Heterochromatin study demonstrating the non‐linearity of fluorometry useful for calculating genomic base composition

TLDR

The study arose from investigations of heterochromatin in the Crepis praemorsa complex, where closely related species share karyotypes but differ in heterochromatin, prompting a curvilinear interpretation of fluorometric data based on ligand binding statistics. The authors present a novel method for calculating whole‑genome base‑pair frequencies. By exploiting the overspecificity of bisbenzimide Hoechst 33342 and mithramycin, they derive coefficients that allow AT proportion to be computed from cytometric fluorescence relative to a reference species. AT frequency differences among species were attributed to heterochromatin, and cytofluorometric genome size correlated positively with heterochromatin content and AT proportion. © 1993 Wiley‑Liss, Inc.

Abstract

Abstract A novel procedure for calculating basepair frequencies in whole genomes is reported. This has been developed during a study of the role of heterochromatin in microevolution. Closely related species of the Crepis praemorsa complex have similar karyotypes but for their heterochromatin. The changes in relative AT frequency between species have been attributed to heterochromatin sequences by in situ banding of chromosomes with two base‐specific fluorochromes. The absolute genome size of species, measured by cytofluorometry, correlated positively with increased karyotypic heterochromatin, as did the proportion of AT bases in the DNA. However, the determination of base content has called for a curvilinear interpretation of data obtained with two base‐specific fluorochromes (bisbenzimide Hoechst 33342 and mithramycin), in contrast to the commonly assumed but erroneous direct relationship between fluorescence intensity and base content. Essentially, the fluorochromes' requirements for a sequence of certain basepairs lead to the notion of Coefficients or' Overspecificity: the result is a simple for mula for calculating the AT proportion in a genome relative to a reference species from cytometric data, taking account of ligand binding statistics. These statistics and probabilities of oligonucleotide binding are essentially the same. © 1993 Wiley‐Liss, Inc.