Improvements in oligodeoxyribonucleotide synthesis: methyl N,N-dialkylphosphoramidite dimer units for solid support phosphite methodology

Kumar, G. Suresh; Poonian, Mohindar S.

doi:10.1021/jo00199a032

Cited by 48 publications

(21 citation statements)

References 5 publications

(8 reference statements)

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“…Therefore, it is not possible to remove the TBDMS protecting group selectively in the presence of a phosphite function. [9] The low yield is attributed to a partial cleavage of the PÀO bond by the F À ion.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Isolation of Homeomorphous Isomers of P-Containing Cryptands

et al. 2000

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The novel isomeric phosphite cryptands 2, 3, and 4 could be synthesized by a simple one-pot tripod capping method starting from bisphenol 1 and PCl3. The assembling of five components led to the formation of a macrobicyclic structure, which probably requires an appropriate preorganization of the reactants. In contrast to the NMR spectra of 2, 3, and 4 in solution, the X-ray structures of 2 and 3 reveal that these molecules have no C3 symmetry in the solid state. In the 31P NMR spectra, both in- and out-P atoms have remarkably different chemical shifts due to a distorted geometry around the in-phosphorus. Phosphorus atoms in the inposition have a decreased reactivity. They are, therefore, more slowly oxidized by cumene hydroperoxide than out-P atoms. A stepwise synthesis was developed for phosphite/phosphatecryptands (5, 7, 9, and 15) via the monoprotected bisphenol 11 and the phosphate 14. In addition, the cylindrical macrotricycle 16 was isolated as a mixture of diastereomers from the crude product of this reaction.

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Section: Resultsmentioning

confidence: 99%

Synthesis and Isolation of Homeomorphous Isomers of P-Containing Cryptands

et al. 2000

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“…The yeast expression vector pYE7(+BamHI) is a derivative of pYE4 (24) with an additional BamHI site. Synthetic oligodeoxyribonucleotides were prepared as described (25). Methods used for transformation ofE.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.

Miyamoto¹,

Chizzonite²,

Crowl³

et al. 1985

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

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ABSTRACTmRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the farst intron and includes the coding exons 2 and 3. The cDNA insert in pMl-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.Activation of the cellular myc gene has been associated with neoplasia in a variety of species. In transformed cells, the c-myc gene can be altered by several mechanisms that result in abnormal or elevated expression of c-myc RNA (1, 2) and, presumably, in increased levels of c-myc protein. Analysis of genomic clones of the human c-myc gene shows that it contains three exons (3). Exon 1 represents a long 5' noncoding leader region, whereas all of the coding sequences are located in exons 2 and 3. The coding sequences are highly conserved among species and with v-myc, the viral oncogene homolog harbored by avian myelocytomatosis virus (4, 5). Immunoprecipitation studies show that myc-specific antisera recognize proteins of =60 kDa in human cells (6-8). The highly conserved nature of the c-myc protein in many vertebrate species suggests that c-myc must supply a crucial function in eukaryotic cells. Such a function has not been elucidated, although studies on v-myc and c-myc indicate that this protein is found predominantly in the nucleus and binds to double-stranded DNA (9-13).Studies on the structure and function of the c-myc gene product have been hampered by the difficulty in obtaining sufficient amounts of protein for biochemical analyses from human cells that express elevated levels of c-myc RNA (unpublished observations). In this report, we describe the isolation and characterization of cDNA clones containing the complete coding region of human c-myc. The cDNA clones were used to express the c-myc gene in heterologous cells under regulated conditions. The c-myc gene products synthesized in Escherichia coli and in Saccharomyces cerevisiae were compared and found to exhibit host-cell-specific modifications. MATERIALS AND METHODSCell Strains. E. coli strain KRR123 was derived from strain RR1 (14) by phage P1 transduction of the lon9 allele from str...

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“…By analogy, the most logical method of achieving a random family of two adjacent nucleotide deletions would be by inefficient oligonucleotide synthesis using dinucleotides (25) Electrophoresis of the product obtained from the uncapped synthesis would be expected to reveal very little full length product and a band containing the mixed population of (n-i) oligonucleotides as the major component in the ladder of truncated heterogeneous species.…”

Section: Strategymentioning

confidence: 99%

A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides

1989

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We describe a method for the generation of random point deletions in any target DNA sequence using synthetic mixed oligonucleotides. A mixed pool of oligonucleotides, which contain single nucleotide deletions randomly distributed throughout the full length, was generated by a modification of the synthesis cycle of an automated DNA synthesiser that allowed the inefficient incorporation of nucleotide monomers during each cycle of synthesis. A family of oligonucleotides was used to prime in vitro synthesis of the complementary strand of a cloned DNA fragment in an M13 vector which had previously been passaged through a dut-, ung-Escherichia coli host. Strong selection for progeny from the newly synthesised strand is provided by transforming the heteroduplex into a dut+, ung+ host. This procedure introduced point deletions at 10-25% efficiency. It has been used to introduce point deletions into operator sequences which bind the yeast regulatory proteins encoded by MATal and MATx2. INTRODUCTIONThe use of synthetic oligonucleotides for the introduction of point substitutions has been an invaluable tool in the study of structure-function relationships of proteins and DNA elements. Recently, several methods have emerged, which use deliberately contaminated oligonucleotides, for the generation of random mutations within a short DNA target sequence. These employ cassette mutagenesis or primer extension type strategies (1)(2)(3)(4)(5)(6)(7)(8) and allows the generation of families of variant proteins or DNA elements which can subsequently be used to perform a complete and detailed analysis of the protein or DNA sequence element under investigation.Another type of analysis, particularly useful for the study of operator elements, is deletion analysis of DNA promoter regions. This has been used to define upstream activation sequences, enhancer sequences or other segments of DNA that are involved in the correct expression or regulation of the gene products. The methods that are available for this type of approach rely on exonucleolytic removal of nucleotides from the ends of linear DNA fragments with exonuclease IH or Bal31 (9, 10), or through oligonucleotide directed deletions (11). A disadvantage with the use of exonucleases is that single and double point

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Improvements in oligodeoxyribonucleotide synthesis: methyl N,N-dialkylphosphoramidite dimer units for solid support phosphite methodology

Cited by 48 publications

References 5 publications

Synthesis and Isolation of Homeomorphous Isomers of P-Containing Cryptands

Synthesis and Isolation of Homeomorphous Isomers of P-Containing Cryptands

Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.

A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides

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