Synthesis Of Β-galactosidase Research Articles

Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe

A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time. The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27 017 Da. The cloned GST gene was expressed and was found to function in S. pombe, Saccharomyces cerevisiae, and Escherichia coli. The plasmid pGT207 encoding the S. pombe GST gene appeared to be able to accelerate the growth of a wild type S. pombe culture. In a culture of S. pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone. The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless β-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000. β-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione. It was also induced by high temperature. These results suggest that the cloned S. pombe GST gene is involved in the oxidative stress response.

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless β-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of β-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.

Optimization of pH, Temperature and Inoculum Ratio for the Production ofβ-D-Galactosidase byKluyveromyces marxianus Using a Lactose-free Medium

The pH, temperature and inoculum ratio for the production of β-galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar-cane molasses (100 g/l) in a lactose-free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by-product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose-free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β-galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7.

Estrogen and estrogen mimics contamination in water and the role of sewage treatment

The yeast estrogen screen was applied to sewage treatment process waters to identify the presence of estrogenic activity and to investigate the fate and behavior of estrogenic substances through treatment. Hydrophobic fractions in the water phase were extracted and concentrated using C18 cartridges for the effective extraction of 17β-estradiol (E2) and other estrogen mimics. Clear dose-dependent elevation in the synthesis of β-galactosidase in the yeast screen was observed with all the samples tested, demonstrating that these samples were estrogenic. However, estrogenic activity tended to reduce during the treatment, especiallyiin the biological process. Quantification results of the yeast estrogen screen in terms of E2 equivalent were compared with actual E2 concentrations measured by an ELISA. E2 occupied 34% of the whole estrogenicity in the raw sewage, while almost 100% in the final effluent. The analyses of the sewage treatment process waters revealed that human estrogens are major causative substances in terms of estrogenic activity in sewage and its treated effluent. Although findings of possible correlation of environmental estrogens with the real impact on human health and the ecosystem are still the focus of scientific debate and investigation, proper management should be established in the sewage treatment process which receives various environmental contaminants.

Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast- Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11 261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless β-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of β-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso- N-acetylpenicillamine.

Is plant biotin holocarboxylase synthetase a bifunctional enzyme?

Holocarboxylase synthetases (HCSs) catalyse the biotinylation of biotin-dependent carboxylases in both prokaryotes and eukaryotes. In Escherichia coli and Bacillus subtilis, the protein also acts as a transcriptional repressor that regulates the synthesis of biotin. Previously, we isolated and characterized a cDNA encoding an Arabidopsis thaliana HCS and subsequently assigned this enzyme form to the chloroplast compartment. To investigate whether or not the Arabidopsis protein may function as a regulator in E. coli, we have expressed the functional plant HCS in a birA-derepressed mutant strain of E. coli devoid of the corresponding E. coli protein and carrying a promoter-less LacZ gene marker inserted into the biotin operon, such that the bio promoter drives the synthesis of β-galactosidase. Our data demonstrate that although the expressed plant HCS efficiently complemented the function of apo-carboxylase biotinylation in E. coli, it proved unable to regulate the expression of the biotin biosynthetic genes.

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli

The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded beta-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased beta-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of beta-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.

Systematic study of gene expression and transcription organization in the gntZ-ywaA region of the Bacillus subtilis genome.

Within the framework of the international project 'The functional analysis of the Bacillus subtilis genome' in Japan and Europe, the gene expression and transcription organization of the gntZ-ywaA region (160 kb) of the B. subtilis genome has been systematically analysed. First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN. No essential gene was found which could not be inactivated. All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium. But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium. The synthesis of beta-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media. The results indicated that 36% of the promoters were inactive when cells were grown in at least one of these two media. Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map. The results indicate that the gntZ-ywaA region contains at least 24 polycistronic operons, including several published ones. The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA-yxnB-asnH-yxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR-yxxB, hutPHUIGM, bgIPH-yxiE, wapA-yxxG, yxiM-deaD, katB-yxiS, yxjCDEF, yxjJI and yxkF-mmsX.

Coupling of a neural filter and a neural controller for improvement of fermentation performance

The noise associated with fermentation processes is normally minimised by a filtering technique. However, sometimes the noise may be beneficial if it is properly regulated. For recombinant β-galactosidase production in a fed-batch fermentation subject to Gaussian disturbances, it is shown that a neural network trained to act as a noise filter can allow disturbances of only a particular variance which maximizes β-galactosidase synthesis. By coupling such a filter with a neural controller, the productivity may be enhanced beyond what is possible with static filtering and either proportional-integral-derivative or neural control.

