Constitutive Synthesis Research Articles

Functional Expression of c-kit by Acute Myelogenous Leukemia Blasts Is Enhanced by Tumor Necrosis Factor-α Through Posttranscriptional mRNA Stabilization by a Labile Protein

The c-kit proto-oncogene encodes a transmembrane glycoprotein identical to the receptor for the recently cloned stem cell factor (SCF). The present study examines constitutive synthesis of transcripts in primary acute myelogenous leukemia (AML) blasts and the effects of recombinant human tumor necrosis factor (TNF)-alpha on c-kit mRNA expression in these cells. The c-kit transcripts were detectable at low levels in 10 of 10 different AML samples investigated. TNF treatment of AML cells was associated with enhanced c-kit mRNA expression in all specimens. Nuclear run-on transcription assays indicated that the c-kit gene was transcriptionally active in all leukemias examined and the rate of transcription was unaffected by exposure to TNF, suggesting posttranscriptional control mechanisms of c-kit mRNA accumulation. In the absence of TNF, the half-life of c-kit transcripts was 2 to 3 hours, while in TNF-treated AML cells, c-kit half-life was found to be 5 to 9 hours. Inhibition of protein synthesis reduced TNF-induced c-kit mRNA expression by Northern blot analysis, but did not affect the rate of c-kit gene transcription. In the presence of inhibition of protein synthesis, the half-life of c-kit transcripts in TNF-induced leukemia cells decreased to 2 to 4 hours. These findings indicate that levels of c-kit mRNA are controlled by a labile protein that is involved in TNF-mediated stabilization of c-kit transcripts. The effects of TNF-alpha also extended to the protein level in that TNF-alpha treatment of primary AMLs was associated with enhanced surface expression of the SCF receptor by some of these cells. While exogenous SCF induced clonogenic growth of all primary AML samples investigated, TNF-alpha failed to stimulate leukemic cells to proliferate. However, the combination of SCF and TNF-alpha resulted in synergistic growth stimulation in seven of nine different AML specimens investigated. The finding of transmodulation of the SCF receptor through posttranscriptional modifications might further contribute to our understanding of the synergistic interplay of TNF-alpha and SCF.

Constitutive synthesis of polyoma antisense RNA renders cells immune to virus infection

Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.

Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae.

We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.

Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae.

We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells.

Constitutive synthesis of tumor necrosis factor in the thymus.

Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown. If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions. To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence. This construct was inserted into the murine genome, yielding 13 transgenic founders. Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro. Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined. Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium. CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells. CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity. The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.

Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent.

Pentoxifylline Inhibits Certain Constitutive and Tumor Necrosis Factor-α-Induced Activities of Human Normal Dermal Fibroblasts

Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.

Constitutive synthesis of insulin-like growth factor-II by primary osteoblast-enriched cultures from fetal rat calvariae

Constitutive synthesis of insulin-like growth factor-II by primary osteoblast-enriched cultures from fetal rat calvariae

Constitutive synthesis of insulin-like growth factor-II by primary osteoblast-enriched cultures from fetal rat calvariae.

While a number of osteotropic hormones regulate insulin-like growth factor-I (IGF-I) synthesis in osteoblast-enriched (Ob) and intact bone cultures, their direct effects on IGF-II production are still unresolved. For example, cAMP stimulators, such as PTH and prostaglandin E2, increase Ob IGF-I transcript and polypeptide levels within the first 24 h of treatment, but have no effect on IGF-II expression. To examine the possibility that other circulating factors could directly modify IGF-II synthesis by osteoblasts, primary rat Ob cultures were briefly treated with a number of polypeptide and steroid hormones known to regulate bone metabolism. Prepro-IGF-II steady state transcripts were assessed by Northern blot analysis, and immunoreactive polypeptide levels (iIGF-II) were examined by RIA. Predominant prepro-IGF-II transcripts of 3.7 kilobases were readily detected in quiescent Ob cultures, and constitutive iIGF-II levels were approximately 2-7 nM throughout the first 24 h of culture. GH, placental lactogen, insulin, cortisol, testosterone, T3, 17 beta-estradiol, and 1,25-dihydroxyvitamin D3 each had no effect on prepro-IGF-II transcripts within 6 h or on iIGF-II polypeptide expression within a 24-h period. These studies indicate that IGF-II synthesis is constitutive in unstimulated primary fetal rat Ob cultures, and that these levels are not directly modulated by short term treatment with a variety of osteotropic hormones.

Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene

The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations.

Effect of interferon-gamma on complement gene expression in different cell types.

We have studied the expression of the complement components C2, C3, factor B, C1 inhibitor (C1-inh), C4-binding protein (C4-bp) and factor H in human peripheral blood monocytes, skin fibroblasts, umbilical vein endothelial cells (HUVEC) and the human hepatoma cell line G2 (Hep G2) in the absence and the presence of interferon-gamma (IFN-gamma). E.l.i.s.a. performed on culture fluids, run-on transcription assays, Northern blot and double-dilution dot-blot techniques confirmed that monocytes expressed all six components, whereas fibroblasts, HUVEC and HepG2 each expressed five of the six components. Fibroblasts and HUVEC did not synthesize C4-bp, and Hep G2 did not produce factor H. In addition to these differences, the synthesis rates of C3, C1-inh and factor H were not the same in all cell types. However, the synthesis rates of C2 and factor B were similar in all four cell types. The half-lives of the mRNAs were shorter in monocytes than in other cell types. Monocyte factor H mRNA had a half-life of 12 min in monocytes, compared with over 3 h in fibroblasts and HUVEC. The instability of factor H mRNA in monocytes may contribute to their low factor H secretion rate. IFN-gamma produced dose-dependent stimulation of C2, factor B, C1-inh, C4-bp and factor H synthesis by all cell types expressing these proteins, but decreased C3 synthesis in all four cell types. Cell-specific differences in the response to IFN-gamma were observed. The increased rates of transcription of the C1-inh and factor H genes in HUVEC were greater than in other cell types, while the increased rate of transcription of the C2, factor B and C1-inh genes in Hep G2 cells was less than in other cell types. IFN-gamma did not affect the stability of C3, factor H or C4 bp mRNAs, but increased the stability of factor B and C1-inh mRNAs and decreased the stability of C2 mRNA. Although these changes occurred in all four cell types studied, the half-life of C1-inh mRNA in monocytes was increased almost 4-fold, whereas the increases in the other cell types were less than 30%. These data show that the constitutive synthesis rates of complement components may vary in the different cell types. They also show that the degree of change in synthesis rates in response to IFN-gamma in each of the cell types often varies due to differences in transcriptional response, sometimes in association with changes in mRNA stability.

Regulation of xylanase activity in cellulomonas sp. IIbc

AbstractThe regulation mechanism governing the xylanolytic activity in the strain Cellulomonas sp. IIbc was studied. High levels of activity were detected during the cultivation on cellulose as the only carbon source. No activity was produced with glucose, xylose or cellobiose cultures, but in the last one, the synthesis was de‐repressed when the sugar concentration dropped to 0.2%. The activity was not inhibited by glucose, cellobiose and xylose up to 1% concentration.A basal constitutive synthesis was detected in nutrient broth cultures. At the same time, xylose and cellobiose acted as inducers of the xylanase activity.

Enhanced resistance to HIV-1 replication in U937 cells stably transfected with the human IFN-beta gene behind an MHC promoter fragment.

