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1.
Nucleic Acids Res ; 36(3): e19, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18208840

ABSTRACT

The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes.


Subject(s)
Alleles , Chromatin/chemistry , DNA Footprinting/methods , Polymerase Chain Reaction/methods , Transcription Factors/metabolism , Animals , Base Sequence , Chromatin/metabolism , Diphosphates/metabolism , Mice , Molecular Sequence Data , NIH 3T3 Cells , Promoter Regions, Genetic , Receptor, Macrophage Colony-Stimulating Factor/genetics , Repetitive Sequences, Nucleic Acid , Robotics
2.
Transplant Proc ; 37(8): 3422-3, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16298615

ABSTRACT

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.


Subject(s)
Cryopreservation/methods , Islets of Langerhans/cytology , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Culture Techniques , Cell Survival , Enzyme Activation , Humans
3.
Biochemistry (Mosc) ; 70(5): 559-67, 2005 May.
Article in English | MEDLINE | ID: mdl-15948709

ABSTRACT

Consideration of epigenetic silencing, perhaps by DNA methylation, led to an epigenetic complementation (EC) model for evolution by gene duplication (Rodin and Riggs (2003) J. Mol. Evol., 56, 718-729). This and subsequent work on genome-wide analyses of gene duplicates in several eukaryotic species pointed to a fundamental link between localization in the genome, epigenetic regulation of expression, and the evolutionary fate of new redundant gene copies, which can be either non- or neo-functionalization. Our main message in this report is that repositioning of a new duplicate to an ectopic site epigenetically alters its expression pattern, and concomitantly the rate and direction of mutations. Furthermore, comparison of syntenic vs. non-syntenic pairs of gene duplicates of different age unambiguously indicates that repositioning saves redundant gene duplicates from pseudogenization and hastens their evolution towards a new development-time and tissue-specific pattern of function.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Evolution, Molecular , Genes, Duplicate , Animals , Humans , Models, Genetic , Mutation , Pseudogenes
4.
Biotechniques ; 34(3): 498-502, 504, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12669699

ABSTRACT

We describe here a quantitative real-time PCR assay for the detection of single-base-pair differences that does not require fluorescently labeled gene-specific probes or complicated primer combinations. Following PCR or RT-PCR of a gene segment that may contain allele-specific differences, 100 pg amplified product are used for a real-time PCR with allele-specific primers and SYBR Green. The use of HEPES buffer at a pH of 6.95 together with AmpliTaq DNA polymerase results in a threshold difference between the correct template and the mismatched template of as many as 20 cycles, depending on the mismatch. Correct matches can be detected in an excess of mismatched template at least at the 0.01 level for the six primer-template matches versus mismatches tested: GC vs. A.C, AT vs. G.T, GC vs. C.C, GC vs. G.G, AT vs. C.T, and GC vs. G.A. Because the initial amplification is separate from real-time detection, conditions can be independently optimized for each step, making the assay particularly suitable for the detection of allele-specific expression in single cells.


Subject(s)
Base Pair Mismatch/genetics , Gene Frequency/genetics , Polymerase Chain Reaction/methods , Sequence Alignment/methods , Animals , Base Sequence , DNA Mutational Analysis/methods , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Sequence Analysis, DNA/methods
5.
Biotechniques ; 33(5): 1090-7, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12449387

ABSTRACT

The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation. The use of near-infrared fluorescent-labeled primers with detection by a LI-COR DNA sequencer provides for safe, nonradioactive detection, similar in sensitivity to the use of 32P-labeled primers but with the additional advantage that high-quality digitized data are obtained directly. Multiplexing can be performed; that is, more than one sequence can be analyzed in a single reaction, and multiple reactions can be processed robotically. Primer sets for exons 5-8 of the tumor suppressor gene, p53, were designed for simultaneous thermal cycling. The improved procedure with infrared detection was used to monitor low-frequency damage (<1 break/kb) and/or repair of UVB, UVC, and chemical methylation. Quantitative data on the linearity of response and reproducibility are described. The coefficient of variation for technical replicates was typically 10%. The methods described here will permit high sample throughput for the detection of DNA damage and repair as well as in vivo protein footprints.