Scale-up and optimization of the low-temperature inducible cspA promoter system.

The performance of the major Escherichia coli cold-shock promoter in directing the synthesis of recombinant proteins at low temperatures was investigated in batch fermentations using a plasmid-encoded transcriptional gene fusion between the cspA promoter region and the lacZ gene. Rapid synthesis of beta-galactosidase was observed when the fermentation broth was chilled to 15 degreesC using a variety of cooling profiles, including one modeling the heat-transfer characteristics of a 60-L pilot plant unit. A linear cooling rate of 0.5 degreesC/min led to optimum recovery yields. For all single-temperature downshift experiments, however, the promoter became repressed 60-120 min after initiation of cooling. Both temperature cycling between 15 and 25 degreesC and stepwise temperature downshifts between 37, 29, 21, and 13 degreesC led to multiple inductions of the cspA promoter. Nevertheless, high-efficiency reinduction was only observed during the first temperature pulse when the former strategy was used and when the cells were held at intermediate temperatures for less than 60 min or more than 120 min in the case of successive downshifts. Promoter repression was abolished in host cells bearing a null mutation in the gene encoding the ribosomal binding factor RbfA, leading to the constitutive and high-level expression of beta-galactosidase for 7 h postshift when shake flask cultures were transferred from 42 to 23 degreesC. The suitability of rbfA cells for cspA-driven recombinant protein production was confirmed in high-density fed-batch fermentations. Our results are consistent with the existence of a cold-shock-induced repressor molecule that must accumulate at a threshold concentration before interfering with the production of proteins placed under cspA transcriptional control.

An optimal model to simulate growth of micro-organism on a dissacharide

The growth of micro-organisms on disaccharides, like lactose, is highly dependant on the enzymic degradation rate of these disaccharides. After degradation of lactose, growth essentially takes place on a mixture of glucose and galactose present in the fermentation broth. The catabolic repression of glucose on the synthesis of β-galactosidase and the uptake of galactose controls growth. This is demonstrated by batch growth experiments on the growth of Lactobacillus bulgaricus on lactose. An optimal model is developed to simulate growth on lactose and on mixtures of glucose, lactose and galactose. The model is able to predict the effect of a glucose pulse during growth on lactose.

Long-term potentiation activates the GAP-43 promoter: selective participation of hippocampal mossy cells.

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing DL-aminophosphonovalerate (APV), an N-methyl-D-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, beta-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of beta-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

Expression of theEscherichia coliThioredoxin Gene Is Negatively Regulated by Cyclic AMP

Regulation of theEscherichia colithioredoxin gene (trxA) was studied usingtrxA-lactranslational fusion constructed in the vector pMC1403. Synthesis of β-galactosidase from thetrxA-lacfusion was found to be repressed in the presence of lactose. A switch of carbon source from glucose to lactose and an addition of cyclic AMP (cAMP) caused a decrease in β-galactosidase synthesis from thetrxA-lacfusion. The repression effect of exogenous cAMP was not observed in thecrpmutant strain. The β-galactosidase synthesis from thetrxA-lacfusion lacking a plausible cAMP-CRP binding site was not lowered in the presence of lactose or in the addition of cAMP. Expression of the chromosomaltrxAgene was reduced by exogenous cAMP. These findings indicate that the expression of thetrxAgene is controlled by cAMP in a negative manner.

Screening for inhibitors of the HMG-CoA reductase promoter in HepG2 cells: Identification of four non-oxysterol inhibitors

The 4.9-kb segment of the Chinese hamster hydroxymethylglutaryl-CoA reductase promoter found in pRedCAT-3 consists of 1.4-kb of the promoter and a 3.5-kb intron. We placed this segment upstream of a reporter gene, lacZ of E. coli. The new construct pRed3lacZ was transfected into human hepatoma cells (HepG2), and a single clone, G52, was isolated. Promoter activity was monitored by measuring β-galactosidase activity in cell lysates in microtiter wells using a fluorogenic substrate, 4-methyl-umbelliferyl β-D-galactoside. The amount of β-galactosidase activity in cell lysates was directly proportional to the initial cell inoculum up to 30,000 cells per well. Fidelity of the reductase promoter in G52 was confirmed by (1) suppression of β-galactosidase synthesis (62.7%) by a sterol mixture consisting of 1.6 × 10−5 M 25-hydroxycholesterol and 3.1 × 10−6 M cholesterol, and (2) increased synthesis (51.3%) of β-galactosidase in the presence of 10−7 M mevinolin, a competitive inhibitor of HMG-CoA reductase activity. Using this cell line, we examined 5,400 compounds and found four compounds, U-9888, U-20685, U-51862, and U-71690, that inhibit the reductase promoter with IC50 values of 13.3, 12.0, 12.3, and 14.3 μM, respectively. In wild-type HepG2 cells, these compounds reduced the synthesis of HMG-CoA reductase by 37, 48, 32, and 22%, respectively. Significantly, none of these compounds inhibit the binding of LDL to its receptor, suggesting an important separation of these two coordinately regulated activities. Furthermore, all four of these inhibitors lie outside of the class of compounds typically defined as classic oxysterols, and thus represent new potential templates for the discovery of HMG-CoA reductase expression regulators. Drug Dev. Res. 40:41–47, 1997. © 1997 Wiley-Liss, Inc.

