Chemical synthesis of a self-complementary octanucleotide, dG-G-T-T-A-A-C-C by a modified triester method

Agarwal, Kan; Riftina, Faina

doi:10.1093/nar/5.8.2809

Cited by 52 publications

(11 citation statements)

References 18 publications

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“…Asterisks indicate the number of additional clones found that had the same recombinant segment. In JM105 (recA+) cells (clones [1][2][3][4][5][6][7][8][9][10][11][12], only the recombinant segment of clone 1 had an inverse orientation, and 94% of the endpoints occurred at Alu I sites. In JM109 (recA-) cells (clones [13][14][15][16][17], four of the five recombinant segments had the inverse orientation, and 20% of the endpoints occurred at Alu I sites.…”

Section: Discussionmentioning

confidence: 99%

Illegitimate recombination induced by benzo[a]pyrene diol epoxide in Escherichia coli.

Kokontis

Vaughan

Harvey

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Duplex DNA oligomer constructs (32 base pairs) were prepared that contained a single benzo[alpyrene (BP) adduct at a specific deoxyadenosine or deoxyguanosine site in either one or both strands. These constructs were inserted into M13 replicative form viral DNA, and the DNA from progeny virus generated by transfection of Escherichia coli was examined by sequence analysis at the site of oligomer insertion. With nonalkylated constructs, and with constructs containing only one BP adduct, no sequence alterations were found in progeny viral DNAs. With constructs containing two BP adducts, one in each strand and closely spaced, some progeny DNAs showed the original oligomer sequence, whereas others exhibited large deletions and illegitimate (nonhomologous) recombination, both of which removed the damaged construct. Increasing the distance between BP adducts in the construct reduced the frequency of recombinant events. These sequence alterations occurred in both recA + and recAhost cells. We speculate that the closely spaced adducts in opposite construct strands cause a rare distortion in DNA structure, which activates the recombinant machinery, and that mutagenic and carcinogenic agents other than polycyclic aromatic hydrocarbons may cause similar DNA distortions, which induce illegitimate recombination.In an earlier report (1), we described the use of short DNA oligomers as targets for alkylation by benzo[a]pyrene diol epoxide (BPDE), the activated metabolite of benzo[a]pyrene (BP) (2), for the study of mutagenesis in Escherichia coli. In our experimental system, E. coli was transfected with phage M13 replicative form (RF) DNA, which was modified to contain a 10-base-pair (bp) BamHI linker treated with BPDE. Sequence analysis of DNA isolated from progeny virus indicated the presence of large deletions at the site of oligomer insertion. It was speculated that the relatively high localized concentration of multiple BP adducts in the linker target might be responsible for the large deletions we observed.To examine this phenomenon in greater depth, we prepared several duplex oligomers having BP adducts at a single site in each DNA strand, but with different spacings between the two adducts. These site-specific constructs were inserted into M13 RF DNA, and, after transfection of host cells, progeny viruses were selected for sequence analysis at the DNA region of oligomer insertion. The results of these analyses are summarized as follows: some progeny viral DNAs contained the original oligomer insert sequence, whereas others were modified exhibiting large deletions and insertions of illegitimate (nonhomologous) recombinant segments, both of which removed the damaged construct. Similar DNA modifications were observed with constructs alkylated either at deoxyadenosine or deoxyguanosine residues, but increasing the distance between BP adducts in the construct reduced the frequency of recombinant events. The induction of deletions and recombinants occurred in both recA + and recA -host cells. MATERIALS AND METHODS

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Section: Discussionmentioning

confidence: 99%

Illegitimate recombination induced by benzo[a]pyrene diol epoxide in Escherichia coli.

