Molecular characterization and tissue distribution of a novel member of the S100 family of EF-hand proteins.

We have isolated from a human prostate cDNA library a cDNA encoding a novel member of the S100 family of EF-hand proteins. The encoded 99-amino acid protein, designated S100Z, is capable of interacting with another member of the family, S100P. S100Z cDNA was cloned into a bacterial expression system, and the S100Z protein was purified to homogeneity from bacterial lysates by a combination of hydrophobic column and gel-filtration chromatography. Direct amino acid sequencing of the 20 N-terminal amino acids confirmed that the sequence of the recombinant protein is identical to the sequence deduced from the cDNA. Low-resolution structural data have been obtained using circular dichroism and fluorescence spectroscopies, and equilibrium analytical centrifugation. These results show that S100Z is a dimeric, predominantly alpha-helical protein. Addition of calcium to a solution of S100Z changes the fluorescence intensity of the protein, indicating that S100Z is capable of binding calcium ions. Analysis of the calcium-binding isotherm indicates the existence of two calcium-binding sites with apparent affinities on the order of 5 x 10(6) and 10(2) M(-1). Binding of calcium results in conformational changes and exposure of hydrophobic surfaces on the protein. Using a PCR-based assay, we have detected differences in the expression level of S100Z mRNA in various tissues. The highest levels were found in spleen and leukocytes. S100Z gene expression appears to be deregulated in some tumor tissues, compared to expression in their normal counterparts.

Why are antibiotics prescribed for patients with acute bronchitis? A postintervention analysis.

BACKGROUND: Despite the findings in controlled trials that antibiotics provide limited benefit in the treatment of acute bronchitis, physicians frequently prescribe antibiotics for acute bronchitis. The aim of this study was to determine whether certain patient or provider characteristics could predict antibiotic use for acute bronchitis in a system where antibiotic use had already been substantially reduced through quality-improvement efforts. METHODS: A retrospective chart review was performed in an academic family medicine training center that had previously instituted a quality-improvement project to reduce antibiotic prescribing for acute bronchitis. Patients who had acute bronchitis diagnosed during an 18-month period and who had no other secondary diagnosis for respiratory distress or a condition that would justify antibiotics were selected from a computerized-record database and included in the study (n = 135). Charts were reviewed to document patient symptoms, physical findings, provider and patient characteristics, and treatment. RESULTS: Thirty-five (26%) patients received antibiotics for their acute bronchitis. Adults were more likely to receive antibiotics than children (34% vs 3%, P < .001). Analysis of 20 different symptoms and physical findings showed that symptoms and signs were poor predictors of antibiotic use. Likewise, no significant differences were found based on prescribing habits of individual providers or provider level of training. CONCLUSION: In a setting where antibiotic use for acute bronchitis had been decreased through an ongoing quality-improvement effort, it did not appear that providers selectively used antibiotics for patients with certain symptoms or signs. Other factors, such as nonclinical cues, might drive antibiotic prescribing even after clinical variation is suppressed.

Does acute bronchitis really exist? A reconceptualization of acute viral respiratory infections.

BACKGROUND: Considerable overlap exists in patient presentations and physical findings in viral upper respiratory tract infections (URIs) and acute bronchitis. Our goal was to determine whether there are any clinical cues that could help physicians differentiate between these 2 conditions. METHODS: We performed a retrospective chart audit on 135 patients who had been given a diagnosis of acute bronchitis and a random sample of 409 patients with URIs over a 2.5-year period. Patient and provider characteristics, patient symptoms, and physical findings were compared with bivariate analyses and then entered into a logistic regression model. RESULTS: In bivariate analyses, a number of demographic variables, symptoms, and signs were associated with acute bronchitis. Multivariate analysis showed that the strongest independent predictors of acute bronchitis were cough (adjusted odds ratio [AOR]=21.12; 95% confidence interval [CI], 6.01-74.26), and wheezing on examination (AOR=12.16; 95% CI, 5.39-27.42). Nausea was the strongest independent predictor that the diagnosis would not be acute bronchitis (AOR=0.01; 95% CI, 0.01-0.85). However, there was considerable overlap between the 2 conditions, and the logistic model explained only 37% of the variation between the diagnoses. CONCLUSIONS: We hypothesize that sinusitis, URI, and acute bronchitis are all variations of the same clinical condition (acute respiratory infection) and should be conceptualized as a single clinical entity, with primary symptoms related to different anatomic areas rather than as different conditions.

Evidence for Gal3p's cytoplasmic location and Gal80p's dual cytoplasmic-nuclear location implicates new mechanisms for controlling Gal4p activity in Saccharomyces cerevisiae.

