10.1101/SQB.1974.038.01.037Access the most recent version at doi: 1974 38: 341-345Cold Spring Harb Symp Quant Biol

 C. V. Hanson and J. E. Hearst 

Drosophila melanogasterCell Line of Bulk Isolation of Metaphase Chromosomes from an In Vitro  

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Bulk Isolation of Metaphase Chromosomes from an In Vitro Cell Line of Drosophila melanogaster

C. V. HANSON AND J. E. HEARST Department of Chemistry, University of California, Berkeley, California 94720

Bulk isolations of metaphase chromosomes have been possible for a decade (see, for example, Chorazy et al., 1963; Cantor and Hearst, 1966; Huberman and Attardi, 1966; Maio and Schildkraut, 1967; Wray et al., 1972). These isolation procedures vary in conditions from pH----3.0 to pH 10.5. Most are accomplished in the presence of milli- molar quantities of Mg++ or Ca ++ or both. Although detergents are sometimes used, they are not re- quired in the majority of reports in the literature. As yet, no procedure has been developed which is successful with all cell sources. The recently de- veloped alkaline conditions of Wray et al. (1972) appear to be superior in inhibiting nuelease activity and therefore in yielding high molecular weight DNA in the isolated chromosomes.

The utility of isolated chromosomes has not been great up to now because the separation of individual chromosome fractions has not been possible and chromosomes have generally been isolated from organisms for which classical genetic information, including point mutations, genetic maps, and transloeation mutants, is not available in detail.

Drosophila melanogaster provides a solution to all of these limitations. Its haploid chromosome number is four. The chromosomes are different enough in size and shape and low enough in number so that separation of chromosomes should be possible. The available genetics and mutants make possible many detailed studies relating DNA struc- ture to the genetic map.

Figure I. Living D. melanogaster cells (Schneider's line no. 3) growing attached to a plastic substrate and visualized by phase-contrast microscopy. The cells are approximately 10 microns in diameter. The small pair of cells without dis- tinguishable nuclei are just completing division.

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342 HANSON AND HEARST

Figure 2. Typical heteroploid D. melanogaster cells (Schneider's line no. 3) grown in suspension and arrested by vin- blastine s u l f a t e as desoribed in the text , The cells were centrifuged onto a cover slip through a hypotonie medium which strips away cytoplasm and spreads chromosomes. The specimen was then fixed with Carnoy's fluid and stained with Giemsa. The average chromosome length is approximately 2 microns.

Drosophila melanogaster Tissue Culture

A cultured cell line of Oregon R strain D. melano. gaster was used for all experiments described below. The established line was the generous gift of Dr. Imogene Schneider (Schneider, 1972). They were grown at 25~ on Schneider's Drosophila medium purchased from the Grand Island Biological Com- pany (GIBCO) and supplemented with 15 ~o heat- denatured fetal calf serum. Cells of Schneider's line #3, Figure 1, were adapted to growth without the vitamin supplement used in the primary culture, and the most rapid cell division was accomplished by addition to the medium of one percent Bacto- peptone rather than the customary one-half percent.

To obtain cells for metaphase arrest and chromo- some isolation, cells shaken from surface cultures were grown in suspension by inoculation of a mini- mum of 5 • 105 cells/ml in 50 to 180 ml of medium per glass bottle. Bottles were new, 500-ml, cylindri- cal screw-cap bottles purchased from GIBCO, and, after inoculation, were constantly rotated horizon- tally on motor-driven rollers at 30 rpm in a 25~ incubator. Logarithmic growth was routinely ob- tained from 5 x 105 to 8 • 10 e cells/ml with a doubling time varying in different cultures between

16 and 24 hr. Some cultures attained densities of 1.5 to 2.0 • 107 cells/ml in stationary phase.

Figures 2 and 3 show the typical results of meta- phase arrest of the suspension cultures. At least 50~o, and as high as 75~o, arrest was routinely achieved by addition of vinblastine sulfate (Velban, Eli Lilly Co.) to a final concentration of 2 to 4/~g/ml to the suspension cultures. The drug was inoculated into a rapidly dividing culture in two equal doses, one-half generation time apart, with harvest oc- curring one generation time after the initial dose.

Despite the wide variety of conditions success- fully employed in the isolation of mammalian chromosomes, none of the existing procedures have proven successful for D. melanogaster. The co- hesiveness of arrested metaphase chromosome spreads in D. melanogaster greatly exceeds that of other cells. Shears sufficient to disperse these spreads under most conditions also damaged chromosome structure.

The two essential features of the successful iso- lation involved a hypotonie swelling of the arrested cells in the presence of 0.05 to 0.10 M sucrose and a precisely controlled pH at 10.0.

