HETEROCHROMATIN STUDY DEMONSTRATING THE NONLINEARITY OF FLUOROMETRY USEFUL FOR CALCULATING GENOMIC BASE COMPOSITION

A novel procedure for calculating base-pair frequencies in whole genom es is reported. This has been developed during a study of the role of heterochromatin in microevolution. Closely related species of the Crep is praemorsa complex have similar karyotypes but for their heterochrom atin. The changes in relative AT frequency between species have been a ttributed to heterochromatin sequences by in situ banding of chromosom es with two base-specific fluorochromes. The absolute genome size of s pecies, measured by cytofluorometry, correlated positively with increa sed karyotypic heterochromatin, as did the proportion of AT bases in t he DNA. However, the determination of base content has called for a cu rvilinear interpretation of data obtained with two base-specific fluor ochromes (bisbenzimide Hoechst 33342 and mithramycin), in contrast to the commonly assumed but erroneous direct relationship between fluores cence intensity and base content. Essentially, the fluorochromes' requ irements for a sequence of certain base-pairs lead to the notion of Co efficients of Overspecificity: the result is a simple formula for calc ulating the AT proportion in a genome relative to a reference species from cytometric data, taking account of ligand binding statistics. The se statistics and probabilities of oligonucleotide binding are essenti ally the same. (C) 1993 Wiley-Liss, Inc.