Subject(s)
DNA Damage , DNA Repair , Polymerase Chain Reaction/methods , Cells, Cultured/radiation effects , DNA Primers , DNA, Neoplasm/isolation & purification , DNA, Neoplasm/metabolism , DNA, Neoplasm/radiation effects , Exons/genetics , Fibroblasts/radiation effects , Genes, p53 , HeLa Cells , Humans , Robotics , Sensitivity and Specificity , Time Factors , Ultraviolet Rays/adverse effects
6.
Cytogenet Genome Res ; 99(1-4): 17-24, 2002.
Article in English | MEDLINE | ID: mdl-12900540

ABSTRACT

X chromosome inactivation and DNA methylation are reviewed, with emphasis on the contributions of Susumu Ohno and the predictions made in my 1975 paper (Riggs, 1975), in which I proposed the "maintenance methylase" model for somatic inheritance of methylation patterns and suggested that DNA methylation would be involved in mammalian X chromosome inactivation and development. The maintenance methylase model is discussed and updated to consider methylation patterns in cell populations that have occasional, stochastic methylation changes by de novo methylation or demethylation, either active or passive. The "way station" model for the spread of X inactivation by LINE-1 elements is also considered, and some recent results from my laboratory are briefly reviewed.


Subject(s)
Cell Differentiation/genetics , Cytogenetics/history , DNA Methylation , Dosage Compensation, Genetic , X Chromosome/genetics , Animals , Histones/metabolism , History, 20th Century , Humans , Japan , Models, Genetic , Sex Chromatin/genetics , United States
7.
Sci STKE ; 2001(77): pl1, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11752648

ABSTRACT

Terminal transferase-dependent PCR (TDPCR) is a versatile, sensitive method for detecting DNA lesions such as those generated by the footprinting agents commonly used to detect in vivo protein-DNA interactions. Data similar to those obtained by ligation-mediated PCR (LMPCR) are obtained, but one advantage of TDPCR is that no special enzymes are needed other than terminal deoxynucleotide transferase, T4 DNA ligase, and thermostable DNA polymerases. A detailed TDPCR protocol is given for using UV photofootprinting to detect in vivo footprints and chromatin fine structure in vertebrate cells. One version of the protocol makes use of nonradioactive labeling by near-infrared fluorochromes and detection by a LI-COR DNA sequencing instrument. Sensitivity similar to that of (32)P-labeling is obtained, but with superior band resolution and quantitation.


Subject(s)
DNA Footprinting/methods , DNA Nucleotidylexotransferase/metabolism , Polymerase Chain Reaction/methods , Animals , Humans , Microscopy, Ultraviolet/methods
8.
Mol Ther ; 4(5): 454-60, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11708882

ABSTRACT

The efficacies of antisense oligonucleotides and ribozymes are greatly dependent on the accessibility of their mRNA targets. Target site accessibility is affected by both RNA structure and the proteins associated along the length of the RNA. To mimic the native state of mRNA for site identification, we have previously used endogenous mRNAs in cellular extracts as targets for defined sequence oligodeoxynucleotides (ODNs) designed to identify both antisense pairing and potential ribozyme cleavage sites. The rationale for this approach is that the specific pairing of an ODN with a mRNA forms a DNA:RNA hybrid that is cleaved by the endogenous RNaseH in the cell extract. To extend the usefulness of this basic approach, we report here the use of semi-random ODN libraries to identify hammerhead ribozyme cleavage sites. Thus, the most accessible sites for antisense and ribozyme base pairing are selected by this approach. A novel feature of the approach described here is the use of terminal transferase-dependent PCR (TDPCR) after reverse transcription to estimate the cleavage efficiency and to precisely determine the RNaseH and ribozyme cleavage sites on mRNAs in cell extracts following treatment with ODN or ribozyme libraries. As a model system, we have targeted the NCOA3 (also known as AIB-1) mRNA in cell extracts. The NCOA3 mRNA encodes a nuclear receptor co-activator that is amplified and over-expressed in a high proportion of breast and ovarian cancers. A highly accessible site on this mRNA was identified, and a ribozyme targeted to this site was demonstrated to effectively downregulate NCOA3 function in cells.


Subject(s)
DNA, Antisense/metabolism , RNA, Catalytic/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Base Pairing , Base Sequence , Binding Sites , DNA, Antisense/genetics , Humans , Nuclear Receptor Coactivator 3 , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Plasmids/genetics , Plasmids/metabolism , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Messenger/chemistry , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease H/metabolism , Substrate Specificity , Transfection , Tumor Cells, Cultured
9.
Genomics ; 74(2): 186-96, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11386755

ABSTRACT

Imprinting of the mouse H19 and Igf2 genes is dependent on the presence of an intervening imprinting control region (ICR) situated 2 kb upstream of H19 and approximately 70 kb downstream of Igf2. Several recent studies have provided substantial evidence that the unmethylated maternal ICR acts as an insulator that prevents activation of Igf2 by a suite of enhancers downstream of the H19 gene. The methylated paternal ICR and H19 promoter have no activity, allowing sole activation of Igf2 expression. We have produced mice in which a duplication of the H19/Igf2 ICR produces, in each generation, two classes of methylation levels that correlated with two Igf2 imprinting phenotypes. One hypermethylated class also shows activation of the normally silent Igf2 gene, whereas the other hypomethylated class shows only slight activation of Igf2, in agreement with methylation's role in ICR function. This study describes a rare, possibly unique type of mutation that induces two distinct phenotypes in each generation.


