cpk1. The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1. Interestingly, crg1 mutants of MATalpha cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.

Cell-type-dependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p-Itc1p chromatin remodelling complex.

In Saccharomyces cerevisiae MATa haploid cells, the a-specific genes are expressed, whereas in the MATalpha haploid and MATa/alpha diploid cell types their transcription is repressed. It is shown in this report that Itc1p, a component of the ATP-dependent Isw2p-Itc1p chromatin remodelling complex, is required for the repression of a-specific genes. It has previously been reported that disruption of the ITC1 gene leads, in MATalpha cells, to an aberrant cell morphology resembling the polarized mating projection of cells responding to pheromone. The activation of the pheromone signalling pathway in itc1 mutants of both mating types was examined and found to be constitutively active in MATalpha itc1 but not in MATa itc1 cells. Furthermore, unlike the wild-type, MATalpha itc1 and MATa/alpha itc1/itc1 cells secrete a-factor and express significant levels of other a-specific genes. The results indicate that the inappropriate a-factor production in a MATalpha context, due to the derepression of the a-specific genes, produces an autocrine signalling loop that leads to the aberrant morphology displayed by MATalpha itc1 cells. It is suggested that the Isw2p-Itc1p complex contributes to maintain the repressive chromatin structure described for the asg operator present in the promoters of a-specific genes.

Roles of sexual cell agglutination in yeast mass mating.

The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia.

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene.

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.

Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37 degrees C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcine urin regulates filamentation and 37 degrees C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our findings indicate that calcineurin plays a conserved role in the control of cell morphology.

Expression of MFA1 and STE6 is sufficient for mating type-independent secretion of yeast a-factor, but not mating competence.

The yeast a-factor mating peptide and its transporter Ste6 are normally expressed only in MATa haploid cells. The a-factor is initially produced as a 36- or 38-residue peptide precursor and must undergo extensive post-translational processing to produce an active 12 amino-acid lipopeptide. To better understand the steps required for Ste6-dependent a-factor transport, we have reconstituted a-factor synthesis and transport in MATalpha haploids and MATa/alpha diploids. Ste6 and a-factor were stably expressed in MATalpha and MATa/alpha cells and the ectopically expressed a-factor was correctly processed. In addition, Ste6 was able to transport a-factor from all cell types, indicating that once expressed no other MATa-specific functions are required. However, despite significant levels of a-factor secretion, MATalpha cells are unable to support efficient mating.

Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching.

Switching of mating type in Saccharomyces cerevisiae is directional; MAT alpha cells recombine to transfer information from HMRa while MATa cells switch using the silent cassette at HML alpha. Genetic analysis recently has defined a 700 bp recombination enhancer approximately 29 kb from the left end of chromosome III that is necessary for directionality. The chromatin structure of this region differs strikingly in a- and alpha-cells. Mat alpha2p organizes a 3.7 kb chromatin domain that opposes interaction of trans-acting proteins with the enhancer. In a-cells lacking the alpha2 repressor, two footprinted regions flank an approximately 100 bp section having a unique DNA structure. This structural signature probably reflects interactions of proteins that result in directional mating type switching.

Periplasmic Bar1 protease of Saccharomyces cerevisiae is active before reaching its extracellular destination.

Saccharomyces cerevisiae MATa and MAT alpha cells secrete a-factor and alpha-factor pheromones. These peptides act on cells of the opposite mating type. They induce physiological changes which allow the formation of diploid cells. MATa strains produce an extracellular protease which cleaves, and thus inactivates the MAT alpha cell-specific alpha-factor pheromone. This pepsin-like enzyme is encoded by the BAR1(SST1) gene and is secreted into the periplasmic space of MATa cells. We found that the Bar1p protease is already active in early compartments of the secretory pathway. Our results indicate that Bar1 protease tolerates large N-terminal extensions of its substrate and does not require Golgi-specific modifications such as outer-chain glycosylation for activity.

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.

Global alterations in chromatin accessibility associated with loss of SIN4 function.

Sin4p is a component of a mediator complex associated with the C-terminal domain of RNA polymerase II and SIN4 is required for proper regulation of several genes in yeast, including the HO endonuclease gene, glucose repressible genes and MATa cell-specific genes. Previous studies indicated that SIN4 may influence transcription through changes in the organization of chromatin. We have examined a specific chromatin structure associated with MATa cell-specific repression in sin4 MATalpha cells to determine if SIN4 is required for nucleosome positioning. Although the loss of SIN4 has no effect on nucleosome location, we find that the sensitivity of bulk chromatin from sin4 cells to micrococcal nuclease digestion is strikingly increased relative to chromatin from isogenic wild-type cells. The nuclease hypersensitivity of chromatin from sin4 cells is not related to gross alterations in histone gene expression or to bulk increases in histone modification. Our experiments suggest that SIN4 directly or indirectly regulates a global aspect of chromatin accessibility, providing a molecular basis for phenotypic similarities between sin4 mutations and mutations in histones.

A role for the actin cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection.

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MAT alpha cells bud in an axial pattern, and MATa/alpha cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MAT alpha cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/alpha cells were defective in the bipolar budding pattern, but MATa and MAT alpha cells still exhibit a normal axial budding pattern. We also observed that MATa/alpha actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/alpha cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/alpha cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins.

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.

Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins. alpha-Agglutinin is a GPI-anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor. Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C. 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9. Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses. One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins. When labeled with [2-3H]inositol, mutants accumulate reduced amounts of radiolabeled GPI-anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains. On the other hand, invertase and acid phosphatase are secreted by all but one mutant. All mutants show an increased sensitivity to calcofluor white and hygromycin B. This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall.

MATa donor preference in yeast mating-type switching: activation of a large chromosomal region for recombination.

During mating-type gene switching in Saccharomyces cerevisiae, DNA at the MAT locus is replaced by sequences copied from one of two unexpressed donor loci, HML or HMR, located near the two ends of the same chromosome and > or = 90 kb from MAT. MATa cells recombine nearly 90% of the time with HML, whereas MAT alpha cells select HMR. MATa donor preference was examined by deleting HML and inserting a donor at other chromosome III locations. MATa activated a large (> or = 40 kb) region near the left end of chromosome III, such that a donor placed at several sites within this domain was strongly preferred over HMR. When inserted outside of this domain, the donor was used equally with HMR. MATa donor preference for HML was abolished by the expression of the negative regulator, MAT alpha 2; however, HML regained its preferred status when the donor was unsilenced. Mating-type-dependent activation of the left end of the chromosome is also observed for other types of recombination that do not involve MAT switching. Spontaneous recombination between two leu2 alleles is 20-30 times higher in MATa than in MAT alpha when one of the leu2 alleles is inserted in place of HML. Transcription in this donor activation region is not affected by mating type. We conclude that MATa donor preference involves a mating-type-regulated change in the accessibility of a large chromosomal domain for recombination.

Mutations affecting donor preference during mating type interconversion in Saccharomyces cerevisiae.

Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to alpha or alpha to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MAT alpha cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MAT alpha background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.