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PubMed1987

Binding of specific glycoconjugates to human brain synaptosomes: studies using glycosylated beta-galactosidase.

Using glycosylated beta-galactosidase (beta-gal) as a glycoprotein model, binding of glycoconjugates to human brain synaptosomes was studied. Out of beta-gal modified with a series of p-aminophenyl alpha- or beta-glycosides, beta-gal modified with p-aminophenyl beta-D-glucopyranoside (beta-D-Glc beta-gal) was bound to the synaptosomes most effectively, then beta-gal modified with beta-D-galactoside and with alpha-D-mannoside. Kinetic studies on the binding of beta-D-Glc beta-gal indicated the presence of saturable binding on human brain synaptosomes. The values of the apparent Km and of the maximal binding velocity were obtained to be 248 +/- 32.9 microM and 43.8 +/- 1.43 fmol/min/mg synaptosome protein, at 4 degrees C and pH 7.5. The specificity of the sugar recognition site proved by the competitive inhibition of the binding of beta-D-Glc beta-gal by bovine serum albumin modified with the same glycoside. The binding of beta-gal modified with beta-D-galactoside was inhibited by treatment of the synaptosomes with trypsin, phospholipase A2, C and D, and with neuraminidase, while the binding of beta-D-Glc beta-gal was inhibited by neuraminidase treatment of synaptosomes. In subcellular fractions of human brain the binding protein was localized mainly in synaptosomes.

Cerebral Cortex↗
PubMed1990

Some properties of beta-D-galactosidase from the adult filarial nematode Setaria digitata.

beta-D-galactosidase (beta-D-galactoside galactohydrolase, E.C. 3.2.1.23) activity was localised in the digestive tract of Setaria digitata. The enzyme extract shows maximum activity in the pH range between 3.5 and 5.0 and at 45 degrees C. The enzyme shows the Km value of 3.636 mM for the substrate 6-bromo-2-naphthyl beta-D-galactoside and Vmax of 28.57 nmol 6-bromo-2-naphthol liberated mg-1 protein min-1. Activation/inhibition of the enzyme by various ions, medicinal plants and drugs has been studied. Polyacrylamide gel electrophoresis revealed that the enzyme exists as single form. The medicinal plants and the drug filarin effectively inhibit the enzyme. The significance of these results are discussed in relation to chemotherapy.

Animals↗
PubMed1988

Demonstration of an endo-beta-galactosidase and an endo-beta-xylosidase that degrade the proteoglycan linkage region.

Purified proteochondroitin sulfate was incubated at pH 4.0 with a rabbit liver lysosomal enzyme fraction. The formed degradation products were isolated by gel filtration followed by reduction with NaB3H4. After acid hydrolysis of the reduced digest, the hydrolysate was passed through Dowex 50W-X8 and 1-X2 columns. The separated neutral sugars were then subjected to paper chromatography, which demonstrated that galactose and xylose had been at the reducing termini of chondroitin sulfate after incubation with the liver lysosomal preparation. These results suggest the presence in the liver lysosomal preparation of an endo-beta-galactosidase and an endo-beta-xylosidase which act at the linkage region of proteochondroitin sulfate.

Animals↗
PubMed1987

Purification and properties of beta-galactosidase from chicken seminal plasma.

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.

Animals↗
PubMed1983

M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of beta-galactosidase alpha-complementation.

A DNA fragment encoding the transposon Tn9 chloramphenicol acetyltransferase gene (cat) was inserted into M13 phage and pUC plasmid cloning vehicles. When the cat gene was inserted in the same orientation as the lacZ gene, two new polypeptides were produced. One polypeptide possessed chloramphenicol acetyltransferase activity, while the other expressed beta-galactosidase alpha-donor activity. Both new polypeptides were translated from a hybrid messenger RNA initiating from the lac promoter. These observations may help explain why not all inserts produce white plaques.

Acetyltransferases↗
PubMed1983

Isolation of a functional exuR-repressor-beta-galactosidase hybrid protein by use of in vitro gene fusions.

