- Email: [email protected]
Separation of large quantities of chromosomes by velocity sedimentation at unit gravity
Cell Researcll
Experitnenlnl
SEPARATION
OF LARGE
VELOCITY J. G. COLLARD.’
126 (1980) 191-197
QUANTlTIES
SEDIMENTATION A.
TULP,’
J. H.
OF CHROMOSOMES AT
HOLLANDER,’
UNIT
F. W.
BAUER’
BY
GRAVITY and
.I. BOEZEMAN’
SUMMARY A chromosome sorting technique is described based on velocity sedimentation at unit gravity which combines simplicity with good resolution. Large quantities of chromosomes (up to IO’“) can be sorted for further biological and biochemical research. Male Indian muntjac cells were first synchronized and subsequently accumulated in mitosis with vinblastine. After hypotonic treatment, mitotic cells were disrupted and the crude chromosomal preparation was layered as a thin film onto a two-step linear sucrose gradient in a specially designed sedimentation chamber (area 127.6 cm’). After 18 h of sedimentation at unit gravity, fractions were collected and the chromosomal composition of the fractions was analyzed by flow cytometry and microscopy.
Separation of large quantities of isolated metaphase chromosomes in different subclasses opens interesting possibilities for physicochemical studies on nucleic acids, for chromosome-mediated gene transfer studies and for localization of virus integration in the genome of cells. In the past, attempts to fractionate isolated metaphase chromosomes have relied upon one or more biochemical or physical parameters such as chromosomal size. density, configuration, electronic charge and DNA content. Most of the separation techniques used are based on swinging bucket centrifugation [ 1, 21 or zonal rotor centrifugation [3-51 on sucrose gradients in which case only zonal rotor centrifugation gives reasonable results. Almost perfect purification of chromosomes has been achieved by fluorescence-activated flow sorting based on the desoxyribonucleic acid (DNA) content of the chromosomes [6. 71. However, this method has the disadvantage that only a limited number of chromosomes can be 13-79IXIX
sorted in reasonable time whereas in addition the expenses of the sorter are extremely high. In this paper we report a chromosome sorting technique based on velocity sedimentation at unit gravity in a specially designed sedimentation chamber. Velocity sedimentation at unit gravity has already been successfully used to separate cells [8,9] and nuclei [lo] that differ in size. Now we used this technique to sort chromosomes of male Indian muntjac cells based on their differences in size. The advantage of this approach is that large quantities of chromosomes can be sorted which are required for biochemical research. The method is simple and easy and results in chromosome fractions of relatively high purity. MATERIALS /solution Indian
mrrnfjd)
AND
METHODS
of tne~aphcise ckrotnosotnes
muntjac were
cells grown
(derived in 75
from a male Mllnritrcus cm? flasks in Dulbecco’s
192
Colltrrlt
et
lil.
Fix. _7. Set (mithramycin
Fig. 1. Diagram of the sedimentation chamber. Hatched, flow deflecting rhomb; small dots, chromosomes “sandwiched” between gradient and overlay liquid; dashed line interphase between gradient and cushion liquid. A, Inlet part: B, cylindrical tube: C, top part: i. inlet; 0. outlet: m. meniscus.
modified Eagle’s medium supplemented with 204 fetal calf serum (FCS) and antibiotics in a humidified CO, incubator at 37°C. Cell cultures were first synchronized with thymidine as described earlier [I I]. whereas the degree of synchronization was checked by pulsecytometry [II]. Briefly, cells were grown to confluency and after that seeded again in a three times lower cell concentration in the presence of excess thymidine (7 mM). After 20 h, the cultures were washed with prewarmed regular medium and the cells were allowed to pass the S and G2 phase of the cell cycle. When the synchronized wave of cells reached mitosis, vinblastine (0. I pg/ml) was added and the cells were accumulated in mitosis during 4 h. In this way a reasonable number of mitotic cells (approx. 40-50s) per culture were obtained, whereas the difference in chromosomal contraction due to variations in exposure to vinblastine was minimalized. Mitotic cells (approx. 50-100~ 10’9 were harvested by shake-off. washed 2 times with phosphate buffered saline (PBS) and resuspended in KCI (0.075 M). After hypotonic treatment in KCI during 30 min at 25”C, cells were resuspended in cold 2 ‘% citric acid plus I mM CaCI,. I mM MgCl? and 0.1 M sucrose (approx. IO’ cells/ ml). Cell disruption was accomplished by shearing the cell suspension twice through a 22 gauge needle at 4 “C. Cell disruption was checked microscopically. The crude chromosome preparation was layered onto the gradient without further purification.
Sedimentation
at unit gruvit!
