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Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form pro-toperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.
Key words: conidiation, fungal cell wall biosynthesis, galactomannan, protein glycosylation, protoperithecia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Only a limited amount of information is available on the composition and structure of the Neurospora cell wall. The Neurospora cell wall has been shown to contain chitin, beta-1,3-glucan having beta-1,6-linked branches and glycoproteins with a galactomannan component. A proteogalactomannan fraction from the glycoprotein layer has been isolated and structurally characterized (Nakajima et al 1984a
, b). The analysis showed that galactomannan polysaccharides were attached to the peptide at aspargine (N-linked) and serine/threonine (O-linked) sites. Nakajima et al (1984b) proposed a structure for the N-linked and O-linked galactomannans. The proposed N-linked galactomannan structure consisted of an alpha-1,6-mannose core with alpha-1,2-linked mannose side chains terminated with beta-1,5-linked galactofuranose residues. The proposed O-linked galactomannan structure consisted of two alpha-1,2-linked man-nose residues attached to the protein followed by two beta-1,5-linked galactofuranose residues. A secreted galactomannan also has been characterized and shown to have a similar structure (Nakajima et al 1982).
The structure of the yeast cell wall has been extensively studied and many of the enzymes involved in its synthesis have been identified (Gemmill and Trimble 1999). In Saccharomyces cerevisiae, the mannan component of the wall contains a long alpha-1,6-mannose core structure with alpha-1,2-linked mannose side chains that are capped with alpha-1,3-linked mannose. A number of enzymes involved in generating the yeast cell wall have been identified.
We have isolated and characterized two Neurospora mutants affected in their morphology, growth properties and in their ability to regulate the entry into the asexual developmental program. The mutations are located in the Neurospora mnt-1 gene, which encodes a putative alpha-1,2-mannosyltransferase that functions in the synthesis of cell wall components. The encoded MNT-1 protein is related closely to the yeast mannosyltransferases that function in yeast cell wall biosynthesis. Similar mannosyltransferases have been found to play critical roles in cell wall biosynthesis in Candida albicans, where they are required for normal hyphal growth and virulence (Buurman et al 1998).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b). It has an aro-9, qa-2, inv, al-2 mat-a genotype and contains multiple transforming copies of a grg-1/tyrosinase chimeric gene construct integrated into its genome. The al-1, arg-5, cot-1, A (FGSC No. 2252), lys-1, al-3, inl, pab-2, A (FGSC No. 4131), and al-3, inl, A (FGSC No. 2308) strains used in the gene mapping experiments were obtained from the Fungal Genetics Stock Center (Kansas City, Kansas). The inl A strain (FGSC No. 1453) was used for the RIP mapping experiment. These strains were maintained on supplemented Vogel’s minimal media. The preparation and supplementation of the media and culturing conditions were as described in Davis and DeSerres (1970).
Mutant isolations and genetic mapping.— –
The GTH-16 strain was derived from an aro-9, qa-2, inv, al-2, a isolate that had been transfected with the pGRG-1/TYR103 plasmid (Kothe et al 1993, Kothe and Free 1998). The strain expresses tyrosinase under the direction of a ccg-1/grg-1 promoter, a promoter that is activated by carbon source deprivation and which gives high level expression of tyrosinase in cells undergoing asexual development (Wang et al 1994). The expressed tyrosinase, which catalyzes the only enzymatically required step in melanin biosynthesis from tyrosine, causes the cells to turn black with melanin. The GTH-16 strain thus lets the investigator visually screen large numbers of mutagenized cells for isolates that have lost the ability to repress asexual development.
The mutagenesis of GTH-16 was accomplished by subjecting conidia to UV light. GTH-16 conidia were subjected to 10 min exposure to a UV light source held at 10 cm from a Petri dish containing 10 mL of a conidial suspension. The UV mutagenesis resulted in a 99.7% killing of the conidia. The mutagenized conidia were plated on a Vogel’s sorbose plate, which imposes a colonial growth pattern on the germinating conidia and represses the entry of the cells into the asexual developmental program. Two days after plating the conidia, 10 mL of sterile 5 mM tyrosine was carefully added to the plate. Colonies that were unable to repress entry into the developmental program were identified by the fact that they turned black with melanin. MC-34 was identified as such a colony.