Cells of the monocyte lineage act as a major reservoir for HIV, and ways of enhancing the resistance of mononuclear phagocytes to HIV replication would be useful for delaying the onset of AIDS in infected individuals. Seif et al. (J. Virol. 65:664, 1991) have recently shown the possibility of obtaining stable antiviral expression (SAVE), directed against three nonretroviral RNA viruses, and normal cell viability in a significant percentage of murine BALB/c 3T3 cells transformed with an IFN-beta expression plasmid under the control of the 0.6-kb XhoII-NruI promoter region of the murine H-2Kb MHC gene. In the present paper, we show that it is possible to establish SAVE in human promonocytic cells. Cells of the human promonocytic U937 line were stably transfected with a human IFN-beta expression plasmid carrying the neo- and human IFN-beta-coding sequences under the control of the H-2Kb promoter fragment previously used in murine cells. After selection with G418, two transformed clones were isolated that released small amounts of human IFN-beta into the culture medium, without affecting the expression of CD4 and leucocyte function-associated Ag-1 differentiation Ag. The presence of construct-derived IFN-beta mRNA was demonstrated by polymerase chain reaction amplification of cDNA, and the level of 2-5A synthetase, one of the major IFN-induced antiviral proteins, was shown to be constitutively increased. These clones were less permissive for HIV-1 than control clones transformed with the neo gene only. The antiviral state could be modulated by anti-IFN-beta antibodies, in that the continuous presence of antibodies in the culture medium abolished the enhanced resistance to HIV-1 replication, whereas the withdrawal of the antiserum restored the antiviral state, indicating that it did indeed result from the constitutive synthesis of human IFN-beta. These results demonstrate the possibility of restricting HIV-1 replication in human promonocytic cells by establishing SAVE. Further exploration of this method as a possible approach to somatic cell gene therapy of HIV infection appears worthwhile.

Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression.

Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4- to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GAL4 regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GAL4 promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression of GAL1 expression. Furthermore, promoter deletions that modestly reduce GAL4 expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL1 expression. These results suggest that GAL4 works synergistically to activate GAL1 expression. Thus, glucose repression of GAL1 expression is due largely to a relatively small reduction of GAL4 protein levels caused by reduced GAL4 transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplified responses to environmental changes.

Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.

Cell cycle-specific expression of the SWI4 transcription factor is required for the cell cycle regulation of HO transcription.

Expression of the HO endonuclease triggers mating-type switching in Saccharomyces cerevisiae. Transcription of the HO gene is start-dependent and restricted to the late G1/early S phase of haploid mother cells. The HO promoter contains 10 copies of a cell cycle-regulated upstream activation sequence, which is activated by SWI4 and SWI6. SWI4 mRNA levels vary at least 10-fold throughout the cell cycle and rise sharply just before the rise in HO mRNA levels. Constitutive synthesis of SWI4 mRNA leads to constitutive synthesis of HO mRNA. These data suggest that the cell cycle regulation of SWI4 mRNA is required for the tight cell cycle regulation of HO transcription. High-level constitutive synthesis of SWI4 also suppresses swi5 and swi6 mutations, suggesting that SWI4 is the predominant activator of HO transcription and that mutations in negative regulators of SWI4 could be isolated as suppressors of swi6 mutations. One recessive suppressor of swi6 (ssx1-1) that allowed high-level expression of SWI4 during alpha-factor arrest and constitutive expression of both SWI4 and HO after release from the arrest was isolated. This result suggests that SSX1 has a negative regulatory role in the cell-cycle regulation of SWI4 mRNA accumulation.

The PH084 Gene of Saccharomyces cerevisiae Encodes an Inorganic Phosphate Transporter

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter.

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

Isolation of mutants of Aspergillus awamori with enhanced production of extracellular xylanase and ?-xylosidase

Plate screening tests were designed for the selection and isolation of mutant strains of the fungus Aspergillus awamori CMI 142717 showing over-production and constitutive synthesis of xylanase and β-xylosidase. Following mutation by N-methyl-N-nitro-N-nitrosoguanidine, nitrous acid and UV (254 nm), two generations of mutants were isolated and cultured in shake fiasks containing glucose, ball-milled oat straw or oat speit xylan as carbon source. Growth of a number of selected mutants in shake flask culture on medium containing oat spelt xylan produced the highest titres of xylanase and β-xylosidase. Thus, xylanase producton by mutant AANTG43 was 132 U/ml when the Somogyl-Nelson (alkaline copper) method of measuring reducing sugar released was used, or 1160 U/ml using the dinitrosalicylic acid method of reducing sugar analysis. These values were 8-fold higher than those produced by the wild type. A 20-fold improvement in β-xylosidase production was produced by mutant AANO19 (3.51 U/ml). The titres for these two enzyme activities are the highest recorded so far in the literature. Mutant AANTG43 also produced high levels of xylanase (49.8 U/ml) in submerged culture in a fermenter and showed a substantial improvement in the overall productivity of enzyme compared to the wild type strain.