Pyrimidine-specific chemical reactions useful for DNA sequencing

A family of mitochondrial RNAs hybridizes specifically to the var1 region on Saccharomyces cerevisiae mitochondrial DNA (Farrelly et al., J. Biol. Chem. 257:6581-6587, 1982). We constructed a fine-structure transcription map of this region by hybridizing DNA probes containing different portions of the var1 region and some flanking sequences to mitochondrial RNAs isolated from var1-containing petites. We also report the nucleotide sequence of more than 1.2 kilobases of DNA flanking the var1 gene. Our primary findings are: (i) The family of RNAs we detect with homology to var1 DNA is colinear with the var1 gene. Their direction of transcription is olil to cap, as it is for most other mitochondrial genes. (ii) Extensive hybridization anomalies are present, most likely due to the high A-T (A-U) content of the hybridizing species and to the asymmetric distribution of their G-C residues. An important conclusion is that failure to detect transcripts from A-T-rich regions of the yeast mitochondrial genome by standard blot transfer hybridizations cannot be interpreted to mean that such sequences, which are commonly supposed to be spacer DNA, are noncoding or lack direct function in the expression of mitochondrial genes.

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“…Sequencing reactions were those of Maxam and Gilbert (16) and Rubin and Schmid (20), modified as previously noted (11).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional analysis of the Saccharomyces cerevisiae mitochondrial var1 gene: anomalous hybridization of RNA from AT-rich regions.

Zassenhaus¹,

Farrelly²,

Hudspeth³

et al. 1983

Mol. Cell. Biol.

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“…KMnO 4 Sensitivity of U or T Paired with Pyrene-The sensitivity of the 5, 6 double bond of pyrimidines to oxidation, leading to cleavage of the glycosidic bond in the presence of piperidine, has long been used in DNA sequencing strategies (26), and as a probe of the solvent accessibility of T in singlestranded and duplex DNA (27). Here, we have used KMnO 4 sensitivity to address the question of whether Y pushes the opposite U or T from the DNA base stack, rendering it more accessible to oxidation.…”

Section: Design and Characterization Of Normal And Pyrene Wedgementioning

confidence: 99%

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

Jiang

Kwon

Stivers

2001

Journal of Biological Chemistry

105

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Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the Nglycosidic bond of 2-deoxyuridine (2-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.

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“…All fragments were 3'-end-labeled using the large Klenow fragment of E. coli DNA polymerase I (22). DNA (approximately 50 pmole of 3' ends) was dissolved in 40 il H20 and 5 jl of buffer stock (0.5 M Tris, 0.05 M MgCl2, 0.1 M mercaptoethanol, 0.5 mg/ml BSA) and then added to dried cx32P-dNTPs (25 iCi of each dNTP, 400 Ci/mnole, Amersham). The Klenow fragment of DNA polymerase I was then added (New England Biolabs, 5 ul of 0.7 units/jil), and the reaction mixture was incubated at 20°C for 30 min.…”

Section: Materials Amd Methods Selection Of Clonesmentioning

confidence: 99%

A complex repeated DNA sequence within the Drosophila transposable element copia

Fouts

Manning

Fox³

et al. 1981

Nucl Acids Res

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A320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analys-is of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the termina repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.

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Pyrimidine-specific chemical reactions useful for DNA sequencing

Abstract: Potassium permanganate reacts selectively with thymidine residues in DNA (1) while hydroxylamine hydrochloride at pH 6 specifically attacks cytosine (2). We have adopted these reactions for use with the chemical sequencing method developed by Maxam and Gilbert (3).

Cited by 321 publications

References 6 publications

Transcriptional analysis of the Saccharomyces cerevisiae mitochondrial var1 gene: anomalous hybridization of RNA from AT-rich regions.

Transcriptional analysis of the Saccharomyces cerevisiae mitochondrial var1 gene: anomalous hybridization of RNA from AT-rich regions.

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

A complex repeated DNA sequence within the Drosophila transposable element copia

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