The use of insertional mutagenesis to generate and isolate GTH-16-derived mutants affected in the ability to repress asexual development had been reported (Kothe and Free 1998a). The T-6 mutant was one of a number of mutants isolated that were unable to repress entry into the asexual developmental program. The T-6 mutant has the pRAL-1 plasmid, the plasmid used in the insertional mutagenesis, inserted into its genome.
Genetic mapping of the mutations in MC-34 and T-6 responsible for the colonial phenotype were carried out with standard mapping experiments (Davis and DeSerres 1970). Because MC-34 and T-6 are unable to elaborate protoperithecia, the female mating structures, and are therefore female sterile, they were used as the male component in the mapping experiments. The colonial growth phenotype was followed as a marker of the mutant phenotype in these mapping experiments.
Isolation of additional mnt-1 mutants using the Neurospora RIP phenomenon.— –
The RIP (repeat induced point mutation) phenomenon is associated with the mating process and can be used to generate null mutants of cloned genes (Selker et al 1989). During the premeiotic phase of mating, Neurospora identifies DNA sequences larger than 1000 base pairs in length that are duplicated in the haploid genome and introduces multiple G/C to A/T transitions within these duplicated sequences.
To generate additional mnt-1 mutants, the GTH-16 strain was transformed with a pRAL-1 plasmid carrying a PCR-generated copy of the entire mnt-1 gene. Individual transformants were isolated and mated with an inl A strain (FGSC No. 1453) to generate mnt-1 RIP mutants. Progeny ascospore were isolated and examined for the mnt-1 phenotype. Several progeny having the colonial mnt-1 phenotype were isolated.
PCR analysis and sequencing.— – All PCR experiments were carried out with primer oligonucleotides designed to amplify genes of interest in the short genomic region identified as containing the mutant gene. The sequences for the oligonucleotides were derived from the published genomic DNA sequence provided by the Neurospora Sequencing Project at the Broad Institute/MIT Center for Genome Research (assembly 3 at www-genome.wi.mit.edu). Neurospora DNA was isolated from MC-34, T-6, and GTH-16 using the Trizol reagent as described in the manufacturer’s instructions (Invitrogen, Carlsbad, California). The amplified genomic DNA regions were sequenced at the DNA sequencing facility at the Roswell Park Division of the State University of New York at Buffalo. Accession numbers for the mnt-1 gene (locus NCU01388.1) are AL4511020, BX294017 [GenBank] and XM326880.
Cell wall analysis.— – Crude cell wall fractions were obtained from cells grown in liquid Vogel’s media supplemented to permit the growth of the mutant cells and their wild-type parent. The cells were harvested on a Buchner funnel, frozen in liquid nitrogen and lyophilized. The frozen cells were ground to a fine powder with a mortar and pestle. The material was resuspended in ice-cold 50 mM sodium acetate buffer, pH 5.0 containing 1% Triton X-100 and subjected to sonication to further disrupt the cells. The cell walls were then obtained by a centrifugation step (5000 x g for 10 min). The isolated cell walls were washed three times by resuspending them in ice cold water and collected by a centrifugation step. The material was lyophilized and samples of the material sent to the Complex Carbohydrate Research Center, University of Georgia (Athens, Georgia) for analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Kothe and Free 1998a). When GTH-16 enters asexual development it expresses tyrosinase, the only enzymatically required step in the biosynthesis of melanin, and turns black.
GTH-16 conidia were mutagenized by exposure to UV light and the surviving conidia were plated on a supplemented Vogel’s sorbose medium (Davis and DeSerres 1970). The sorbose medium causes the cells to grow in a tight colonial mode and represses the onset of asexual development. Mutants affected in the ability to repress asexual development are easily obtained by screening the Vogel’s sorbose-containing plates for black colonies. MC-34 was one such colony. The formation of a halo of melanin by MC-34 growing on a glucose-containing agar medium, which normally represses asexual development and the expression of the chimeric ccg-1/grg-1 tyrosinase gene, is shown (FIG. 1).