A diagram of the sedimentation chamber is shown in fig. I. The chamber consists of three parts made of perspex, viz. an inlet part; a cylindrical tube with a diameter of 12.7 cm and a height of 5 cm: a top part, L-\/I
Cd/
Ret
126 , IYh’llj
of
chromosome\ stained). ~750.
~)f the
Indian
muntjac
which can be removed t’or cleaning purpose,. The top part consists of a cylindrical cone into which a ~rotational-symmetric) flow deflector i\ glued tsee the hatched rhomb in fig. I) to three small perspex pods. The distance between Bow deflector and wall of the top cone is 2 mm. The chamber is filled with l5? sucrose via the inlet. where glass beads I@ I cm) are present to prevent turbulence. The outlet is connected to a gradient mixer. A linear sucrose gradient ranging from l5-7.5c”r sucrose is introduced into the chamber by letting cushion liquid drip out from i: 200 ml can be administered within 30 min. Next. a steep gradient t7.5-5cJ < sucrose) of 20 ml is introduced into the chamber within 5 min. 4 disposable open syringe is connected to the outlet with a small silicon tube and the sample (5-10 ml, containing crude chromosome preparation plus 0.1 M sucrose) is pipetted into the syringe tube. The sample is introduced into the chamber within I min followed by an overlay of citric acid (25 ml). Next, the meniscus is lowered until the band of chromosomes has reached the cylindrical part of the device (surface 126.7 cm’). An extremely thin film of undisturbed chromosome material is obtained with a thickness of 0.U.8 mm only. Chromosomes are then allowed to sediment at unit gravity during 18 h at 4°C. After this cushion liquid is introduced via the inlet and the content of the chamber is fractionated manually via the outlet within 3 min. Fractions I5 ml) were analyzed by flow cytometry. Specification of gradient layers: Overlay, 29 citric acid t2 mm thick): chromosomes in 3.4cC sucrose (0.5 mm thick): linear sucrose gradient 5-7.5 “r ( I .6 mm thick): linear sucrose gradient 7.5-154 (12.7 mm thick,: cushion 115~; sucrose. Gradient solutions contained citric acid (2%). and I mM CaCI, and I mM MgCI?.
Flop* cytometry
crnd rnicroscop~
The chromosomal composition of the fractions was analyzed with a Phywe pulse cytophotometer (ICP I I. equipped with a sheath flow cell) after staining the chromosomes with a mixture of mithramycin and ethidium bromide. The chromosomes of the 5 ml fractions were centrifuged (3 000 rpm: 30 mitt) or allowed to sediment to the bottom of the tube for at least 2 weeks at I g at 4°C. After removing the supematant. chromosomes were resuspended in a 0.01 M Tris buffer pH 7.4. containing 0.01 M NaCI: 0.01 M MgCI,: I2 mg/l mithramycin and 5 mg/l ethidium bromide and analyzed by flow cytometry. The relative number of chromosomes in the fractions was determined by
Chromosome
Fin. 3. Abscissa: Channel no .x=rel. amount of DNA/ chromosome; or¬e: distribution,As), no. of chromosome/channel (X 109. Flow cytometric measurement of unfractionated muntjac chromosomes. For explanation see text. measuring the number of chromosomes in a small volume of a capillar connected to the flow cytometer. To visualize the chromosomes sorted in the different fractions, samples were collected on nucleopore filters (0.4 pm), stained with mithramycin and studied by fluorescence microscopy. Permanent slides were made by adding a drop of the chromosome fraction on a microscopic slide. After drying and fixation with methanol : acetic acid (3 : I), the slides were washed in aqua dest, stained with Giemsa. dried and mounted.
