Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus K1L (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3' end of the K1L gene and the adjacent M2L gene deleted and replaced with the beta-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the K1L gene was used to insert the HBs gene into vAbT33. The M2L negative K1L positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The K1L gene selection system allows the isolation of recombinants arising at frequencies as low as 1/100,000. It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3' end of the K1L gene. The K1L gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the K1L gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5' end of the M2L gene transcripts. Although the insertion lengthened the 3' end and lowered the accumulation of K1L transcripts it altered neither the virulence nor the immunogenicity of the recombinant.
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PMID:Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences. 842 36

We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector. DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease. To express a fusion protein of beta-galactosidase linked with the HTLV-1 protease in Escherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of a lacZ gene directed by a lambda phage Pr promoter and designated pUEX-pro. By Western blot analysis using anti-beta-galactosidase antibody, a bigger molecular size band than that of the control beta-galactosidase molecule was observed in E. coli cells transformed with pUEX-pro but not with control pUEX2, suggesting that the particular fusion protein was successfully expressed. This recombinant protease protein in the E. coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1 gag precursor polyprotein. The gag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme. Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously. Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.
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PMID:Human T-cell leukemia virus type 1 protease protein expressed in Escherichia coli possesses aspartic proteinase activity. 843 41

Two reporter genes: the firefly Photinus pyralis luciferase gene and the Escherichia coli beta-galactosidase gene were used for construction and characterization of the five unique recombinant vaccinia strain LIVP viruses expressing these genes in three nonessential regions of the virus genome. We give comparative characteristics of beta-galactosidase and luciferase activities in experiments of transient expression and expression dynamics of reporter genes by different stable recombinant viruses. Both genes are expressed with high efficiency independent on the sites of virus genome localization. It is shown that the TK-, HA- and N-regions of vaccinia virus DNA may be used for inserting foreign genes.
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PMID:[Analysis of reporter gene expression at different segments of the vaccinia virus genome]. 848 70

The adenovirus precursor to the terminal protein (pTP), expressed in a vaccinia virus expression system or in native adenovirus, was assayed for its ability to interact with the nuclear matrix. Biochemical function was measured by determining the relative amount of pTP protein or of adenovirus DNA that remained associated with the nuclear matrix after extensive washing. pTP was retained on the matrix whereas beta-galactosidase was not, as assayed by quantitative immunoblot analysis. Nuclear matrix isolated from adenovirus-infected HeLa cells retained bound adenovirus DNA even when washed with 1 M guanidine hydrochloride; this interaction could be inhibited by added purified pTP protein. Analogous experiments with matrix isolated from HeLa cells infected with a recombinant vaccinia virus that expressed pTP showed a similar retention of pTP protein; this association could also be inhibited by added pTP protein. Binding of pTP to nuclear matrix isolated from uninfected cells was saturable, with an apparent Kd of 250 nM and an estimated 2.8 x 10(6) sites for pTP binding per cell nucleus. The association of pTP with matrix is postulated to help direct adenovirus replication complexes to the appropriate locale within the nucleus.
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PMID:Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro. 849 57

We have developed a novel technique using vaccinia virus for highly efficient introduction of foreign genes into viable cells of brain slice to study function of nervous system. Hippocampal slices of adult guinea pigs were infected with a vaccinia virus carrying a gene for beta-galactosidase (beta-gal) as a reporter gene. Expression of beta-galactosidase was first detected after 5 hours and reached maximum after 16 to 24 hours. Light microscopic observation revealed that beta-galactosidase was expressed uniformly in the CA1-CA3 pyramidal cell layer, granule cell layer of dentate gyrus and glial cells. Serial sections of infected slices showed that cells were uniformly infected throughout the thickness of the slice. Thus, the vaccinia virus system provides a convenient gene transfer tool for studying brain function in cultured slice system.
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PMID:A vaccinia virus vector for efficiently introducing into hippocampal slices. 851 65

