ALG3 Research Articles

A Comprehensive Analysis of Gene Expression Profiles in a Yeast N-Glycosylation Mutant

Although protein N-glycosylation is critical to many cell functions, its downstream targets remain largely unknown. In all eukaryotes, N-glycosylation utilizes the lipid-linked oligosaccharide (LLO) precursor, whose synthesis is initiated by the ALG7 gene. To elucidate the key signaling and metabolic events affected by N-glycosylation, we performed genomewide expression profiling of yeast cells carrying a hypomorphic allele of ALG7. DNA microarrays showed that of more than 97% of known or predicted yeast genes, 29 displayed increased expression while 23 were repressed in alg7 mutants. Changes in transcript abundance were observed for a and α mating-type genes, for genes functioning in several mitogen-activated protein kinase (MAPK) cascades, as well as in phosphate, amino acid, carbohydrate, mitochondrial and ATP metabolism. Therefore, DNA microarrays have revealed direct and indirect targets, including internal feedback loops, through which N-glycosylation affects signaling and metabolic activities and is functionally linked with cellular regulatory circuitry.

Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase.

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.

Reduced Heparan Sulfate Accumulation in Enterocytes Contributes to Protein-Losing Enteropathy in a Congenital Disorder of Glycosylation

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE.

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.

Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.

Carbohydrate deficient glycoprotein syndrome type IV: deficiency of dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase.

Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins. Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl-phosphate mannose on to the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-dolichol. The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full-length LLOs. N-glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full-length oligosaccharides and because of the incomplete utilization of N-glycosylation sites. The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene.

Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus.

The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites. Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-(3)H]glucosamine and [2-(3)H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.

The Dictyostelium discoideum beta-1,4-mannosyltransferase gene, mntA, has two periods of developmental expression.

The precise roles of protein glycosylation in multicellular development are poorly understood. We have characterized the mntA gene from Dictyostelium discoideum which encodes the beta-1,4-mannosyltransferase enzyme that catalyzes the reaction: GDP-Man + dolichol-PP-GlcNAc2 --> dolichol-PP-GlcNAc2-Man + GDP. This gene has a central role in the synthesis of the lipid-linked oligosaccharide precursor which becomes the core of all asparagine-linked (N-linked) glycans. The mntA gene contains a single small intron and encodes a 493 aa protein with a predicted molecular size of 56 kDa. It is located 5' to the repE gene on chromosome IV and is transcribed in the opposite orientation to repE with which it shares a 585 bp of upstream intergenic region. The predicted mntA gene product shares 38% homology with the S. cerevisiae ALG1 gene product. The MntA protein has a region homologous to the putative dolichol-binding region in the yeast ALG1 protein, but it is located in a different part of the molecule. Northern analysis revealed that the expression of the mntA gene is regulated during multicellular development with two periods of mRNA accumulation. The mntA gene product has a classical endoplasmic reticulum retention motif, and is the first Dictyostelium gene encoding a protein that is active in this organelle. The identification of this gene will allow expanded studies of the role of N-linked glycans in multicellular development.

Deregulation of the First N-Glycosylation Gene, ALG7, Perturbs the Expression of G1 Cyclins and Cell Cycle Arrest inSaccharomyces cerevisiae

The evolutionarily conserved ALG7 gene encodes the dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT) and functions by initiating the dolichol pathway of protein N-glycosylation. InSaccharomyces cerevisiae,ALG7 has been shown to play a role in cell proliferation. The yeast α-factor-induced cell cycle arrest in G1 occurs, in part, by downregulation of CLN1 and CLN2. The function of ALG7 in G1 arrest was examined inalg7mutants containing diminished GPT activity. In wild type, CLN1 and CLN2 mRNAs were rapidly downregulated, while inalg7mutants, these transcripts were only transiently repressed before becoming greatly augmented. Analyses of DNA contents and budding indices showed thatalg7mutants resumed cycling when wild type cells remained arrested. Thus, deregulation of ALG7 interferes with cell cycle arrest by preventing a sustained downregulation of CLN1 and CLN2 mRNAs. These results provide a molecular insight into the role of ALG7, and protein N-glycosylation in general, in proliferation.

The dual role of mRNA half-lives in the expression of the yeast ALG7 gene.

