Regulated overproduction of α-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis

Summary High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called SwARS1. The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5–10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned -amylase gene from S. occidentalis secreted about five times more -amylase than cells without additional copies of the -amylase gene. Both the chromosomal copy and the plasmid-carried copies of the -amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.     

Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active -amylase (76 250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae -amylase acted by endo-hydrolysis on glucose polymers containing -1,4 and -1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH₂-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro-Glu-Ser-Val-Thr-Gly. The L. kononenkoae -amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Molecular cloning and characterization of a Candida tsukubaensis α-glucosidase gene in the yeast Saccharomyces cerevisiae

Summary The molecular cloning of an -glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cerevisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing -1,2, -1,3, -1,4 and 1,6 linked, as well as aryl and alkyl, d-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an -glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2–4.6, a temperature optimum of 58°C and is readily inactivated at pasteurization temperature (60°C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X--d-glucoside to detect the expression of the cloned -glucosidase in S. cerevisiae transformants, was developed.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Molecular properties of the lys1 + gene and the regulation of α-aminoadipate reductase in Schizosaccharomyces pombe

The -aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1 ⁺ gene and the regulation of the encoded -aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-EcoRI fragment of S. pombe DNA, containing a functional lys1 ⁺ gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented minimal or YEPD media was less than the activity of cells grown in minimal mediu. The AAR of S. pombe was more sensitive to feedback inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the -aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.     

Interspecific actions of α mating pheromones on the a mating-type cells of three Saccharomyces yeasts

Summary The a mating pheromones synthesized in three Saccharomyces yeasts (S. cerevisiae, S. kluyveri, and S. exiguus) displayed interspecific actions on the a cells of all three species despite the fact that the amino acid sequences of all three pheromones are different. Mating between species, however, did not occur. The interspecifie pheromone — a cell reaction was not necessarily more effective than the interspecific one. Deceased on March 28, 1987     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

Contractile protein gene expression in serum-free cultured adult rat cardiac myocytes

The effects of two adhesion substrates (serum and laminin) and time in culture on the expression of genes encoding myosin heavy chain (MHC) isoforms and -skeletal actin were analysed in myocytes isolated from adult rat heart and maintained in serum-free culture. Relative messenger ribonucleic acid (mRNA) abundances were quantitated by dot-blot analysis. Gene expression was not influenced by the substrate used. Time in culture induced a decrease in total mRNA abundance and an up-regulation of -MHC and -skeletal actin genes. It is proposed that atrophy of adult myocytes is associated with a pattern of gene expression similar to the fetal program.     

Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-α antibody

The role of tumor necrosis factor- in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor- and in eight chimpanzees after intravenous administration of endotoxin in the absence or presence of concurrent treatment with a neutralizing anti-tumor necrosis factor- monoclonal antibody. Injection of tumor necrosis factor- in humans elicited a significant, instantaneous (after 15 min) increase in the plasma concentrations of both types of soluble tumor necrosis factor receptors. In chimpanzees, treatment with the anti-tumor necrosis factor- antibody completely neutralized endotoxin-induced tumor necrosis factor- activity. The release of soluble tumor necrosis factor receptors was strongly (80–90%) inhibited in the presence of the neutralizing antibody. Our results indicate that tumor necrosis factor- is a prime mediator of endotoxin-induced release of its own soluble receptors.     

Purification and genetic control of α-pheromone-inactivating glycoproteins in the yeast Saccharomyces cerevisiae

Summary Two kinds of a-mating-type-specific proteins inactivating pheromone ( factor) were purified from heat shock extract of MATa cells. Their molecular weights were estimated to be 400,000 and 200,000 by gel filtration. Both proteins were detected in MATa SST1 cells but not in MAT SST1, MATa sst1-1 and MATa/MAT SST1/SST1 cells. In addition, the proteins were detected in mat2-1 SST1 cells but not in mat1-2 SST1 cells. From these results, it is concluded that these proteins are synthesized under the control of the SST1 gene and responsible for the Barrier action of MATa cells. The relationship of these proteins to the secreted Barrier protein having a higher molecular weight is discussed.Abbreviations Mr. molecular weight - PBS 10 mM phosphate buffer, pH 5.5 - SDS sodium dodecyl sulfate     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Expression of transforming growth factor alpha in human tissues: Immunohistochemical study and Northern blot analysis