Genetics and in vitro studies have shown that the direct interaction between Gal3p and Gal80p plays a central role in galactose-dependent Gal4p-mediated GAL gene expression in the yeast Saccharomyces cerevisiae. Precisely how Gal3p-Gal80p interaction effects induction is not clear. It has been assumed that Gal3p interacts with Gal80p in the nucleus upon galactose addition to release Gal80p inhibition of Gal4p. Although Gal80p has been shown to possess nuclear localization signal (NLS) peptides, the subcellular distribution of neither Gal80p nor Gal3p was previously determined. Here we report that Gal3p is located in the cytoplasm and apparently excluded from the nucleus. We show that Gal80p is located in both the cytoplasm and the nucleus. Converting Gal80p into a nucleus-localized protein (NLS-Gal80p) by exogenous NLS addition impairs GAL gene induction. The impaired induction can be partially suppressed by targeting Gal3p to the nucleus (NLS-Gal3p). We document a very rapid association between NLS-Gal3p and Gal80p in vivo in response to galactose, illustrating that the nuclear import of Gal80p is very rapid and efficient. We also demonstrate that nucleus-localized NLS-Gal80p can move out of the nucleus and shuttle between nuclei in yeast heterokaryons. These results are the first indication that the subcellular distribution dynamics of the Gal3 and Gal80 proteins play a role in regulating Gal4p-mediated GAL gene expression in vivo.

Urine free light chains in SLE: clonal markers of B-cell activity and potential link to in vivo secreted Ig.

As a marker of in vivo B-cell activity, urine levels of free light chain (FLC) were measured twice weekly by radioimmunoassay (RIA) and correlated with disease activity over periods of 5-10 months in seven patients with systemic lupus erythematosus (SLE). In addition, RIA-measured urine albumin was used to track glomerular injury, and alpha1-microglobulin (alpha1-M) levels, 28- to 32-kDa protein, provided control measurements on excretion of low-molecular-weight proteins. As controls, urine FLC levels were obtained from healthy normals and in subjects with acute pharyngitis, sickle-cell anemia, and acute sepsis or pneumonia. The control results showed that with acute sepsis/pneumonia had marked increases in urine FLC, while pharyngitis and sickle-cell controls had normal FLC levels. In SLE, active patients receiving intravenous cyclophosphamide and high-dose steroids exhibited highly increased urine FLC that fluctuated widely during therapy and fell to normal range levels with disease remission. During active SLE, urine albumin often was increased, while alpha1-M levels remained in normal range. In contrast to the increased FLC of active disease, inactive patients on low-dose maintenance therapy had predominantly normal FLC levels throughout the collection period. These results support our hypothesis that longitudinal levels of urine FLC can be used to track disease-related B-cell activity in SLE. Furthermore, we suggest that the urine FLC of active SLE would share LC idiotype with the clonal associated in vivo secreted Ig, and thus permit the identification of these antibodies that are targeted to the culprit immunogen(s) responsible for the pathogenesis of SLE.

Vectors allowing amplified expression of the Saccharomyces cerevisiae Gal3p-Gal80p-Gal4p transcription switch: applications to galactose-regulated high-level production of proteins.

The Gal4, Gal80, and Gal3 proteins of Saccharomyces cerevisiae constitute a galactose-responsive regulatory switch for GAL gene promoters. The low cellular levels of these proteins have hampered mechanistic studies and limit the utility of the GAL gene promoters for high-yield production of endogenous and exogenous proteins. We have constructed two new vectors, pMEGA2 and pMEGA2-DeltaURA3, that increase the level of the Gal4p-Gal80p-Gal3p switch proteins under conditions that preserve the Gal3p-Gal80p-Gal4p stoichiometries required for normal switch function. Cells carrying pMEGA2 show 15- to 20-fold more Gal4p and 30- to 40-fold more Gal3p and Gal80p than cells lacking pMEGA2. These high levels of Gal4p, Gal80p, and Gal3p do not perturb the integrity of galactose-inducible regulation. Cells that carry pMEGA2 exhibit normal galactose-induction kinetics for the chromosomal MEL1 gene expression and normal, albeit slower, log-phase growth. Insertion of the MEL1 gene into pMEGA2 provides a 24- to 30-fold increase in the Mel1 protein. Cells carrying a 2-microm-based URA3-selectable plasmid containing a GAL1pro:lacZ reporter gene and a second plasmid, pMEGA2-DeltaURA3, produce 12-fold more beta-galactosidase than cells carrying only the GAL1pro:lacZ reporter plasmid. The performance of the MEGA plasmids in providing amplified production of the Gal3, Gal80, and Gal4 proteins should prove useful in investigations of the mechanistic aspects of these transcription switch proteins and in work aimed at achieving high-level, galactose-regulatable production of proteins in yeast.

