- Email: [email protected]
Chapter 19 Preparation of Mammalian Metaphase Chromosomes for Autoradiography
Chapter 19 Preparation of Mammalian Metaphase Chromosomes for Autoradiography D. M. PRESCOTT
AND
M. A. BENDER
Biology Division, Oak Ridgu National Oak Ridgs, Tmnusses
I. Introduction . . . . . . . . 11. Procedure. . . . . . . . . 111. Examples and Variation of Technique References . . . . . . . .
. . . . . . . . . . . 381 . . . . . . . . . . . 382 . . . . . . . . . . . 383
. . . . . . . . . . .
384
I. Introduction I n autoradiographic studies on labeling of deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , and proteins of mammalian chromosomes with tritiated precursors, we devised a procedure for preparing cytoplasm-free metaphase chromosomes. The method has the following advantages: (1) several hundred to several thousand sets of chromosomes can be affixed to a single slide, (2) the chromatids are well separated and autoradiographic resolution of label between sister chromatids is excellent, (3) no cytoplasmic layer is present between the chromosomes and the autoradiographic emulsion, thereby increasing the sensitivity and resolution of the autoradiographic detection of incorporated label, and (4) autoradiographic determination of labeled RNA and protein in chromosomes is possible because of the absence of “background1’ radioactivity from nonchromosomal RNA and protein. The method has been described in principle in a previous paper (Prescott and Bender, 1961);several improve&Operatedby Union Carbide Corporation for the United States Atomic Energy Commission. 381
382
I). M. PRESCOTT AND M. A. BENDER
ments have been added in the account below, and a few illustrations of autoradiographic results with chromosomes are shown in three accompanying figures. The method has been used mainly with the Chinese hamster “fibroblast”-line CHEF 125 (Prescott and Eender, 1962a), but it works equally well with several other mammalian cell types.
11. Procedure The cells are cultured in 55 mm Petri dishes, and colchicine is added to M ) several hours before isolation of chromosomes. the culture (to Just before the isolation, the medium is decanted and the cells washed once with balanced salt solution. The solution is then replaced by hypotonic saline (10 to 15% of full-strength, phosphate-buffered Hank’s balanced salt solution) at room temperature. An important part of these steps is the complete removal of serum proteins from the culture. After 3 or 4 minutes in the hypotonic medium, cells arrested in metaphase become dislodged from the glass (Axelrad and McCulloch, 1958). They are then concentrated in the center by very gently swirling the medium. The cells remain in good condition in the hypotonic medium at room temperature for about 1 hour. While the Petri dish is being observed under a dissecting microscope, several hundred to several thousand metaphase cells in about 0.5 p1 of medium are drawn into the tip of a braking pipette (see Stone and Cameron, this volume, Chapter 8, for description of braking pipettes) controlled by mouth through a rubber tube. The tip of the pipette is dipped into a reservoir of glacial acetic acid, and an equal volume of the acid is drawn up. After 5 to 10 seconds the entire contents are expelled onto a clean microscope slide over which a drop of 3:l (100% alcoho1:acetic acid) fixative has been allowed to spread a few seconds earlier. [We have not found it necessary to use “subbed” slides for autoradiography when using either NTB, NTB-2, or NTB-3 (Kodak) liquid emulsion.] After a few seconds the drop of acetic acid and medium spreads, leaving behind individual, isolated chromosomes and well-spread, partial, and complete complements of chromosomes uncontaminated by cytoplasm. Just as the chromosomes are about to dry, they are rinsed with another drop of the 3:l fixative. The fixative accumulates along the edges of the slide and should be wiped off with gauze to prevent reflowing of the fixative over the chromosomes. The slides are air-dried and, at this point, are ready for emulsion-coating.
19. CHROMOSOME
PREPARATION FOR AUTORADIOGRAPHY
383
111. Examples and Variation of Technique A set of chromosomes from a third metaphase after labeling with thymidine-H3 is shown in Fig. 1. The radioactivity is restricted to one
FIG.1. A complement of chromosomes from a Chinese hamster cell isolated at the third metaphase after incubation in thymidine-Ha. Autoradiographic resolution between chromatids is good. Evidence of sister chromatid exchanges during previous cell cycles are indicated by arrows. For a full discussion of the experiments, see Prescott and Bender (1963) and Marin and Prescott (1963).