RESULTS
AND
DISCUSSION
The set of chromsomes of male Indian muntjac are shown in fig. 2. The cells contain two autosomes no. 1; two autosomes no. 2; one autosome no. 3; one X+3 composite chromosome and the Y chromosome. The X+3 chromosome is composed of the X chromosome (short arm) and autosome no. 3 (long arm). The two arms are joined by a long neck region of constitutive heterochromatin [7]. Flow cytometric measurements of isolated unfractionated muntjac chromosomes show that the individual chromosomes appear in different peaks according to their different DNA content (fig. 3). The autosomes no. 1, the X+3 chromosomes, the autosomes no. 2 and the chromosomes no. 3 appear around
sorting
at 1 g
193
channel nos 94, 65, 59 and 46, respectively. as indicated in fig. 3. The small peaks around channel nos 30 and 22 represent pieces of the relatively fragile X+3 chromosomes and represent, respectively, the X chromosomes plus the long neck region of heterochromatin (fig. 3, X+) and the X chromosomes only (fig. 3, X). The Y chromosomes have been lost in the background of the measurement (fig. 3, Ytb). The shape of the flow cytometric graph and the relative difference in DNA content between the individual chromosomes are in good agreement with the results of Carrano et al. [7]. Fig. 4 shows the chromosome composition of the different fractions obtained after sorting at 1 g. Note that the graphs only give information on the relative chromosome composition of the fractions and not on the absolute number of chromosomes in the fraction. When compared with the unfractionated chromosomes [I], the first fractions 12 and 14 contain mainly fragments of the X+3 chromosomes and presumably the Y chromosomes in the background of the measurement. Going to higher fraction numbers, an increase in the percentage of larger chromosomes can be observed, whereas at the same time the smaller chromosomes disappear. For example, fraction 24 contains mainly the autosomes no. 2 and the X+3 chromosomes, whereas fractions 30 and 32 consists mainly of the large autosomes no. 1. The enrichment of different chromosomes in the fractions can easily be calculated by measuring the surface area of the different peaks in the cytofluorometric measurements of the fractions. Because of their large size and distinct difference in morphology, muntjac chromosomes are easy to identify in the microscope. Microscopic analysis of the sorted chromosomes in the different fractions fully agrees with the flow cytometric measure-
40
80
120
40
80
12
120
40
80
120
40
80
120
40
80
120
20
i-
40
80
170
22
I L 4P
40
80
120
40
80
120
16
80
120
Fig. 1. A6sc~isstrt channel nos .r=rel. amount of DNA/ chromosome; ortiinoret distribution .fi.rj. rel. no. of chromosomes/channel. Flow cytometric graphs of muntjac chromosomes fractionated at 1 g. No. I repre-
sents the unfractionated chromosome>: nob. 12-32 the fraction numbers. Note that the graphs represent the rel. chromosomal composition of the fractions and not the absolute number of chromosomes in the fractions.
merits as shown in fig. 5. No clumps of chromosomes were seen. The chromosomes in fig. 5 correspond with the flowcytometric measurements of fraction 16,22. 26 and 30. respectively. For example fig. 5~ shows the high enrichment of the X+3 chromosomes in fraction 26, whereas fig. Stl
shows that virtually only the large autosomes number 1 are present in fraction 30. As mentioned in Materials and Methods the crude chromosomal preparation was layered onto the gradient. Further purification is not necessary since cell debris remain on top of the gradient whereas non-
Cllromosonw
sorting iit 1 g
195
Fig. 5. Chromosomes in fractions 16 3 ‘--3 7 36 - and 310, respectively (see fig. 4): mithramycin stained. x400.
disrupted cells. nuclei and clumps of chromosomes sediment rapidly into the cushion fluid on the bottom of the gradient. According to our previous experience with the separation of intact cells and nuclei, recoveries of 90% and more are routineously realized in the I g sedimentation method [9, lo]. It is expected that the same holds true for isolated single chromosomes. Based on the crude muntjac chromosome preparation which contains generally approx. 70-80% of the chromosomes in single form one can calculate that the recovery will be around 60-70 %. Fig. 6 shows a three-dimensional plot of the sorted muntjac chromosomes at 1 g. In fig. 6 the relative chromosome compositions per fraction are scaled on unit area and multiplied by the estimated number of chromosomes per fraction. In fact this
figure gives the total distribution of all the different chromosomes in the gradient and the correct information to decide which fractions can be used for further biochemical studies. Velocity sedimentation at unit gravity has already proved to be quite useful for the separation of different kinds of lymphoid cells [8], for separation of cells according to the phases of the cell cycle [9] and for separation of nuclei according to ploidy [lo]. The above described results demonstrate that this technique is also suitable for the separation of chromosomes according to size. In order to avoid differences in contraction of the isolated chromosomes due to the variations in exposure to vinblastine. cells were first synchronized. Otherwise large but strongly contracted chromosomes would sediment together with smaller less
20
40
60
80
100
120
i.
Kg. 6. (A) Two-dimensional distribution ,/Lr. y) of chromosomes in channel .Y and fraction .v. The distribution is composed of the multiplications of the conditional distributions f(.r 1.v) (the separate histograms scaled on unit area). andj(y) (the estimated number of chromosomes in each fraction). (B) Marginal distribution f; ji.\. 1~). the summation of the c&ditional distribution of the different fractions proiected in one plane (cf C). (C) One-dimensional distiibution ,Q). the histogram of the unfractionated sample.
