scholarly journals Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

1986 ◽  
Vol 6 (1) ◽  
pp. 97-104 ◽  
Author(s):  
R Ivarie ◽  
J A Morris
Keyword(s):  
Dna Methylation ◽  
Genomic Dna ◽  
Human Tumor ◽  
Tumor Cell Line ◽  
Optimal Dose ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Wild Type ◽  
Reversion Frequency ◽  
Enzymatic Methylation ◽  
Silent Allele

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation

1986 ◽  
Vol 6 (1) ◽  
pp. 97-104
Author(s):  
R Ivarie ◽  
J A Morris
Keyword(s):  
Dna Methylation ◽  
Genomic Dna ◽  
Human Tumor ◽  
Tumor Cell Line ◽  
Optimal Dose ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Wild Type ◽  
Reversion Frequency ◽  
Enzymatic Methylation ◽  
Silent Allele

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).

Effect of low temperature on genomic DNA methylation in Nile tilapia (Oreochromis niloticus)

2013 ◽  
Vol 37 (10) ◽  
pp. 1460 ◽  
Author(s):  
Huaping ZHU ◽  
Maixin LU ◽  
Zhanghan HUANG ◽  
Fengying GAO ◽  
Xiaoli KE ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Low Temperature ◽  
Oreochromis Niloticus ◽  
Genomic Dna ◽  
Nile Tilapia ◽  
Nile Tilapia Oreochromis Niloticus

Inhibition by dihydrorifampicin of RNA polymerases I and II isolated from nuclei of Rous sarcoma cells and human tumor cells HEp-2

1979 ◽  
Vol 44 (9) ◽  
pp. 2722-2736 ◽  
Author(s):  
Jindřich Kára ◽  
Zdeněk Hostomský
Keyword(s):  
Tumor Cells ◽  
Human Tumor ◽  
Tumor Cell Line ◽  
Selective Inhibition ◽  
Rna Polymerases ◽  
Embryonic Cells ◽  
Human Tumor Cells ◽  
Ribonucleic Acids ◽  
Rous Sarcoma ◽  
Sarcoma Cells

Dihydrorifampicin, a rifampicin derivative hydrogenated at the 18-19 carbon atoms of the aliphatic ansa chain of the rifampicin molecule, inhibits the enzymatic activity of RNA polymerases I and II, isolated from the nuclei of avian tumor cells (Rous sarcoma) and from the human tumor cell line HEp-2. The RNA polymerases from these tumors have been separated and partially purified by chromatography on DEAE Sephadex A-25 and characterized by the sensitivity to α-amanitin. The [3H]UMP-labeled ribonucleic acids synthesized in the isolated nuclei of Rous sarcoma cells in the presence and absence of DHR were analyzed by sedimentation analysis in sucrose density gradients. It was found that the synthesis of rRNAs and mRNAs is very significantly inhibited by dihydrorifampicin, whereas the synthesis of tRNAs is much less inhibited at the same DHR concentration (100μg/ml). The observed cytostatic effect of DHR on the growth of human tumor cells HEp-2 and embryonic cells in culture is apparently mediated by the selective inhibition of RNA polymerases I and II in human and avian cells. The relationship between the molecular structure of DHR and its affinity to RNA polymerases of eukaryotic cells is discussed.

scholarly journals DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Vanessa Lakis ◽  
◽  
Rita T. Lawlor ◽  
Felicity Newell ◽  
Ann-Marie Patch ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Neuroendocrine Tumors ◽  
Methylation Profile ◽  
Pancreatic Neuroendocrine Tumors ◽  
Wild Type ◽  
Genomic Information ◽  
Chromosome 11 ◽  
Dna Methylation Profile ◽  
Methylation Patterns ◽  
Recurrent Patterns

AbstractHere we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.

scholarly journals Mutants of downy mildew resistance in Lactuca sativa (lettuce).

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 867-874
Author(s):  
P A Okubara ◽  
P A Anderson ◽  
O E Ochoa ◽  
R W Michelmore
Keyword(s):  
Molecular Marker ◽  
Downy Mildew ◽  
Genomic Dna ◽  
Spontaneous Mutation ◽  
Multigene Families ◽  
Downy Mildew Resistance ◽  
Bremia Lactucae ◽  
Wild Type ◽  
Mildew Resistance ◽  
Recessive Mutations

Abstract As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Theo A Schuurs ◽  
Eveline A M Schaeffer ◽  
Joseph G H Wessels
Keyword(s):  
Gene Silencing ◽  
Genomic Dna ◽  
Type Strain ◽  
Cytosine Methylation ◽  
Wild Type Strain ◽  
Schizophyllum Commune ◽  
Southern Analysis ◽  
Transcriptional Level ◽  
Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

scholarly journals Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

2009 ◽  
Vol 117 (2) ◽  
pp. 217-222 ◽  
Author(s):  
Letizia Tarantini ◽  
Matteo Bonzini ◽  
Pietro Apostoli ◽  
Valeria Pegoraro ◽  
Valentina Bollati ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Particulate Matter ◽  
Promoter Methylation ◽  
Genomic Dna ◽  
Inos Promoter
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