Microfluorometric Detection of Deoxyribonucleic Acid Replication in Human Metaphase Chromosomes

Abstract

DNA replication in eukaryotic organisms is semiconservative (1), initiating at discontinuous foci (2) and terminating in a temporal sequence characteristic of the location of the DNA segment in metaphase chromosomes (3). Biochemical and cytological studies have correlated late DNA replication with both genetic inactivity and the persistence of chromosome condensation throughout most of the cell cycle (4, 5). Regions with these properties are termed heterochromatic and have been shown to correspond to regions of metaphase chromosomes staining intensely either with quinacrine or quinacrine mustard, or with Giemsa mixtures after any of a variety of pretreatments (6, 7). However, previous correlation of late DNA replication with banded regions of metaphase chromosomes has been limited by the resolution usually realized of the autoradiographic techniques used, and the chromosomebanding stains do not effectively differentiate the facultative heterochromatic X chromosome from its active homologue. More precise correlation of DNA replication with chromosome location in individual metaphases could be achieved were it possible to detect DNA synthesis at the level of resolution of optical techniques.Direct light-microscopic visualization of DNA replication might use DNA base analogues with distinctive absorptive or fluorescent properties. However, available base analogues do not, at present, make such an approach practical. As ai alternative method, one might use an indirect phenomenon, Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Address reprint requests to author at: Children's Hospital Medical Center, 300 Longwood Ave., Boston, Mass. 02115. 3395 such as the environmental sensitivity of fluorescent chromosome stains, using them to report the incorporation of nucleotide analogues containing substituents, such as heavy atoms (8), perturbing dye luminescence.Towards this purpose, we have examined several dyes, including the compound 33258 Hoechst [2-[2-(4-hydroxyphenyl)-6-benzimidazolyl]-6-(1-methyl4-piperazyl)-benzimidazol * 3 HClJ, which has previously been used to demonstrate constitutive heterochromatin in rodent metaphase chromosomes (9). We observed that the fluorescence of a sample of 33258 Hoechst, kindly supplied by Dr. H. Loewe, Hoechst, is much less efficient when the dye is bound to poly(dA-BrdU) than when bound to poly(dA-dT). This paper introduces a method based on this observation. Reduction in the fluorescence intensity of 33258 Hoechst bound to chromatin, upon incorporation of BrdU, has been used to follow DNA synthesis in interphase nuclei and metaphase chromosomes. MATERIALS AND METHODSCell Growth. Peripheral human leukocytes were cultured in Eagle's minimal essential medium (MBA) with 2 mM Lglutamine and 20% fetal-calf serum (GIBCO), to which crude phytohemagglutinin was added. BrdU and thymidine, added to cultures grown at 370, as described in the figure legends, were obtained from Sigma and their tritiated derivatives from New England Nuclear Corp. Uridine was also obt...

Staining properties of a benzimidazol derivative “33258 hoechst” and a simplified staining method for chromosome banding

“…Chromosome banding with "33258 Hoechst" under various conditions were reported by several authors (Hilwig and Gropp, 1972;Natrajan et aL, 1973;Seth and Gropp, 1973;Latt, 1973;Kim, 1974;Raposa and Natarajan, 1974;Holmquist, 1975;Jalal et al, 1975). Natarajan et al (1973) described that with the manipulation of the staining intensity with this dye, only bands corresponding to C-bands or those corresponding to both C-and Q-bands were obtained.…”

“…Fluorescence efficiency of "33258 Hoechst" bound to poly (dA-BrdU) is less than one fifth of the dye bound to poly (dA-dT) (Latt, 1973;Latt, 1974). Substitution of chromosomal DNA by BrdU provided a degree of resolution for the replication of chromosomal DNA by staining with "33258 Hoechst" (Latt, 1973;Latt, 1974;Morita et al, in prep.…”

“…Recently, Hilwig and Gropp (1972) found that this dye bound preferentially itself to chromosome segments composed of constitutive heterochromation or sites of repetitious DNA's in mammals. Furthermore, a banding pattern similar to those of Q-banding was demonstrated under suitable conditions (Natarajan et al, 1973;Latt, 1973), then this compound has gained increasing popularity among cytogenetists (Paris Conference (1971), Supplement (1975)). …”

“…3) Demonstation of DNA replicating regions (Latt, 1973;Dutrillaux, 1975;Epplen et al, 1975;Morita et al, in prep.). Before harvesting cells 5-bromo-2'-deoxyuridine (BrdU) was added to medium, giving a final concentration of 100 t~g/ml, corresponding to different phases of the cell cycle.…”

Improved BrdUrd‐Hoechst bivariate cell kinetic analysis by helium‐cadmium single laser excitation

“…Due to the presence of BrdUrd in DNA the intensity of emitted Hoechst 33258 fluorescence is decreased (10). Although the underlying biophysical and biochemical mechanisms of this quenching effect are not fully elucidated, it represents a powerful tool for flow cytometric cell cycle analysis (2,5,6,8,9,11-15).…”