Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Gene delivery into neuronal and glial cells by using a replication-deficient adenovirus vector: prospects for neurological gene therapy

We have used a recombinant adenovirus vector (E1−) expressing β-galactosidase to explore a novel mechanism with which to transfer genes into cells of the central nervous system (CNS). The replication-deficient adenovirus vector expressing β-galactosidase (RAd35) was propagated on a permissive helper cell line (293 cells). High level protein expression from the human cytomegalovirus immediate early promoter (hCMV IE) was obtained in a target cell population of RAd35 infected cultured neuronal and glial cell lines. Light microscopy showed that over 50% of the glial cells studied expressed β-galactosidase. Following retinoic acid treatment, RAd35 infected cell lines ND7/23, NG108 and NTera2, showed β-galactosidase expression in up to 90% of the cells. In addition, these cells showed morphological evidence of differentiation into neurons. This pattern of β-galactosidase expression was also observed in primary rat cerebella granule neuron cultures. In vivo studies were performed in Balb/c mice following direct intracranial injections of RAd35 into the brain. Cell sections showed a localised staining in the brain at the site of injection of the virus. Non-replicating adenovirus vectors are therefore highly efficient systems for delivering a transgene into brain cells. However, their broad cell tropism may limit their applications for genetic disorders in which a specific cell type is to be targeted for gene therapy. To address this problem, we have constructed adenovirus vectors which contain specific neuronal promoters and are currently assessing in vitro expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.     

On-off regulation of gene expression from tryptophan promoter with cross-flow filtration

Summary Recombinant plasmid containing β-galactosidase gene fused to trp promoter (pMCT98) and that containing cloned trp repressor gene (pRLK13) were introduced into Escherichia coli C600. The bacterium was cultivated in a jar-fermetor equipped with a cross-flow filtration apparatus to attain the on-off regulation of the gene expression by controlling tryptophan concentration in the medium. In logarithmic growth phase, the cross-flow filtration was started. Tryptophan concentration dropped to a low level within 1 h and an efficient expression of β-galactosidase gene was started. By this twostage cultivation, very high biomass was achieved (final OD₅₇₀: 150) and the amount of produced β-galactosidase was about 10% of total cellular proteins.     

Heterologous expression of a gene encoding a thermostable β-galactosidase from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable β-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The β-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant β-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-β-d-galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant β-galactosidase were 5.8–6.0 and 70°C, respectively. The enzyme retained 90% of its activity when heated at 70°C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65°C.     

Construction and Expression of Food-Grade β-Galactosidase Gene in <Emphasis Type="Italic">Lactococcus Lactis</Emphasis>

Recombinant Lactococcus lactis MG1363/pMG36e-lacZ exhibiting high β-galactosidase activities were constructed by us in the previous study. However, erythromycin resistance present in these recombinants restricted their practical application in food preparation. This study was conducted to delete the gene coding for erythromycin resistance present in recombinant L. lactis, as a result of which these bacteria express food-grade β-galactosidase. In this study, the recombinant plasmid pMG36e-lacZ was digested with restriction enzymes BclI and HpaI and the food-grade plasmid FGZW was rebuilt. FGZW was transformed into Escherichia coli JM109 and L. lactis MG1363. Erythromycin resistance, enzyme activity determination, gene sequencing and SDS-PAGE analysis indicated that these new recombinant bacteria lost erythromycin resistance and its relevant gene but still expressed β-galactosidase activities, although a decrease in the expression of β-galactosidase of these new strains was observed. The β-galactosidase food-grade expression system was successfully constructed and it could provide a new solution for the management of lactose intolerance. These results might promote the usage of gene-modified microorganisms and related technology in the food sector, which has the highest priority for food safety.     

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and β -galactosidase expression by Saccharomyces cerevisiae

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne β -galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Δμ) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited Δμ -dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. Δμ decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or Δμ, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased Δμ values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Δμ may have conferred was counteracted by an increased R value. Increased β-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity. Received 08 July 1996/ Accepted in revised form 14 January 1997     

The Fate of Myoblasts Following Transplantation into Mature Muscle

Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme β-galactosidase (β-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (β-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and/β-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest β-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell trans-plantation and for the application of such studies to cell therapy and cell-mediated gene therapy.     

The β-Galactosidase Activity in <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> CBS6556 Decreases by High Concentrations of Galactose

In this paper we report on the effect of different concentrations of lactose and galactose in the production of β-galactosidase by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the β-galactosidase activity. Therefore, maximum induction of β-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those informations can help to design low cost medium with higher β-galactosidase productivity by K. marxianus cells. Received: 8 August 2001 / Accepted: 15 October 2001     

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, σ^S, or σ³⁸) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS′–′lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that β-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its β-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of β-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS–lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.     