contracted chromosomes. This does hardly occur in our experiments since fractions with a low fraction number are free from large chromosomes. However. some overlap of different chromosomes in the fractions can be seen, probably due to still minor differences in contraction between chromosomes. as could be seen in the fluorescence microscope. In addition, the marginal difference in size between the individual chromosomes plays a role. For example, the difference in size (and DNA content) between the X+3 chromosomes and the autosomes no. 2 is apparently too small to allow a clear-cut separation of these chromosomes (fig. 6). In the experiments described above. citric acid was used for isolation and fractionation of the muntjac chromosomes,
mainly because chromosomal htructure ib better preserved at low pH. However. if chromosomes, isolated and fractionated at neutral pH, are required the same procedure can be followed with buffers at neutral pH. In our hands the resolution of the separation at neutral pH was somewhat lower when compared with the separation in citric acid, probably due to changes in chromosomal morphology during the sedimentation. In the experiments described here, approx. 5x IO” chromosomes were applied to the sedimentation chamber. Larger quantities can easily be separated in sedimentation devices having appropriate dimensions. In fact. we did already 1 g sorting experiments with approu. IO”’ rat chromosomes in a chamber with an effective surface of 1255 cm”. Velocity sedimentation at unit gravity sorts muntjac chromosomes with a relatively high purity. Preliminary results with Chinese hamster chromosomes indicate that with these chromosomes also a considerable enrichment of the individual chromosomes in the different fractions could be obtained [I?, 131. At present. the resolving power of 1 g sorting of human. rat and mouse chromosomes is under investigation. For most biochemical studies. fractions highly enriched in particular chromosome will be sufficient. If, however, pure chromosome fractions are required, I g sedimentation followed by flow sorting can be done. Chromosomes of a single type can be sorted at a conservative rate of 300 chromosomeslsec [7]. This enables the collection of IO” purified chromosomes in about 1.5 h. Sorting at 1 g usually results in a 5-10 times enrichment of a particular chromosome in a certain fraction. If these pre-enriched fractions are used, the sorting speed of individual chromosomes can be increased by a factor 5-10.
Chromosome The advantage of the present sedimentation device are its low costs and easy technical manipulation. Others may prefer zonal rotor separation with its inherent rapidity. However, we have found previously [14] that separation of diploid stromal from diploid parenchymal nuclei is much better using the unit gravity technique then that achieved by zonal rotor separation [1.5]. The same may be true for the separation of chromosomes. Preliminary experiments with Chinese hamster chromosomes separated at unit gravity show that the resolving power is better than that reported before [4], using zonal rotor centrifugation. However, this can also be due to the difference in chromosome preparation technique which influences the resolving power of both techniques. Thanks are due to Drs J. B. A. Kipp and J. A. Aten for the flow cytometric measurements in the beginning of these experiments and to Mr C. Murre and Mrs M. Bamhoom for expert technical assistance.
REFERENCES I. Mendelsohn. J. Moore. D E & Salzman, N P, J mol bio132 (1968) 101.
Printed
in Sweden
sorting nt 1 g
197
Stubblefield, E & Wray, W, Cold Spring Harbor symp quant biol38 (1974) 835. 3. Burki, H J, Regimbal, T J & Mel. H C, Prep biothem 3 (1973) 157. 4. Stubblefield, E, Cram, S & Deaven, L. Exp cell 94 (1975) 464. 5. Benz, R D & Burki. H J. Exp cell res II2 (1978) 143. 6. Gray. J W, Carrano, A V, Steinmetz. L L. Van Dilla, M A, Moore II. D H. Mayall. B H & Mendelsohn. M L, Proc natl acad sci US 72 (1975) 1231. 7 Carrano. A V. Grav. J W. Moore II. D H, Minkler, J L. Mayall. B H, Van Dilla, M A & Mendelsohn, M L, J histochem cytochem 24 (1976) 348. 8. Bont, W S. de Vries, J E, Geel. M, van Dongen, A & Loos. H A, J immunol methods 29 (1979) I. 9. Tulp. A & Welagen, J J M N, Europ j cancer I2 (1976) 519. 10. Tulp, A. Welagen, J J M N & Emmelot, P. Biochim biophys acta 45 I ( 19761567. II. Collard, J G & Temmink. J H M, J cell biol 68 (1976) 101. 12. Collard, J G, Tulp, A, Murre, C, Kipp, J B A. Aten J A &z Bauer. F W. Acta pathol microbial Stand (1979). In press. 13. Bont, W, Gray, J W, Tulp, A. de Vries, J E & Cram. S. Submitted for nublication. 1-I. Tulp,’ A, Welagen, J J M N & Westra, J G. Chem-biol interactions 23 (1978) 293. 15. Johnston. I R, Mathias. A P,‘Pennington, F & Ridge, D. Nature 220 (1968) 668. 7 b.
