Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst ...DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.
Non-CpG methylation occurring in the context of CNG sequences is found in plants at a large number of genomic loci. However, there is still little information available about non-CpG methylation in ...mammals. Efficient methods that would allow detection of scarcely localized methylated sites in small quantities of DNA are required to elucidate the biological role of non-CpG methylation in both plants and animals. In this study, we tested a new whole genome approach to identify sites of CCWGG methylation (W is A or T), a particular case of CNG methylation, in genomic DNA. This technique is based on digestion of DNAs with methylation-sensitive restriction endonucleases
EcoRII-C and
AjnI. Short DNAs flanking methylated CCWGG sites (tags) are selectively purified and assembled in tandem arrays of up to nine tags. This allows high-throughput sequencing of tags, identification of flanking regions, and their exact positions in the genome. In this study, we tested specificity and efficiency of the approach.
ABSTRACT BspLUII III, an isomer of FinI (1) and BsmFI (2), was found to cleave DNA at two points 10, 11 and 14, 15 bp in the different strands away from the recognition site, and in the presence of ...SAM it exhibits the adenine specific methyltransferase activity.
Two site-specific endonucleases have been purified from soil thermophilic bacteria Bacillus species LU11 by chromatography on blue--Sepharose, hydroxyapatite and heparin--Sepharose.
Fragments of the 16S rRNA of Thermus thermophilus representing the 3′ domain (nucleotides 890–1515) and the 5′ domain (nucleotides 1–539) have been prepared by transcription in vitro. Incubation of ...these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3′ RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3′ domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5′ RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA95, 999–1003, these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state.
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