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  • 1
    ISSN: 1573-4927
    Keywords: X chromosome ; phosphoglycerate kinase gene ; LINES
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1b allele causes the appearance of mouse PGK-1b enzyme activity. We describe here cloning of mousePgk-1a, an allele ofPgk-1 which encodes an enzyme, PGK-1a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1b andPgk-1a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1b and PGK-1a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1b andPgk-1a including part of an L1 repeated element unique toPgk-1a.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-4927
    Keywords: X chromosome ; phosphoglycerate kinase gene ; LINES
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1b allele causes the appearance of mouse PGK-1b enzyme activity. We describe here cloning of mousePgk-1a, an allele ofPgk-1 which encodes an enzyme, PGK-1a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1b andPgk-1a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1b and PGK-1a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1b andPgk-1a including part of an L1 repeated element unique toPgk-1a.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Plasmid DNA can be efficiently transfected into embryonal carcinoma cells but it is difficult to isolate clones of cells stably expressing genes present on the transfected plasmids. Even in clonal populations derived from transfected cells, the introduced genes are expressed in some but not all cells. Cotransfection with a region of thePgk-1 gene results in more efficient, stable cotransformation due to increased numbers of copies of the transfected plasmids integrated into the genomic DNA. ThePgK-1 genomic sequences did not allow the plasmid DNA to replicate autonomously but seemed to enhance the ligation of transfected plasmids before their integration into the host genome. Our results suggest a model in which the plasmid DNAs are able to integrate and subsequently excise from the host genome by recombination events enhanced by transcription through the tandemly repeated sequences of the transfected plasmids.
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  • 4
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding theE. coli β-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the β-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.
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  • 5
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Introduction of recombinant genes into mammalian cells in culture has been an important procedure in establishing the molecular mechanisms of various cellular processes. The efficiency with which plasmid borne recombinant genes are expressed following stable integration into genomes of embryonal carcinoma cells is low. Using the P19 embryonal carcinoma cells as recipients, we found that constructs carrying the promoter and intragenic regions of the murinePgk-1 gene were expressed with high efficiency. This elevated expression was associated with increased numbers of copies of the transfected plasmid DNA stably associated with the genomes of recipient cells. The elevated plasmid copy numbers may result from enhanced ligation of transfected plasmids because cotransfected plasmids were also integrated in increased numbers. The enhanced integration and expression of transfected plasmids required active transcription through an intragenic region ofPgk-1, perhaps resulting in more recombinogenic plasmid DNAs.
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  • 6
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast-like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast-like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle-specific isoform of myosin heavy-chain mRNA in immortal lines of these fibroblast-like cells. These results suggest that these fibroblast-like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.
    Additional Material: 9 Ill.
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  • 8
    ISSN: 1058-8388
    Keywords: X-linked gene ; β-galactosidase ; Pgk-lacZ ; glycolysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: 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 β-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 β-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. © 1994 Wiley-Liss, Inc.
    Additional Material: 15 Ill.
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