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). In addition to having an altered gross morphology as judged by a visual inspection of the colony, the mutant has an altered hyphal morphology as seen under the microscope. The MC-34 mutant grows as an array of dichotomously branching hyphae with a morphology that is reminiscent of hyphal cells undergoing asexual development (FIG. 2) (Springer and Yanofsky 1989). The normal wild-type vegetative hyphae produced by the GTH-16 parent strain is shown (FIG. 2). We were unable to find any wild-type vegetative hyphae in samples of the MC-34 mutant.
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Another interesting phenotypic aspect of MC-34 is that the strain is unable to function as a female in a mating with a strain of the opposite mating type. Examination of MC-34 growing on a supplemented cornmeal agar medium shows that MC-34 does not elaborate protoperithecia (female mating structures). Cornmeal agar induces the formation of protoperithecia (Davis and DeSerres 1970). The inability to enter into the sexual developmental stage, as defined by the production of protoperithecia, explains why MC-34 is female sterile.
Isolation and characterization of the T-6 mutant.— –
The T-6 mutant was isolated from an insertional mutagenesis experiment. In Neurospora most transformation events are the result of nonhomologous recombination between an incoming plasmid and the genomic DNA. These events occur in what would appear to be random positions in the genome and can be used effectively to mutagenize and tag genes. The GTH-16 strain was used as the parental strain for the isolation of the T-6 mutant. The use of insertional mutagenesis to obtain mutants from GTH-16 had been described (Kothe and Free 1998a, b). Briefly, the GTH-16 strain has, in addition to multiple integrated copies of the grg-1/tyrosinase/hygromycin vector, mutations in the aro-9 and qa-2 genes. These two genes respectively encode anabolic and catabolic dehydroquinases. Dehydroquinase catalyzes a required step in the aromatic amino acid biosynthetic pathway. As a consequence of having mutations in the aro-9 and qa-2 genes, the strain is an auxotroph for aromatic amino acids and p-amino-benzoic acid. GTH-16 was transformed with the pRAL-1 plasmid, which contains a copy of the qa-2 gene and therefore transforms GTH-16 from auxotrophy to prototrophy (Akins and Lambowitz 1985). The cells were plated on unsupplemented Vogel’s sorbose plates to select for transformants. Transformants that had lost the ability to repress the onset of asexual development were isolated by a visual inspection of the plates for colonies that were expressing tyrosinase and had turned black with melanin. T-6 produces a melanin halo when grown on a glucose-containing (asexual development repressing) agar medium (FIG. 1).
Initial characterization of the T-6 mutant showed it to have a tight colonial phenotype. It was unable to produce conidia and, in all aspects of its gross morphology, was indistinguishable from the MC-34 mutant (FIG. 1). Like MC-34, the T-6 mutant is unable to form protoperithecia and is therefore unable to function as a female. At a microscopic level T-6 hyphae have the dichotomously branching morphology reminiscent of asexually developing cells (FIG. 2). Analysis of T-6 hyphae demonstrates that T-6 does not produce hyphae having the characteristics expected of vegetative hyphae.
To determine whether the mutations in T-6 and MC-34 were able to complement one another, a forced heterokaryon was prepared between the T-6 mutant (an aromatic amino acid auxotroph) and an MC-34 mutant that was obtained as an auxotrophic progeny (inositol auxotroph) from an MC-34 mapping experiment. The forced heterokaryons grew with the characteristic colonial morphology, indicating that the mutations in T-6 and MC-34 were unable to complement each other. Both mutations were readily complemented in other forced heterokaryons. We conclude that MC-34 and T-6 are allelic mutations.
