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

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 of Overspecificity: the result is a simple formula 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. Key terms: DNA amount, AT%, mithramycin, bisbenzimide Hoechst, ethidium bromide, fluorochrome banding, Crepis praemorsa complex Constitutive heterochromatin is a particular structure of eukaryote chromatin, differing from the euchromatin by its replication and condensation, frequently containing highly repeated sequences (1). It is revealed by several staining procedures (13) and may arise by transformation of euchromatin andlor by multiplication of repeated sequences (23). Addition of supplementary heterochromatic sequences has nucleotypic effects such as increase of cell cycle length or other phenomena linked to nuclear organization (20). The degree of multiplication of, for instance, a GC-rich region may be sufficient to produce a significant change in the measure of overall genomic GC. Results notably from (30) suggest that modification of heterochromatin is a rapid process relative to biological evolution: in closely related species one would therefore expect to find a close link between quantity and type of heterochromatin and quantity and composition of DNA.We have used fluorometric procedures to assess genome size and GC content in a complex of closely related plant species of Crepis, modifying the classical and often erroneous interpretation of fluorescence ratios to a n interpretation based upon binding statistics for base-specific dyes. GC-or AT-rich sequences were also sought at the chromosome level by f luorochromebanding.

Lack of correlation between AT frequency and genome size in higher plants and the effect of nonrandomness of base sequences on dye binding

“…The base sequence of Pseudomonas aeruginosa was downloaded from the website of the Pseudomonas Genome Project http://www.pseudomonas.com/, all remaining sequences from the website of The Institute of Genomic Research (TIGR) http://www.tigr.org/ n DAPI ϭ n HO ⅐ ln͑DF_DAPI͒/ln͑DF_HO͒ (11) which follows from eq. (10) in Godelle et al (9).…”

“…Doležel et al (6) showed that base-specific fluorescence is not proportional to the base content, but rather follows a curvilinear relation. Provided that the bases in the genome are distributed randomly, a formula is given for the relation between base frequency and fluorescence intensity (8,9). It is based on the condition that only a certain number, n, of consecutive bases of the same type (AT or GC) is able to bind a dye molecule.…”

“…It is based on the condition that only a certain number, n, of consecutive bases of the same type (AT or GC) is able to bind a dye molecule. For Hoechst 33342 (HO), this number has been found to be n ϭ 5 (8,9), whereas a range of n ϭ 3-4 is proposed for 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 10). Using this formula, a comparison between DAPI and HO should give an unambiguous value of n for DAPI.…”

Evaluation of the nuclear DNA content and GC percent in four varieties of Fagus sylvatica L

“…In Angiosperms, haploid genome size varies from less than one picogram (pg) (Arabidopsis thaliana: 0.15 pg) [7] to more than 100 pg (Fritillaria assyriaca: 127 pg) [18]. Among techniques used for genome studies, flow cytometry is extremely rapid and convenient: it allows accurate determinations of nuclear DNA content [13] and of AT/GC base composition in a genome [15]. Favre and Brown [ 12] developed a fast and simple flow cytometry protocol for [14] with 0.5 % Triton X-100 and sodium metabisulfite (10 mM) as an antioxidant.…”

Cytometry and plant sciences: A personal retrospective

“…Accurate analysis of plant genome sizes also requires that the method of fluorochromatic staining be insensitive to basepair composition and/or the state of chromatin compaction. ISAC member Spencer Brown at Gif-sur-Yvette did pioneering work in this area (33,34).…”