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)

Physiological properties of mutants of Escherichia coli defective in glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, or enolase are described. Introduction of a lesion in any one of the reversible steps catalyzed by these enzymes impaired both the glycolytic and gluconeogenic capabilities of the cell and generated an obligatory requirement for a source of carbon above the block (gluconeogenic) and one below (oxidative). A mixture of glycerol and succinate supported the growth of these mutants. Mutants lacking glyceraldehyde 3-phosphate dehydrogenase and glycerate 3-phosphate kinase could grow also on glycerol and glyceric acid, and enolase mutants could grow on glycerate and succinate, whereas double mutants lacking the kinase and enolase required l-serine in addition to glycerol and succinate. Titration of cell yield with limiting amounts of glycerol with Casamino Acids in excess, or vice versa, showed the gluconeogenic requirement of a growing culture of E. coli to be one-twentieth of its total catabolic and anabolic needs. Sugars and their derivatives inhibited growth of these mutants on otherwise permissive media. The mutants accumulated glycolytic intermediates above the blocked enzyme on addition of glucose or glycerol to resting cultures. Glucose inhibited growth and induced lysis. These effects could be substantially overcome by increasing the osmotic strength of the growth medium and, in addition, including 5 mM cyclic adenosine 3',5'-monophosphate therein. This substance countered to a large extent the severe repression of beta-galactosidase synthesis that glucose caused in these mutants.
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PMID:Properties of Escherichia coli mutants deficient in enzymes of glycolysis. 41 Jul 89

A functional analysis of the Aspergillus nidulans 3-phosphoglycerate kinase pgk promoter was undertaken using gene fusions to the lacZ gene of Escherichia coli, and introducing these into a beta-galactosidase-deficient strain of A. nidulans. Expression of a particular gene fusion in transformed strains depends upon the site of integration of the vector into the genome, and when specifically targeted to the catabolic quinate dehydrogenase qutE (selective marker) locus is directly proportional to its copy number. The analysis of transformed strains with single copies of pgk promoter deletion--lacZ fusions at the qutE locus identified three constitutive, positively acting sequence elements in the pgk gene. Sequence located between -161 and -120 nucleotides relative to the transcript start site +1, and including an element with a seven-out-of-eight nucleotide match (AAGCAAAT; -131 to -124) to the consensus eukaryotic octamer sequence ATGCAAAT, is essential for expression, and deletion of the complete 41-nucleotide sequence abolishes transcription. Sequence encompassing codons 14 to 183 and including the two introns of pgk contributes approximately one-third of the total activity, and far upstream sequence 5' to position -638 contributes approximately a further one-third total activity. In addition, sequence located -638 to -488 nucleotides, which includes an apparent consensus feature of A. nidulans glycolytic genes, affects carbon source-dependent regulation of expression. This region is required for an approximately 50% increase in pgk expression when A. nidulans is grown on gluconeogenic compared with glycolytic carbon sources.
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PMID:Functional analysis of the expression of the 3'-phosphoglycerate kinase pgk gene in Aspergillus nidulans. 160 65

A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons. UAA, UAG and UGA in Saccharomyces cerevisiae. The assay employs a 3-phosphoglycerate kinase-beta-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is disrupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring beta-galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi-] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [psi+] broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA greater than UAG greater than UGA.
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PMID:Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay. 190 59

A series of expression plasmids was constructed to compare the usefulness of various promoters for the synthesis of a given protein in the Saccharomyces cerevisiae. The plasmids pMBL212, -213, -214, -215 and -216 can be used to synthesize the protein of interest directly as a non-fused protein or, if the protein is difficult to detect, indirectly as an enzymatically active beta-galactosidase fusion protein. The plasmids were employed to identify which yeast promoter and strain are suitable for the synthesis of poliovirus protein VP2. It was concluded that the GAL7 and PGK promoters in combination with strain X904 can be used for efficient synthesis of a VP2 in the form of a N-terminally fused VP2-beta-galactosidase protein.
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PMID:Construction of expression plasmids for Saccharomyces cerevisiae: application for synthesis of poliovirus protein VP2. 312 75

The ability of the bacteriophage 434 operator/repressor system to function in a eukaryotic cell has been explored. An idealized 434 operator was placed at various positions in the PGK promoter of Saccharomyces cerevisiae: within the upstream activator sequence, close to the TATA box, and downstream of the transcription-initiation site. Expression of the 434 cI gene from a 2 microns-based plasmid resulted in significant repression of gene expression from constructs containing the altered promoters linked to a beta-galactosidase reporter gene. Attempts to use a variant of the 434 repressor that has the binding specificity of the P22 repressor (434P22) were unsuccessful, due to a severely inhibitory effect of this gene-product on the growth of the yeast cells.
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PMID:The bacteriophage 434 operator/repressor system in yeast. 749 31