The integrative plasmids with the expressive marker gene for beta-galactosidase were constructed for insertional inactivation of nonessential genes E7R and D8L of vaccinia virus located in the central region of the viral genome. Inactivation of the D8L gene in the strains WR and LIVP results in smaller viral plaques in the culture of chicken embryo cells, decreases the viral ability to propagate in mouse brain and has no effect on the size and character of damage in intracutaneous infection of rabbits. Inactivation of E7R gene did not affect the known biological properties of the virus. The existence of the nonessential genes in the central region of the viral genome, inactivation of which does not result in viral attenuation, can be used for increase of antigenic activity of the live attenuated vaccines.
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PMID:[Insertion mutants of the vaccinia virus. The effect of inactivating E7R and D8L genes on the biological properties of the virus]. 851 49

This study examines the suitability of replication-defective adenovirus vectors for engineering recombinant vaccines. The immunological abilities and limitations of E1-deleted adenoviruses containing the lacZ gene (Ad-beta-gal) were investigated by examining the humoral and cellular immune responses to the beta-galactosidase protein. BALB/c mice (H-2d) were given in a single injection of recombinant adenovirus. The cytotoxic T lymphocyte (CTL) response of spleen cells was evaluated. Recognized target cells were H-2d-derived tumor cells transfected by the lac Z gene, or incubated with the 876-884 beta-galactosidase peptide known to be restricted by the Ld molecule of the major histocompatibility complex. A long-lasting beta-galactosidase-specific cytotoxic T cell response was obtained. By contrast, CTL from mice immunized with the Ld-restricted peptide were less specific for the endogenous epitope presented by the transfectants expressing beta-galactosidase. Ad-beta-gal-immunized mice were also protected against an intra-cerebral challenge with a recombinant vaccinia virus expressing the lac-Z gene. These results suggest that Ad-beta-gal-induced CTL have protective abilities in vivo. The induction of beta-galactosidase-specific T helper lymphocytes and humoral IgG responses were also examined. A proliferative response occurred only late after immunization and the primed T lymphocytes produced interleukin-2, but no interleukin-4. A humoral IgG response to the beta-galactosidase protein was detected 15-30 days after a single immunization and remained stable for 6 months without boosting. Lastly, we followed the evolution of the immune response over the course of successive immunizations. The magnitude and kinetics of the cellular and humoral responses were similar to those obtained after a single immunization. Consistent with these observations, an adenovirus-specific neutralizing antibody response was detected as early as the second immunization. Thus, a single immunization with a replication-defective adenovirus recombinant vector induces long-lasting humoral and cellular immune responses specific to the transgene product.
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PMID:Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. 856 39

A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, beta-galactosidase (beta-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding beta-gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag and the costimulatory molecule B7-1, designated B7-1 beta-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor-bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines.
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PMID:IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines: enhancement by simultaneous B7-1 expression. 861 61

The G1 and G2 glycoproteins of La Crosse virus, a member of the Bunyavirus genus of the Bunyaviridae, are encoded as a single open reading frame (ORF) in the viral middle-sized RNA segment. The primary product from this ORF is processed, either cotranslationally or shortly after translation, into the two glycoproteins and a nonstructural protein, NSm, of unknown function. We have expressed La Crosse glycoproteins using vaccinia vectors and studied their processing and localization. When expressed in the native G2-NSm-G1 configuration, both G1 and G2 targeted to the Golgi apparatus as shown by their colocalization with wheat germ agglutinin and acquired resistance to endoglycosidase H. When expressed independently, G2 was targeted to the Golgi apparatus but G1 was retained in the endoplasmic reticulum, indicating that a G1-G2 association is required for Golgi targeting of G1. In contrast to results with other members of the Bunyaviridae, we found that expression of G1 and G2 from separate vectors did not lead to the transport of the G1-G2 complex to the Golgi. However, disruption of the NSm region with a foreign sequence did not interfere with transport of the complex. When a portion of the beta-galactosidase gene was inserted in frame into NSm, the glycoproteins derived from this construct were processed and targeted properly and were capable of mediating cell-to-cell fusion.
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PMID:Analysis of the intracellular transport properties of recombinant La Crosse virus glycoproteins. 866 99

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.
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PMID:Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases. 866 47


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