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Biomedical subjects

G D Ginder

Publications and source records attributed to G D Ginder.

43 records · Page 3Linked to original sources
PubMed1983

In vivo demethylation of chicken embryonic beta-type globin genes with 5-azacytidine.

We have studied the in vivo effects of the methyl-blocking cytotoxic pyrimidine analog 5-azacytidine upon DNA methylation and expression of the normally inactive embryonic beta-type globin genes in adult chickens. We find that 5 daily treatments with intravenous 5-azacytidine is capable of converting the normally completely methylated embryonic rho gene to a greater than 75% demethylated state as assayed by the restriction endonucleases HpaII, MspI, and HhaI. However, this change in methylation is not accompanied by activation of the embryonic rho gene at the level of globin protein or globin RNA. Although our analysis does not exclude a few copies of embryonic globin RNA per cell, this result is still in contrast with the results seen with the fetal gamma-globin genes in baboons and humans [17; see also other chapters in this volume]. Hence demethylation by 5-azacytidine treatment alone is not sufficient to activate the chicken embryonic beta-globin genes. We propose that these seemingly disparate results can be reconciled by a model in which certain genes in specific cell types (eg, human and baboon gamma-globin in adult erythroid cells, chicken ev-1 in MSB cells, mouse metallothionein-1 in lymphoblastoid cells) are in a poised state such that removal of specific methyl cytosines results in a high level of transcription. In contrast other genes (including chicken rho and epsilon embryonic globin genes in adult erythroid cells) are in a suppressed state that overrides activation by demethylation. Alternatively, 5-azacytidine could activate certain genes by a mechanism totally independent of DNA methylation. For instance, selection of specific early erythroid cell types could account for the results seen in baboon fetal globin gene activation. The availability of various eukaryotic DNA transformation systems, characterization of eukaryotic DNA methylases, and development of large-scale cultures of erythropoietic precursor cells should facilitate detailed examination of these various states of gene activation and allow the elucidation of the precise role of DNA methylation in the regulation of globin gene switching.

Animals↗
PubMed1979

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.

Animals↗
PubMed1978

A new method for the purification and identification of covalently closed circular DNA molcules.

A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.

DNA, Circular↗
PubMed1975

Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes.

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.

Adult↗
PubMedDate not supplied

Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines.

BACKGROUND: Normal mammary epithelial cells express mainly gap junction connexin 26 (Cx26) that is either reduced or absent in breast cancers. Since connexin gene mutations are rare we examined if Cx26 gene repression is related to hypermethylation. MATERIALS AND METHODS: Five breast epithelial cell lines were examined for Cx26 mRNA expression and hypermethylation. Treatment with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-CdR), was carried out to determine if Cx26 gene expression could be upregulated. RESULTS: Cx26 expression was easily detectable in an immortalized human mammary epithelial cell line (MCF-10) and markedly diminished (MDA-MB231) or undetectable in (MCF-7, BT-20, T47-D) breast cancer cell lines. Hypermethylation of the Cx26 5' region was observed in MCF-10 and MCF-7 cells. Treatment with 5-Aza-CdR resulted in slight or no induction in Cx26 expression in breast cancer cell lines. CONCLUSIONS: Hypermethylation is unlikely to be a major mechanism for Cx26 gene repression in human mammary cancer cell lines.

Antimetabolites, Antineoplastic↗