Summary The expression of transforming growth factor alpha (TGF-) was examined in various human tissues and the fetus, using immunohistochemistry and Northern blot analysis. TGF- immunoreactivity was detected mainly in the epithelial cells of the digestive tract, liver, pancreas, kidney, thyroid, adrenal, skin, mammary gland and genital organs. In the digestive tract, epithelial cells with regenerative change or hyperplastic change showed strong immunoreactivity to TGF-. Peripheral nerve, vessels, megakaryocytes and macrophages in the lung and spleen were also positive for TGF-. By Northern blot analysis the expression of TGF- mRNA was confirmed in the digestive tract, salivary gland, thyroid, kidney and mammary gland. In the human fetus, the nerve tissues, liver, adrenal and kidney were positive for TGF-. Strong immunoreactivity to TGF- was observed in the hepatocytes of the fetus. These findings indicate that TGF- is produced by a variety of nonneoplastic cells in both adult and fetal tissues.     

Presence of two isoforms of Na,K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the ₁ isoform of the catalytic subunit, whereas the collecting duct expresses an ₃-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC₅₀ 5.10^–6 M) which is recognized by an anti- ₃ antibody and another moiety of lower affinity for ouabain (IC₅₀ 5.10^–4 M) which is recognized by an anti- ₁ antibody. Whether these two subpopulations correspond to different isoforms of the subunit of Na,K-ATPase ( ₁ and ₃-like) remains to be determined.     

Proteinase — Antiproteinase imbalance in meningitis: Determination of α 1 proteinase inhibitor (α 1PI), elastase — α-1PI complex,and elastase inhibition capacity in cerebrospinal fluid

Summary Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness.Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions.To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase 1-proteinase inhibitor (elastase- 1PI), 1-proteinase inhibitor ( 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-214 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month–15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months–15 years. The concentration of elastase- 1PI in the BM group (median 552 g/l) was significantly higher than in either the NBM group (median 30 g/l,p<0.01) or the control group (median 30 g/l,p<0.01). Similarly, the 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l,p<0.025) or the control group (median 6.3 mg/l,p<0.001). The concentration of elastase- 1PI shows a significant correlation with the duration of the infectious symptoms before admission to the hospital (r=0.51,p<0.02), but not with the number of neutrophil granulocytesr=0.23, p=0.21).Free elastolytic capacity in CSF could be demonstrated in 4 patients: 1 with BM, 2 with NBM, and 1 with pertussis pneumonia and enzephalitis.The measured insufficiency of the proteinase-antiproteinase system may indicate high-risk patients in need of additional anti-inflammatory therapy, e.g., with corticosteroids, during the initial phase of meningitis.Abbreviations 1PI 1-proteinase inhibitor, 1-antitrypsin - elastase- 1PI complex elastase- 1-proteinase inhibitor complex - EIC elastase inhibition capacity - BM group: bacterial meningitis - NBM group: non-bacterial meningitis - CSF cerebrospinal fluid     

Molecular cloning of the DAC2/FUS3 gene essential for pheromone-induced G1-arrest of the cell cycle in Saccharomyces cerevisiae

Summary Mating pheromones, known as a and -factors, arrest the division of cells of opposite mating types, and a respectively, in Saccharomyces cerevisiae. I have cloned the DAC2 gene, which is required for both pheromone-induced division-arrest and cell-fusion during conjugation. The constructed dac2::LEU2 null mutation leads to defects in both pheromone-induced divisionarrest and cell-fusion during conjugation; it also suppresses the growth defect caused by the gpal mutation (a mutation in the subunit of the S. cerevisiae G protein). These results indicate that DAC2 may be the same gene as FUS3, which was recently isolated by Elion et al. (1990) as a gene essential for cell-fusion during conjugation. The dac2::LEU2 null mutant also showed morphological alterations in response to mating pheromones. I show here that the DAC2 product plays an essential role in both the division-arrest signalling pathway of the yeast pheromone response and in cell-fusion during conjugation.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.     