Received August 14, 1979 Revised version received October 9. 1979 Accepted October I I, 1979
Es/? Cdl
Re\
126 (IWO,
Experitnenlnl
SEPARATION
OF LARGE
VELOCITY J. G. COLLARD.’
126 (1980) 191-197
QUANTlTIES
SEDIMENTATION A.
TULP,’
J. H.
OF CHROMOSOMES AT
HOLLANDER,’
UNIT
F. W.
BAUER’
BY
GRAVITY and
.I. BOEZEMAN’
SUMMARY A chromosome sorting technique is described based on velocity sedimentation at unit gravity which combines simplicity with good resolution. Large quantities of chromosomes (up to IO’“) can be sorted for further biological and biochemical research. Male Indian muntjac cells were first synchronized and subsequently accumulated in mitosis with vinblastine. After hypotonic treatment, mitotic cells were disrupted and the crude chromosomal preparation was layered as a thin film onto a two-step linear sucrose gradient in a specially designed sedimentation chamber (area 127.6 cm’). After 18 h of sedimentation at unit gravity, fractions were collected and the chromosomal composition of the fractions was analyzed by flow cytometry and microscopy.
Separation of large quantities of isolated metaphase chromosomes in different subclasses opens interesting possibilities for physicochemical studies on nucleic acids, for chromosome-mediated gene transfer studies and for localization of virus integration in the genome of cells. In the past, attempts to fractionate isolated metaphase chromosomes have relied upon one or more biochemical or physical parameters such as chromosomal size. density, configuration, electronic charge and DNA content. Most of the separation techniques used are based on swinging bucket centrifugation [ 1, 21 or zonal rotor centrifugation [3-51 on sucrose gradients in which case only zonal rotor centrifugation gives reasonable results. Almost perfect purification of chromosomes has been achieved by fluorescence-activated flow sorting based on the desoxyribonucleic acid (DNA) content of the chromosomes [6. 71. However, this method has the disadvantage that only a limited number of chromosomes can be 13-79IXIX
sorted in reasonable time whereas in addition the expenses of the sorter are extremely high. In this paper we report a chromosome sorting technique based on velocity sedimentation at unit gravity in a specially designed sedimentation chamber. Velocity sedimentation at unit gravity has already been successfully used to separate cells [8,9] and nuclei [lo] that differ in size. Now we used this technique to sort chromosomes of male Indian muntjac cells based on their differences in size. The advantage of this approach is that large quantities of chromosomes can be sorted which are required for biochemical research. The method is simple and easy and results in chromosome fractions of relatively high purity. MATERIALS /solution Indian
mrrnfjd)
AND
METHODS
of tne~aphcise ckrotnosotnes
muntjac were
cells grown
(derived in 75
from a male Mllnritrcus cm? flasks in Dulbecco’s
192
Colltrrlt
et
lil.
Fix. _7. Set (mithramycin
Fig. 1. Diagram of the sedimentation chamber. Hatched, flow deflecting rhomb; small dots, chromosomes “sandwiched” between gradient and overlay liquid; dashed line interphase between gradient and cushion liquid. A, Inlet part: B, cylindrical tube: C, top part: i. inlet; 0. outlet: m. meniscus.
modified Eagle’s medium supplemented with 204 fetal calf serum (FCS) and antibiotics in a humidified CO, incubator at 37°C. Cell cultures were first synchronized with thymidine as described earlier [I I]. whereas the degree of synchronization was checked by pulsecytometry [II]. Briefly, cells were grown to confluency and after that seeded again in a three times lower cell concentration in the presence of excess thymidine (7 mM). After 20 h, the cultures were washed with prewarmed regular medium and the cells were allowed to pass the S and G2 phase of the cell cycle. When the synchronized wave of cells reached mitosis, vinblastine (0. I pg/ml) was added and the cells were accumulated in mitosis during 4 h. In this way a reasonable number of mitotic cells (approx. 40-50s) per culture were obtained, whereas the difference in chromosomal contraction due to variations in exposure to vinblastine was minimalized. Mitotic cells (approx. 50-100~ 10’9 were harvested by shake-off. washed 2 times with phosphate buffered saline (PBS) and resuspended in KCI (0.075 M). After hypotonic treatment in KCI during 30 min at 25”C, cells were resuspended in cold 2 ‘% citric acid plus I mM CaCI,. I mM MgCl? and 0.1 M sucrose (approx. IO’ cells/ ml). Cell disruption was accomplished by shearing the cell suspension twice through a 22 gauge needle at 4 “C. Cell disruption was checked microscopically. The crude chromosome preparation was layered onto the gradient without further purification.
Sedimentation
at unit gruvit!