The Gal3p-Gal80p-Gal4p transcription switch of yeast: Gal3p destabilizes the Gal80p-Gal4p complex in response to galactose and ATP.

The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation of GAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

Genetic and carbon source regulation of phosphorylation of Sip1p, a Snf1p-associated protein involved in carbon response in Saccharomyces cerevisiae.

The SIP1 gene of Saccharomyces cerevisiae is a carbon-catabolite-specific negative regulator of GAL gene transcription and acts as a multicopy suppressor of growth defects associated with impaired Snf1p protein kinase activity. The Sip1 protein is known to undergo phosphorylation when associated in vitro with the Snf1 protein kinase. We have carried out in vivo studies of the genetic and carbon control of Sip1p phosphorylation. Metabolic labeling reveals phosphorylation of Sip1p under both carbon catabolite-repressing and non-repressing conditions and in both SNF1 wild-type and snf1-deletion cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot assay, we detect apparent changes in Sip1p phosphorylation states in response to changes in carbon source. At least one dephosphorylation of Sip1p occurs with a shift from non-repressing carbon source to repressing carbon source. The MIG1 gene, acting through SNF1-dependent and SNF1-independent pathways, is required for some Sip1p phosphorylations. REG1 appears to be required for at least one dephosphorylation of Sip1p, whereas SSN6 appears to be required for at least one phosphorylation of Sip1p. These results reveal new complexities in carbon response signaling, and may reflect the involvement of the Sip1 protein in the same complex as the Mig1 and Ssn6 proteins.

SIP1 is a catabolite repression-specific negative regulator of GAL gene expression.

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

Comprehensive pharmaceutical services for pediatric patients.

The development of comprehensive pharmaceutical services for pediatric patients at a tertiary-care teaching hospital is described. A team of three staff pharmacists, a clinical specialist, and supportive personnel was formed. A pediatric pharmacy, operating from 0800 to 2100 daily, was created in a separate area of the central pharmacy to focus on potential problems with pediatric dosage calculations and drug administration. The team staffs the pediatric pharmacy for 75% of its day and evening shifts. The pharmacy prepares the 24-hour unit dose supply for each nursing unit, processes new orders, and provides drug information and problem-solving services. Clinical services are provided by the decentralized pharmacist, a rotating member of the team who makes rounds each day to the pediatric nursing units to review patient charts, provide medication information, and answer questions. The pediatric clinical specialist conducts educational programs, provides consultations, maintains reference materials, monitors pharmacokinetic evaluations, reviews medication communication forms, and assists in developing medication administration procedures. The creation of a pediatric pharmacy and a pediatric pharmacy team that coordinates both dispensing and clinical functions has made it possible to provide comprehensive pharmaceutical services to pediatric patients.

Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon.

The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.

Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains.

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.

The mechanism of inducer formation in gal3 mutants of the yeast galactose system is independent of normal galactose metabolism and mitochondrial respiratory function.

Saccharomyces cerevisiae cells defective in GAL3 function exhibit either one of two phenotypes. The gal3 mutation in an otherwise normal cell causes a 2-5-day delay in the galactose triggered induction of GAL/MEL gene transcription. This long term adaptation (LTA) phenotype has been ascribed to inefficient inducer formation. The gal3 mutation causes a noninducible phenotype for GAL/MEL transcription if cells are defective in Leloir pathway function, in glycolysis or in respiratory function. It was recently shown that multiple copies of the intact GAL1 gene partially suppress the LTA phenotype of gal3 cells. Here we report that constitutively expressed GAL1 restored gal3 mutants to the rapidly inducible phenotype characteristic of wild-type cells and conferred rapid inducibility to gal3 gal10, gal3 gal7 or gal3 rho- strains that are normally noninducible. As shown by immunoblot analysis, the GAL1-mediated induction exhibits phosphorylation of the GAL4 protein, suggesting a mechanism similar to GAL3-mediated induction. Altogether our results indicate that the deciding factor in the inducibility of the GAL/MEL genes in gal3 strains is the Gal3p-like activity of Gal1p. Based on the above we conclude that inducer formation does not require normal metabolism of galactose nor does it require mitochondrial respiratory function. These conclusions vitiate previous explanations for gal3 associated long-term adaptation and noninducible phenotypes.

GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator.

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

Phosphorylated forms of GAL4 are correlated with ability to activate transcription.

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.

Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible.

The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.