Genetic mapping of the MC-34 and T-6 mutations.— –
Mapping of the MC-34 mutation occurred in a number of mapping experiments. The MC-34 strain (aro-9, qa-2, inv, al-2, a) first was mated to an al-1, arg-5, cot-1, A isolate (FGSC No. 2252). Sixty-seven progeny were analyzed for the segregation of the MC-34 colonial phenotype as well as for the cot-1, inv, arg-5 and A/a mating type markers. The MC-34 phenotype segregated independently of the cot-1, arg-5, and A/a mating type markers but did show linkage to the inv marker. The data suggested that the MC-34 mutation was approximately 30 centimorgans from the inv (invertase) marker on linkage group V. To further examine where on linkage group V the MC-34 mutation occurred, MC-34 was mated to a lys-1, al-3, inl, pab-2, A isolate (FGSC No. 4131). Data from 187 progeny showed that the MC-34 mutation was approximately 1 centimorgan from the inl (inositol) marker on the right arm of linkage group V. We were unable to map the MC-34 relative to the nearby al-3 gene in this mating because of the presence of the al-2 marker in MC-34. By mating a MC-34-containing isolate that lacked the al-2 marker present in the original MC-34 (obtained as a progeny from a genetic cross) with an al-3, inl, A strain (FGSC No. 2308), we were able to carefully map the MC-34 mutation relative to inl and al-3 in a three point cross. Data from 1746 progeny of this mating showed that the MC-34 mutation was centromere proximal to both al-3 and inl. We found that MC-34 mapped 0.7 cm to the left of al-3 and 1.0 cm to the left of inl (FIG. 3). Similar mapping experiments with T-6 showed that it also mapped to a location approximately 1 cm from the inl marker. In all these mapping experiments the described colonial morphology segregated as a single gene trait. We found a normal ratio of mutant and wild-type progeny and no other phenotypes, or modifiers of the colonial phenotype, were observed.
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). The encoded 393 amino acid protein is closely related to yeast cell wall biosynthetic mannosyltransferases. Lussier et al (1999) showed that the region having the highest level of homology between the members of the mannosyltransferase family is located near the carboxyl terminus of the protein and suggested that this region may contain the mannosyltransferase active site. This region is lost as a result of the mutation in MC-34.
The T-6 mutant was generated by insertional mutagenesis, and efforts to PCR amplify the mannosyltransferase gene from T-6 with the mannosyltransferase primers failed to give a product. If the mannosyltransferase gene in T-6 had an insertion of the pRAL-1 plasmid, the region between the two primers could be large and difficult to obtain with PCR amplification. Thus we used primers from within the pRAL-1 plasmid paired with mannosyltransferase primers to see whether we could PCR amplify a fragment of DNA from T-6 containing a pRAL-1 insertion site in the mannosyltransferase gene. We were successful in obtaining such an amplified gene product with the primer from the 3' end of the mannosyltransferase gene and a primer from pRAL-1. Sequencing the PCR product demonstrated that a pRAL-1 insertion had occurred in the mannosyltransferase gene within a glutamic acid codon (GAG) at amino acid position 251 (FIG. 3). This insertion would result in the production of a truncated mannosyltransferase gene product lacking the presumptive active site near the carboxyl terminus of the protein.
We have opted to name the mannosyltransferase gene mutated within MC-34 and T-6 the mnt-1 gene. BLAST searches (Altschul et al 1997) indicate the Neurospora mnt-1 gene has 40–49% identity with the KTR1, KRE2/MNT1, KTR2, KTR4, KTR3 and YUR1 genes from Saccharomyces cerevisiae. It has a similar level of identity with putative mannosyltransferase genes from Candida albicans and Schizosaccharomyces pombe. The Saccharomyces cerevisiae genes have been studied extensively and shown to be involved in the addition of alpha-1,2-linked mannose residues to O-linked or to N-linked oligosaccharides (Hausler et al 1992, Hausler and Robbins 1992, Hill et al 1992, Lussier et al 1993, Lussier et al 1999, Romero et al 1997). The KTR1 and KRE2/MNT1 gene products have overlapping specificity and function in the synthesis of O-linked oligosaccharides (Lussier et al 1997). The KRE2/MNT1, KTR1, KTR2 and YUR1 gene products have been localized to the yeast medial Golgi (Chapman and Munro 1994, Lussier et al 1995, Lussier et al 1996).