A diagram of the sedimentation chamber is shown in fig. I. The chamber consists of three parts made of perspex, viz. an inlet part; a cylindrical tube with a diameter of 12.7 cm and a height of 5 cm: a top part, L-\/I
Cd/
Ret
126 , IYh’llj
of
chromosome\ stained). ~750.
~)f the
Indian
muntjac
which can be removed t’or cleaning purpose,. The top part consists of a cylindrical cone into which a ~rotational-symmetric) flow deflector i\ glued tsee the hatched rhomb in fig. I) to three small perspex pods. The distance between Bow deflector and wall of the top cone is 2 mm. The chamber is filled with l5? sucrose via the inlet. where glass beads I@ I cm) are present to prevent turbulence. The outlet is connected to a gradient mixer. A linear sucrose gradient ranging from l5-7.5c”r sucrose is introduced into the chamber by letting cushion liquid drip out from i: 200 ml can be administered within 30 min. Next. a steep gradient t7.5-5cJ < sucrose) of 20 ml is introduced into the chamber within 5 min. 4 disposable open syringe is connected to the outlet with a small silicon tube and the sample (5-10 ml, containing crude chromosome preparation plus 0.1 M sucrose) is pipetted into the syringe tube. The sample is introduced into the chamber within I min followed by an overlay of citric acid (25 ml). Next, the meniscus is lowered until the band of chromosomes has reached the cylindrical part of the device (surface 126.7 cm’). An extremely thin film of undisturbed chromosome material is obtained with a thickness of 0.U.8 mm only. Chromosomes are then allowed to sediment at unit gravity during 18 h at 4°C. After this cushion liquid is introduced via the inlet and the content of the chamber is fractionated manually via the outlet within 3 min. Fractions I5 ml) were analyzed by flow cytometry. Specification of gradient layers: Overlay, 29 citric acid t2 mm thick): chromosomes in 3.4cC sucrose (0.5 mm thick): linear sucrose gradient 5-7.5 “r ( I .6 mm thick): linear sucrose gradient 7.5-154 (12.7 mm thick,: cushion 115~; sucrose. Gradient solutions contained citric acid (2%). and I mM CaCI, and I mM MgCI?.
Flop* cytometry
crnd rnicroscop~
The chromosomal composition of the fractions was analyzed with a Phywe pulse cytophotometer (ICP I I. equipped with a sheath flow cell) after staining the chromosomes with a mixture of mithramycin and ethidium bromide. The chromosomes of the 5 ml fractions were centrifuged (3 000 rpm: 30 mitt) or allowed to sediment to the bottom of the tube for at least 2 weeks at I g at 4°C. After removing the supematant. chromosomes were resuspended in a 0.01 M Tris buffer pH 7.4. containing 0.01 M NaCI: 0.01 M MgCI,: I2 mg/l mithramycin and 5 mg/l ethidium bromide and analyzed by flow cytometry. The relative number of chromosomes in the fractions was determined by
Chromosome
Fin. 3. Abscissa: Channel no .x=rel. amount of DNA/ chromosome; or¬e: distribution,As), no. of chromosome/channel (X 109. Flow cytometric measurement of unfractionated muntjac chromosomes. For explanation see text. measuring the number of chromosomes in a small volume of a capillar connected to the flow cytometer. To visualize the chromosomes sorted in the different fractions, samples were collected on nucleopore filters (0.4 pm), stained with mithramycin and studied by fluorescence microscopy. Permanent slides were made by adding a drop of the chromosome fraction on a microscopic slide. After drying and fixation with methanol : acetic acid (3 : I), the slides were washed in aqua dest, stained with Giemsa. dried and mounted.