FIG.2. A complement of chromosomes from a Chinese hamster cell isolated at the first metaphase after incubation in Ha-labeled amino acids.
384
D. M. PRESCOTT A N D M. A. BENDER
Fro. 3. A complement of chromosomes from a Chinese hamster cell isolated at the first metaphase after incubation in uridine-Ha. The uridine-Ha was present during the Gz period, and the radioactivity represents incorporation into RNA only.
chromatid in those chromosomes still containing radioactivity. Since the segregation of labeled DNA is semiconservative, some chromosomes are no longer labeled a t the third metaphase but a few sister chromatid exchanges have occurred (see Prescott and Bender, 1963). Treatment with the acetic acid takes out much of the protein and RNA from the chromosomes. This loss of material was greatly decreased by substituting a short fixation with neutral formalin for the glacial acetic acid treatment and disrupting the cells with 3:l fixative. The sequence of steps was modified as follows: Living cells are drawn up into the tip of a braking pipette in the usual 0.5 pliter of medium. An equal volume of 10% neutral formalin is drawn up and allowed to remain in the pipette for 60 seconds. Next about 1 pliter of 100% alcoho1:acetic acid fixative (3:l) is drawn into the pipette and the whole mixture immediately expelled onto a clean slide covered a few seconds previously with a drop of the alcohol :acetic acid fixative. Subsequent steps are as described for the acetic acid method. Complements of chromosomes in the first metaphase after labeling with H3-amino acids or uridine-H3 are shown in Figs. 2 and 3, respectively. For a brief description of these experiments see Prescott and Bender (1962b).
REFERENCES Axelrad, A. A., and McCulloch, E. A. (1958). Stain Technol. 33, 67. Marin, G., and Prescott, D. M. (1963). J . Cell Biol., in press. Prescott, 1). M., and Bender, M. A. (1961). Exptl. Cell Res. 25, 222.
19.
CHROMOSOME PREPARATION FOR AUTORADIOGRAPHY
385
Prescott, D. M., and Bender, M. A. (1962a). Exptl. Cell Res. 26, 260. Prescott, D. M., and Bender, M. A. (1962b). Abstr. 2nd Ann. Meeting Am. SOC.Cell Eiol., San Francisco p. 146. Preecott, D. M., and Bender, M. A. (1963). Exptl. Cell Res. 29,430.
AND
M. A. BENDER
Biology Division, Oak Ridgu National Oak Ridgs, Tmnusses
I. Introduction . . . . . . . . 11. Procedure. . . . . . . . . 111. Examples and Variation of Technique References . . . . . . . .
. . . . . . . . . . . 381 . . . . . . . . . . . 382 . . . . . . . . . . . 383
. . . . . . . . . . .
384
I. Introduction I n autoradiographic studies on labeling of deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , and proteins of mammalian chromosomes with tritiated precursors, we devised a procedure for preparing cytoplasm-free metaphase chromosomes. The method has the following advantages: (1) several hundred to several thousand sets of chromosomes can be affixed to a single slide, (2) the chromatids are well separated and autoradiographic resolution of label between sister chromatids is excellent, (3) no cytoplasmic layer is present between the chromosomes and the autoradiographic emulsion, thereby increasing the sensitivity and resolution of the autoradiographic detection of incorporated label, and (4) autoradiographic determination of labeled RNA and protein in chromosomes is possible because of the absence of “background1’ radioactivity from nonchromosomal RNA and protein. The method has been described in principle in a previous paper (Prescott and Bender, 1961);several improve&Operatedby Union Carbide Corporation for the United States Atomic Energy Commission. 381
382
I). M. PRESCOTT AND M. A. BENDER
ments have been added in the account below, and a few illustrations of autoradiographic results with chromosomes are shown in three accompanying figures. The method has been used mainly with the Chinese hamster “fibroblast”-line CHEF 125 (Prescott and Eender, 1962a), but it works equally well with several other mammalian cell types.
