Cloning of calf thymus satellite I DNA in Escherichia coli

In DNA of the dinoflagellate Crypthecodinium cohnii, 38% of the thymine is replaced by the modified base 5-hydroxymethyluracil, and approximately 3% of the cytosine is replaced by 5-methylcytosine. Both of the modified bases are non-randomly distributed in the DNA. Determinations of 3' nearest neighbors show that HOMeU is preferentially located in the dinucleotides HOMeUpA and HOMeUpC. Pyrimidine tract analysis shows that HOMeU is also greatly enriched in the trinucleotide purine-HOMeU-purine. As in other eukaryotes, methylcytosine in C. cohnii DNA occurs predominantly in the dinucleotide MeCpG. By analysis of restriction endonuclease digestion patterns of C. cohnii total DNA and ribosomal DNA, we have found that the central CpG dinucleotides in the sites for the enzymes Hpa II (CCGG) and Hha I (GCGC) are extensively methylated in both total DNA and ribosomal DNA. Results of digestion with Ava I, however, indicated that not all CpG dinucleotides in the sequence CCTCGGAG are methylated in C. cohnii DNA.

Section: Resultsmentioning

confidence: 99%

Ordered distribution of modified bases in the DNA of a dinoflagellate

Steele

Rae

1980

“…2). The fragment in this band has been shown to originate from satellite I (14). Typical yields with our isolation procedure were about 30 pg of fragment DNA per mg original DNA.…”

Section: Digestion Of the Major Ecori Fragmentmentioning

confidence: 90%

“…Bovine satellite I, density 1.714 g/cm3 (12), which is cut by EcoRI into segments of about 1400 base pairs (13)(14)(15)(16), is very much undermethylated in sperm DNA compared to thymus DNA. The difference in methylation of the fragments from both tissues is large enough to suggest that reduced methylation of all highly repeated sequences could account for all or most of the difference in the level of methylation between total sperm and somatic cell DNA.…”

Section: Introductionmentioning

confidence: 99%

Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

Sturm

Taylor

1981

Genomic DNA of calf thymus contains 1.5 times as much 5-methylcytosine as similar sperm DNA, but the major EcoRI repeat fragment from satellite I of thymus contains ten times as much 5-methylcytosine as the corresponding fragment from sperm DNA. Restriction enzyme analyses of the total DNA and the satellite I fragment show that three HpaII sites in the fragment are completely unmethylated in sperm but fully methylated in thymus DNA. Undermethylation of many sites in the satellite DNAs can probably account for the lower level of methylation of sperm DNA rather than hemimethylation as previously suggested. These results are also discussed in relation to maintenance and de novo (initiation-type) methylases.

“…6). The plasmid had been constructed previously from the Col El -Ap vector (pSF 2124) and the 1400 base pair repeat unit of Calf thymus satellite I (20). The separation of the small satellite fragment (Mr: 0.93 x 106) in fraction 22-25 from an excess of the larger vector fragment (Mr: 7.35 x 106) and incompletely digested hybrid plasmid DNA in fraction 29-34 is absolutely complete as can be seen from electrophoresis pattern in the insert of Figure 6.…”

Section: Resultsmentioning

confidence: 99%

Base specific fractionation of double stranded DNA: affinity chromatography on a novel type of adsorbant

Bûnemann

Müller

1978

A material suitable for base-pair-specific "affinity chromatography" of double stranded DNA is described. The synthesis of this material involves two successive polymerization reactions yielding solid particles of cross-linked bisacrylamide to which base-pair-specific dyes are covalently attached by spacers of polyacrylamide chains of different length. Materials with immobilized A.T-specific malachite green or G.C-specific phenyl neutral red were used for base-pair-specific fractionation of sheared DNA from bacterial sources, as well as of higher molecular weight Calf thymus DNA or defined fragments of coliphage lambda DNA. The excellent resolving power of this method of "affinity chromatography" of nucleic acids is comparable only to DNA fractionation in buoyant density gradients in the presence of base-pair-specific ligands. A great advantage of this material is its simple handling and its chemical stability, which allows repeated re-use of the same column.