Rapid identification of human chromosomes by TV-techniques

759

199

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...

“…Quinacrine mustard and modified Giemsa staining and photomicrography followed established techniques (6,7). Autoradiography.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Microfluorometric Detection of Deoxyribonucleic Acid Replication in Human Metaphase Chromosomes

Latt

1973

759

199

“…Metaphase chromosomes are especially amenable to energy transfer analysis, because quenching of donor fluorescence by added acceptor in different chromosomal regions can be detected microscopically. The results can then be interpreted in the context of metaphase chromosome banding patterns, which constitute reproducible structural subdivisions of chromosomes (6,7).…”

mentioning

confidence: 99%

Enhancement of banding patterns in human metaphase chromosomes by energy transfer

Sahar¹,

Latt²

1978

Intermolecular energy transfer between appropriately chosen pairs of dyes can be used to induce or enhance banding patterns in human metaphase chromosomes. Intermolecular transfer of electronic excitation energy, which depends on the inverse sixth power of the separation between donor and acceptor dyes (1), has been extensively used to examine proximity relationships in well-defined biochemical systems (2)(3)(4)(5) Values for R0, the critical distance for 50% efficient energy transfer between different dye pairs, were calculated from the * To whom reprint requests should be sent. 5650The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…The frequency of these exchanges in individual chromosomes generally increases with metaphase chromosome length. Within a given chromosome, the majority of exchanges appear to be either in interband regions [defined by quinacrine banding patterns (20) ] or very near band-interband junctions. Unlike the translocations and quadriradial figures induced by mitomycin C (14-16), the sister chromatid exchanges are not preferentially located on chromosomes no.…”

mentioning

confidence: 99%

Sister Chromatid Exchanges, Indices of Human Chromosome Damage and Repair: Detection by Fluorescence and Induction by Mitomycin C

Latt

1974

382

Sister chromatid exchanges in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with the Jisbenzimidazole dye 33258 Hoechst. These exchanges are mucbh more frequent than chronosome or chromatid breaks and appear to be partly but not entirely due to 5-broxpodeoxyuridine incorporation. Sister