Kokontis

Vaughan

Harvey

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…RESULTS AND DISCUSSION Short, synthetic oligonucleotides can be synthesized relatively easily by either chemical (11,14,15,(21)(22)(23) or enzymatic methods (24). Such oligonucleotides have been used as primers for DNA (25) and RNA (26) sequence determinations, as hybridization probes to isolate cloned genes (27) and as detectors of specific mRNAs (28).…”

Section: Methodsmentioning

confidence: 99%

Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligodeoxynucleotide primer.

Sood¹,

Pereira²,

Weissman³

1981

Proc. Natl. Acad. Sci. U.S.A.

346

107

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We have isolated a cDNA clone for one of the HLA-B locus alloantigens by hybridization with a 30-nucleotidelong DNA probe. The probe was isolated from a reverse transcriptase (RNA-dependent DNA nucleotidyltransferase)-catalyzed cDNA synthesis reaction on poly(A)-mRNA in which an oligonucleotide (5'-32P)dC-T-T-C-T-C-C-A-C-A-ToH served as a primer and in which dideoxynucleoside triphosphates were used to reduce the size and heterogeneity of the cDNA products. The desired cDNA clone was isolated from a library of recombinant cDNA clones in the plasmid pBR322. The partial nucleotide sequence ofthe cDNA clone corresponds to the amino acid sequence of HLA-B7 antigen. The approach described in this paper is extremely sensitive and maybe useful in cloning other genes for which the corresponding mRNA is present at low levels. This cDNA clone is nearly full length and can be used to isolate and to study the genes within the HLA region and to obtain expression of HLA-B peptides in cells.

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“…The key intermediates in this synthesis are the fully protected deoxyribonucleoside S'-p-chlorophenyl /3-cyanoethyl phosphotriesters, d-[(MeO)2Tr]NpCE, which are required in large quantities and high purity. These protected monomers were synthesized by using a modification of an approach originally described by Agarwal & Riftina (1978). This procedure involves phosphorylation of suitably protected 5'-0-(dimethoxytrityl)deoxyribonucleosides with a small excess of p-chlorophenyl bis(triazolide), followed by esterification with hydroacrylonitrile.…”

Section: Resultsmentioning

confidence: 99%

Preparation of a decadeoxyribonucleotide helix for studies by nuclear magnetic resonance

Miller¹,

Cheng

Dreon

et al. 1980

Biochemistry

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A self-complementary decadeoxyribonucleotide d-CpCpApApGpCpTpTpGpG was chemically synthesized by a procedure based on the phosphotriester approach. This procedure was carefully monitored and appropriately modified to ensure the purity of oligomer components at each step of the synthetic scheme. Extensive use was made of both analytical and preparative high-pressure liquid chromatography to purify and characterize the decamer and its constituent oligonucleotides. The final product (1318 A257 units or 16.5 mumol) was obtained in high purity and sufficient quantity for extensive physical studies by UV, CD, and NMR spectroscopy. Our preliminary results show that at a strand concentration of 1.3 X 10(-5) M and in 0.10 M sodium chloride and 0.01 M sodium phosphate buffer, pH 7.0, the decamer duplex has a Tm at 47 degrees C. The CD spectrum of this decamer duplex is similar to that of B-form DNA. All the resonances of the nonexchangeable base protons of the decamer are well resolved in the 1H NMR spectrum, when the single-stranded form was examined by using a 360-MHz spectrometer and when the duplex form was examined by using a 600-MHz spectrometer. These base proton resonances have been tentatively assigned by using the incremental assignment technique. Although the decamer duplex serves as a substrate for AluI restriction endonuclease, it is not cleaved by HindIII endonuclease.

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Chemical synthesis of a self-complementary octanucleotide, dG-G-T-T-A-A-C-C by a modified triester method

Cited by 52 publications

References 18 publications

Illegitimate recombination induced by benzo[a]pyrene diol epoxide in Escherichia coli.

Illegitimate recombination induced by benzo[a]pyrene diol epoxide in Escherichia coli.

Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligodeoxynucleotide primer.

Preparation of a decadeoxyribonucleotide helix for studies by nuclear magnetic resonance

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