Possible functions of the c-mos proto-oncogene during spermatogenesis were investigated through perturbations of its expression in transgenic mice. Two promoters, one from the pre-meiotic male germ cell-specific mouse phosphoglycerate kinase 2 gene, and the other from the post-meiotic male germ cell-specific rat RT7 gene were used to direct expression of c-mos. Northern blot analysis of testis RNA from transgenic PGK-c-mos mice indicated elevated levels of c-mos RNA in spermatocytes and spermatids compared to controls. No transgene expression was detected in any other tissue examined, suggesting that the mouse PGK2 promoter, like the previously used human PGK2 promoter, confers correct cell-specific expression onto c-mos. The promoter from a newly characterized rat gene, RT7, was shown to direct expression specific to post-meiotic spermatids. Transgenic mice carrying an RT7-lacZ construct displayed immunoreactive bacterial beta-galactosidase as well as enzyme activity in round spermatids. The cellular specificity for beta-galactosidase expression observed in RT7-lacZ transgenic animals was in agreement with endogenous RT7 transcript expression. Northern blot analysis of testis RNA of RT7-c-mos transgenic mice showed elevated levels of c-mos in spermatids, but not in other cells or tissues examined. Western blot analysis demonstrated elevated levels of p43c-mos in spermatids of both PGK-c-mos and RT7-c-mos transgenic animals, but only PGK-c-mos transgenics had increased p43c-mos levels in spermatocytes. Both RT7-c-mos and PGK-c-mos transgenic mice are fertile and show no tendency toward transformation. RT7-c-mos mice have no discernible phenotype associated with the c-mos overexpression in spermatids. However, PGK-c-mos transgenic males exhibited a significant increase in germ cell number, as determined by cell counts using total germ cells and germ cells fractionated by centrifugal elutriation. Because mitotic divisions of germ cells occur prior to PGK-c-mos transgene expression, our observations suggest that c-mos overexpression in spermatocytes causes an alteration in cell-cell interactions.
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PMID:Cell interactions in testis development: overexpression of c-mos in spermatocytes leads to increased germ cell proliferation. 773 67

The ability to direct foreign gene expression from the herpes simplex virus type 1 (HSV-1) genome during an acute or latent infection is a subject of increasing importance in the utilization of HSV vectors for gene therapy. Little is known about the types of transcription factors present in neurons or about whether different neuronal populations within a ganglion vary in their complement of these factors. With respect to HSV-1 latency, it is not known how or why the latency-associated transcript (LAT) promoter is able to function continually during latency while all other viral promoters are inactive. To further studies of these two phenomena, we constructed seven recombinant viruses with various promoter constructs driving expression of the lacZ reporter gene. Each construct was inserted into HSV-1 at the glycoprotein C locus, and recombinant viruses were evaluated for the ability to express beta-galactosidase during acute and latent viral infections in murine dorsal root ganglia. During acute infection of murine dorsal root ganglia, the activities of the promoters varied over a wide range. Constructs containing the murine metallothionein promoter (MT1), the phosphoglycerate kinase promoter, the Moloney murine leukemia virus long terminal repeat (LTR), or the region upstream of and including the HSV LAT core promoter (LAT) were active during the acute but not the latent phase of infection. The addition of transcription factor binding sites present in the upstream LAT region to the MT1 and LTR promoters (LAT-MT1 and LAT-LTR, respectively) significantly increased acute-phase expression. Despite these high initial rates of transcription, of all the promoter constructs only LAT-LTR was able to remain transcriptionally active after the establishment of a latent state. Thus, the Moloney murine leukemia virus LTR provides a DNA element which functions to prevent promoter inactivation during latency. An analogous HSV long-term-expression element is evidently not present in the upstream LAT promoter, indicating that the HSV long-term-expression function is provided by a region outside of that which gives high-level neuronal expression during the acute phase of infection.
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PMID:Long-term promoter activity during herpes simplex virus latency. 793 97

Pgk-1 is an X-linked gene encoding 3-phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis. The regulatory sequences of the Pgk-1 gene were used to drive the E. coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk-lacZ transgene carried on autosomes. The levels of expression of Pgk-1 varied from one adult tissue to another and the transgene was similarly regulated. However, in situ staining of the beta-galactosidase encoded by the transgene indicated extensive cell-to-cell variability in its level of expression. A reproducible subset of cells stained darkly for the transgene product. Some of these beta-galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk-1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis. Although Pgk-1 is X-linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells. Thus, the Pgk-1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell.
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PMID:Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice. 799 75

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1,251 base pairs encoding approximately 47-kDa polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alk1 encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis beta-galactosidase) was tested. Expression of P450alk1 gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining beta-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.
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PMID:Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene. 808 86

In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells. We investigated the feasibility of preparing a glioma vaccine through retrovirus-mediated gene transduction. Glioma cells were cultured primarily from surgically resected tumor tissues of six patients. We obtained more than 1000-fold proliferation of cultures within eight weeks in all six cases. In vitro infection with a recombinant retrovirus GKlacZ carrying an Escherichia coli beta-galactosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells. Further enrichment (approximately 90%) of transduced cells was possible by employing repeated infections or using vectors with neo' marker gene. Two cytokine genes, granulocyte-macrophage colony-stimulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors. The transduced glioma cells produced high levels of both cytokines. We also evaluated simultaneous introduction of two genes with double-expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK). Although the cells transduced with double-expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product. The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation. Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients.
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PMID:Efficient retrovirus-mediated cytokine-gene transduction of primary-cultured human glioma cells for tumor vaccination therapy. 914 Jan 15


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