Construction of a flocculent brewer's yeast strain secreting Aspergillus nigerβ-galactosidase

One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting β-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular β-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by flocculent cells. Received: 13 January 2000 / Accepted: 23 January 2000     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

β-Galactosidase-deficient mouse as an animal model for GM1-gangliosidosis

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Improvement of Germ Line Transmission by Targeting β-galactosidase to Nuclei in Transgenic Mice

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate β-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing β-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing β-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when β-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic β-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting β-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.     

Sizing the Fusobacterium nucleatum genome by pulsed-field gel electrophoresis

Abstract The β-galactosidase (β-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli . Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum . Its sequence is very similar to a Leuconostoc lactis β-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis , was four-fold higher in cells grown in lactose compared to those grown in glucose. The presence of the β-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.     

Molecular thermal telemetry of free-ranging adult Drosophila melanogaster

The expression of two temperature-sensitive reporter genes, hsp70 and an hsp70-LacZ fusion, in free-ranging adult Drosophila melanogaster indicates that natural thermal stress experienced by such small and mobile insects may be either infrequent or not severe. Levels of the heat-shock protein Hsp70, the major inducible Hsp of Drosophila, were similar in most wild Droso- phila captured after warm days to levels previously reported for unstressed flies in the laboratory. In a transgenic strain transformed with an hsp70-LacZ fusion (i.e., the structural gene encoding bacterial β-galactosidase under control of a heat shock promoter), exposure to temperatures ≥32°C in the laboratory typically resulted in β-galactosidase activities exceeding 140 mOD₄₅₀ h^–1μg^–1 soluble protein. Flies caged in sun frequently had β-galactosidase activities in excess of this level, whereas flies caged in shade and flies released and recaptured on cool days did not. Most flies (>80%) released on warm, sunny days had low β-galactosidase activities upon recapture. Although the balance of recaptured flies had elevated β-galactosidase activities on these days, their β-galactosidase activities were <50% of levels for flies caged in direct sunlight or exposed to laboratory heat shock. These data suggest that even on warm days most flies may avoid thermal stress, presumably through microhabitat selection, but that a minority of adult D. melanogaster undergo mild thermal stress in nature. Both temperature-sensitive reporter genes, however, are limited in their ability to infer thermal stress and demonstrate its absence. Received: 14 July 1999 / Accepted: 21 December 1999     

β-Galactosidase activity in cultured cotton cells (Gossypium Hirsutum L.): A comparison between cells growing on sucrose and lactose

Summary Cotton callus and suspension cultures developed from a cotton variety susceptible toXanthomonas malvacearum (E. F. Sm.) Dow, were grown on chemically defined media that contained one of the carbohydrate sources: 3% sucrose, 3% lactose, 3% maltose, 3% fructose, and 3% glucose. All cells were maintained on a medium with sucrose as the carbohydrate and subsequently transferred to media containing the above carbohydrates. Sucrose was the best carbon source for a high growth rate; however, cells growing on glucose, which was almost as good as sucrose, and cells growing on lactose did not turn brown when they reached the stationary phase of growth. A crude extract from callus tissue growing on lactose has a fivefold increase in β-galactosidase [EC 3.21.23] activity as compared with the extract from callus tissue growing on sucrose. When callus tissue growing on lactose was transferred tomedium containing sucrose, β-galactosidase activity decreased to the level as measured in cells maintained on sucrose. Callus cells growing on a lactose medium showed staining when treated with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside in which very heavy granular stains appeared. Cells growing on sucrose did not show the histochemical staining. These observations suggest that β-galactosidase is induced in cotton callus tissue that has been transferred from a medium containing sucrose to a medium containing lactose. This is journal article J-3704 of the Oklahoma Agricultural Experiment Station. The research was supported in part by a Presidential Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station.     

Transduction of Dendritic Cells by DNA Viral Vectors Directs the Immune Response to Transgene Products in Muscle Fibers

Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as β-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate β-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.     

酪氨酸羟化酶基因载体－pCMVTH 质粒的构建及在大鼠骨骼肌内的表达

为用转基因方法治疗巴金森氏病大鼠模型,本研究采用分子克隆技术,将合成多巴胺的关键酶－酪氨酸羟化酶（ＴＨ）的基因,克隆进入以巨细胞病毒ＣＭＶ为启动子的载体质粒内,经限制性内切酶定位分析证实该重组的ＤＮＡ质粒的可靠性。携带ＴＨ基因的ＰＣＭＶＴＨ质粒以ＬＩＰＯ－ＦＥＣＴＩＮ介导,在培养的原代骨骼肌细胞中高效表达。本研究为进一步用转基因的细胞植入脑内以治疗巴金森氏病打下一定基础。