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Figure 3. The metaphase spread of a single, line-3 cell prepared as in Figure 2. This typical diploid karyotype contains two pairs of large autosomes, two X chromosomes, a single number 4 autosome, and a neo-chromosome originating during culturing of the cell line. Fluorescent staining indicates that the neo-chromosome does not contain the missing chromo- some 4.

F igure 4. D. mdanogaster chromosomes isolated as described in the text. The homogenate has been purified only by separation from interphase nuclei by means of selective filtration. Large and small autosomes and sex chromosomes are visually distinguishable in the isolate.

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344 HANSON AND HEARST

Figure 5. Highly magnified micrograph of several dispersed isolated chromosomes prepared as in Figure 4. Two large autosomes and one X chromosome are readily distinguishable, along with the likelihood that chromosome 4 is represented by one or more of the small dot-shaped bodies.

Metaphase Chromosome Isolation The chromosome isolation buffer contained 0.05 sucrose, 0.33 M 2-methyl, 2,4 pentanediol (hexy-

lene glycol), 0.0013 M CaClu, and 0.001 M cyclo- hexylamino propane sulfonic acid ("CAPS," Cal- biochem) t i t rated to pH 10.0 by addition of solid Ca(OH)2. The pH was adjusted prior to addition of hexylene glycol, and the buffer was used within one day (Wray et al., 1972).

The cells were washed twice at room temperature in 3-4 volumes of buffer. The cell pellet after the wash was resuspended in a one-half volume of buffer, cooled to 0~ and an equal volume of cold buffer containing 1 .5~ Nonidet P-40 (Shell Oil Ltd., London) was added. Immediate mechanical homogenization was accomplished at 0~ with 20 to 30 strokes in a cold, siliconized glass Dounce homogenizer fitted with a size " B " (tight) pestle.

The contaminating interphase nuclei were re. moved from homogenates by several passages through a 3-micron Nuclepore filter (General Electric Co.). A single filter can accommodate the homogenate from approximately l0 s cells. Figures 4 and 5 show the resulting chromosome preparation after centrifugal pelleting at 3000 rpm for 30 rain.

Applications for Isolated D. melanogaster Metaphase Chromosomes

While the availability of this unfractionated ma- terial will contribute to the comparative biochem- istry of eukaryotie chromosomes, this isolation is more importantly motivated by the many applica- tions for pure single-chromosome fractions. There is ample precedent for expecting the separation of some or all of the chromosomes from such a simple karyotype, either by differential velocity sedi- mentation or by more novel means, ranging from selective filtration to countercurrent distribution. Once separate chromosome fractions are obtained, one may explore, by direct extraction, the possible existence of categories of protein or nucleotide sequences which are specific to the different chromo- somes. A study of interehromosomal nucleotide sequence homologies in general, and of highly re- i terated sequences in particular, should shed light on current models for control of gene expression. The var ie tyof heterochromatin content of Drosphila chromosomes might also facilitate a physical study of heterochromatization utilizing the separate chromosomes. Finally, by exploiting available translocation mutations, the isolation of a specific

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D. M E L A 2 i O G A S T E R C H R O M O S O M E S 345

subehromosomal segment of a eukaryot ic genome m a y u l t ima te ly be possible.

Acknowledgments

This research was suppor ted in par t by a D a m o n R u n y o n Pos tdoc tora l Fel lowship and by U.S. Publ ic Hea l th Service Gran t GM-11180 and GM- 15661.

References

CANTOR, K. P. and J. E. HEARST. 1966. Isolation and partial characterization of metaphase chromosomes of a mouse ascites tumor. Proc. Nat. Acad. Sci. 55: 642.

CHORAZY, M., A. BENDICH, E. BORENFREUND, and D. J . HVTcHr~soN. 1963. Studies on the isolation of meta- phase chromosomes. J . Ge//. B/o/. 19: 59.

HUBERMAN, J. and G. ATTARDI. 1966. Isolation of mete- phase chromosomes }'rom Hela cells. J . Cell. B/ol. 31 �9 95.

MAIO, J. J. and C. L. SCHILDKRAUT. 1967. Isolated mam- malian metaphasc chromosomes. I. General character- istics of nucleic acids and proteins. J. Mol. B/o/. 24: 29.

SCHNEIDER, I. 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morph. 27: 353.

WRAY, W., E. STUBBLEFIELD, and R. HUMPHREY. 1972. Mammalian metaphase chromosomes with high mo- lecular weight DNA isolated at pH 10.5. Nature New Biol. 238: 237.

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