Journal: Nucleic Acids Research

Article Title: Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana

doi: 10.1093/nar/gkw067

Figure Lengend Snippet: DNA methylation is involved in telomere homeostasis in Arabidopsis thaliana . ( A ) Southern blot analysis showing the pattern of DNA methylation of telomeric repeats in cmt3, met1, axe1–5 (left panel), hdt4–1 and attrb2–1 mutants (right panel). DNA blots of methylation-sensitive restriction enzyme-digested gDNA probed with [TTTAGGG] 70 repeat sequences. M, MspI; H, HpaII; B, BstNI; E, EcoRII. Arrowheads represent the different signals detected by probe compared to wild-type. Small and large white bar indicate the high and low molecular weight ranges of the gDNAs, respectively, affected by restriction enzyme digestion. ( B ) Chop-PCR in subtelomeric and ITSs regions using McrBC endonuclease. 4R-300, -600, -800 and -2100 indicate the different regions on the subtelomeric regions closed to telomere in the right arm of chromosome IV. The 60S RP gene was used as a reference for quantitative comparisons. −, +; McrBC-untreated/ -treated.

Article Snippet: A total of 10 μg of genomic DNA was digested with the methylation sensitive restriction enzymes, 20 U of MspI, HpaII, BstNI and EcoRII (Fermentas, Thermo Scientific) and then separated by electrophoresis in a 0.8% agarose gel.

Techniques: DNA Methylation Assay, Southern Blot, Methylation, Molecular Weight, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: GFP expression during selection with puromycin. Cells plated on puromycin containing medium were sorted for GFP expression at the indicated times. The percentage of cells positive for GFP expression in pI-GFP transfected cells (GFP expressors) is given in black. The percentage of GFP expression in pI-EcoGFP transfected cells (M.EcoRII–GFP fusion expressors) is given in grey. After each round of sorting, equal numbers of GFP positive cells were re-plated for continued growth in the presence of puromycin.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: Expressing, Selection, Transfection

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Fluorescence detection of GFP and the M.EcoRII–GFP fusion in HK293 cells. Each of the two constructs was transfected into HK293 and subjected to long-term growth under puromycin selection. GFP fluorescence was easily detected with fluorescence microscopy when either construct was used. A set of micrographs is depicted for expression of the M.EcoRII–GFP fusion: ( A ) light microscopy and ( B ) fluorescence microscopy.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: Fluorescence, Construct, Transfection, Selection, Microscopy, Expressing, Light Microscopy

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Effect of C m CWGG methylation at a promoter that is unmethylated at CG sites. Bisulfite treated DNA from the APC promoter was amplified cloned and sequenced as described in Materials and Methods. The 279 bp amplicon contains 19 CG dinucleotide pairs. It contains three CCWGG sites, one in the forward primer, and two downstream from the forward primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the three CCWGG sites in each clone. ( A ) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. ( B ) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: Methylation, Amplification, Clone Assay, Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Comparison of RNA expression levels from the APC gene and the hTERT gene in cells expressing GFP alone, or the M.EcoRII–GFP fusion. C t values obtained at each fold dilution of input RNA are plotted. Two independent RNA preparations were used, one preparation was tested in duplicate and one preparation was tested in triplicate. P -values from χ 2 analyses are listed below each set of data points. Each value is > > 0.05, indicating that there is no significant difference in the level of RNA expression when comparing the two cell lines, for either of the two genes tested. ( A ) Data for the hTERT gene. closed squares, cells expressing the M.EcoRII–GFP fusion. Closed diamonds, cells expressing the GFP alone. ( B ) Data for the APC gene. Closed squares, cells expressing the M.EcoRII–GFP fusion. Closed diamonds, cells expressing the GFP alone.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: RNA Expression, Expressing

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Florescence cell sorting quantification of cells expressing GFP and the M.EcoRII–GFP fusion in human HK293 cells. Each of the two constructs was transfected into HK293 and subjected to long-term growth under puromycin selection. Fluorescence cell sorting at the emission maximum for GFP quantifies the number of cells expressing each protein. ( A ) Quantification of cells expressing the GFP construct. ( B ) Quantification of cells expressing the M.EcoRII–GFP fusion. In each panel background fluorescence is given in black and cellular GFP fluorescence above background is given in grey. Total counts given by the stippled line indicate that not all positive events were collected in the sort.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: FACS, Expressing, Construct, Transfection, Selection, Fluorescence

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Effect of C m CWGG methylation at a promoter that is heavily methylated at CG sites. Bisulfite treated DNA from the SERPINB5 promoter was amplified, cloned and sequenced as described in Materials and Methods. The 372 bp amplicon contains 19 CG dinucleotide pairs. It also contains two CCWGG sites, one in the reverse primer and one downstream from the reverse primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the two CCWGG sites in each clone. ( A ) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. ( B ) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: Methylation, Amplification, Clone Assay, Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Detection of M.EcoRII activity from the M.EcoRII–GFP fusion expression vector. After 21 days of selection with 0.5 µM puromycin, fluorescent cells were harvested and genomic DNA was prepared. The DNA was digested with the isoschizomers R.EcoRII and R.BstNI. In this pair, R.EcoRII is unable to cleave the C m CWGG site when the internal cytosine is methylated. While R.BstNI is unaffected by cytosine-5 methylation at this site.

Article Snippet: RNA was isolated from 10 week HK293 cells from cells expressing GFP and M.EcoRII–GFP, by use of an RNAqueous™ Kit, (Ambion, Austin, TX).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Selection, Methylation

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Effect of C m CWGG methylation at a promoter that is unmethylated at CG sites. Bisulfite treated DNA from the APC promoter was amplified cloned and sequenced as described in Materials and Methods. The 279 bp amplicon contains 19 CG dinucleotide pairs. It contains three CCWGG sites, one in the forward primer, and two downstream from the forward primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the three CCWGG sites in each clone. ( A ) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. ( B ) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.

Article Snippet: Five micrograms of uncut GFP and 5 µg of cut EcoRII–GFP 293 genomic DNA was then bisulfite treated using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA), according to the manufacturer's instructions.

Techniques: Methylation, Amplification, Clone Assay, Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Effect of C m CWGG methylation at a promoter that is heavily methylated at CG sites. Bisulfite treated DNA from the SERPINB5 promoter was amplified, cloned and sequenced as described in Materials and Methods. The 372 bp amplicon contains 19 CG dinucleotide pairs. It also contains two CCWGG sites, one in the reverse primer and one downstream from the reverse primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the two CCWGG sites in each clone. ( A ) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. ( B ) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.

Article Snippet: Five micrograms of uncut GFP and 5 µg of cut EcoRII–GFP 293 genomic DNA was then bisulfite treated using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA), according to the manufacturer's instructions.

Techniques: Methylation, Amplification, Clone Assay, Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

doi: 10.1093/nar/gki920

Figure Lengend Snippet: Detection of M.EcoRII activity from the M.EcoRII–GFP fusion expression vector. After 21 days of selection with 0.5 µM puromycin, fluorescent cells were harvested and genomic DNA was prepared. The DNA was digested with the isoschizomers R.EcoRII and R.BstNI. In this pair, R.EcoRII is unable to cleave the C m CWGG site when the internal cytosine is methylated. While R.BstNI is unaffected by cytosine-5 methylation at this site.

Article Snippet: Five micrograms of uncut GFP and 5 µg of cut EcoRII–GFP 293 genomic DNA was then bisulfite treated using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA), according to the manufacturer's instructions.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Selection, Methylation