Detection Of Β-galactosidase Activity Research Articles

Amperometric quantification of total coliforms and specific detection of Escherichia coli.

The quantitative determination of total and fecal coliforms, as indicators of fecal pollution, is essential for water quality control. We developed a sensitive, inexpensive amperometric enzyme biosensor based on the electrochemical detection of beta-galactosidase activity, using p-amino-phenyl-beta-D-galactopyranoside as substrate, for determining the density of coliforms, represented by Escherichia coli and Klebsiella pneumoniae. The specific detection of E. coli was achieved using an antibody-coated electrode that specifically binds the target bacteria. Amperometric detection enabled the determination of 1000 colony-forming units/mL within 60-75 min. Preincubation for 5-6 h further increased the sensitivity more than 100-fold. The present experimental setup allowed the simultaneous analysis of up to eight samples, using disposable screen-printed electrodes.

Identification of novel elements which regulate the cell-type specificity of Dictyostelium 7E gene expression

Previously, we have identified the Dictyostelium 7E gene promoter and shown that it is capable of driving expression in the same temporal and cAMP responsive manner as the endogenous gene during development. Furthermore, we have mapped the corresponding transcriptional regulatory sequences within the promoter. In the present study we used the lacZ reporter gene system to examine the role of 7E promoter elements in regulating cell-type specific expression during Dictyostelium morphogenesis. In situ detection of β-galactosidase activity revealed that expression was induced within anterior prestalk cells at approximately 18 h of development. Subsequently, we found that promoter activity was independently regulated in subpopulations of prestalk cells. Element(s) upstream of position − 532 were necessary for expression in pstA cells while more proximal elements (located downstream of position − 426) were capable of directing expression in pstO cells. Deletion of a G-rich element (‘GGT’ box; 5′-GGT GAT GA-3′) located between positions − 159 and − 152 resulted both in a loss of expression in pstA cells and aberrant expression in the prespore zone. Furthermore, the spatial organisation of reporter gene expression directed by this construct during culmination delineated a population of cells that have not been previously defined. These data suggest that the 7E gene is independently regulated in subpopulations of prestalk cells during development.

Phase-Specific Elements of the stringGene Regulatory Region in Drosophila melanogaster

The phases of the reporter gene expression controlled by different fragments of the string(stg) gene regulatory region were determined in Drosophilaneuroblasts by detection of β-galactosidase activity and radioautography. In the D10 and D22 lines carrying the constructs pstgβ-E4.9 and pstgβ-E5.3, respectively, the reporter gene activity was detected in the G1 phase of the cell cycle. In the D12 and D20 lines (pstgβ-E6.4 and pstgβ-E2.6), no periodic expression was observed. The regulatory regions of the stgfrom lines D10 and D22 and that of Drosophilagene cyclin Dshared consensus aagaactttg, which was also expressed in the G1 phase. The phase-specific expression of the cell-cycle genes was compared in a model for the mitotic-wave cells of eye imaginal disk and neuroblasts of the nerve ganglia.

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli.

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.

Expression of stylar incompatibility in the Andean clonal tuber crop oca ( Oxalis tuberosa Mol . , Oxalidaceae )

Long-, mid-, and short-styled clonal accessions of oca (Oxalis tuberosa) were intercrossed in a complete diallelic design. Pollen tube growth in styles was monitored in all diallelic crosses. Pollen fertility was estimated by two tests: staining of pollen grains with aceto-carmine and detection of β-galactosidase activity by the substrate X-Gal. The two methods of pollen fertility estimation were equally useful to detect fertility levels. Pollen originating from short stamens had the highest fertility (85%) and pollen from long stamens had the lowest fertility (70%). Pollen fertility was high throughout, but its degree varied with the stylar morph on which the pollen was formed. Long-styled accessions had the highest rates of fertile pollen. Differences in pollen fertility at different anther levels in the same style morph were also apparent. Pollen grain diameter of the six morph-anther level combinations was inversely correlated with pollen fertility. Pollen grains from long stamens were the largest and pollen grains from short stamens were the smallest. Neither pollen fertility nor pollen grain size had an influence on pollen tube growth in the style or on fruit and seed set. Pollen tubes growing within the styles were inhibited at a different level for each of the 18 cross combinations in the diallel. Although legitimate crosses had greatest pollen tube growth, some of the illegitimate inter- and intramorph crosses had equally high scores. Of all illegitimate crosses, mid-styled seed parents had the lowest level of stylar incompatibility. Fruit and seed set were highly correlated with the extent of pollen tube growth in the style. The number of pollen tubes entering ovules in a flower was in good agreement with the number of seeds produced per fruit. Therefore, it is concluded that stylar incompatibility is the major determinant of limited seed formation in oca even in the most successful legitimate cross combinations.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.

Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and β-Galactosidases

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pKa ≈ 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pKa values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A β-galactosidase substrate was also prepared based on 6,8-difluoro-4-methylumbelliferone. This substrate, 6,8-difluoro-4-methylumbelliferyl β-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl β-d-galactopyranoside (MUG), for the detection of β-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and β-galactosidase activity, respectively, at neutral and acid pH.

Spatial and temporal expression of an Antennapedia/lac Z gene construct integrated into the endogenous Antennapedia gene of Drosophila melanogaster.

In order to study the regulation of spatial and temporal expression of the homeotic gene Antennapedia (Antp) in Drosophila melanogaster, we have constructed fusion genes which contain Antp sequences linked to the reporter gene lac Z of Escherichia coli. In one case of P-element transformation, a fusion gene construct integrated into the endogenous Antp gene close to one of the two promoters (P1). The spatial expression from the reporter gene in this transformant line, as analysed by the detection of β-galactosidase activity, was found to exactly mimic the normal expression from the P1 promoter of the Antp gene. We have used this unique transformant as a tool for studying the expression of the P1 promoter in embryonic, larval and adult development. Parallel lines transformed with the same fusion gene construct did not confer a correct P1 pattern of expression. The position in the genome was, therefore, crucial for the expression pattern of the reporter gene. Experiments aiming at the detection of autoregulatory control of Antp gene expression were designed. The results did not, however, support models of positive or negative autoregulation of P1 expression by Amp protein.

Improved methods for the detection of β-galactosidase activity in colonies ofEscherichia coliusing a new chromogenic substrate: VBzTM-gal (2-(2-(4-(β-d-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate)

The use of a new substrate (2-(2-(4-(β-d-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluence-4-sulphonate (VB-zTM-gal) is described for the detection of β-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 μm) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4°C for several days, and this identified the expression of β-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium of Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms is polluted water.