RESULTS
AND
DISCUSSION
The set of chromsomes of male Indian muntjac are shown in fig. 2. The cells contain two autosomes no. 1; two autosomes no. 2; one autosome no. 3; one X+3 composite chromosome and the Y chromosome. The X+3 chromosome is composed of the X chromosome (short arm) and autosome no. 3 (long arm). The two arms are joined by a long neck region of constitutive heterochromatin [7]. Flow cytometric measurements of isolated unfractionated muntjac chromosomes show that the individual chromosomes appear in different peaks according to their different DNA content (fig. 3). The autosomes no. 1, the X+3 chromosomes, the autosomes no. 2 and the chromosomes no. 3 appear around
sorting
at 1 g
193
channel nos 94, 65, 59 and 46, respectively. as indicated in fig. 3. The small peaks around channel nos 30 and 22 represent pieces of the relatively fragile X+3 chromosomes and represent, respectively, the X chromosomes plus the long neck region of heterochromatin (fig. 3, X+) and the X chromosomes only (fig. 3, X). The Y chromosomes have been lost in the background of the measurement (fig. 3, Ytb). The shape of the flow cytometric graph and the relative difference in DNA content between the individual chromosomes are in good agreement with the results of Carrano et al. [7]. Fig. 4 shows the chromosome composition of the different fractions obtained after sorting at 1 g. Note that the graphs only give information on the relative chromosome composition of the fractions and not on the absolute number of chromosomes in the fraction. When compared with the unfractionated chromosomes [I], the first fractions 12 and 14 contain mainly fragments of the X+3 chromosomes and presumably the Y chromosomes in the background of the measurement. Going to higher fraction numbers, an increase in the percentage of larger chromosomes can be observed, whereas at the same time the smaller chromosomes disappear. For example, fraction 24 contains mainly the autosomes no. 2 and the X+3 chromosomes, whereas fractions 30 and 32 consists mainly of the large autosomes no. 1. The enrichment of different chromosomes in the fractions can easily be calculated by measuring the surface area of the different peaks in the cytofluorometric measurements of the fractions. Because of their large size and distinct difference in morphology, muntjac chromosomes are easy to identify in the microscope. Microscopic analysis of the sorted chromosomes in the different fractions fully agrees with the flow cytometric measure-
40
80
120
40
80
12
120
40
80
120
40
80
120
40
80
120
20
i-
40
80
170
22
I L 4P
40
80
120
40
80
120
16
80
120
Fig. 1. A6sc~isstrt channel nos .r=rel. amount of DNA/ chromosome; ortiinoret distribution .fi.rj. rel. no. of chromosomes/channel. Flow cytometric graphs of muntjac chromosomes fractionated at 1 g. No. I repre-
sents the unfractionated chromosome>: nob. 12-32 the fraction numbers. Note that the graphs represent the rel. chromosomal composition of the fractions and not the absolute number of chromosomes in the fractions.
merits as shown in fig. 5. No clumps of chromosomes were seen. The chromosomes in fig. 5 correspond with the flowcytometric measurements of fraction 16,22. 26 and 30. respectively. For example fig. 5~ shows the high enrichment of the X+3 chromosomes in fraction 26, whereas fig. Stl
shows that virtually only the large autosomes number 1 are present in fraction 30. As mentioned in Materials and Methods the crude chromosomal preparation was layered onto the gradient. Further purification is not necessary since cell debris remain on top of the gradient whereas non-
Cllromosonw
sorting iit 1 g
195
Fig. 5. Chromosomes in fractions 16 3 ‘--3 7 36 - and 310, respectively (see fig. 4): mithramycin stained. x400.
disrupted cells. nuclei and clumps of chromosomes sediment rapidly into the cushion fluid on the bottom of the gradient. According to our previous experience with the separation of intact cells and nuclei, recoveries of 90% and more are routineously realized in the I g sedimentation method [9, lo]. It is expected that the same holds true for isolated single chromosomes. Based on the crude muntjac chromosome preparation which contains generally approx. 70-80% of the chromosomes in single form one can calculate that the recovery will be around 60-70 %. Fig. 6 shows a three-dimensional plot of the sorted muntjac chromosomes at 1 g. In fig. 6 the relative chromosome compositions per fraction are scaled on unit area and multiplied by the estimated number of chromosomes per fraction. In fact this
figure gives the total distribution of all the different chromosomes in the gradient and the correct information to decide which fractions can be used for further biochemical studies. Velocity sedimentation at unit gravity has already proved to be quite useful for the separation of different kinds of lymphoid cells [8], for separation of cells according to the phases of the cell cycle [9] and for separation of nuclei according to ploidy [lo]. The above described results demonstrate that this technique is also suitable for the separation of chromosomes according to size. In order to avoid differences in contraction of the isolated chromosomes due to the variations in exposure to vinblastine. cells were first synchronized. Otherwise large but strongly contracted chromosomes would sediment together with smaller less
20
40
60
80
100
120
i.
Kg. 6. (A) Two-dimensional distribution ,/Lr. y) of chromosomes in channel .Y and fraction .v. The distribution is composed of the multiplications of the conditional distributions f(.r 1.v) (the separate histograms scaled on unit area). andj(y) (the estimated number of chromosomes in each fraction). (B) Marginal distribution f; ji.\. 1~). the summation of the c&ditional distribution of the different fractions proiected in one plane (cf C). (C) One-dimensional distiibution ,Q). the histogram of the unfractionated sample.