Subject(s)
DNA Methylation , Gene Targeting , Genomic Imprinting , Proteins/genetics , Alleles , Animals , Blotting, Southern , CpG Islands , Fathers , Female , Insulin-Like Growth Factor II/genetics , Male , Mice , Models, Genetic , Mothers , Mutagenesis, Site-Directed , Mutation , Phenotype , RNA, Long Noncoding , RNA, Messenger/metabolism , RNA, Untranslated/genetics
10.
Biochemistry ; 40(13): 4096-105, 2001 Apr 03.
Article in English | MEDLINE | ID: mdl-11300790

ABSTRACT

We have developed a new genomic sequencing method for detecting, with resolution at the nucleotide level, the interstrand DNA cross-links induced by 4,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cross-links (diadducts) initially formed are converted into monoadducts by alkali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR). After alkali reversal, but not before, the DNA strands can be separated and used as templates for gene-specific primer extension, which is the first step in the TD-PCR procedure. The converted psoralen adducts block primer extension, and the prematurely terminated single-stranded products are then amplified by TD-PCR and visualized on a sequencing gel. Adducts formed by angelicin, a psoralen derivative that forms only monoadducts, were also investigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vitro-treated DNA revealed that the binding of transcription factors inhibited both psoralen cross-linking and angelicin monoadduct formation in the c-JUN and c-FOS promoters in living human cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of general use for study of in vivo protein-DNA interactions and DNA repair.


Subject(s)
Cross-Linking Reagents/metabolism , DNA Adducts/metabolism , Nucleosomes/metabolism , Transcription Factors/antagonists & inhibitors , Trioxsalen/metabolism , Base Sequence , Binding Sites/genetics , Cell Line , Cross-Linking Reagents/chemistry , DNA Adducts/antagonists & inhibitors , DNA Footprinting , DNA Nucleotidylexotransferase/metabolism , Furocoumarins/metabolism , Humans , Intercalating Agents/metabolism , Molecular Sequence Data , Nucleosomes/genetics , Polymerase Chain Reaction/methods , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Trioxsalen/analogs & derivatives , Trioxsalen/chemistry
12.
Nat Biotechnol ; 18(10): 1108-11, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11017053

ABSTRACT

Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein-DNA footprints. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually <200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (>350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5'-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam-Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.


Subject(s)
DNA Footprinting/methods , DNA Ligases/metabolism , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Animals , Automation , CHO Cells , Cricetinae , DNA Damage/genetics , DNA Methylation , DNA Primers/genetics , DNA Repair/genetics , Fluorescent Dyes , Genes, p53/genetics , HeLa Cells , Humans , Infrared Rays , Phosphoglycerate Kinase/genetics , Promoter Regions, Genetic/genetics , Reproducibility of Results , Robotics , Sensitivity and Specificity , Time Factors , Ultraviolet Rays
13.
Nucleic Acids Res ; 28(18): 3594-9, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10982881

ABSTRACT

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.


Subject(s)
DNA-Cytosine Methylases/metabolism , DNA/metabolism , Gas Chromatography-Mass Spectrometry/methods , Alkaloids/metabolism , Azocines , Cloning, Molecular , DNA Methylation , Nitrogen Isotopes , Quinolizines , Substrate Specificity
14.
Mol Ther ; 2(1): 26-38, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10899825

ABSTRACT

Modulation of gene expression by catalytic RNA requires accessible ribozyme cleavage sites in the target mRNA, and accessibility is determined by the secondary and tertiary structure of the target RNA, as affected by its interactions with cellular proteins. As we previously reported, an oligonucleotide-scanning approach using antisense oligonucleotides can be used to determine RNA accessibility in cell extracts. To test whether this method can be used to improve selection of ribozyme target sites, we designed ribozymes corresponding to the sites identified by oligonucleotide scanning and have evaluated their catalytic activities, first in cell extracts and then in transduced cell lines. As a target we used the mRNA of murine DNA (cytosine-5)-methyltransferase 1 (MTase). For intracellular studies, the ribozyme genes were inserted downstream of a Pol III tRNAVAL promoter, which in turn was cloned in the U3 region of a retroviral vector. We find that the efficiency of the ribozymes both in cell extracts and in vivo corresponds with the relative effectiveness predicted by the oligonucleotide-scanning assay. The best ribozyme causes a 70-80% reduction in the MTase mRNA levels in NIH 3T3 cells that are stably transduced with the retroviral constructs. This reduction in mRNA levels is accompanied by a small decrease in the methylation of repetitive intercisternal A particle DNA elements. Ribozyme expression also increased several-fold the reactivation frequency of a methylation-silenced green fluorescent protein (GFP) transgene. Both the reduction in methylation and reactivation of GFP were roughly equivalent to the effects obtained by treating NIH 3T3 cells with 2.5 microM 5-azacytidine, which gives an effect of about 10% of maximum. These results confirm the validity of the cell extract approach for ribozyme site selection and provide a potentially useful ribozyme for future study of DNA methyltransferase function.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Oligonucleotides/metabolism , RNA, Catalytic/metabolism , RNA, Messenger/metabolism , 3T3 Cells , Animals , Blotting, Northern , Blotting, Southern , CpG Islands/genetics , Gene Silencing , Genes, Intracisternal A-Particle , Genes, Reporter , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Methylation , Mice , Models, Genetic , Nucleic Acid Conformation , Plasmids/metabolism , Promoter Regions, Genetic , RNA, Catalytic/genetics , Retroviridae/genetics , Ribonucleases/metabolism , Transcription, Genetic , Transduction, Genetic
15.
Nucleic Acids Res ; 28(7): 1656-64, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10710433