The yeast ALG7 gene functions by initiating the synthesis of the dolichol-linked oligosaccharide precursor and plays an important role in the control of protein N-glycosylation. The levels of ALG7 multiple transcripts are modulated by the physiological status of the cell and environmental cues, and deregulation of their abundance is deleterious to several cellular functions. Since ALG7 mRNAs are unstable, we investigated the role of these transcripts' half-lives in determining their steady-state levels. Using a temperature-sensitive RNA polymerase II mutant, we demonstrate that increased stability was the primary determinant of higher ALG7 mRNA abundance in response to glucose limitation or treatment with tunicamycin. In contrast, at the G1/G0 transition point, changes in the decay rates were inversely related to ALG7 transcript accumulation: the decreased abundance of ALG7 mRNAs following exit from the mitotic cycle was associated with lengthening of the decay rates, while their increased accumulation after growth stimulation correlated with decreased stability. This suggests that, depending on the circumstance, mRNA half-lives can either directly determine the level of ALG7 transcript accumulation or oppose regulatory changes at other control levels.

Stepwise assembly of the lipid-linked oligosaccharide in the endoplasmic reticulum of Saccharomyces cerevisiae: identification of the ALG9 gene encoding a putative mannosyl transferase.

The core oligosaccharide Glc3Man9GlcNAc2 is assembled at the membrane of the endoplasmic reticulum on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the oligosaccharyl transferase complex. Based on the synthetic phenotype of the oligosaccharyl transferase mutation wbp1 in combination with a deficiency in the assembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus encodes a putative mannosyl transferase because deletion of the gene led to accumulation of lipid-linked Man6GlcNAc2 in vivo and to hypoglycosylation of secreted proteins. Using an approach combining genetic and biochemical techniques, we show that the assembly of the lipid-linked core oligosaccharide in the lumen of the endoplasmic reticulum occurs in a stepwise fashion.

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae alg3-1 mutant is described as defective in the biosynthesis of dolichol-linked oligosaccharides (Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). Man5GlcNAc2-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. In this study, we describe the cloning of the ALG3 locus by complementation of the temperature sensitive growth defect of the alg3 stt3 double mutant. The isolated ALG3 gene complements both the defect in the biosynthesis of lipid-linked oligosaccharides of the alg3-mutant and the under-glycosylation of secretory proteins. The inactivation of the nonessential ALG3 gene results in the accumulation of lipid-linked Man5GlcNac2 and protein-bound carbohydrates which are completely Endo H resistant. The ALG3 locus encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence.

Expression of the First N-Glycosylation Gene in the Dolichol Pathway, ALG7, Is Regulated at Two Major Control Points in the G1 Phase of the Saccharomyces cerevisiae Cell Cycle

The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene. To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle. We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of α-factor. ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START. In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by α-factor treatment later in G1. Importantly, deregulated expression of ALG7 resulted in an aberrant α-factor response. Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START.

Diminished activity of the first N-glycosylation enzyme, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), gives rise to mutant phenotypes in yeast

The enzyme which initiates the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), is encoded by the ALG7 gene. Essential for viability, ALG7 has been evolutionarily conserved and shown to be involved in a variety of functions. ALG7 is an early growth-response gene in yeast, and downregulation of ALG7 expression results in diminished N-glycosylation and secretion of Xenopus oocyte proteins. We have now investigated the consequences of diminished GPT activity in yeast using mutant ALG7 genes with deletions in the 3′ untranslated region (3′ UTR). We show that a 2.5- to 4-fold reduction in GPT activity gave rise to distinct phenotypes, whose severity was inversely related to the level of GPT activity. These phenotypes included hypersensitivity to tunicamycin, enlarged cell size, extensive aggregation, lack of a typical stationary (G 0) arrest, and defective spore germination. We conclude that yeast cells are sensitive to GPT dosage, and that attenuation of GPT activity interferes with various functions in the yeast life cycle.

Developmental Regulation and Tissue-Specific Expression of Hamster Dolichol-P-Dependent N-Acetylglucosamine-1-P Transferase (GPT)

The first enzyme in the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-phosphate transferase, GPT, has been implicated in the development of a wide variety of eukaryotes. GPT is encoded by ALG7, an early growth response gene, whose expression has been shown to affect the extent of N-glycosylation and secretion of proteins. To initiate the molecular characterization of ALG7 involvement in mammalian growth and differentiation, we have used the postnatally developing hamster submandibular gland (SMG) as an experimental paradigm. In this study we report that the ALG7 gene was differentially expressed during postnatal development and in terminally differentiated adult tissues. Throughout development, GPT activity paralleled ALG7 mRNA levels, suggesting that the amount of functional enzyme was determined by modulation of transcript abundance.