The exuR regulatory gene of Escherichia coli codes for a repressor molecule that controls negatively the expression of the exu regulon genes involved in the hexuronate degradation. The exuR gene was fused to the lacZ gene in vitro on plasmid cloning vectors developed by Casadaban et al. [J. Bacteriol. 143 (1980) 971-980]. The exuR-lacZ fusion gene allows the production of hybrid molecules possessing in a single polypeptide beta-galactosidase activity as well as the capacity to bind in vivo specifically to the operators of the exu operons. However, the affinity of ExuR hybrid protein for the exu operators seems to be reduced in comparison with the native repressor since, even overproduced in the cell, the chimeric polypeptide was not able to completely repress the expression of the exu operons. The other functions of the ExuR repressor, i.e., repression of the uxuAB operon and inactivation by the inducers, were not retained in the chimeric protein. This particular chimaera of Mr about 120 000 was purified by chromatography on phosphocellulose from strains carrying the fusion plasmids. The purified polypeptide was able to repress in vitro the synthesis of an exu gene product in a cell-free transcription and translation system. The implications of the repressor activity in the chimeric protein are discussed. The NH2-terminus of ExuR repressor probably recognizes the exu operators specifically, and this region represents less than 20% of the entire ExuR molecule.

Bacterial Proteins↗
PubMedDate not supplied

One-step purification of hybrid proteins which have beta-galactosidase activity.

A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described. Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95%. The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.

DNA, Recombinant↗
PubMed1985

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase).

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.

Amino Acid Sequence↗
PubMed1986

Vaccinia virus vectors utilizing the beta-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expression.

Plasmids were constructed fusing vaccinia transcriptional regulatory sequences (promoters) to the lacZ gene of Escherichia coli. These recombinant plasmids were used to compare relative promoter strengths in transient expression assays and to construct recombinant vaccinia viruses producing beta-galactosidase (beta Gal). Viruses synthesizing beta Gal were determined by utilizing the chromogenic substrate, 5-bromo-4-chloro-3-indoyl-beta-D-galactoside to form blue plaques. A recombinant virus producing beta Gal was then used to select a second recombinant virus. This was accomplished via in vivo recombination replacing the lacZ gene with a sequence coding for the gp85 protein of Friend murine leukemia virus. The recombinant virus was selected by its inability to form blue plaques under appropriate conditions.

Chromosome Mapping↗
PubMed1986

Expression in Escherichia coli of synthetic human interleukin-1 alpha genes encoding the processed active protein, mutant proteins, and beta-galactosidase fusion proteins.

We have synthesized, cloned, and expressed the coding region for the C-terminal 159 amino acids (aa) of the human active interleukin polypeptide hormone IL-1 alpha. The sequence was assembled in stages and includes preferred Escherichia coli codons and unique restriction sites. The coding region was cloned on a multicopy plasmid vector adjacent to signals for transcription and translation that directed synthesis of 6% of total E. coli protein as IL-1 alpha. Active IL-1 alpha mutants that have a C-terminal additional eleven aa and that have N-terminal deletions of six and fourteen aa are described. Plasmids expressing beta-galactosidase fusion proteins with various parts of IL-1 alpha at their N-termini were constructed.

Base Sequence↗
PubMed1987

A low-copy-number vector utilizing beta-galactosidase for the analysis of gene control elements.

A low-copy-number vector, pFZY1, with the multiple restriction site linker of M13mp18 inserted upstream from a promoterless beta-galactosidase (beta Gal)-coding lacZ gene has been constructed to provide a convenient and accurate system to analyze regulatory elements in vivo. The plasmid contains the oriF replication origin without the par locus and is present in the cell in one to two copies per genome. It is retained in the host by the presence of ampicillin, and each inserted promoter yielded consistent values of beta Gal activity under all the conditions tested. A series of tetracycline resistance (TcR) promoter fragments and lac promoter fragments have been compared in pFZY1 and the high-copy-number pKO-vector series. The transcriptional activity measured for different fragments containing the same TcR promoter varied within a six-fold range among the several constructs tested. Regulation of the wild-type lac promoter and mutants in pFZY1 was similar to that observed for lac promoters in the chromosome while their regulation in pKO-1mp18 was significantly affected by the high copy number, as expected.

Base Sequence↗
PubMed1988

Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes.

