Progress toward an amplifiable metabolic label for DNA: conversion of 4-thiothymidine (4sT) to 5-methyl-2′-deoxycytidine and synthesis of a 4sT phosphorodiamidate prodrug

The ability to metabolically label DNA in a way that produces a latent change from one nucleobase to another would create a signal that can be amplified by PCR — this in turn would allow studies of newly synthesized DNA using high-throughput sequencing. To function as an amplifiable metabolic label, a nucleotide analogue would need to be taken up by cells and incorporated into cellular DNA; after purification of DNA, it could be converted into a different nucleobase with a different base pairing pattern. We selected 4-thiothymidine (4sT) as a candidate metabolic label: 4sT is readily taken up by a large number of polymerases in vitro, and we present a method that allows 4sT to be converted into 5-methyl-2′-deoxycytidine (5mC) after incorporation into DNA. Encouraged by these results, we treated cells with 4sT nucleoside; however, we found that 4sT is not incorporated into DNA in bacterial, yeast, or mammalian cells to useful levels under the conditions we tested. A phosphorodiamidate prodrug of 4sTMP was successfully synthesized but did not measurably improve incorporation into cellular DNA.

Patterns of metabolic activity in cytoarchitectural area SII and surrounding cortical fields of the monkey

The pattern of [14C]2-deoxyglucose (2-DG) labeling evoked by tactile stimuli was determined in cerebral cortical cytoarchitectural area SII and the fields that surround it (including area 7b, the retroinsular field (Ri), and the granular insular region (Ig) for a series of nine monkeys (macaca fascicularis). In all animals and for all tactile stimuli, the cortical labeling most frequently occurred in the form of patchlike aggregates of metabolically active neurons. Individual patches typically included laminae II-V, were most densely labeled in the central layers, and possessed limited tangential width. Analysis of the relations between patches of label in adjacent sections revealed that the metabolically active neurons form three-dimensional aggregates (termed modules or strips), which can extend for several millimeters. It is hypothesized that these metabolic modules may correspond to information-processing units within the cerebral cortex. Two-dimensional reconstructions of the 2-DG label in the hemispheres ipsilateral and contralateral to the somatic stimuli reveal that the strips of high metabolic activity are interspersed with regions of substantially less activity. In all cortical regions examined in this study, the strips were oriented roughly from anterior to posterior. Systematic changes in the place of the somatic stimulus led to systematic changes in the cortical location of the strips of metabolic label. Conversely, animals subjected to nearly identical tactile stimuli produced very similar patterns of metabolic activity. Comparison of the distribution of metabolic activity in area SII of the hemispheres ipsilateral and contralateral to the stimulus demonstrated that although the amount of labeling in SII ipsilateral to the stimulus was typically less than that present in SII of the contralateral hemisphere, it was both substantial and topographically highly organized. The labeling in the cytoarchitectural zones surrounding SII (i.e., 7b, Ri, and Ig), although clearly stimulus related, occupied extensively overlapping regions in all experiments even though the body regions stimulated were in widely different locations. As a result, a relative lack of topographical organization within these cortical fields is indicated.

Radiolsotopes as metabolic labels for Glossina (Diptera: Glossinidae). I. Laboratory determination of the relationship between radioisotope metabolism, respiration and temperature

The turnover of the radioisotope 137Cs is shown to be of a one-component nature and to be positively correlated with temperature for males of both Glossina morsitans Westw. and G. pallidipes Aust. A direct correlation with oxygen consumption is also demonstrated, and the relation for both species may be described by the same regression line. The feasibility of using 137Cs as a metabolic label for tsetse is therefore confirmed, and it is suggested that the predictive nature of the relationship between radioisotope turnover and metabolism may extend beyond closely related species.