The potential dolichol recognition sequence of beta-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside as acceptor.

The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1-->4GlcNAc beta 1-->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.

Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells

We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11. The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency. A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2. The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa. This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene. The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein. Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin. Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.

The sequence and transcript heterogeneity of the yeast gene ALG1, an essential mannosyltransferase involved in N-glycosylation.

The yeast gene ALG1 encodes a mannosyltransferase which participates in the formation of the lipid-linked precursor oligosaccharide for N-glycosylation by catalyzing the formation of dolichol pyrophosphate (Dol-PP)-GlcNAc2Man from GDP-Man and Dol-PP-Glc-NAc2. The DNA region including the ALG1 gene was sequenced and found to contain an open reading frame of 1347 bases. The predicted ALG1 protein contained 449 amino acids (51.9 kDa) with a hydrophobic region near the amino terminus, suggesting it was an integral membrane protein, and four potential sites for N-glycosylation. Disruption of the ALG1 open reading frame by insertion of the URA3 gene showed that the ALG1 gene was essential for viability. Northern analysis and transcript protection showed that the ALG1 gene was transcribed into two classes of messages which differed by about 100 bases at their 5' end and shared a common 3' end. In actively growing cells the short transcript predominated, but as growth slowed the long transcript was more prevalent. The short transcript was predicted to encode the ALG1 protein while the long transcript was predicted to encode a 74-amino acid protein of unknown function. Disruption of the 74-amino acid reading frame, which ended 42 bases upstream of the potential start codon for the ALG1 protein, showed it was not essential for viability.

Amplification and molecular cloning of the hamster tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase gene. The hamster and yeast enzymes share a common peptide sequence.

The first step in the assembly of the dolichol-linked oligosaccharides required for asparagine-linked glycosylation in eukaryotes is catalyzed by a tunicamycin-sensitive, dolichol phosphate-dependent N-acetylglucosamine-1-phosphate transferase (GPT). A fragment of the gene encoding the enzyme from Chinese hamster ovary (CHO) cells was partially cloned and characterized by a novel strategy. By stepwise selection, CHO cells were made 80-fold resistant to tunicamycin and found to have 10-fold elevated levels of GPT activity. Using a cloned segment of the yeast ALG-7 gene, which encodes the putative GPT from yeast, an amplified gene was identified by Southern blotting of the CHO DNA and a 6.6-kilobase segment of the gene was molecularly cloned. A family of RNA molecules in the 2.0-2.2-kilobase range identified with a probe from this gene was overexpressed in the resistant cells. The cloned DNA revealed a 24-amino acid residue sequence that was 92% conserved with the corresponding yeast sequence.

Protein glycosylation in yeast: transcript heterogeneity of the ALG7 gene.

The first enzyme in the lipid-linked oligosaccharide biosynthetic pathway, UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (UDP-N-acetyl-D-glucosamine:dolichyl-phosphate-N-acetyl- D-glucosaminephosphotransferase, EC 2.7.8.15), is encoded by the ALG7 gene. We show that this gene is essential for cell growth, since a null mutation constructed with standard gene disruption techniques results in cell lethality. The ALG7 gene is transcribed into two major messages, approximately 1.38 and 1.56 kilobase pairs (kbp) in size, and this heterogeneity has been mapped to the 3' untranslated region. Two sets of tripartite sequences implicated in transcription termination begin 15 bp and 256 bp past the translation stop codon, TGA. The ratios of the two major transcripts change with gene dosage, with the longer mRNA becoming more abundant in cells containing higher levels of the ALG7 gene. Changes in transcript ratios are also observed in mutants defective in lipid-linked sugar-donor biosynthesis. In addition, there is 5' heterogeneity in the ALG7 mRNAs. The transcripts start at four initiation sites located within a 20-bp region. Two potentially functional TATA elements have been identified at positions -157 and -139, which may be involved in initiation from multiple sites. These features point to numerous factors that may be involved in the regulation of the expression of the ALG7 gene.