11. Procedure The cells are cultured in 55 mm Petri dishes, and colchicine is added to M ) several hours before isolation of chromosomes. the culture (to Just before the isolation, the medium is decanted and the cells washed once with balanced salt solution. The solution is then replaced by hypotonic saline (10 to 15% of full-strength, phosphate-buffered Hank’s balanced salt solution) at room temperature. An important part of these steps is the complete removal of serum proteins from the culture. After 3 or 4 minutes in the hypotonic medium, cells arrested in metaphase become dislodged from the glass (Axelrad and McCulloch, 1958). They are then concentrated in the center by very gently swirling the medium. The cells remain in good condition in the hypotonic medium at room temperature for about 1 hour. While the Petri dish is being observed under a dissecting microscope, several hundred to several thousand metaphase cells in about 0.5 p1 of medium are drawn into the tip of a braking pipette (see Stone and Cameron, this volume, Chapter 8, for description of braking pipettes) controlled by mouth through a rubber tube. The tip of the pipette is dipped into a reservoir of glacial acetic acid, and an equal volume of the acid is drawn up. After 5 to 10 seconds the entire contents are expelled onto a clean microscope slide over which a drop of 3:l (100% alcoho1:acetic acid) fixative has been allowed to spread a few seconds earlier. [We have not found it necessary to use “subbed” slides for autoradiography when using either NTB, NTB-2, or NTB-3 (Kodak) liquid emulsion.] After a few seconds the drop of acetic acid and medium spreads, leaving behind individual, isolated chromosomes and well-spread, partial, and complete complements of chromosomes uncontaminated by cytoplasm. Just as the chromosomes are about to dry, they are rinsed with another drop of the 3:l fixative. The fixative accumulates along the edges of the slide and should be wiped off with gauze to prevent reflowing of the fixative over the chromosomes. The slides are air-dried and, at this point, are ready for emulsion-coating.
19. CHROMOSOME
PREPARATION FOR AUTORADIOGRAPHY
383
111. Examples and Variation of Technique A set of chromosomes from a third metaphase after labeling with thymidine-H3 is shown in Fig. 1. The radioactivity is restricted to one
FIG.1. A complement of chromosomes from a Chinese hamster cell isolated at the third metaphase after incubation in thymidine-Ha. Autoradiographic resolution between chromatids is good. Evidence of sister chromatid exchanges during previous cell cycles are indicated by arrows. For a full discussion of the experiments, see Prescott and Bender (1963) and Marin and Prescott (1963).
FIG.2. A complement of chromosomes from a Chinese hamster cell isolated at the first metaphase after incubation in Ha-labeled amino acids.
384
D. M. PRESCOTT A N D M. A. BENDER
Fro. 3. A complement of chromosomes from a Chinese hamster cell isolated at the first metaphase after incubation in uridine-Ha. The uridine-Ha was present during the Gz period, and the radioactivity represents incorporation into RNA only.
chromatid in those chromosomes still containing radioactivity. Since the segregation of labeled DNA is semiconservative, some chromosomes are no longer labeled a t the third metaphase but a few sister chromatid exchanges have occurred (see Prescott and Bender, 1963). Treatment with the acetic acid takes out much of the protein and RNA from the chromosomes. This loss of material was greatly decreased by substituting a short fixation with neutral formalin for the glacial acetic acid treatment and disrupting the cells with 3:l fixative. The sequence of steps was modified as follows: Living cells are drawn up into the tip of a braking pipette in the usual 0.5 pliter of medium. An equal volume of 10% neutral formalin is drawn up and allowed to remain in the pipette for 60 seconds. Next about 1 pliter of 100% alcoho1:acetic acid fixative (3:l) is drawn into the pipette and the whole mixture immediately expelled onto a clean slide covered a few seconds previously with a drop of the alcohol :acetic acid fixative. Subsequent steps are as described for the acetic acid method. Complements of chromosomes in the first metaphase after labeling with H3-amino acids or uridine-H3 are shown in Figs. 2 and 3, respectively. For a brief description of these experiments see Prescott and Bender (1962b).
REFERENCES Axelrad, A. A., and McCulloch, E. A. (1958). Stain Technol. 33, 67. Marin, G., and Prescott, D. M. (1963). J . Cell Biol., in press. Prescott, 1). M., and Bender, M. A. (1961). Exptl. Cell Res. 25, 222.
19.
CHROMOSOME PREPARATION FOR AUTORADIOGRAPHY
385
Prescott, D. M., and Bender, M. A. (1962a). Exptl. Cell Res. 26, 260. Prescott, D. M., and Bender, M. A. (1962b). Abstr. 2nd Ann. Meeting Am. SOC.Cell Eiol., San Francisco p. 146. Preecott, D. M., and Bender, M. A. (1963). Exptl. Cell Res. 29,430.