Isolation of additional mnt-1 mutants.— –
To verify that the MC-34 and T-6 mutations within the mnt-1 gene were responsible for the mutant phenotype, additional mnt-1 mutants were generated with the RIP phenomenon. The RIP phenomenon generates multiple G/C to A/T transition mutations in genes that are present in duplicate copies during the mating process (Selker et al 1989). A plasmid with a full-length copy of the mnt-1 gene was used to transform GTH-16 cells to generate transformants with an endogenous mnt-1 gene and a duplicate plasmid-contained copy of the gene. Individual transformants were isolated and crossed to generate RIP mutant progeny. Among the progeny obtained from these matings were some having a phenotype that was indistinguishable from the phenotype observed in the MC-34 and T-6 mutants. PCR sequencing of the mnt-1 gene from three of these mutant progeny demonstrated that, in each case, the gene had a number of G/C to A/T transition mutations. In two of the sequenced RIP mutant genes the mutations included stop codons. In the third sequenced RIP mutant gene, we found 29 nucleotide changes leading to 13 amino acid changes in the encoded protein. These mnt-1 RIP mutants verify that mutations within the mnt-1 gene give rise to the described colonial phenotype.
Analysis of MC-34, T-6 and parental cell walls.— – To determine whether the cell wall structure of the Neurospora mnt-1 mutants was altered, cell walls were isolated from MC-34, T-6 and their wild-type parent, GTH-16. Crude total cell walls were analyzed for the presence of constituent sugars at the Complex Carbohydrate Research Center at the University of Georgia. The glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry of per-O-trimethylsilyl derivatives of monosaccharide methyl glycosides produced from the cell wall samples by acidic methanolysis. The mass fraction of the cell walls attributed to carbohydrate was dramatically different in the mutant and wild-type cell walls. The wild-type cell wall contained 67% of its mass as carbohydrate. The MC-34 and T-6 cell walls contained respectively only 41% and 46% of their mass as carbohydrate. The mutant cells clearly were much less effective in the incorporation of carbohydrates into their cell walls than the wild-type parent. The composition of the incorporated carbohydrate also differed. The carbohydrate composition of the wild-type parent cell wall consisted of 68% glucose, 20% mannose, 10% galactose and 2% N-acetylglucamine. In contrast the carbohydrate composition of the MC-34 cell wall consisted of 85% glucose, 9% mannose, 3% galactose and 2% N-acetylglucosamine. The carbohydrate composition of the T-6 cell wall was 78% glucose, 13% mannose, 6% galactose and 2% N-acetylglucosamine. As is evident from the data, the MC-34 and T-6 mutant cell walls contain reduced levels of mannose and galactose. The amount of mannose per mg of total cell wall material are reduced respectively by 72% and 55% in MC-34 and T-6 when compared with the parental, wild-type cell wall. The amount of galactose per mg cell wall material similarly are reduced by 82% and 59% in the MC-34 and T-6 cell walls.
Although the Neurospora cell wall has not been studied as extensively as the yeast cell wall, it is known to contain chitin polymers, glycoprotein and glycan. Glycan consists of beta-1,3-linked glucose residues with beta-1,6-linked branches. Nakajima et al (1984a, b) isolated a hot water extractable proteogalactomannan fraction from the Neurospora cell wall and characterized its composition and structure. The proteogalactomannan contained both N-linked and O-linked polysaccharides. The proposed structure of the N-linked polysaccharides consisted of a long alpha-1,6-linked mannose backbone with alpha-1,2-linked mannose side chains. The side chains were terminated by the presence of multiple beta-1,5-linked galactofuranose residues. The proposed structure for the O-linked polysaccharides consisted of two alpha-1,2-linked mannose residues attached to the serine or threonine of the peptide, followed by one or two beta-1,5-linked galactofuranoses. Because the galactose is terminal to the mannose in these structures, mutations affecting the synthesis of the mannose portion of the galactomannan would be expected to affect the amount of both mannose and galactose in the cell wall. Based on the cell wall composition data, we infer that the mnt-1 mutations affect the synthesis and composition of the galactomannan component of the Neurospora cell wall.
