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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.
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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.
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  • 3
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The C86 line of female embryonal carcinoma cells contains one active and one inactive X chromosome. Following methylnitrosourea mutagenesis, a clone called C86AGM2 was isolated that carries a mutated hprtgene on the active X chromosome. This hprtm allele encodes an HPRT enzyme that has less than 1% normal enzyme activity, is thermolabile, and has an altered isoelectric point. Following treatment with drugs that demethylate DNA, the hprt+ gene from the inactive X chromosome in C86AGM2 cells became active as determined by the appearance of HPRT activity with the thermodenaturation and electrofocusing characteristics of the normal enzyme. No expression of this hprt+ gene occurred if C86AGM2 cells were induced to differentiate prior to DNA demethylation. Stable lines of C86AGM2 cells expressing both the hprtm and hprt+ genes did not inactivate either gene following differentiation.
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  • 4
    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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  • 5
    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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  • 6
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The mammalian genome contains two genes encodingphosphoglycerate kinase; the pgk-1gene is X-linked and is expressed in all cells except sperm, while the pgk-2gene is expressed exclusively in sperm cells. The mouse genome contains no pseudogenes derived from pgk-2.On the other hand, the genomes of Balb/c and C3H/He strain mice contain six other regions with sequences homologous to those of pgk-1cDNA. These pgk-related sequences are likely derived from the pgk-1gene by retroposition because all are located on autosomal chromosomes and because none appear to be interrupted by introns. Two of the presumed pseudogenes contain sequences homologous to all regions of the pgk-1cDNA while the other four genomic regions were truncated at the 5′, 3′, or both ends. One of the truncated pseudogenes was sequenced. Its pgk-related sequence was not flanked by direct repeats, suggesting that loss of the 5′ and/or 3′ ends of this retrogene may have occurred following its integration into the genome. Our evidence suggests that pgk-1-derived retroposons arose initially more than 100 million years ago and have continued to arise until so recently that some are unique to different mouse strains.
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  • 7
    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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  • 8
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The extent of methylation of DNA sequences upstream and within the two X-linked genes,Pgk-1 andHprt, was analyzed in male and female somatic cells and in female embryonal carcinoma cells carrying either two active X chromosomes (Xa) or one active and one inactive X chromosome (Xi). Sites upstream and within the first intron of bothPgk-1 andHprt were heavily methylated on the Xi in somatic cells and in embryonal carcinoma cells with an Xi. Reactivation of this Xi was accompanied by extensive demethylation of these sites. In female embryonal carcinoma cells with two active X chromosomes, one X inactivates during differentiation in culture; however, methylation did not occur during differentiation, consistent with the idea that DNA methylation does not play a role in the initiation of X inactivation but may be involved in maintaining inactivation of those genes on the Xi.
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  • 9
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract TheXist gene resides on the X chromosome and is expressed in female but not male somatic cells. In female cells, only theXist allele on the inactive X chromosome is transcribed. We investigated the expression ofXist in diploid P10 female embryonal carcinoma cells that have two active X chromosomes.Xist RNA was present in these P10 cells. The X chromosomes in P10 cells carry differentXist alleles whose transcripts can be distinguished by restriction digestion of their cDNAs. Both alleles were expressed. Clones of P10 cells that had lost an X chromosome did not expressXist from the remaining allele. ThusXist is expressed in cultured cells developmentally arrested prior to X chromosome inactivation, indicating that theXist transcript is not always derived from an inactive X chromosome. Therefore,Xist expression per se cannot be a sufficient signal to inactivate an X chromosome.
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  • 10
    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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