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene ( polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.

Effect of hypoxia duration on the oxygen-dependent production of a recombinant protein, β-galactosidase, by an animal cell line, F6D2, with a hypoxia-inducible enhancer.

Expression of specific genes is a strategy of animal cells for adaptation to oxygen deficiency and the mechanism underlying the hypoxic activation of gene expression may be useful for efficient production of recombinant proteins by animal cells, because oxygen is a limiting factor in animal cell cultures. We prepared an animal cell line harboring the plasmid in which expression of a reporter gene, β-galactosidase, is controlled by an enhancer responsible for the hypoxic activation of gene transcription. The purpose of this paper is to understand this hypoxic production of recombinant proteins quantitatively by a mathematical model originally developed based on the following hypotheses; 1 lacZ (the reporter gene) is transcribed after HIF-1 protein complex is bound to the hypoxic enhancer, 2. β-galactosidase synthesis rate is limited at the transcription of lacZ, 3. HIF-1 is an inactive form under a normal oxygen concentration, 4. Oxygen works as a repressor in the synthesis of HIF-1 protein, 5. Both β-galactosidase and HIF-1 are decomposed according to the first order reaction. The effects of hypoxic duration as well as oxygen concentration on the β-galactosidase production were successfully predicated by the model.

Activation and regeneration of whole cell biocatalysts: Initial and periodic induction behavior in starvedEscherichia coli after immobilization in thin synthetic films

Activation and regeneration of whole cell biocatalytic activity via initial and subsequent induction of the lacZ gene was investigated in starved Escherichia coli using a novel synthetic biofilm. Stationary-phase bacteria were entrapped in 10-80 mum thick multi-layer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. The E. coli were placed in a defined starvation medium containing essentially no nitrogen or carbon source and induced initially using lactose or isopropylthiogalactoside (IPTG). Subsequent inductions were performed with IPTG. Comparison studies with suspended bacteria showed that when IPTG was the initial inducing agent, induction kinetics are linear for both immobilized and suspended cells. After induction with lactose, however, a lag time is noted for suspended cells, but not for E. coli in the biofilm. Biocatalytic activity was successfully regenerated by re-inducing starved suspended cells 1-3 days after an initial induction with lactose. This regeneration was demonstrated in the synthesis of additional active beta-galactosidase. However, immobilized cells could be re-induced for at least 17 days after the initial induction, and viability in the synthetic biofilms remained greater than 90%, demonstrating that periodic induction is a valuable method for extending the life of whole cell biocatalysts.

Characterisation of carbon dioxide-inducible genes of the marine bacterium, pseudomonas sp. S91

Characterisation of two genes in Pseudomonas sp. S91 that are responsive to carbon dioxide is reported. These were identified by random transposon mutagenesis leading to fusion of the Escherichia coli lacZ reporter gene to the genes of interest. Expression of the genes' promoters was quantified by measuring the reporter gene product, β-galactosidase. β-Galactosidase synthesis was induced when cells were exposed to 10% CO 2 on solid media or during growth in aqueous phase when the culture density was greater than 1 at 610 nm, in either rich or minimal media. Induction of β-galactosidase synthesis was not due to: increased alkalinity, onset of stationary phase, build up of soluble metabolites in the culture supernatant, or cell density-dependent signalling. The CO 2-inducible gene fusions were not induced by other environmental conditions that are known to stimulate global regulators of environmental gene expression. Benzole acid (2 mM) induced β-galactosidase synthesis in one of the mutants indicating the CO 2 response may involve the intracellular CO 2 partial pressure/bicarbonate ion concentration/pH equilibrium.

RcsC-mediated induction of colanic acid by secretion of streptokinase in Escherichia coli K-12

The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.