It should be noted that the mnt-1 mutations do not render the cell walls devoid of mannose and galactose. This suggests that there are other mannosyltransferases that function in Neurospora cell wall biosynthesis. In Saccharomyces cerevisiae the KRE2/MNT1 family of mannosyltransferases contains nine members. These mannosyltransferases have been shown to have overlapping specificities and to function at various steps in the synthesis of O-linked and N-linked oligosaccharides (Lussier et al 1997). A search of the Neurospora genome shows that it contains, in addition to the mnt-1 gene, four other closely related presumptive mannosyltransferase genes. It is probable that one or more of these other presumptive mannosyltransferase genes provides for the synthesis of the residual galactomannan found in the cell walls of the mnt-1 mutants. However, given the severity of the mnt-1 phenotype, and the major reduction in cell wall carbohydrate content associated with the phenotype, we conclude that the mnt-1 gene clearly plays a critical role in Neurospora cell wall biosynthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Neurospora mnt-1 gene encodes a 393 amino acid protein. The protein would be expected to be a type 2 membrane protein, with a short eight amino acid cytoplasmic N terminal region, a transmembrane region, and a larger C terminal region in the lumen of the secretory pathway organelles (FIG. 3). The proposed start of translation site, intron sequences and codon usage is typical of Neurospora genes (Edelman and Staben 1994). The gene has a start of translation site (CACAATGG) that matches the Neurospora consensus start of translation site. The coding region has three introns (75, 77 and 57 nucleotides in length) that closely adhere to the consensus intron 5', branch site and 3' sequences. The gene has the codon bias of having pyrimidines in the wobble base site that is characteristic of Neurospora genes.
BLAST searches demonstrate that the Neurospora MNT-1 protein is related closely to members of the Kre2p/Mnt1p family of yeast alpha-1,2-mannosyltransferases. The yeast genome contains nine members of the gene family, and a number of the members have been shown to have alpha-1,2-mannosyltransferase activity. The Neurospora MNT-1 is closely related to the Kre2p/Mnt1p and Ktr1p, two proteins that have overlapping specificity for the addition of alpha-1,2-linked mannose residues to O-linked oligosaccharides (Lussier et al 1997). Our data showing a reduction in the levels of galactomannan from the Neurospora cell wall suggests that MNT-1 functions in the addition of mannose residues to the galactomannan structure. Closely related putative mannosyltransferase genes are found in the genomes of other recently sequenced filamentous and dimorphic ascomycetes, such as Magnaporthe grisea, Fusarium graminearum, Aspergillus nidulans, Chaetomium globosum, Coccidioides immitis and species of Candida. These genomes contain 3–5 mannosyltransferase genes, with one of the genes being closely related to the Neurospora mnt-1 gene.
Analysis of the yeast Kre2p/Mnt1p family has shown that the most highly conserved sequence, which may be the mannosyltransferase active site, consists of the amino acid sequence YNLCHFWSNFEI (Lussier et al 1999). The Neurospora mnt-1 encoded protein has a close match to this putative active site, YSTCHFWSNFEI, between amino acids 281–292 (FIG. 3). The Saccharomyces cerevisiae mannosyltransferases are in the medial Golgi apparatus. Research on the intracellular localization of Kre2p, Ktr4p and Ktr6p demonstrated that a cytosolic N terminal consensus sequence of (F/L)LSK(R/K)(I/L)(L/A)(K/R) functioned to localize these proteins to the medial Golgi apparatus (Lussier et al 1995, Lussier et al 1997). The Neurospora N terminal sequence MAIARPVR is related to the consensus yeast sequence in placement of hydrophobic and charged amino acids. We have no data on the location of the Neurospora MNT-1 protein or on the importance of the N terminal sequence in protein targeting. One interesting aspect of the MC-34 and T-6 mutants is that both of them were isolated in screening experiments designed to isolate mutants that had lost the ability to repress entry into the asexual developmental program. There is no obvious explanation of why the loss of the mannosyltransferase affects the ability of the cells to repress the asexual developmental program. Entry into the asexual developmental program is repressed by the presence of glucose in the medium. One possible explanation for the results is that a cell surface component that plays a critical role in evaluating the presence of glucose in the environment is modified in a mnt-1-dependent manner and is unable to function in the absence of the modification.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors made equal contributions to this work. 
2 Corresponding author. E-mail: free{at}buffalo.edu.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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