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PubMed1982

Ultrafast sequencing of oligodeoxyribonucleotides by FAB-mass spectrometry.

A fully instrumental method is described for the bidirectional sequencing of oligodeoxyribonucleotides. The method makes use of the negative ion fragmentation patterns of fast atom bombardment mass spectrometry. It is less time consuming than any other sequencing procedure known to date. Since one sequencing run takes as little as one hour, this new method is anticipated to cut down considerably the time required for the controlled synthesis of oligodeoxyribonucleotides of (currently) up to ten nucleotide units in length.

Base Sequence↗

PubMed1975

Total synthesis of a structural gene for the human peptide hormone angiotensin II.

Seven oligonucleotide chains containing between 6 and 11 nucleotide units were synthesized. The segments were phosphorylated by T4 polynucleotide 5'-hydroxyl-kinase and joined by T4 polynucleotide synthetase (ATP) to give the double-stranded DNA consisting of 33 base pairs. The DNA sequence was deduced from the known peptide sequence according to the genetic code.

Angiotensin II↗

PubMedDate not supplied

An efficiently transcribed human tRNA(Val) gene variant produces a stable pre-tRNA: repair of the processing defect by in vitro mutations.

A tRNA(CACVal) gene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNA(CACVal) gene by four base exchanges: T residues in place of C25, C62, and C66 create G:U pairs, and an A instead of G65 creates an A:C mismatch in the corresponding RNA transcript. The tRNA(Val) gene variant in pHtV4 is efficiently transcribed in HeLa cell nuclear extracts; however, the resulting pre-tRNA is processing-deficient, i.e., neither its 5'- nor its 3'-flanking sequences are removed to generate mature tRNA. Reversion of all four point mutations in pHtV4 by oligonucleotide-directed mutagenesis yielded a functional tRNA(CACVal) gene within the flanks of pHtV4, the pre-tRNA of which was processed to mature tRNA. Construction of a chimeric tRNA(Val) gene and site-directed mutagenesis of the tRNA(Val) gene in pHtV4, respectively, followed by transcription and processing studies showed that each of the four mutations contributes to the processing defect of the pHtV4-derived pre-tRNA. Moreover, this revealed that G:U pairs, which are common in all tRNAs, can impair pre-tRNA processing and therefore do not occur in certain positions in eukaryotic tRNAs.

Base Sequence↗