Isolation of Metaphase Chromosomes From Hela Cells

Abstract

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of R N A for each milligram of DNA. The R N A bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA. I N T R O D U C T I O NMany possible biochemical and biophysical approaches to the study of chromosomes in higher organisms have been hindered, until recently, by the lack of suitable procedures for large-scale isolation of chromosomes. Although the methods for isolation of interphase chromosomes, or "chromatin," which have been developed in recent years (1, 2) are satisfactory for certain purposes, a definite need still exists for a procedure which will allow large-scale isolation of morphologically intact metaphase chromosomes. Metaphase chromosomes are an indispensable complement to interphase chromosomes for the general study of chromosome structure. In addition, metaphase chromosomes have the unique advantage of being so condensed that they can be distinguished microscopically both from each other and from contaminating nonchromosomal material. Consequently, one is not limited to studying the average properties of all chromosomes; one can also examine single types of chromosomes.According to our experience, in the isolation of metaphase chromosomes by most previously published methods (3-5), morphological damage to some of the chromosomes cannot be avoided and only partial purification of the chromosomes from cell debris can be achieved. We report here a method for the rapid preparation, in milligram quantities, of fairly pure, morphologically intact metaphase chromosomes from HeLa cells. We also report the results of studies on the chemical composition of isolated chromosomes. M A T E R I A L A N D M E T H O D S Cultivation of CellsHeLa $3 cells (6) were grown in suspension culture in a modified Eagle's medium (7) supplemented with 5% calf serum. For accumulation of metaphase cells, partial synchrony was induced by lowering the culture temperature to 4°C for 1 hr and then returning it to 37°C (8). Ten to 11 hr later, colchicine was added to a final concentration of 0.5 to 1 X 10 -5 M. The cells were harvested by centrifugation 9 to 10 hr after colchicine addition and washed 3 times in 0.137M NaC1, 0.005M KC1, 0.007M NaH2PO4, 0.025 m Tris, pH 7.4. This procedure routinely produced about 30% metaphase cells. 95 on

Factors for Giemsa-Band Formation in Air-Dried Mammalian Chromosomes

“…It is likely that histories play a structural role and involved in the coiling of DNA into the compact form of the chromosome (Cole 1962;Richards and Pardon, 1970;Luie and Dixon 1973). According to Huberman and Attardi (1966), human chromosomes include about twice as much proteins at metaphase as at interphase. It seems likely that such increased proteins which consist of both histones and non-histon proteins play a role on maintenance of a chromosomal configuration at metaphase.…”

Synchronization and Isolation of Metaphase Chromosomes in Cultured Chinese Hamster Don Cells

“…Simultaneously, the isolated chromosomes with excellent preservation of morphology and biological activities are very useful materials for studying the fine structure and physico-chemical properties of chromosomes (see Salzman et al 1966;Cantor and Hearst 1966;Huberman and Attardi 1966;Schildkraut 1967, 1969;Mendelsohn et al 1968;Wray and Stubblefield 1970).…”

“…In recent years, many investigators have reported the isolation of metaphase chromosomes from mouse ascites tumor (Cantor and Hearst 1966), mouse lymphocytic leukemia cells (Chorazy et al 1963) and cultured mammalian cells (Somers et al 1963;Huberman and Attardi 1966;Maio and Schildkraut 1966;Salzman et al 1966;Burkholder and Mukherjee 1970). Isolated metaphase chromosomes with excellent preservation of morphology and biological activities are very useful for analyzing the fine structure and physico-chemical properties of chromosomes.…”

MASS PREPARATION OF NUCLEI FROM THE LARVAL SALIVARY GLANDS OF DROSOPHILA HYDEI

“…Vacuum-dried bovine serum albumin was used as the standard. RNA was determined by the modification of the Schmidt and Thannhauser procedure used by Huberman and Attardi (9). The concentration of the standard RNA solution was determined spectrophotometrically.…”

“…For DNA measurements, the suspension was pelleted, and the residue was washed with ethanol-ether (3:1). This residue was well suspended in 0.5 M HC10 4 (9). DNA and RNA were determined as described for the gland analyses.…”