ABSTRACT

We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin [J. Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)]. When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step. The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer. This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR. Band patterns obtained are reproducible and quantifiable. We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage. Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.


Subject(s)
RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Base Sequence , Cell Line , DNA Nucleotidylexotransferase , DNA Primers/genetics , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , RNA/chemistry , RNA/genetics , RNA Splicing , RNA, Catalytic/metabolism , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data
16.
Nucleic Acids Res ; 28(7): E25, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10710442

ABSTRACT

We describe here a very sensitive technique for RNA structure analysis and the determination of transcription start sites and demonstrate its use for mapping the start site of the imprinted Snrpn gene in individual hippocampal neurons. The method is adapted from reverse transcription-terminal transferase-dependent PCR (RT-TDPCR) to include amplification of the antisense sequence by in vitro transcription just prior to the final PCR step. The method should be useful for analysis of all genes for which variation in promoter usage and/or differences in RNA secondary structure may be specific to a given cell type or developmental stage.


Subject(s)
Neurons/metabolism , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribonucleoproteins, Small Nuclear/genetics , Animals , DNA , DNA Nucleotidylexotransferase/metabolism , Hippocampus/chemistry , Hippocampus/cytology , Mice , Molecular Sequence Data , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA/chemistry , RNA/metabolism , Transcription, Genetic
18.
Nucleic Acids Res ; 26(7): 1807-11, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9512556

ABSTRACT

We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.


Subject(s)
DNA Adducts/analysis , DNA Damage , DNA Footprinting/methods , DNA Nucleotidylexotransferase/metabolism , DNA/radiation effects , Polymerase Chain Reaction/methods , RNA, Untranslated , Transcription Factors/genetics , Animals , Base Sequence , DNA/chemistry , DNA Primers , Mice , Promoter Regions, Genetic , RNA, Long Noncoding , Sensitivity and Specificity , Ultraviolet Rays , X Chromosome
19.
Novartis Found Symp ; 214: 214-25; discussion 225-32, 1998.
Article in English | MEDLINE | ID: mdl-9601020

ABSTRACT

DNA methylation of the X chromosome is reviewed and discussed, with emphasis on the partial methylation seen in the mouse X-linked Pgk1 promoter region. A new study of partial methylation is presented in which the methylation of CpG site H3 in the mouse Igf2 upstream region was quantitatively measured during growth of subcloned cells in tissue culture. Before subcloning the average methylation level was 50%. After subcloning, methylation was highly variable in early stage clones. With continued passage, clones initially having high methylation lost methylation, whereas clones initially having low methylation gained methylation. By about the 25th generation, all clones had returned to a steady-state methylation level of 50%. These findings are discussed in the context of epigenetic mechanisms and epigenetic fidelity. Interpretation of the results is made according to a model that assumes stochastic methylation and demethylation, with rate parameters influenced by local chromatin structure. A second type of study is reported in which we have measured chromatin accessibility differences between the active X chromosome (Xa) and the inactive X chromosome (Xi). We found that Xa/Xi differences in accessibility to DNase I are surprisingly labile. Relatively infrequent DNA nicks rapidly eliminate differential accessibility.


Subject(s)
DNA Methylation , X Chromosome , Animals , Humans , Mice
20.
Nucleic Acids Res ; 26(2): 684-6, 1998 Jan 15.
Article in English | MEDLINE | ID: mdl-9421536

ABSTRACT

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.


Subject(s)
DNA Primers , DNA Replication , RNA, Untranslated , X Chromosome , Alleles , Animals , Base Composition , Cells, Cultured , DNA/analysis , DNA/chemistry , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , RNA, Long Noncoding , Transcription Factors/genetics
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