Immunohistochemical studies on oncogene products (EGF-R,c-erbB-2) and growth factors (EGF,TGF-α) in human breast cancer: their relationship to oestrogen receptor status,histological grade,mitotic index and nodal status

Summary In this investigation, 83 human mammary carcinomas were examined for the expression of oestrogen receptor (ER), epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF), transforming growth factor alpha (TGF-), c-erbB-2, histological grade, mitotic index and nodal status, all of which are reportedly prognostically significant factors (Bloom and Richardson 1957; Baak et al. 1985; Wright et al. 1989). ER expression was biochemically recognized in 43.4% of mammary carcinomas, and EGF-R, EGF, TGF- and c-erbB-2 were histochemically recognized in 25.3, 14.5, 27.7 and 18.0% of mammary carcinomas examined respectively, using conventional sections of buffered formalin-fixed, paraffin-embedded tissue and monoclonal or polyclonal antibodies. There were significant relationships between negative ER and positive EGF-R or TGF-; positive EGF-R and TGF-; positive EGF-R and c-erbB-2; and positive c-erbB-2 and TGF-. The single changes which were the negative ER and the positive c-erbB-2 correlated with histological grade and mitotic index. Co-expression of EGF-R and TGF- correlated with positive nodal status. Therefore, the present investigation indicates that the negative ER, single expression of c-erbB-2 and co-expression of EGF-R and TGF- are important markers which contribute indirectly to prognosis, which reconfirms previous findings on the former two while adding the new finding that immuno-histochemical demonstration of expression of EGF-R and TGF- may provide useful information for selecting the appropriate treatment.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

Comparison of the eIF-2α Homologous Proteins of Seven Ranaviruses (Iridoviridae)

The -subunit of the eukaryotic initiation factor 2 (eIF-2) is a key component of the translation machinery of the cell. In response to cellular stress such as viral infections, eIF-2 is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis. The importance of eIF-2 as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR. Thus, Vaccinia virus encodes K3L protein, which resembles eIF-2 and acts as a pseudo-substrate inhibitor of PKR. Nucleotide sequencing of the genome of epizootic haematopoietic necrosis virus (EHNV), a member of the genus ranavirus of Iridoviridae, has revealed an eIF-2 equivalent gene. We have cloned and sequenced eIF-2 genes of several iridoviruses of fishes and frogs. The eIF-2 open reading frames and deduced proteins of the iridoviruses investigated exhibit a high degree of homology of both nucleotide and amino acid sequences. At the N-terminus, the iridoviral eIF-2 shows significant homology to the N-termini of cellular initiation factor 2- of various species, to full-length poxviral eIF-2 proteins, and to the S1 domain of ribosomal proteins. Comparison of amino acid sequences of corresponding iridoviral proteins with eIF-2 homologous proteins of poxviruses and eukaryotes has revealed a high conservation of motifs. A phylogenetic analysis of eukaryotic eIF-2 and poxvirus and iridovirus eIF-2 sequences has demonstrated the relationship of these iridoviruses. In order to investigate the role of the eIF-2 equivalent, respective genes have been expressed in prokaryotic and eukaryotic (insect, fish and chicken cell) systems. The iridoviral eIF-2 protein has a molecular weight of 31 kDa and is cytoplasmic. The cellular and viral protein synthesis of iridoviruses is probably regulated by a mechanism similar to that of Vaccinia virus. Frog-virus 3, the type species of the genus ranavirus of Iridoviridae, has a unique translational efficiency and, moreover, down-regulates the cellular protein synthesis of infected cells.