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L W McLaughlin

Publications and source records attributed to L W McLaughlin.

82 records · Page 5Linked to original sources
PubMed1982

The effect of acceptor oligoribonucleotide sequence on the T4 RNA ligase reaction.

In order to make efficient use of T4 RNA ligase in a program involving chemical-enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High-performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5')pp(5')Cp or A(5')pp(5')UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Conversely, examination of the four nucleoside 3',5'-bisphosphate donors pUp, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.

Base Sequence↗
PubMed1981

Nucleotide sequence of 5S ribosomal RNA from Aspergillus nidulans and Neurospora crassa.

The nucleotide sequences of 5S rRNA molecules isolated from the cytosol and the mitochondria of the ascomycetes A. nidulans and N. crassa were determined by partial chemical cleavage of 3'-terminally labelled RNA. The sequence identity of the cytosolic and mitochondrial RNA preparations confirms the absence of mitochondrion-specific 5S rRNA in these fungi. The sequences of the two organisms differ in 35 positions, and each sequence differs from yeast 5S rRNA in 44 positions. Both molecules contain the sequence GCUC in place of GAAC or GAUY found in all other 5S rRNAs, indicating that this region is not universally involved in base-pairing to the invariant GTpsiC sequence of tRNAs.

Aspergillus nidulans↗
PubMedDate not supplied

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA.

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.

Bridged Bicyclo Compounds↗
PubMedDate not supplied

Oligonucleotides tethering Hoechst 33258 derivatives: effect of the conjugation site on duplex stabilization and fluorescence properties.

A series of DNA conjugates have been prepared in which two different derivatives of Hoechst 33258 have been tethered to a sequence containing a 5'-GAATTC-3' target site. The two derivatives differ only in the length of the tether between the DNA and the Hoechst fluorophore. By using a DNA backbone labeling protocol, one in which the Hoechst dye is tethered to an internucleotide phosphoramidate residue, it was possible to easily vary the site of attachment with respect to the A-T rich binding site. When tethered outside the GAATTC sequence, little if any helix stabilization results upon hybridization of the conjugate to its complementary sequence. As the site of conjugation is moved to one end of the target sequence and finally within the AATT sequence, more effective helix stabilization results. When tethered between the two A residues, or between the A and T residue, a delta Tm of at least +20 degrees C is observed. Upon hybridization and formation of the B-form DNA, binding by the tethered Hoechst dye results, and the bound dye becomes brightly fluorescent. Upon a simple titration of the single-stranded conjugate with the complementary target sequence the quantum yield enhancement for hybridization only appears to be 5-7-fold at best. These fluorescence effects, generally less dramatic than those observed with other sequences, result from an increase in quantum yield for the single-stranded conjugate relative to the free Hoechst 33258. Heating the single-stranded conjugate reduces the inherent fluorescence of the single-stranded conjugate to a level comparable with that of the free Hoechst dye. In experiments monitoring absorbance vs temperature, a cooperative transition is observed for the single-stranded conjugate. Both the high quantum yield observed for the single-stranded conjugate and the observed thermally induced transition suggest that the single-stranded conjugate can dimerize (at the GAATTC site), mediated by the groove-binding fluorophore.

Base Composition↗