contracted chromosomes. This does hardly occur in our experiments since fractions with a low fraction number are free from large chromosomes. However. some overlap of different chromosomes in the fractions can be seen, probably due to still minor differences in contraction between chromosomes. as could be seen in the fluorescence microscope. In addition, the marginal difference in size between the individual chromosomes plays a role. For example, the difference in size (and DNA content) between the X+3 chromosomes and the autosomes no. 2 is apparently too small to allow a clear-cut separation of these chromosomes (fig. 6). In the experiments described above. citric acid was used for isolation and fractionation of the muntjac chromosomes,
mainly because chromosomal htructure ib better preserved at low pH. However. if chromosomes, isolated and fractionated at neutral pH, are required the same procedure can be followed with buffers at neutral pH. In our hands the resolution of the separation at neutral pH was somewhat lower when compared with the separation in citric acid, probably due to changes in chromosomal morphology during the sedimentation. In the experiments described here, approx. 5x IO” chromosomes were applied to the sedimentation chamber. Larger quantities can easily be separated in sedimentation devices having appropriate dimensions. In fact. we did already 1 g sorting experiments with approu. IO”’ rat chromosomes in a chamber with an effective surface of 1255 cm”. Velocity sedimentation at unit gravity sorts muntjac chromosomes with a relatively high purity. Preliminary results with Chinese hamster chromosomes indicate that with these chromosomes also a considerable enrichment of the individual chromosomes in the different fractions could be obtained [I?, 131. At present. the resolving power of 1 g sorting of human. rat and mouse chromosomes is under investigation. For most biochemical studies. fractions highly enriched in particular chromosome will be sufficient. If, however, pure chromosome fractions are required, I g sedimentation followed by flow sorting can be done. Chromosomes of a single type can be sorted at a conservative rate of 300 chromosomeslsec [7]. This enables the collection of IO” purified chromosomes in about 1.5 h. Sorting at 1 g usually results in a 5-10 times enrichment of a particular chromosome in a certain fraction. If these pre-enriched fractions are used, the sorting speed of individual chromosomes can be increased by a factor 5-10.
Chromosome The advantage of the present sedimentation device are its low costs and easy technical manipulation. Others may prefer zonal rotor separation with its inherent rapidity. However, we have found previously [14] that separation of diploid stromal from diploid parenchymal nuclei is much better using the unit gravity technique then that achieved by zonal rotor separation [1.5]. The same may be true for the separation of chromosomes. Preliminary experiments with Chinese hamster chromosomes separated at unit gravity show that the resolving power is better than that reported before [4], using zonal rotor centrifugation. However, this can also be due to the difference in chromosome preparation technique which influences the resolving power of both techniques. Thanks are due to Drs J. B. A. Kipp and J. A. Aten for the flow cytometric measurements in the beginning of these experiments and to Mr C. Murre and Mrs M. Bamhoom for expert technical assistance.
REFERENCES I. Mendelsohn. J. Moore. D E & Salzman, N P, J mol bio132 (1968) 101.
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Stubblefield, E & Wray, W, Cold Spring Harbor symp quant biol38 (1974) 835. 3. Burki, H J, Regimbal, T J & Mel. H C, Prep biothem 3 (1973) 157. 4. Stubblefield, E, Cram, S & Deaven, L. Exp cell 94 (1975) 464. 5. Benz, R D & Burki. H J. Exp cell res II2 (1978) 143. 6. Gray. J W, Carrano, A V, Steinmetz. L L. Van Dilla, M A, Moore II. D H. Mayall. B H & Mendelsohn. M L, Proc natl acad sci US 72 (1975) 1231. 7 Carrano. A V. Grav. J W. Moore II. D H, Minkler, J L. Mayall. B H, Van Dilla, M A & Mendelsohn, M L, J histochem cytochem 24 (1976) 348. 8. Bont, W S. de Vries, J E, Geel. M, van Dongen, A & Loos. H A, J immunol methods 29 (1979) I. 9. Tulp. A & Welagen, J J M N, Europ j cancer I2 (1976) 519. 10. Tulp, A. Welagen, J J M N & Emmelot, P. Biochim biophys acta 45 I ( 19761567. II. Collard, J G & Temmink. J H M, J cell biol 68 (1976) 101. 12. Collard, J G, Tulp, A, Murre, C, Kipp, J B A. Aten J A &z Bauer. F W. Acta pathol microbial Stand (1979). In press. 13. Bont, W, Gray, J W, Tulp, A. de Vries, J E & Cram. S. Submitted for nublication. 1-I. Tulp,’ A, Welagen, J J M N & Westra, J G. Chem-biol interactions 23 (1978) 293. 15. Johnston. I R, Mathias. A P,‘Pennington, F & Ridge, D. Nature 220 (1968) 668. 7 b.
Received August 14, 1979 Revised version received October 9. 1979 Accepted October I I, 1979
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