Archive for the ‘Demethylation’ Category

A Gene-Specific Promoter in Transgenic Mice: DISCUSSION(5)


We assume that Pgk-2 demethylation is related to gene expression, specifically, potentiation of gene expression. Demethylation is the earliest potential regulatory event we have observed prior to the initiation of endogenous Pgk-2 gene transcription. The appearance in the Pgk-2 promoter of DNase I-hypersensitive sites, tissue-specific protein-DNA interactions, and the initiation of transcription itself all occur 5-10 days after the demethylation event. flovent inhaler

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A Gene-Specific Promoter in Transgenic Mice: DISCUSSION(4)

The Pgk-2/CAT transgenes underwent the same stage-specific demethylation that the endogenous Pgk-2 gene undergoes in prospermatogonia. Thus the Pgk-2 core promoter region appears to contain a unique cis-acting signal that confers tissue-, stage-, and cell type-specific demethylation on the CAT reporter gene in a manner exactly analogous to that occurring in the endogenous Pgk-2 gene. That this signal is unique to the Pgk-2 promoter is indicated by the absence of any stage-specific demethylation of the Tf/ CAT transgene (Fig. 6B). buy cipro

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A Gene-Specific Promoter in Transgenic Mice: DISCUSSION(3)


Thus, the same sites in the CAT sequence that showed testis-specific demethylation in Pgk-2/CAT transgenes showed ubiquitous hypomethylation in TfCAT transgenes in all tissues tested, including the testis. Hypomethylation of this transgene occurred in the testis despite the fact that this construct is not expressed in spermatogenic cells. This is once again consistent with a role for hypomethylation in potentiating gene expression. Thus genes bearing strong CpG islands appear to remain in a state of constitutive hy-pomethylation such that they are poised to be expressed in a large variety of tissues, if not all. The specific parameters of different CpG islands that make one stronger or weaker than another with respect to inducing maintenance of hy-pomethylation of flanking sequences in the testis, or any other tissue, are yet to be defined. Such parameters could include the total size of the CpG island, the total G+C content of each, the frequency of CpG dinucleotides within each, and/or the number of islands and their proximity, if more than one island is present. buy ventolin inhalers

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A Gene-Specific Promoter in Transgenic Mice: DISCUSSION(2)

Demethylation of sites within the 5′ half of the coding sequence of the Pgk-2 gene is presumed to represent the 3′ extent of a domain of demethylation that is centered over the 5′-flanking regulatory region. Analogous testis-specific demethylation of transgenes at sites within the coding sequence of the CAT reporter gene linked to either the core promoter alone (188 Pgk-2/CAT), or the core promoter plus enhancer region (515 Pgk-2/CAT) of the Pgk-2 gene, indicates that the cis-acting signal for testis-specific demeth-ylation resides in the Pgk-2 core promoter region. That the 188 Pgk-2/CAT transgene undergoes demethylation, but is not subsequently expressed, demonstrates that this demeth-ylation signal is distinct from the tissue-specific enhancer signal that has been mapped to a region 5′ to the core promoter. This result also shows that demethylation alone is not sufficient to initiate transcription but may be part of a requisite process of potentiation of the gene that normally precedes transcriptional activation. buy yasmin online

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A Gene-Specific Promoter in Transgenic Mice: DISCUSSION(1)


Differential expression of tissue-specific genes during mammalian development is achieved through a complex hierarchy of regulatory mechanisms. The well-documented correlation between expression and the degree of hypo-methylation of DNA in a large number of tissue-specific genes suggests that demethylation plays a role in this process. Many genomic DNA sequences are at least partially methylated in the genome of the spermatozoon and in the very early (1- to 8-cell stage) embryonic genome. During subsequent stages in the blastula, this methylation is lost, such that a relatively unmethylated ‘‘ground state’’ is attained. This is followed by de novo methylation of most non-CpG island-bearing genes at or just prior to gastrula-tion. Then, as specific cell lineages become allocated, demethylation occurs in a gene- and tissue-specific manner, such that many tissue-specific genes become hypomethy-lated specifically in the cell types in which they are, or will be, expressed. canadian family pharmacy

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A Gene-Specific Promoter in Transgenic Mice: RESULTS(6)

Disruption of the Sp1-Binding Site Did Not Affect Demethylation of Pgk-2/CAT Transgenes

Previous results have shown that in certain CpG island-bearing genes, demethylation is regulated by Sp1-binding sites; and Sp1 is known to be required for normal transcriptional activation from the Pgk-2 promoter. To determine whether the Sp1-binding site in the non-island-containing Pgk-2 promoter is involved in regulating testis-specific demethylation of transgenes, we generated three lines of mice carrying a 515 Pgk-2/CAT transgene with a mutated Sp-1-binding site (GC box) in the core promoter (mSp1/1, mSp1/2, and mSp1/5) (Fig. 1D). All three lines showed reduced levels of testis-specific expression of the CAT transgene (data not shown), confirming that the Sp1-binding site is important for normal expression of the Pgk-2 gene. Methylation of this construct in both testis and liver was analyzed by Southern blotting (Fig. 7). Like the 515 Pgk-2/CAT and 188 Pgk-2/CAT transgenes, the mSp1 Pgk-2/CAT transgene was demethylated in the testis and methylated in liver. This suggests that neither the GC box sequence itself, nor binding of Sp1 to that sequence, acts as the signal for testis-specific demethylation of the Pgk-2/ CAT transgenes. buy ortho tri-cyclen online

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A Gene-Specific Promoter in Transgenic Mice: RESULTS(5)


To determine whether the gene- and tissue-specific hy-pomethylation of Pgk-2/CAT transgenes observed in testes of adult transgenic mice results from a similar developmentally staged demethylation event, we first used Southern blot analysis to show that the 188 Pgk-2/CAT transgene is partially demethylated in DNA isolated from whole testis tissue from newborn mice (Fig. 6A). This partial demethylation is presumed to reflect a combined signal from testicular somatic cells in which the transgene is fully methylated and germ cells in which the transgene is un-methylated. We then used PCR to analyze methylation at Hpa II sites in the 188 Pgk-2/CAT and Tf/CAT transgenes in DNA isolated from purified populations of prospermatogonia at 18.5 dpc and pachytene spermatocytes from adult mice (Fig. 6B). ventolin inhalers

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