The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process. Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences. Moreover, the synthetic sequences cloned are often altered versions of those originally designed. We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame. The test is the reversal of the beta-galactosidase alpha-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors. We begin with a modified vector defective in alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation. The alpha-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment. The alpha-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites. Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy. We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.

Amino Acid Sequence↗
PubMed1988

A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product.

A plasmid-cloning vector system has been constructed which allows the production of fusion proteins with beta-galactosidase at the N terminus, followed by a recognition sequence for the site-specific protease, collagenase, and the foreign protein at the C terminus. A multicloning site allows the insertion of foreign genes in any translational reading frame. Fusion proteins were isolated by affinity chromatography on APTG-Sepharose. The foreign protein was released from the fusion product by collagenase cleavage. The vector was successfully utilized for the production of Escherichia coli single-stranded (ss) DNA-binding protein (SSB protein). The proteolytically released SSB protein resisted elution from an ss DNA-cellulose column with 1 M NaCl.

Bacterial Proteins↗
PubMed1989

Introns in the 3'-untranslated region can inhibit chimeric CAT and beta-galactosidase gene expression.

The expression of a cyc::cat [cytochrome c/chloramphenicol acetyltransferase (CAT)] chimeric gene was stimulated 100-fold by the inclusion of a cyc intron in the 5'-untranslated region. In contrast, a single intron in the 3'-untranslated region was at best only slightly stimulatory, and surprisingly, inhibited expression of cat when an intron was also included in the 5'-untranslated region. This inhibition was independent of the identity of the downstream intron, occurring when either the simian virus 40 (SV40) small t intron or a cyc intron was located downstream from the cat coding region. Analysis of CAT mRNA levels, using a riboprobe spanning the 5' end of the CAT message, revealed that the stimulatory effect of a 5'-noncoding region intron were manifest at both the protein and RNA levels, whereas the inhibitory effects of 3'-noncoding region introns were detectable only at the protein level. The effects of intron position on chimeric gene expression were observed in both primate and rodent cell lines and also when the beta-galactosidase coding region was substituted for that of cat. Therefore, the common placement of an intron in the 3'-noncoding region is not the most beneficial to the expression of cyc chimeric genes. The position of introns within a transcriptional unit is a major factor to be considered when optimizing the efficiency of animal cell expression vectors.

Animals↗
PubMed1989

Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum.

We show that a fusion gene, containing the promoter and 5'-noncoding region of a Dictyostelium discoideum actin 6 gene linked to the Escherichia coli beta-galactosidase (beta Gal) gene (lacZ), directs the production of functionally active beta Gal in D. discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression. We illustrate this by fusing lacZ to the promoter of the prespore-specific gene, D19, and localizing expressing cells in migrating slugs. Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.

Amino Acid Sequence↗
PubMed1990

Development of a beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

A versatile beta-galactosidase alpha-complementation system for Bacillus subtilis was developed, which can be used for molecular cloning in this Gram+ organism. The cloning system, which is based on the highly efficient host-vector system 6GM-pHP13, offers several advantages over previously described systems: (1) convenient direct selection of recombinants; (2) the cloning of large heterologous DNA fragments with high efficiency; and (3) the availability of six unique target sites: SphI, NdeI, NheI, BamHI, SmaI and EcoRI.

Bacillus subtilis↗
PubMed1990

A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression.

For analysis of chimeric mice made by injecting embryonic stem (ES) cells into host blastocysts, it is very desirable if the ES cells have a good cell marker that can distinguish them from host cells. It is ideal if the marker can be easily visualized in every type of cell and tissue throughout the embryogenesis. We tried to produce such ES cell lines by introducing an E. coli beta-galactosidase (beta-gal) gene construct by electroporation. One of the transformant lines (MS1-EL4) showed beta-gal activity in every undifferentiated stem cell. After being induced to differentiate in vitro, cells with various morphologies showed beta-gal activity. We also detected beta-gal activity in a wide variety of tissue elements in solid tumors made by injecting the MS1-EL4 cells into syngeneic mice. Then we produced chimeric embryos by injecting the MS1-EL4 cells into blastocysts and recovering the embryos at various developmental stages. We found that the MS1-EL4 cells contributed to various tissues and expressed beta-gal activity, including not only descendants of the inner cell mass but also the trophectoderm-derived extraembryonic ectoderm.

Animals↗